The P2X7 receptor mRNA and proteins in guinea-pig dorsal root ganglia (DRG) were studied by using RT-PCR and immunohistochemistry. The co-localization of P2X7 receptor with four cytochemical markers, the neurofilament protein NF200, S100, substance P and isolectin B4 (IB4) binding glyco-conjugates, were also examined. It was found that P2X7 receptor immunoreactivity (P2X7R-IR) was present mostly in large- and medium-sized DRG neurons (62%±9% and 36%±6% respectively in all P2X7R-IR neurons). All the P2X7R-IR neurons were also NF200 and S100 immunopositive. However, in a small number of NF200 or S100 immunopositive neurons no P2X7R-IR was detectable. All the IB4-positive or substance P-immunopositive neurons had no P2X7R-IR. These results demonstrate that P2X7 receptors are expressed in a large subpopulation of DRG neurons and they may play a role in the transduction of specific peripheral sensory signals.
In this study, we studied the effect of glycogen synthase kinase-3 (GSK-3) overactivation on neurofilament phosphorylation in cultured cells. After N2a cells were treated with the specific inhibitor (wortmannin) of phosphoinositol-3 kinase (PI-3K) or treated with wortmannin and the specific inhibitor (LiCl) of glycogen synthase kinase-3 (GSK-3), GSK-3 activity and neurofilament phosphorylation were detected by using GSK-3 activity assay, Western blots and immunofluoresence. Our results showed that after treatment of N2a cells with wortmannin for 1 h, overactivation of GSK-3 caused a reduced staining with antibody SMI32 and an enhanced staining with antibody SMI31. When N2a cells were treated with wortmannin and LiCl, the activity of GSK-3 was reduced substantially. At the same time, the phosphorylation of neurofilament was also reduced. The study demonstrated that overactivation of GSK-3 induced hyperphosphorylation of neurofilament and suggested thatin vitro overactivation of GSK-3 resulted in neurofilament hyperphosphorylation and this may be the underlying mechanism for Alzheimer's disease.
The specific anti-tumor immune response induced by mouse bone marrow dendritic cells (DCs) transfected with recombinant adenovirus carrying mutant k-ras genes was investigated. DCs were generated from mouse bone marrow in the presence of rmGM-CSF (3.3 ng/mL) and rmIL-4 (1.3 ng/mL) and detected by FACS, and then transfected with the recombinant adenovirus encoding mutant k-ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The results showed that DCs had dendritic veiled morphology. BmDCs highly expressed B7-1 (80%), B7-2 (77%), MHC II (70%), CD11c (65%), CD40 (70%) and CD54 (96%) with FACS, and no significant difference in the expression was observed before and after the transfection (P>0.05). The DCs transfected by mutant k-ras gene could significantly stimulate lymphocytes proliferation as compared with those transfected by Ad-c or non-modified DCs (P<0.05). DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific cytotoxicity against Lewis lung cancer in Ad-k-ras/12-transduced DC group was significantly higher than those in the control, vector and non-transfected DCs groups (P<0.05). It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.
Primary cell culture, techniques of gene transfection, gelatin zymography, and Western blot were used to investigate the effect of hypoxia on the secretion of MMP-2 and MMP-9 in pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), and the role of HIF-1, Our results showed that (1) after exposure to hypoxia for 24 h, the protein content and activity of MMP-2 in the PAEC medium as well as these of MMP-2 and MMP-9 in PASMC medium (P<0. 01) decreased significantly in contrast to those in normoxic group (P′<0.05); (2) after transfection of wild type EPO3′-enhancer, a HIF-1 decoy, the content and activity of MMP-2 and MMP-9 in hypoxic mediums became higher than those in normoxic group (P<0.01), while transfection of mutant EPO3′-enhancer didn't affect the hypoxia-induced down-regulation. It is concluded that hypoxia could inhibit the secretion and activity of MMP-2 and MMP-9 in PAEC and PASMC, which could be mitigated by the transfection of EPO3′-enhancer and that HIF-1 pathway might contribute to hypoxia-induced down-regulation of MMP-2 and MMP-9.
