2,3,5,4′-tetra-hydroxystilbene-2-O-glucoside (THSG), the water-soluble active components extracted from dried tuber root ofPolygonum multi florum (Polygonaceae), can promote the release of nitric oxide (NO) from vascular endothelial cells and has strong antioxidation. The post-conditioning's protection of THSG on cardiac ischemia-reperfusion injury and the mechanism were investigated. After reperfusion for 3 h following occlusion of rat left anterior descending coronary artery (LAD) for 30 min. SαT recovery speed, arrhythmia and cardiac infarct size were observed. The ischemic size and infarct size was identified by using Evans blue and TTC staining methods re-spectively. The results showed that the infarct size in THSG 7. 5 mg/kg postconditioning group was significantly decreased from 43.6%±9.1% in mode group to 16.5%±6.5% (P<0.01). SαT recovery was quicker and the incidence of arrhythmia (55.6% vs 100%,P<0.05) was significantly lower than in control group. The infarct size in THSG+glybenclamide group was greater than in THSG group, but equivalent to that in control group (46.8%±9.8% vs 43.6%±9.1%,P>0.05), SαT recovery speed slower and the incidence of arrhythmia also lower (33.3% vs 100%,P<0.01), suggesting that glybenclamide could abolish the effects of THSG postconditioning reducing the cardiac infart size. It was concluded that THSG administration before reperfusion could effectively alleviate the cardiac reperfusion injury and possessed the postconditioning effects of reducing cardiac infarct size, which might be related with the K_ATP channel opening.

The different effects of capsaicin on I_A and I_K currents in pain-conduct neurons of trigeminal ganglia (TG) were investigated. In cultured TG neurons of rats, whole-cell patch clamp techniques were used to record the I_A and I_K before and after capsaicin perfused. Results revealed that 1 μmol/L capsaicin could inhibit the amplitude of I_A by 48.2% (n=10,P<0.05), but had no inhibitory effect on I_K (n=7,P>0.05). Ten μmol/L capsaicin could significantly inhibit the amplitude of I_A by 93.2% (n=8,P<0.01), but only slightly inhibit the amplitude of I_K by 13.2% (n=7,P<0.05). Neither 1 μmol/L nor 10 μmol/L capsaicin had effects on the active curve of I_A and I_K. It was concluded that capsaicin could selectively inhibit the I_A current, and this effect might involve in the analgesic mechanisms of capsaicin.

The possibility that a recombinant protein vaccine based on xenogenic homologous FGFR-1 of chicken induces production of autoantibodies against self-FGFR-1 in BALB/c mice was examined by using ELISA, Western blot analysis and ELISPOT assay respectively. Autoantibodies against mouse FGFR-1 were identified by Western blot analysis and ELISA. Compared with the two control groups, the number of APBCs, which were detected by ELISPOT assay, was significantly increased in the spleens of mice immunized with cFR1 (P<0.05). IgG1 and IgG2b, which were detected by ELISA, were the major subclasses and were substantially increased in response to chicken FGFR-1 when compared with control group. The recombinant chicken FGFR-1 protein used as a vaccine can induce autoantibodies against self-FGFR-1 in mice and provide a basis for the active immunotherapy of tumor angiogenesis.

The proliferating cell nuclear antigen (PCNA) gene expression was blocked and retinal pigment epithelium (RPE) proliferation was inhibited by using antisense oligonucleotides (AS-ODN) mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy (PVR). RPE cells cultured in vitro were transfected with synthetic fluoresence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN (0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides (S-ODN with the same concentrations as AS-ODN) mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by ³H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression of PCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density (AOD) was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN (0.28 and 1.12 μ mol/L) markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant (P<0.05 and P<0.01, repectively) as compared with blank control group. AOD was well correlated with CPM (r=0.975). It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

To isolate and culture adipose stromal cells (ASCs), and study the effect of cytokines secreted by ASCs on endothelial cells, human adipose tissue was digested with collagenase type I solution and ASCs were derived by culture. The cells surface phenotype was examined by flow cytometry. ELISA was used to detect the secretion of VEGF, HGF, SDF-1α and RT-PCR was employed to detect the expression of their mRNA. Then the ASC medium was utilized to culture human umbilical vein endothelial cells ECV304. Cells were counted by hemacytometer to determine the proliferation and Annexin V / PI was employed for the examination of the apoptosis rate of ECV304. ASCs were derived by culture and expressed CD34, CD105 while they did not express CD31 or CD45. ASCs secreted cytokines such as VEGF, HGF and SDF-1α so the ASC medium could stimulate proliferation and counteract apoptosis of endothelial cells (P<0.05). Bcl-2 mRNA was also found to be up-regulated in the endothelial cells. It is concluded that ASCs can secrete cytokines and has significant effect on the proliferation of endothelial cells and apoptosis.

Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs (siRNAs) targeted wild type APP (APPwt) and Swedish mutant APP (APPswe). One siRNA targeting APPwt and the other siRNA targeting APPswe were designed. All these siRNAs were endogenously expressed by siRNAs expressing plasmids. COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector. The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein (GFP). It was found that the siRNAs silenced APPwt and APPswe to different degrees. siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration of APP gene.

