The purpose of this study was to construct expression vectors of idiotype (Id) SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24–25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.

In order to investigate the effects of aconitine on [Ca²⁺] oscillation patterns in cultured myocytes of neonatal rats, fluorescent Ca²⁺ indicator Fluo-4 NW and laser scanning confocal microscope (LSCM) were used to detect the real-time changes of [Ca²⁺] oscillation patterns in the cultured myocytes before and after aconitine (1.0 μmol/L) incubation or antiarrhythmic peptide (AAP) and aconitine co-incubation. The results showed under control conditions, [Ca²⁺] oscillations were irregular but relatively stable, occasionally accompanied by small calcium sparks. After incubation of the cultures with aconitine, high frequency [Ca²⁺] oscillations emerged in both nuclear and cytoplasmic regions, whereas typical calcium sparks disappeared and the average [Ca²⁺] in the cytoplasm of the cardiomyocyte did not change significantly. In AAP-treated cultures, intracellular [Ca²⁺] oscillation also changed, with periodic frequency, increased amplitudes and prolonged duration of calcium sparks. These patterns were not altered significantly by subsequent aconitine incubation. The basal value of [Ca²⁺] in nuclear region was higher than that in the cytoplasmic region. In the presence or absence of drugs, the [Ca²⁺] oscillated synchronously in both the nuclear and cytoplasmic regions of the same cardiomyocyte. It was concluded that although oscillating strenuously at high frequency, the average [Ca²⁺] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. The observations indicate that aconitine induces the changes in [Ca²⁺] oscillation frequency other than the Ca²⁺ overload.

The influence of exercise at high temperature on adult males’ routine blood indexes and biochemical indexes and the expression of HSP72 in peripheral blood lymphocytes (PBLs) was studied in order to provide theoretical ground for health supervision of adults receiving exercise at high temperature. 180 adult males were selected and divided into exercise group and control group, in which the exercise group was subdivided into subgroup 1 and subgroup 2 receiving exercise at high temperature in the afternoon and in the morning, respectively. Peripheral venous blood was phlebotomized before and after the exercise to examine routine blood indexes and blood biochemical indexes. The expression levels of HSP72 in PBLs were detected by flow cytometry. The results showed that the routine blood indexes and biochemical indexes in each group were within the range of normal values of male adults. There was no significant difference between each exercise group and control group in indexes before exercise. After exercise, the expression levels of HSP72 in PBLs in exercise groups were higher than those before exercise, and HSP72 expression levels in subgroup 1 were obviously higher than those in subgroup 2 and control group. The contents of ALT, urea, Na⁺, Cl⁻, Ca²⁺ and K⁺ in subgroups 1 and 2 were lower than those in control group, but CK level was higher than in control group (P<0.05). The contents of Na⁺ and Cl⁻ in subgroup 1 were relatively lower than those in subgroup 2 (P<0.05). It was concluded that while receiving exercise at high temperature, adult males’ HSP72 levels in PBLs could be increased and the biochemical indexes changed. Attention should be paid to health supervision to avoid obvious body injuries at high temperature.

The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with _pIRES₂-EGFP-^S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with _pIRES₂-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with _pIRES₂-EGFP-^K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor (NGF) β subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF β subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGF. The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF β was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and faciliate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF β subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.

The aim of this study was to determine if the potassium aspartate and magnesium (PAM) prevent reperfusion-induced ventricular arrhythmias (RIVA) in ischemia-reperfusion (IR) rabbit heart. Thirty rabbits were randomly divided into control, ischemia and PAM groups. Arterially-perfused rabbit left ventricular preparations were made, and transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded in the whole process of all experiments. In control group rabbit ventricular wedge preparations were continuously perfused with Tyrode’s solution, and in ischemia group and PAM groups the perfusion of Tyrode’s solution was stopped for 30 min. Then the ischemia group was reperfused with Tyrode’s solution and the PAM group with Tyrode’s solution containing 2.42 mg/L PAM, respectively. ECG, QT interval, transmural repolarization dispersion (TDR) and action potentials from epicardium and endocardium were simultaneously recorded, and the RIVA of the wedge preparation was observed. Compared with control group, TDR and incidence of RIVA were significantly increased in ischemia group (P<0.05). The incidence of RIVA in control, ischemia and PAM group was 0/10, 9/10 and 1/10, respectively. Compared with ischemia group, TDR and incidence of RIVA were significantly reduced in PAM group (P<0.05). Potassium aspartate and magnesium significantly reduce TDR and prevent ventricular arrhythmia in ischemic rabbit heart.