To explore the expression of heme oxygenase-1 (HO-1) in the peripheral blood mononuclear cells (PBMCs) and its relationship with pulmonary ventilation function in asthmatic patients, 18 asthmatic patients and 18 healthy subjects were selected. HO-1 protein and mRNA levels in PBMCs were measured by immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Blood carbon monoxide Hb (COHb), serum total IgE and pulmonary ventilatory function were observed. Our results showed that the percentage of cells positive for immunohistochemical staining of HO-1 were significantly higher in asthmatic patients (41.72±7.44)% than that in with healthy subjects (10.45±4.36)% (P<0.001) and the optical density of PBMC HO-1 mRNA was higher in asthmatic patients (26.05±4.14) than that in healthy subjects (10.82±4.26) (P<0.001). The relation analysis showed that PBMC HO-1 protein and mRNA levels had significantly negative relation with FEV1%, PEFR, MEFR50%, respectively (r=−0.51–0.89,P<0.05–0.001, respectively) and a positive relation with COHb and serum total IgE (r =0.48–0.85, 0.05–0.001, respectively). It is concluded that the expression of PBMC HO-1 protein and mRNA increased significantly in asthmatic patients, and HO-1 may play a significant role in the pathogenesis of asthma. The expression of HO-1 may bear a relation with severity of asthma.
To study the expression of DNA repair enzyme hMTH1 mRNA and protein in hepatocellular carcinoma (HCC) tissues, tissues adjacent to the cancers, normal liver cells and hepatoma cell lines, and to investigate their function in the progress of HCC, semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was employed to examine the expression of hMTH1 mRNA in matched HCC tissues (HT)/surrounding tissues (HST) of HCC, normal liver cell L02 and hepatoma cell lines SMMC7721, HepG2. hMTH1 protein was detected in corresponding HT as well as their HST by immunohistochemistry. Our results showed that the expression level of hMTH1 mRNA in HT was higher than that in HST (t=2.424,P<0.05). The expression level of hMTH1 mRNA in two hepatoma cell lines was higher than that in normal liver cell line (F=6.810,P<0.01). The expression of hMTH1 mRNA in SMMC7721 was similar to that in HepG2. hMTH1 protein was 88.2% (15 of 17) positive in HT and 82.4% (14 of 17) in HST. The protein level of hMTH1 in HT was correspondingly higher than in their HST (t=2.618,P<0.05). It is concluded that hMTH1 mRNA and protein were over-expressed in HCC and hepatoma cell lines. It may be one of the key events during the carcinogenesis, progression of HCC and may promote the malignant growth. These results suggest that hMTH1 plays a role in HCC and may be a candidate marker for the diagnosis of HCC.
To investigate the effects of ATRA, acitretin and tazarotene on the growth and apoptosis of human tongue squamous cell carcinoma cell line Tca8113. The effect of retinoids on growth of Tca8113 cellsin vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis with double staining with Annexin V-FITC and PI, and active caspase-3 analysis with the staining of FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody were made by flow cytometer. Streptavidin-biotin complex (SABC) immunocytochemical assays were employed for the detections of Bax/Bcl-2 proteins expressions. Our results showed that the retinoids inhibited growth of Tca8113 cells in a dose- and time-dependent manner with maximal inhibition 24 h after treatment of 10−5 mol/L. 10−5 mol/L retinoids altered cell cycle distribution of Tca8113 cells, revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. 10−5 mol/L retinoids significantly induced apoptosis of Tca8113 cells (allP<0.05), elevated the cells population with detectable active caspase-3 (P<0.05 for all), increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly (allP<0.05). Acitretin played a most prominent role among the retinoids. It is concluded that the inhibition of cell cycle progress of Tca8113 cells by ATRA, acitretin and tazarotene is one of the possible mechanisms for proliferation arrest of Tca8113 cells elicited by the retinoids. The retinoids mediate apoptosis in Tca8113 cells that may be caspase-dependent through mitochondria pathway. High concentration retinoids inhibit growth of Tca8113 cellsin vitro by interfering with proliferation and inducing apoptosis of cells. Acitretin may be an alternative medicine for the prevention and treatment of tongue squamous cell carcinoma.