The relationship between Bcl-2, Bax, Fas, caspase-3 and development of hemangioma and the molecular mechanism was investigated. By using immunohistochemical S-P method, proliferating cell nuclear antigen was detected. According to the classification of Mulliken in combination with PCNA expression, 27 cases were identified as proliferating hemangioma and 22 cases as involutive hemangioma. Five normal skin tissues around the tumor tissue served as controls. By using immunohistochemical technique, the expression of Bcl-2, Bax, Fax and Caspase-3 was detected. The cells expressing Bcl-2, Bax, Fax and cappase-3 were identified as hemangioma endothelia by immunohistochemical staining of VIII factor. The average absorbance (A) and average positive area rate of Bcl-2, Bax, Fas and caspase-3 expression were measured by using HPIAS-2000 imaging analysis system. The results showed that the expression of Bcl-2 in the endothelia of proliferating hemangioma was significantly higher that in involutive degenerative hemangioma endothelia and vascular endothelia of normal skin tissue (P<0.01). The expression of Bax, Fas and Caspase-3 in the endothelia of involutive hemangioma was obviously higher than in the endothelia of proliferating hemangioma and normal skin tissue (P<0.01). The expression of BAx and Fas in endothelia of proliferating hemangioma was higher than in those of normal skin tissue (P<0.05). It was suggested that Bcl-2, Bax, Fas and caspase-3 might be involved in the development and involution of hemangioma. Bcl-2 could promote the growth of hemangioma by inhibiting apoptosis of endothelia. Bax, Fas and caspase-3 promote the switch of hemangioma from proliferation to involution by inducing the apoptosis of hemangioma endothelia.

In order to explore the value of p63, smooth muscle actin (α-SMA) and cytokeratin 5/6 (CK5/6) in the differential diagnosis of ductal lesions of breast, 88 tissue specimens of ductal lesions of breast were collected and examined histologically by HE staining. By using immunohistochemistry, the expression of p63, α-SMA and CK5/6 was detected. The results showed that in 38 cases of benign breast lesions, the proliferating cells were all positive for p63 and α-SMA. In 19 cases of ductal carcinoma in situ (DCIS) and 7 cases of intraductal papillary carcinoma, α-SMA positive cells formed a layer of continuous embroider-shaped structure and the p63 positive cells formed a layer of evenly separated embroider-shaped structure around the ducts. There was no cross-reaction between p63 and interstitial myofibroblasts and vascular smooth muscle cells. In 38 cases of benign breast lesions, the positive rate of CK5/6 expression was 100 %. In 5 cases of atypical ductal hyperplasia, there were few positive cells in the ducts. In 19 cases of CDIS, no tumor cells expressed CK5/6. In 19 cases of invasive ductal carcinoma, almost no CK5/6 was detectable. It was suggested that p63 could serve as a novel specific marker for the identification of breast myoepithelial cells. CK5/6 is of value in differentiating ductal proliferation of varying degrees, especially in the differentiation between cancerous and non-cancerous changes. Simultaneous detection of p63, CK5/6 and α-SMA can help increase the diagnostic accuracy of breast diseases.

A persistent infection model was established after human hepatoma cells infected by Japanese encephalitis viruses were subcultured for several times. Viral titers of mutant viruses in persistently infected cells were examined by plaque methods using BHK cells. Nucleotides of the E coding region of two wild and two mutant viruses were amplified by RT-PCR. PCR products were sequenced by ABI-PRSM™310 sequencing system. Compared to JaGAr-01 wild strains, four amino acids were replaced (E61Tyr→Asp, E219His→Tyr, E384Val→Glu, E418Pro→Ala) in the E sequence of JaGAr-01 persistently-infected mutant strains. Eleven amino acid replacement (E51Arg→Ser, E61Tyr→Asp, E83Lys→Glu, E123Ser→Arg, E209Arg→Lys, E227Pro→Ser, E276Asp→Ser, E290Arg→Lys, E387Lys→Arg, E418Leu→Pro, E454Arg→Gly) was also noted when we compared the E sequence between persistently infected Nakayama and its wild strains. A lot of similarities of amino acid sequence between mutant strains JaGAr-01 and Nakayama were also noted. It was concluded that geno-variation existed in E region of mutant viruses and the mutant protein encoded by E region, especially the mutation of E61 (Tyr→Asp) may contribute to the maintenance of the persistent infection of Japanese encephalitis virus.

The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs) were explored. CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34⁺ and CD34⁻ from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined. EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34⁺ and CD34⁻ and total MNC led to a significant increase in the expansion of CD34⁺ cells as compared with CD34 enrichment (P<0.05). There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis (P>0.05). These differentiated EPCs were positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34⁺ cells accounted for (68.2±6.3) % of attaching (AT) cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34⁺ and CD34⁻ or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.