The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-κB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-κB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-κB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF protein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-κB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-KB, subsequently mediate TF gene expression and protein synthesis. NF-κB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.

The inhibitory effects of parthenolide (PTL) on angiogenesis induced by multiple myeloma (MM) cells in vitro and the mechanism were investigated. Human MM line RPMI8226 cells were cultured in vitro. The effects of MM culture supernatant on the migration and tubule formation ability of human umbilical vein endothelial cells (HUVECs) treated with PTL were observed. By using Western blot, the expression of p65 and IκB-α in MM cells was detected. RT-PCR was used to assay the expression of VEGF, IL-6, MMP2 and MMP9 mRNA in MM cells. ELISA was used to measure the levels of VEGF and IL-6 in MM cell culture supernatant. The expression of MMP2 and MMP9 in MM cells was examined by immunohistochemistry. (1) In 3.5, 5.0, 7.5 and 10 μmol/L PTL groups the number of migrated cells was 310±56, 207±28, 127±21 and 49±10 respectively, which was significantly different from that in positive control group (598±47) (P<0.01). In 3.5 and 5.0 μmol/L PTL groups the areas of capillary-like structures were 0.092±0.003 and 0.063±0.002 mm², significantly less than in positive control group (0.262±0.012 mm²) (P<0.01), but in 7.5 and 10 μmol/L PTL groups no capillary-like structures were found; (2) After treatment with different concentrations of PTL for 48 h, the expression of p65 protein was gradually decreased, while that of IκB-α was gradually enhanced with the increased concentration of PTL; (3) After treatment with 3.5, 5.0, 7.5 and 10 μmol/L PTL for 48 h, the VEGF levels in the supernatant were 2373.4±392.2, 1982.3±293.3, 1247.0±338.4 and 936.5±168.5 pg/mL respectively, significantly different from those in positive control group (2729±440.0 pg/mL) (P<0.05). After treatment with 7.5 and 10 μmol/L PTL, the IL-6 levels in the culture supernatant were 59.6±2.8 and 41.4±9.8 pg/mL respectively, significantly lower than in positive control group (1287.3±43.5 pg/mL) (P<0.05); (4) RT-PCR revealed that PTL could significantly inhibit the expression of VEGF and IL-6 mRNA in MM cells, but not influence the expression of MMP2 and MMP9 mRNA.; (5) Immunohistochemistry indicated that PTL had no significant effects on the expression of MMP2 and MMP9 protein in MM cells. It was concluded that the abilities of the culture supernatant of MM cells treated with PTL to induce endothelial cells migration and tubule formation were significantly reduced, suggesting PTL could obviously inhibit the angiogenesis induced by MM cells. PTL could decrease NF-kappaB activity and significantly suppress the expression of VEGF and IL-6 mRNA and protein, which might contribute to the mechanism by which PTL inhibited the angiogenesis induced by MM cells.

The changes of proto-oncogene c-fos and c-jun mRNA expression in angiotensin II (Ang II)-induced hypertrophy and effects of sodium tanshinone IIA sulfonate (STS) in the primary culture of neonatal rat cardiomyocytes were investigated. Twelve neonatal clean grade Wistar rats were selected. The cardiomyocytes were isolated, cultured and divided according to different treatments in the medium. The cardiomyocyte size was determined by phase contrast microscope, and the rate of protein synthesis was measured by [³H]-Leucine incorporation. The c-fos and c-jun mRNA expression in cardiomyocytes was detected by reverse transcription polymerase chain reaction (RT-PCR). It was found after cardiomyocytes were treated with Ang II for 30 min, the c-fos and c-jun mRNA expression in cardiomyocytes was increased significantly (P<0.01). After treatment with Ang II for 24 h, the rate of protein synthesis in Ang II group was significantly increased as compared with control group (P<0.01). After treatment with Ang II for 7 days, the size of cardiomyocytes in Ang II group was increased obviously as compared with control group (P<0.05). After pretreatment with STS or Valsartan before Ang II treatment, both of them could inhibit the above effects of Ang II (P<0.05 or P<0.01). It was suggested that STS could ameliorate Ang II-induced cardiomyocyte hypertrophy by inhibiting c-fos and c-jun mRNA expression and reducing protein synthesis rate of cardiomyocytes.