To investigate the function of dendritic cells (DC) in patients with unstable angina, 10 mL of blood was drawn from 30 subjects, 15 patients diagnosed as having unstable angina and 15 healthy subjects were included in an observation and a control groups respectively. The mononuclear cells were separated from the peripheral blood and cultured in RPMI1640 supplemented with recombinant human granulocyte/macrophage-colony stimulating factor (rh GM-CSF) and recombinant human interleukin-4 (rh IL-4) to induce dendritic cells. The shape and ultrastructure of DC was examined with electronic microscope. The phenotype of DC was analyzed with FACS and the alloantigen presenting capacity of DC was evaluated by mixed lymphocyte reaction (MLR). The expression rate of CD86 of DC in patients with unstable angina was (40.7±3.6)%, which was obviously higher than that of normal DC (29.6±2.5%) (P<0.001). The capacity of the DCs in unstable angina patients to induce allogenic T cells (OD 2.73±1.10), was significantly higher than that of the normal DC (OD:0.9±0.21) (P<0.005). It is suggested that the function of DC in patients with unstable angina is increased, which may play an important role in the initiation of immune reaction in the plaque.
To study the effect of of lidocaine and amiodarone on the transmural heterogeneity of ventricular repolarization in isolated rabbit hearts model of sustained global ischemia and to explore the mechanisms underlying the antiarrhythmic activity of lidocaine and amiodarone, rabbits were randomly divided into 4 groups: control group, ischemia group, lidocaine group and amiodarone group. By the monophasic action potential (MAP) recording technique, MAPs of epicardium, midmyocardium and endocardium were simultaneously recorded across the left ventricular free wall in rabbit hearts perfused by low-flow ischemia (2.5 mL/min) in Langendorff method to study the transmural dispersion of repolarization (TDR) and arrhythmic induced by ischemia. Our results showed that TDR of three myocardial layers in ischemia group were significantly lengthened after ischemia. TDR was increased from 17.5±3.9 ms to 31.2±4.6 ms at the time that concided with the onset of sustained ventricle arrhythmic. Amiodarone could decrease TDR, but lidocaine could increase TDR at initial ischemia, and no significant difference was found at other ischemia time points. 5 cases had ventriclar arrhythmia in ischemia group (62.5%), but no case in lidocaine group (P<0.01) and only 1 case in amiodarone group had ventrilar arrhythmia (P<0.01). No significant difference was found between amiodarone group and lidocaine group. It is concluded that TDR of of three myocardial layers increases significantly at ischemia and it is closely associated with development of ventricular arrhythmia, and amiodarone could decrease TDR, but lidocaine could increase TDR at initial ischemia and has no effects at other ischemia time points.
To explore the anticancer effect of curcumin on human B cell non-Hodgkin's lymphoma and compare its effects on human B cell non-Hodgkin's lymphoma cells and normal peripheral blood mononuclear cells (NPBMNCs). MTT assay was used to study the effect of curcumin on the growth of Raji cells and NPBMNCs. The effect of curcumin on the apoptosis of Raji cells and NPBMNC were studied by flow cytometry and TDT-mediated dUTP nick and labeling (TUNEL). The effect of curcumin on the cell cycle of Raji cells were examined by propidium iodide staining flow cytometry. The results showed that curcumin strongly inhibited proliferation of Raji cells, 24 h IC50 for Raji cells was 22.8±1.82 μmol/L and curcumin induced Raji cell apoptosis in a time- and dose-dependent manner. Raji cells treated with curcumin showed G0/G1 or G2/M phase increase and S phase decrease. However, curcumin did not demonstrate apparent proliferation inhibition and apoptosis induction in NPBMNCs. It was concluded that curcumin is able to inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Morever, curcumin has low toxicity on NPBMNCs but can selectively induce apoptosis in Raji cells.
The apoptosis and the expression of tumor suppressor gene p53 in hypercholesterolemia (HC)-induced renal injury were investigated in rats. A high cholesterol diet (HCD)-induced HC rat model was made and serum lipid, urinary protein excretion (UPE) and N-aceto-β-D-glucosidase (NAG) were measured. The levels of malondialdehyde (MDA), as an index of lipid peroxidation, in renal cortex and serum were compared between the two diet groups. Apoptosis and p53 expression were determined by TUNEL and immunohistochemistry, respectively. In the HCD-induced HC group, serum total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) as well as triglyceride (TG) were significantly increased, while the level of high density lipoprotein-cholesterol (HDL-C) decreased. Meanwhile, increased excretions of UPE and NAG in urine were observed, which were accompanied with a decrease in urinary creatinine clearance (Ccr) and indicated both glomerular and tubular damages. In addition, apoptotic cell death coexisted in the kidney, as revealed by increased TUNEL positive cells. Finally, an increase in p53 expression was observed in tubuli, but not in glomeruli. Both TUNEL positive cells and p53 expression were found to be correlated to the level of renal cortical MDA (r=0.817,P<0.01 andr=0.547,P<0.01, respectively). The major manifestation of HCD-induced renal injury is apoptosis. The lipid peroxidation is a critical event to induce DNa damage and p53 is involved in the pathogenesis of lipid-induced renal injury.
To study the efficacy and the mechanism of colquhoumia root (Tripterygium hypoglaucum (Le, vL)Hutch) in the treatment of mesangial proliferation glomerulonephritis (MsPGN), SD rats were injected with anti-thymocyte serum (ATS) to make MsPGN model (anti-Thy1 model). The rats were then divided into 3 groups: normal control group, anti-Thy1 model group and treatment group. Histopathological (HE, PAS), immunohistochemical, RT-PCR technique and computer imaging analysis system were used to evaluate mesangial matrix production, the expression of TGF-β1 protein and mRNA in the tissues of kidney. Our result showed that proteinuria and the ratio of extracellular matrix/glomerular capillaries are (ECM/CA) were increased significantly in model group. The expression of both TGF-β1 protein and mRNA in glomeruli was much higher in model group than in control group (P<0.01). After the treatment with Colquhoumia root, proteinuria, ECM/CA and the expression of both TGF-β: protein and mRNA in glomeruli were significantly decreased in treatment group as compared with those in model group. It is concluded that Colquhoumia root is effective in reducing proteinuria and mesangial matrix proliferation in MsPGN and it may achieve these effects by inhibiting the expressions of TGF-β1 protein and mRNA of mesangial cells.
To observe the effects of calcium dobesilate on the expression of glomerular tissue inhibitor of metalloproteinase 1 (TIMP1), collagen IV, and ultrastructure of glomerular basement membrane in diabetic rats, rats model of diabetes was established by unilateral nephrectomy and intraperitoneal injection of 1% STZ (55 mg/kg), and rats were administered calcium dobesilate 100 mg/kg (DD group) or distilled water (DM group) respectively. 12 weeks later, the changes in the renal ultrastructure and creatinine clearance rate (Cer) were examined in each group. The expression of glomerular TIMP1 and collagen IV were studied by immunohistochemical staining. Our results showed that after 12 weeks, the Cer in DD group increased and was significantly higher than that in DM group. Electron microscopy showed that thickness of glomerular capillary basement membrane (GBM) in Group DD was less than that of DM group. No hyperplasia of collagen fibers was found, and the distance between the holes of endothelial cells in DD group was not as even as that in the normal group, but more even than that of DM group, and podocyte processes was still in order. Immunohistochemical staining of glomeruli showed that expression of TIMP1 and collagen IV in DD group were significantly less than those of DM group DM. It is concluded that calcium dobesilate can improve diabetic nephropathy by inhibiting the over- accumulation of collagen IV and calcium dobesilate may also contribute to diabetes by inhibiting the expression of TIMP1.
In order to provide morphological data and theoretical basis for pig-to-human hepatic xenotransplantation, the difference in morphological parameters and vessel wall structural factors between human and porcine hepatic portal vein was studied. From human subjects and pigs of varying ages, hepatic portal veins were collected, paraffin-embedded and cut into sections. The histological structures were stained with HE, and elastin, collagen and smooth muscles were stained with Weigert, Aniline blue and orange G, respectively. Morphological parameters and relative contents of structural components were determined under microscopy and by computer image analysis system, respectively. The results showed that histological structures of human and porcine hepatic portal vein wall were similar. Caliber, wall thickness, lumen and wall area in pigs increased with age, all in linear correlation to months. Morphological parameters of 6-month-old pigs were similar to those of human. In pigs, collagen content increased gradually with months, elastin content remained relatively stable, smooth muscle content reached the peak at the 3rd month, and collagen/elastin (C/E) rose gradually. The contents of collagen and elastin in porcine hepatic portal vein wall were lower, while the content of smooth muscle was higher than in human, and C/E at the 5th and 6th month was similar to that in human. It is concluded that morphological parameters and contents of structural components of porcine hepatic portal vein vary with age. At the 6 month, its caliber, wall thickness, lumen and wall area are similar to those of human. There are differences in contents of structural components between human and pigs. However, in terms of C/E, mechanic properties of pigs at the 5th and 6th month mimic those of human, hence inosculation is viable in xenotrans-plantation between pigs and human.