The effect of atorvastatin on warfarin-induced aortic medial calcification and systolic blood pressure (SBP) of rats induced by warfarin was studied. Thirty healthy and adult rats were randomly divided into Warfarin group (n=10), Atorvastatin group (n=10) and normal control group (n=10). Caudal arterial pressure of rats was measured once a week, and 4 weeks later, aorta was obtained. Elastic fiber, collagen fiber and calcium accumulation in tunica media of cells were measured by Von Kossa staining. The results showed that warfarin treatment led to elevation of systolic blood pressure and aortic medial calcification. The chronic treatment also increased collagen, but decreased elastin in the aorta. However, the atorvastatin treatment had adverse effects. It was concluded that treatment with atorvastatin presented evidence of blood pressure lowing and calcification reducing. These data demonstrate that atorvastatin protected aortic media from warfarin-induced calcification and elevation of systolic blood pressure.

The effects of tanshinone IIA (TSN) on transforming growth factor β1 (TGFβ1) signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10⁻⁶, 10⁻⁵, 10⁻⁴ mol/L TSN respectively. The mRNA levels of fibronectin (FN) were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively (P<0.01, P<0.01), and the protein expression of phosphorylated Smad2/3 (p-Smad2/3) increased by 7 times at the end of TGFβ1 stimulation (P<0.01). TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10⁻⁶ mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression (both P>0.05). After pretreatment with 10⁻⁵ and 10⁻⁴ mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively (P<0.05, P<0.01), the FN protein levels were decreased by 40% and 44% respectively (P<0.05, P<0.05), and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively (P<0.05, P<0.01). The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal interstitial fibroblasts.

Glomerulosclerosis, defined as phenotype transition of mesangial cell and deposition of extracelluar matrix, remains a chronic disease with excessive morbidity and mortality. The molecular mechanism underlying the suppression of mesangial cell activation is not fully understood. Since activation of peroxisome proliferators-activated receptor γ (PPARγ) has been proposed to decrease the effects of transforming growth factor-β (TGF-β) on glomerulosclerosis, we examined here whether and how telmisartan, an angiotensin II type 1 receptor blocker with PPARγ-modulating activity, inhibited TGF-β-induced glomerulosclerosis in rat glomerular mesangial cells. Protein levels of PPARγ were detected by Western blot. Activation of PPARγ response element (PPRE) was analyzed by luciferase assays. Deposition of extracelluar matrix was tested by confocol laser scanning. The results showed that telmisartan, but not valsartan, another angiotensin II type 1 receptor blocker, up-regulated PPARγ protein levels in a dose-dependent manner (P<0.05). Activation of PPRE, represented by luciferase activity, was also increased with higher concentration of telmisartan in a dose-dependent manner (P<0.05). Furthermore, telmisartan inhibited TGF-β-induced α-smooth muscle actin expression and collagen IV secretion in mesangial cells. GW9662, an inhibitor of PPAR-γ, blocked the inhibitory effects of telmisartan on TGF-β-induced glomerulosclerosis in mesangial cells. Our study indicates a benefit of telmisartan as a PPARγ agonist against TGF-β-induced mesangial cells activation in renal glomerulus. It may provide possibility that telmisartan works as a potential agent against diabetic nephropathy and hypertensive renal disease.