To determining the postmortem interval (PMI) through quantitative analysis of the DNA degradation of cell nucleus in human brain and spleen by using image analysis technique (IAT). The brain and spleen tissues from 32 cadavers with known PMI were collected, subjected to cell smear every 1 h within the first 5–36 h after death, stained by Feulgen-Vans staining, Three indices reflecting DNA in brain cells (astrocytes) and splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray (AG) were measured by employing the mage analysis instrument. The results showed that IOD and AOD declined and AG increased with the prolongation of dead time within 5–36 h. A correlation between the PMI and gray parameters (IOD, AOD and AG) was identified and the corresponding regression equation was obtained. The parameters (IOD, AOD and AG) were proved to be effective quantitative indicators for accurate estimation of PMI within 5–36 h after death.
This study studied the use of ERCP and nasobiliary tube in the diagnosis of fungal infection of biliary tract and the efficacy of combined use of local administration via nasobiliary tube and intravenous antifungal treatment for severe biliary tract fungal infection. 5 patients in our series, with age ranging from 47 to 68 y (mean 55.8), were diagnosed as having mixed bacterial and fungal infection of biliary tract as confirmed by smear or/and culture of bile obtained by ERCP and nasobiliary drainage. Besides routine anti-bacteria therapy, all patients received local application of fluconazole through nasobiliary tube and intravenous administration of fluconazole or itraconazole in terms of the results ofin vitro sensitivity test. The mean duration of intravenous fluconazole or itraconazole was 30 days (24–40 days), and that of local application of fluconazole through nasobiliary drainage tube was 19 days (8–24 days). During a follow-up period of 3–42 months, all patient's fungal infection of biliary tract was cured. It is concluded that on the basis of typical clinical features of biliary tract infection, fungal detection of smear/culture of bile obtained by ERCP was the key for the diagnosis of fungal infection of biliary tract. Local application antifungal drug combined with intravenous anti-fungal drugs might be an effective and safe treatment for fungal infection of biliary tract.
This study examined the postoperative plasma endotoxin level, plasma endotoxin inactivation capacity and clinical outcome after administration of an enteral diet supplemented with glutamine, arginine and ω-3-fatty acid in patients undergoing gastrointestinal operations on an prospective, randomized and double-blind design. 40 patients undergoing gastrointestinal operations were randomized into two groups, with each having 20 patients. One group received standard enteral nutrition and the other was fed the formulation supplemented with glutamine, arginine and ω-3-fatty acid. The two groups were isonitrogenous. The infusion was started from day 1 after surgery and continued for 7 days. Blood samples were collected on the morning of day 1 before operation and on the morning of 1, 4 and 7 day(s) after operation and analyzed for plasma endotoxin level and endotoxin inactivation capacity (EIC). Our study found no differences between the two groups on plasma endotoxin level. After surgery a rapid reduction in plasma endotoxin inactivation capacity was observed in both groups, a significant recovery of the plasma endotoxin inactivation capacity was observed on morning of day 4 after surgery in the study group (0.12±0.02 EU/mL and 0.078±0.022 EU/mL respectively,P<0.01). Shortened hospital stay was observed in the experimental group (11.7±2.0 days in the control group and 10.6±1.2 days in the experimental group respectively,P=0.03). It is concluded that perioperative parenteral nutrition supplemented with glutamine, arginine and ω-3-fatty acid ameliorated postoperative immunodepression but without direct effect on endotoxemia.
The regulating mechanism in hepatic injury caused by obstructive jaundice (OJ) was examined in this study. Rat hepatocytes were harvested byin situ collagenase perfusion and subjected to primary culture. The heptocytes were pre-treated with various concentrations of protein kinase C (PKC) agonist PMA and its inhibitor chelerythrine and cultured for 20 min. After the treatment, 50 μmol/L glycochenodeoxycholate (GCDC) was added and the cells were cultured for an additional 24 h. Cells were then detected by flow cytometry (FCM) and TUNEL. After hepatocytes were treated with different concentrations of fructose and 100 μM GCDC, the cells were examined by FCM and TUNEL. Experimental obstructive jaundice (BDL) was induced by double ligation of the bile duct. After BDL, the rats were fed with or without fructos and sacrificed 3, 7, 14 and 21 days after the ligation. The apoptotic status was observed in liver of all rats with TUNEL and PKC protein in liver of OJ was studied by immunohistochemical method. Our results showed that PMA increased GCDC-induced apoptosis and chelerythrine decreased GCDC-induced apoptosis in a concentration-dependent manner. After the treatment with fructose of different concentrations, 100 μM GCDC decreased the apoptotic rate and the apoptotic rate decreased with the increase of fructose concentration. The apoptotic rate of liver was related to the time of OJ. Without the treatment of fructose, PKC and apoptosis index (AI) were highest 14 days after the bile duct ligation. With the treatment of fructose, apoptosis index (AI) and PKC were decreased from the 14th day after the bile duct ligation. It is concluded that PKC is involved in the regulation of apoptosis in the liver cells with OJ and plays important roles in the development and progression of liver injury caused by OJ. Fructose can protect hepatocytes in the bile salt-induced apoptosis by regulating PKC.
This study was designed to investigate the cardioprotective effects of preconditioning with 3-nitropropionic acid, an inhibitor of mitochondrial succinate dehydrogenase. 16 isolated rat hearts were randomly divided into two groups, a treatment group and a control group. The rats of the treatment group were treated intraperitoneally with 3-nitropropionic acid (3-NPA, 4 mg/kg) and the rats of the control group were treated with saline. 24 h after the treatment, the isolated hearts were mounted on a Langendorff apparatus. After 30 min, the hearts were subjected to 30-min ischemia and 60-min reperfusion. The HR, LVDP and ±dp/dtmax were measured at pre-ischemia and 30 min, 60 min after the reperfusion. Coronary effluent was collected 15 min after the reperfusion for the determination of CK and LDH. At the end of the 60-min reperfusion the heart was removed for the determination of myocardial SOD and MDA. Our results showed that in the 3-NPA group LVDP and ±dp/dtmax recovered significantly better, myocardial MDA, CK and LDH were significantly lower and the myocardial SOD was significantly higher than in the control group. It is concluded that chemical preconditioning by 3-nitropropionate has cardioprotective effects against ischemia-reperfusion injury.
To study the effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on PC-3M cell line, PC-3M cell line was incubated with gradient concentrations of TRAIL for 4–24 h. Annixin-V fluorescence staining and TUNEL method were employed to detect the apoptosis of PC-3M cells. The morphology of apoptotic PC-3M cells was observed by electron microscopy. The relationship between TRAIL concentrations and the percentage of apoptotic cells was evaluated by flow cytometry. The proliferation inhibitory ratio was calculated by using MTT colorimetry. Our results showed that apoptosis of PC-3M cells could be induced by treatment with TRAIL for at most 4 h. The results of flow cytometry and MTT colorimetry demonstrated a time- and concentration-dependent relationship between cell apoptosis rate and TRAIL concentration. It is concluded that apoptosis of PC-3M cells can be induced by TRAIL. Because of the selective killing effect of TRAIL on tumor cells, it may become a potential alternative for the treatment of advanced prostate cancer.
To compare the expression level of metastasis associated-1 (MTA1) gene in high and low metastatic human osteosarcoma cell lines and examine the relationship of MTA1 expression and the metastasis potentiality of osteosarcoma cells the expression of MTA1 in MG-63 osteosarcoma cell lines with high and low metastasis potential was detected by semiquantitative TR-PCR. Boyden chamber invasion assay was used to evaluate the invasive capacityin vitro in two osteosarcoma cell lines. The low metastasis MG-63 cells were transfected with MTA1 full-length cDNA expression plasmid by lipofectamine and the changes of MTA1 expression andin vitro invasion potential were examined after the transfection. Our results showed that MG63 cell line with high metastasis potential expressed significantly higher MTA1 than that of MG63 cells with low metastasis as reavealed by RT-PCR. The invasion potential of low metastasis MG63 cell line was increased after MTA1 gene transfection. It is concluded that there may be a relationship between MTA1 and invasive potentiality of human osteosarcoma cells, and the mechanism of MTA1 in osteosarcoma metastasis and its possible role in associated gene therapy deserve further study.