CD4⁺CD25⁺ regulatory T cells (Tregs) and the expression of their molecular markers (GITR, Foxp3) in peripheral blood of the patients with systemic lupus erythematosus (SLE) were investigated in order to reveal the pathogenesis of SLE on the cellular and molecular levels. The level of Tregs in peripheral blood was detected by flow cytometry. The expression levels of GITR and Foxp3 mRNA in peripheral blood mononuclear cells (PBMCs) were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). The level of IL-6 in the plasma was measured by ELISA. Comparisons were made among 3 groups: the active SLE group, the inactive SLE group, and normal control group. The level of Tregs in the active SLE group and the inactive SLE group was significantly lower than in the normal control group (P<0.01). The level of Tregs in the active group was lower than in the inactive group with the difference being not significant (P>0.05). The level of Tregs in SLE patients was significantly negatively correlated with the disease active index in SLE (SLEDAI) (r=−0.81, P<0.01). The expression levels of GITR mRNA in PBMCs of the active SLE group and the inactive SLE group were significantly higher than in the normal control group (P<0.05), and those of Foxp3 mRNA in SLE patients of both active and inactive SLE groups were significantly lower than in the normal control group (P<0.05). There was no significant difference in the expression of GITR and Foxp3 mRNA between the active SLE group and inactive SLE group (P>0.05). The plasma levels of IL-6 in both the inactive SLE group and active SLE group were significantly higher than in the normal control group (P<0.01). The plasma level of IL-6 in the active SLE group was significantly increased as compared with that in the inactive SLE group (P<0.05), and the plasma level of IL-6 in SLE was significantly positively correlated with SLEDAI scores (r=0.58, P<0.01) and significantly negatively correlated with the ratio of CD4⁺CD25⁺ cells/CD4⁺ cells (r=−0.389, P<0.05). It was concluded that the levels of Tregs and Foxp3 mRNA in peripheral blood of SLE patients were decreased and the levels of GITR mRNA and plasma IL-6 were increased. The Tregs and their molecular markers GITR, Foxp3 as well as the plasma IL-6 might play an important role in the pathogenesis of SLE.

The aim of this study was to investigate the effect and possible mechanism of all-trans retinoic acid (ATRA) on liver fibrosis induced by common bile duct ligation (CBDL) in rats. Fifty-three female Wistar rats were randomly divided into 5 groups: sham operation group (group J, 5 animals) and groups A, B, C and D (12 animals in each group). The rats in groups A, B, C and D were subjected to CBDL to induce liver fibrosis, while those in group J to sham operation. From the 3rd week the rats in groups B, C and D respectively received daily administration of ATRA via gastric tube at three different doses [0.1, 1.5 and 7.5 mg/kg body weight (BW)]. Animals were sacrificed at 6th week. Rats’ liver tissues were observed for pathologic changes under a light microscope. The protein levels of type I collagen (COL I), matrix metalloproteinase-2 (MMP2), MMP13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) in liver tissues were determined by immunohistochemical techniques. The expression levels of TGF-β1 and CTGF mRNA in liver tissues were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that loss of normal hepatic architecture and formation of obvious fibrosis were observed in group A, while ATRA treatment for 4 weeks notably alleviated the pathological changes of hepatocytes. The expression of COL I and TIMP-1 proteins in group A was increased, while decreased in ATRA-treated CBDL groups (P<0.05). ATRA (1.5 and 7.5 mg/kg BW) reduced the expression levels of COL I protein more greatly than that of 0.1 mg/kg BW (P<0.05). ATRA treatment increased the protein levels of MMP2 and MMP13. The expression levels of TGF-β1 and CTGF mRNA in group A were increased. In comparison with group A, the mRNA levels of TGF-β1 and CTGF in ATRA-treated CBDL groups were significantly decreased (P<0.05). It was concluded that ATRA could inhibit CBDL-induced liver fibrosis in rats by suppressing the expression of TGF-β1 and CTGF so as to diminish the inhibition of TIMP-1 on MMP2 and MMP13 and increase the activity of MMP2 and MMP13.