To evaluate the effect of wild-type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells, p53 gene expression in transfected cells was detected by RT-PCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells (inhibition rate (IR): (79.60±5.69) %). The killing effect of irradiation alone on U251 cells was not strong (IR: (17. 06±4.35) %, (17.39±1.67) %, (18.73±4.68) %) and increased with the irradiation doses (3, 6, 9 Gy). When combined treatment of wild-type p53 gene transfection and irradiation was used, the effect was significantly increased (IR: (80.60±5.35) %, (90.30±1.67) %, (91.30±2.01) %). The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. The rate induced by irradiation increased (4.61%, 4.84%, 5.40%) with the irradiation doses (3, 6, 9 Gy). The apoptosis rate was also significantly increased (17.80%, 20.03%, 22.34%) after combined treatment of p53 and irradiation with different doses (3, 6, 9 Gy). It is concluded that wild-type p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.
To investigate the reliability and feasibility of human papillomavirus (HPV) DNA test in cervical scraping smears with polymerase chain reaction (PCR), 131 cases of cervical scraping specimens were collected, and the positive rates and accuracy of HPV infection were determined in normal subjects and cervical cancer patients. GP5+/GP6+ and E7 primer pairs designed for detecting HPV L1 and HPV type 16 E7 were tested in this study. Our results showed that positive rates of HPV DNA in normal population and cervical cancer patients were 32.99% and 73.53% respectively and there was significant difference between them (P<0.001). In normal subjects, detection rates of HPV DNA with GP5+/GP6+ and E7 primer pairs were 27.84% and 16.49% respectively, with statistically significant difference between them (P>0.05). However the detection rates in cervical cancer patients were 38.24% and 67.65% for the two markers, with a significant difference found between them (P<0.05). It is concluded that HPV DNA test with PCR for cervical scraping smears was feasible. GP5+/GP6+ primer pairs may be a useful probe to screen HPV infection in normal population, but they are not sensitive enough in cervical cancer patients. It is suggested that high risk type HPV DNA test was very useful in population with high risk of cervical cancer.
To investigate the protective effect of genistein on endotoxin-induced acute lung injury in rats, and explore the underlying mechanisms, 32 male Sprague-Dawley rats were randomly divided into 4 experimental groups: saline control, genistein alone, lipopolysaccaride alone, and genistein pretreatment. Each treatment group consisted of eight animals. Animals were observed for 6 h after LPS challenge, and the wet/dry (W/D) weight ratio of the lung and bronchoalveolar lavage fluid (BALF) protein content were used as a measure of lung injury. Neutrophil recruitment and activation were evaluated by BALF cellularity and myeloperoxidase (MPO) activity. RT-PCR analysis was performed in lung tissue to assess gene expression of ICAM-1. The histopathological changes were also observed using the HE staining of lung tissue. Our results showed that lung injury parameters, including the wet/dry weight ratio and protein content in BALF, were significantly higher in the LPS alone group than in the saline control group (P<0.01). In the LPS alone group, a larger number of neutrophils and greater MPO activity in cell-free BAL and lung homogenates were observed when compared with the saline control group (P<0.01). There was a significant increase in lung ICAM-1 mRNA in response to LPS challenge (P<0.01, group L versus group S). Genistein pretreatment significantly attenuated LPS-induced changes in these indices. LPS caused extensive lung damage, which was also lessened after genistein pretreatment. All above-mentioned parameters in the genistein alone group were not significantly different from those of the saline control group. It is concluded that genistein pretreatment attenuated LPS-induced lung injury in rats. This beneficial effect of genistein may involves, in part, an inhibition of neutrophilic recruitment and activity, possibly through an inhibition of lung ICAM-1 expression.
To establish a simple, sensitive and effective technique for the identification of six common dermatophytes, polymerase chain reaction (PCR) and PCR0restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene were used. The DNA of 6 common dermatophytes was amplified by primer dPsD1 and then primers dPsD2. The products generated by dPsD2 were digested with restrictiion enzymeHinc II andHinf I separately. A DNA fragment of about 3390 bp was amplified by using primer dPsD1 from the genomic DNA of each dermatophyte species. The product of dPsD2 was 2380 bp and the restriction profiles ofHinc II andHinf I were between 58–1670 bp. By using PCR-RFLP, all of the 6 dermatophytoses were diagnosed to species level and no obvious difference identification betweenHinc II andHinf I. It is concluded that the PCR-RFLP identification of dermatophytes byHinc II orHinf I is efficient and rapid in clinical practice.