The effect of target-directed regulation of the uncoupling protein-2 (UCP-2) gene expression on the ischemia-reperfusion injury of hepatocytes under different conditions was investigated. The expression plasmid and RNAi plasmid targeting UCP-2 gene were constructed and transfected into normal hepatocytes and fatty liver cells, respectively. The expression of UCP-2 mRNA was detected by real time PCR. The cells were divided into normal cell group (NCG), group of normal cells transfected with empty vector (EVNCG), group of normal cells transfected with expression plasmid (EPNCG), fatty liver cell group (FCG) and group of fatty liver cells transfected with RNAi plasmid (RPFCG). The ischemia-reperfusion model in vitro was established. One, 6, 12 and 24 h after reperfusion, Annexin V/PI flow cytometry was used to measure cell necrosis rate, apoptosis rate and survival rate. Simultaneously, the intracellular ATP, ROS and MDA levels were determined. The results showed that 1, 6, 12 and 24 h after ischemia-reperfusion, the intracellular ROS, MDA and ATP levels and cell survival rate in EPNCG were significantly lower, and cell necrosis rate significantly higher than in NCG and EVNCG, but there was no significant difference in apoptosis rate among NCG, EVNCG and EPNCG (P>005). Six, 12 and 24 h after reperfusion there was no significant difference in ROS, MDA levels and apoptosis rate between FCG and RPFCG (P>0.05), but the ATP level and survival rate of cells in RPFCG were higher than in FCG (P<0.05). It was concluded that down-regulation of the UCP-2 gene expression in steatotic hepatocytes could alleviate the ischemia-reperfusion injury of liver cells.

The effect of rosiglitazone as the ligand of peroxisome proliferator-activated receptor γ (PPARγ) inhibiting the TLR4 expression in ischemic lobes in partial hepatic ischemia/reperfusion injury (IRI) in BABL/C mice and the action of rosiglitazone inhibiting the TLR4 receptor-mediated inherent immune response were investigated. The model of the mouse partial hepatic ischemia/reperfusion injury was established. All the animals were randomly divided into 3 groups: rosiglitazone group, vehicle (dimethylsulphoxide, DMSO) group and sham operation group. The hepatic samples were collected when mice were sacrificed 0, 2, 4 and 6 h after reperfusion following 1 h ischemia to analyze the acute phase of hepatic IRI. The dynamic expression of TIR4 mRNA was detected quantitatively by real-time-PCR, and the levels of TNF-α, IL-10 and ALT in portal vein were determined in all groups. After restoration of blood supply, the expression of TLR4 mRNA in ischemic lobes was detected in 0, 2, 4 and 6 h after reperfusion following 1 h ischemia in rosiglitazone group and vehicle group. The most intensive expression of TLR4 mRNA was present at 4 h after reperfusion in ischemic lobes in vehicle group. As compared with vehicle group, the expression of TLR4 mRNA in ischemic lobes in rosiglitazone group was significantly decreased at 4 h after reperfusion. The level of IL-10 in portal vein was markedly up-regulated in rosiglitazone group as compared with vehicle group. Contrarily, the levels of TNF-α and ALT in portal vein were markedly down-regulated in rosiglitazone group as compared with vehicle group at every time point in mouse partial hepatic IRI model. Rosiglitazone could alleviate the hepatic IRI by inhibiting TLR4 receptor-mediated inherent immune response.

The regulation of hypoxic response elements on the expression of vascular endothelial growth factor (VEGF) gene transfected to primary cultured rat skeletal myoblasts under hypoxic environment was investigated. pEGFP-C3-9HRE-CMV-VEGF vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western blot and fluorescence microscope under different oxygen concentrations and different hypoxia time. The results showed that in hypoxia group, the VEGF gene bands were seen and with the decrease of oxygen concentrations and prolongation of hypoxia time, the expression of VEGF mRNA was obviously increased. Under hypoxic environment, the expression of VEGF protein in the transfected myoblasts was significantly increased. EGFP was expressed only under hypoxic environment but not under normoxic environment. It was concluded that hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene under hypoxic and normoxic environment, which could enhance the reliability of gene therapy.

The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinβ1 (INTβ1) and focal adhesion kinase (FAK) by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was analyzed. The results showed that INTβ1 was expressed in 46 cases (93.9%) and FAK in 36 cases (73.5%), respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.

The inhibitory effect of niacinamide on tumor necrosis factor-α (TNF-α) induced annulus fibrous (AF) degradation was assessed, and the mechanism of the inhibition was investigated. Chiba’s intervertebral disc (IVD) culture model was established. Forty-eight IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups (12 IVDs in each group), and various concentrations of niacinamide and TNF-α were added to the medium for intervention: negative control group, niacinamide control group (0.5 mg/mL niacinamide), degeneration group (10 ng/mL TNF-α), and treatment group (0.5 mg/mL niacinamide and 10 ng/mL TNF-α). After one week’s culture, AFs were collected for glycosaminoglycan (GS) content measurement, safranin O-fast green staining, and immunohistochemical staining for type I, II collagen and cysteine containing aspartate specific protease-3 (Caspase-3). It was found that the GS content in treatment group was increased by about 48% as compared with degeneration group (t=16.93, P<0.001), and close to that in niacinamide control group (t=0.71, P=0.667). Safranine O-fast green staining exhibited higher staining density and better histological structure of AF in the treatment group as compared with the degeneration group. Immunohistochemical staining for both Type I and II collagen demonstrated that lamellar structure and continuity of collagen in treatment group were better reserved than in degeneration group. Positive staining rate of Caspase-3 in AFs of negative control group, niacinamide control group, degeneration group and treatment group was 3.4%, 4.3%, 17.9% and 10.3% respectively. The positive rate in treatment group was significantly lower than in degeneration group (P<0.01). It was concluded that niacinamide could effectively alleviate TNF-α induced destruction and synthesis inhibition of matrix ingredients in AFs. The inhibition may be related with reduction of expression of Caspase-3. Thus, niacinamide is of potential for IVD degeneration clinical treatment.

The expression of calcium epithelium TRPV5, alcium binding protein Calbindin-D28k and Na⁺/Ca²⁺ exchanger NCX1 was detected in renal distal convoluted tubule, and their effects on urine calcium reabsorption and the possible pathogenic mechanism in idiopathic hypercalciuria (IH) were investigated. Genetic hypercalciuric stone-forming (GHS) rats were chosen as animal models to study urine calcium reabsorption and IH. The cognate female and male rats that had maximal urine calcium were matched to breed next generation. Twelve GHS rats and 12 normal control (NC) SD rats were selected. Western blot and real time quantitative PCR were used to detect the protein and gene expression of TRPV5, Calbindin-D28k and NCX1 respectively. The expression levels of TRPV5 protein and mRNA in GHS rats were significantly lower than in NC rats (P<0.05). Western blot revealed that the expression levels of Calbindin-D28k in GHS rats and NC rats were 0.49±0.02 and 0.20±0.01 respectively, with the difference being significant between them (P<0.05). By using real time quantitative PCR, it was found that there was no significant difference in Calbindin-28k mRNA expression levels between GHS rats and NC rats (P>0.05). There was no significant difference in the NCX1 expression between GHS rats and NC rats (P>0.05). It was suggested that TRPV5 and Calbindin-D28k might play an important role in urine calcium reabsorption and IH, but they differently contributed to the pathogenesis: The down-regulation of TRPV5 decreases urine calcium reabsorption, directly leading to loss of the urine calcium and resulting in hypercalciuria, and the increased Calbindin-D28k expression could relieve, neutralize and decrease intracellular Ca²⁺ concentration to maintain calcium balance. NCX1 is not the key protein in urine calcium reabsorption.

The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl⁻ channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare’s serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl⁻ channels are involved in regulation of follicle development and luteum formation.