The purpose of this study is to define the appearance of normal epiphyseal and metaphyseal marrow and normal changes of marrow due to fatty conversion on Gadolinium (Gd)-enhanced MR Imaging. Unenhanced and enhanced T1-weighted MR imaging were performed in proximal and distal femoral ends of 8 healthy piglets at the ages of 2, 4, 6 and 8 weeks, respectively. The changes with age in signal intensity and enhancement ratio of the epiphyseal and metaphyseal marrow with age were examined. The correlation of MRI characteristics with histological findings was studied. Our study showed that marrow of the metaphysis and of periphery of the 2nd ossification center were well vascularized hematopoietic marrow and had great enhancements. The enhancement ratio of metaphysis was greater than that of epiphyseal marrow and both enhancement ratios degraded gradually with age. The central regions of the epiphyseal ossification center and of the diaphysis were of fatty marrow and had little enhancement. It is concluded that on Gd-enhanced MR imaging the hematopoietic marrow of metaphysis and of periphery of the 2nd ossification center had greater enhancement than that of fatty marrow of central region of the 2nd ossification center. All of their enhancements decreased gradually with age.
To assess the value of echocardiography for detection of the flow-dependent epicardial coronary vasodilation, the changes in internal diameter of the left anterior descending coronary arteries (LAD) induced by reactive hyperemia were studied by echocardiography in 12 health anesthetized open-chest dogs. Reactive hyperemia was induced by brief occlusion of the left anterior descending coronary artery for 30 s followed by rapid release. The two- dimensional images of the left anterior descending coronary artery before and after reactive hyperemia with and without intracoronary infusion of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) were investigated. The internal diameter of LAD was measured and its percent change induced by reactive hyperemia was calculated. Our results showed that the internal diameter of LAD was 2.23±0.19 mm before intracoronary infusion of L-NAME (baseline). The internal diameter of LAD significantly increased to 2.52±0.24 mm (P<0.01) after reactive hyperemia at baseline, and the percent change in internal diameter of LAD was (13.10±3.59)%. The internal diameter of LAD before and after reactive hyperemia under the condition of intracoronary infusion of L-NAME was not different from that before reactive hyperemia at baseline. The percent change in internal diameter of LAD was (1.07±2.97)%, and it was significantly lower than that at baseline (P<0.001). We are led to conclude that the change in internal diameter of LAD responding to reactive hyperemia was detected sensitively by echocardiography, and this change was associated with endothelium-derived nitric oxide.
To investigate the gene mutation in a pedigree with X-linked ichthyosis (XLI) and to explore the relationship between the mutation and its clinical manifestations, genomic DNA of affected members, the normal member of the pedigree and 50 unrelated normal members was extracted with a whole blood genomic DNA extraction kit and the DNA was used as a template for the polymerase chain reaction (PCR)-mediated amplification of exon 1 and exon 10 of the STS gene. hHb6 (human hair basic keratin) gene was used as the internal control. Our results showed that the STS gene was deleted in affected members in the pedigree with X-linked ichthyosis. The normal member of the pedigree and 50 unrelated normal members had no such deletion. The proband and his mother had products in the internal control after PCR amplification. The blank control had no product. It is concluded that deletion of the STS gene existed in this pedigree with X-linked ichthyosis, and it is responsible for the unique skin lesions of X-linked ichthyosis.
To study the bioequivalence of a kind of progesterone sustained release suppository, a randomized cross-over study was conducted in 12 rabbits. A single rectal dose of 2.75 mg/kg progesterone sustained released suppository (tested formulation, T) and progesterone suppository (reference formulation, R) was administered; a multiple dose of 2.75 mg/kg was given up to seven times with an interval of 8 h. Concentrations in serum were determined by a competitive enzyme immunoassay. The main parameters of T were: for single and multiple doses, Cmax was 48.8±11.8 ng/mL and 43.5±9.4 ng/mL, Tmax was 0.5±0.3 h and 0.4±0.3 h, AUC(0–24 h) was 362.4±143 ng·h·mL−1 and 310.6±70.3 ng·h·mL−1, respectively. The relative bioavailability of T to R were (104.2±13.4)% and (111.4±19.1)%, respectively. Statistical analysis showed that the two formulations were bioequivalent and T had sustained released feature.