The effects of NO-Fluvastatin on proliferation of human lens epithelial cells (HLECs) and the action mechanism were investigated. Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. The expression of cell cycle regulatory proteins CyclinE mRNA and P21^waf1 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). MTT staining colorimetry showed that HLECs proliferation was markedly inhibited by NO-Fluvastatin and the effect was dependently related to time (24, 48 and 72 h) and dosage (1, 5 and 20 μmol/L). Flow cytometry revealed that NO-Fluvastatin could significantly block HLECs in the G₀/G₁ phase, resulting in the increased cells in the G₀/G₁ phase and decreased in the S phase (P<0.05). RT-PCR showed that NO-Fluvastatin could obviously inhibit the CyclinE mRNA expression and induce the P21^waf1 mRNA expression as compared with the negative control groups (P<0.05). This experiment suggested that NO-Fluvastatin could suppress the proliferation of HLECs by regulating cell cycle regulatory proteins (inhibiting the expression of CyclinE mRNA and inducing the expression of P21^waf1 mRNA), resulting in the arrest of HLECs in the G₀/G₁ phase, which can offer theory basis for NO-Fluvastatin in treating posterior capsular opacification in clinic practice.

The accommodative function before and after laser in situ keratomileusis (LASIK) was observed, and the effect of LASIk on accommodation was investigated. In a prospective clinical trial, 48 myopic patients (96 eyes) subject to bilateral LASIK in Refractive Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (China) from March 2006 to June 2006 were selected and studied. Refractions, accommodative range, amplitude of accommodative response and high frequency component (HFC) of accommodative microfluctuations were measured with NEDIK-730A before and one week and 30 days after operation. Dominant and non-dominant eyes were determined by hole-in-card method. It was found that all of the operative eyes showed an uncorrected visual acuity of 0.8 or better one week postoperatively, and 1.0 or better 30 days postoperatively. Compared with those preoperatively, accommodative range and HFC had no significant difference at first week and 30th day after operation in both dominant eyes and non-dominant eyes (P>0.05), but there was a significant difference in the amplitude of accommodative response/accommodative stimulus ratio (A/S) after operation (P<0.01), and no significant difference was found in accommodation between one week and 30 days postoperation. No ocular dominance’s change was noted. There was no significant difference in accommodative function between dominant eyes and non-dominant eyes. It was suggested that LASIK produced no significant effect on accommodation.

This study investigated the contents and distribution of collagen V (Col V) in skin lesions of the patients with systemic sclerosis (SSc) and its roles in the pathogenesis. The contents and distribution for α1 chain of collagen type I, III and V [α1 (I), α1 (III) and α1 (V)] in skin lesions of 36 patients with SSc (9 cases of mild fibrosis, 14 moderate, and 13 severe) were detected by using immunohistochemical SP method. Six cases of normal skin tissues served as controls. The results showed that there was diffuse distribution for three kinds of collagens in dermis. The deep staining α1 (I) and α1 (III) masses or bands were seen in reticular layer, while α1 (V) was distributed more homogeneously. From control to weak, moderate and severe fibrosis stages, α1 (I), α1 (III) and α1 (V) showed a gradually increased trend in skin lesions (P<0.05). α1 (V) was obviously elevated in skin lesions at early stage and persisted in whole fibrotic process and risen in greater contents, while α1 (I) and α1 (III) were to go higher late and were apparently elevated at moderate and late stages. Compared with α1 (I), α1 (V) took leading increase at early stage in skin lesions (P<0.01), and had more elevated contents than α1 (III) at moderate and late stages. The fibrotic changes in dermal reticular layer occurred earlier than those in papillary layer, and the abnormalities of α1 (V)/α1 (I) ratio appeared before α1 (III)/α1 (I) ratio. It was concluded that a lot of α1 (V) began to deposit in greater contents prior to α1 (I) and α1 (III) at early stage in SSc and persisted in whole fibrotic process. The changes of α1 (V) contents in reticular layer occurred earlier than those in papillary layer, and it suggested that the fibrosis in reticular layer appeared earlier.

The aim of this study was to investigate the in vitro cytotoxicity of polyphosphoester polymer used as a novel injectable alveolar bone substitutes for controlled delivery of tetracycline. Cell culture medium was exposed to the polymer (0.01–10 mg/mL) for 24 h. The L-929 mouse fibroblasts were then exposed to the treated cell culture medium for 24 h. Finally, cell viability and growth were assessed by using MTT assay and Alamar Blue assay. No significant cytotoxicity of the polyphosphoester against L-929 mouse fibroblasts was observed at a concentration up to 10 mg/mL (P>0.05). The two evaluation methods showed no significant differences (P>0.05). This study suggests that polyphosphoester does not demonstrate any significant toxic effects to cells in vitro and has the potential to be used both as a medical device and as scaffolds in tissue engineering applications.

In order to investigate the clinical significance of ^99mTc-Tetrofosmin (TF) scintigraphy in the evaluation of lung cancer and mediastinal lymphoid node involvement, 33 patients with pulmonary neoplasmas were subjected to both ^99mTc-TF scintigraphies and CT scans in one week before their operations or puncturations. All the images were judged visually and the emission images were analyzed with semi-quantitative methods in addition. The results of each group were compared. There was marked difference in target/non-target (T/N) ratio between the lung cancer group and the benign lesion group (P<0.001). Moreover, in the lung cancer group, T/N ratio in tomographies was significantly higher than that in planar images (P<0.01). The sensitivity and accuracy of semi-quantitative analysis in ^99mTc-TF SPECT were significantly higher than those of CT in the diagnosis of pulmonary neoplasmas (P<0.05 and P<0.01 respectively), so was the sensitivity of ^99mTc-TF SPECT vs CT in the diagnosis of mediastinal lymphoid node metastasis (P<0.05). It was also found that epidermoid squamous cell carcinomas and adenocarcinomas had a higher T/N ratio than in small cell carcinomas (P<0.05), and 2 h washout rate (WR) of adenocarcinomas was higher than that of epidermoid squamous cell carcinomas (P<0.05). In conclusion, ^99mTc-TF scintigraphy showed a favorable diagnostic accuracy in appraising lung cancers and mediastinal lymph node metastases. Furthermore semi-quantitative technology can improve the accuracy, and is potential to offer some information about histological type of the cancer tissue. Therefore, ^99mTc-TF scintigraphy will be a useful tool in the diagnosis and staging of lung cancer.

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein (EGFP) gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction (UTMD) and polyethylenimine (PEI). SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.

In order to compare GnRH agonist with antagonist protocol for the same patient during controlled ovarian stimulation cycles, the in vitro fertilization and embryo transfer (IVF-ET) outcome was retrospectively studied in 81 patients undergoing 105 agonist protocols and 88 antagonist protocols. The results showed that there was no statistically significant difference in duration of ovarian stimulation, number of ampoules, oocytes retrieved, serum estradiol (E₂) and progesterone (P) levels, thickness of endometrium, the zygote-and blastocyst-development rate between GnRH agonist and antagonist protocols (P>0.05). High quality embryo rate was higher in antagonist protocols, but there was no significant difference between two protocols. Implantation rate and clinical pregnant rate were significantly higher in antagonist protocol (15.82% and 30.26%, respectively) than in agonist protocol (5.26% and 10.64% respectively (P<0.05). It was concluded GnRH antagonist protocol probably improved the outcome of pregnancy of older patients with a history of multiple failure of IVF-ET in a GnRH protocol.

The present study aimed to compare the clinical efficacy of a 0.4% carbomer gel and 1% carboxymethyl cellulose (CMC) containing artificial tears in treatment of dry eye patients. Sixty subjects with mean age of 45.89 years who had symptoms and signs of dry eye were enrolled in this prospective, investigator-masked and stratified random sampling study. The subjects were divided into two parallel groups with 30 subjects (60 eyes) in each group. One group received carbomer gel, and the other group received 1% CMC containing artificial tears. Subjects received the drops 3 to 4 times or more per day for 3 months. At the first visit time, the precorneal residence time of these two drops was measured. The efficacy was assessed by comparing the subjective symptoms (ocular dryness, foreign body sensation, burning sensation and pain), and the objective test results of tears breakup time, Schirmer’s test and corneal fluorescein staining prior to the study and after the treatment. As a result, the ocular residence time of carbomer gel was significantly longer than that of 1% CMC (P<0.001). Most of the primary subjective symptoms and objective test results were improved after treatment in both carbomer gel group and 1% CMC group. As to the improvement of each symptom and objective test result, carbomer gel was more effective than 1% CMC group (P<0.01). In conclusion, carbomer gel had longer precorneal residence time and was more effective than 1% CMC in the treatment of patients with dry eyes.