The changes of expression and function of MDM2 and P53 by MDM2 specific antisense oligonucleotides were investigated in HL60 cells. Cells were divided into control group, AS group (MDM2 specific antisense oligonucleotides group), cisplatin group, and combined treatment group. FCM analysis and Western blot and RT-PCR were used to estimate apoptosis and the expression of MDM2 and P53. Our results showed that the transfection of MDM2 specific antisense oligonucleotides obviously inhibited MDM2 expression (P<0.01) and increased the expression of P53 (P<0.05). Apoptosis rate were reduced by MDM2 specific antisense oligonucleitides and cisplatin (P<0.01). It is concluded that MDM2 specific antisense oligonucletides can inhibit the expression of MDM2, induce the expression of P53 and increase the apoptosis of leukemia cells after chemotherapy.

To observe the dynamic changes of the TGF-β₁ expressed in the infarct and non-infarcted region of rat heart during the ventricular remodeling (day 3, 7, 28, 180), myocardial infarction rat model was made and relationship between the cytokine and indicator of myocardial remodeling was analyzed. After the detection of hemodynamic parameter was performed by the Powerlab devices, the size of myocardial infarction and the morphology change was detected by TTC and HE, respectively. The relative levels of mRNA of TGF-β₁, collagen type I, III, and fetal gene beta-MHC were detected by RT-PCR. The distribution of TGF-β₁ protein in the myocardium was detected by immunohistochemistry. The results showed that the size of infarction was higher than that of the sham operated groups in the infarcted group (44.5±0.5 vs 0). The difference in hemodynamic parameters between the infarcted group and sham operated group was significant (P<0.01). HE staining showed that inflammatory cells were accumulated in the infarcted region at the beginning of the 3^rd day, which lasted 4 weeks. Then, it decreased gradually. β-MHC in the non-infarcted region rose from the 3^rd day, reaching its peak at the 4^th week, and it decreased gradually. The ratio of the collagen type I/III showed similar changes as compared with the sham operated groups (P<0.01). And the relative mRNA levels in the non-infarcted group were significantly higher than that in the infarcted and sham operated group (P<0.01) at day 180. Linear regression analysis indicated that the TGF-β₁ was positively correlated with the ventricular remodeling. It was concluded that the cytokine TGF-β₁ participates in the process of the myocardial remodeling, which could be a strategy in the interference of myocardial remodeling.

To compare the efficacy, safety, and tolerability of intravenous moxifloxacin with those of a commonly used empirical antibiotic regimen, cefoperazone and azithromycin in the treatment of community acquired pneumonia (CAP) in adult patients requiring initial parenteral therapy, 40 patients with CAP were divided into two groups, a moxifloxacin group (n=20) and a control group (n=20), which were treated for 7 to 14 days. The patients in the moxifloxacin group were intravenously given 400 mg of moxifloxacin (Avelox^R) once a day. Patients in the control group were administered 2.0 g of cefoperazone twice a day and azithromycin 0.5 g once a day. Clinical, bacteriological, and laboratory examinations were performed before the treatment, and at the end of the treatment. Our results showed that there was no significant difference in the clinical efficacy rate between two treatment groups at end of therapy (90 % for moxifloxacin, 95 % for cefoperazone plus azithromycin) (P>0.05). The bacteriologic eradication rate at the end of treatment was 90% in the moxifloxacin group and 80% in the cefoperazone-plus-azithromycin group, whereas there was no significant difference between the two groups (P>0.05). In addition, both drugs were well-tolerated in this trial, with the number of drug-related adverse events being comparable. It is concluded that moxifloxacin is an effective and well-tolerated treatment for CAP and was equivalent to the commonly used empirical treatment of cefoperazone plus azithromycin. Moxifloxacin is likely to offer clinicians an alternative for reliable empirical CAP treatment in the face of increasing antibiotic resistance.

To investigate the role and mechanisms of apoptosis and apoptosis signaling pathway in 5/6 nephrectomy rat model (SN_X), the mRNA and protein levels of caspase-3,-8,-9 and apoptosis were detected by in situ end labeling (TUNEL), immunohistochemistry, RT-PCR, Western-blotting 1, 2, 4, 8, 12, 16, 26 and 40 weeks after 5/6 nephrectomy rat model was made respectively. The rats in the model group developed glomerular sclerosis and renal interstitial fibrosis. The number of the apoptototic cells in glomeruli, renal tubule and renal interstitium was remarkably higher in the model group than that in the control group (P<0.05, P<0.01). Changes of mRNA and protein level of caspase-3,-8,-9 had the same tendency and was up-regulated wavily in the rat model compared with the control group (P<0.05). Peaks in model appeared on the 4th and the 40th week respectively. The growth amplitude of caspase-9 was remarkably higher than that of caspase-8. It is concluded that the development of 5/6 nephrectomy rat model was correlated with the apoptosis of glomeruli, renal tubule and renal interstitium. Both of death receptor and mitochondria signaling path ways are involved in the process and the latter might play a primary role.

To evaluate the application of nueroelectrophysiological tests in early diagnosis of sub-clinical neuropathy in diabetes mellitus (DM), The routine nerve conductive velocity (NCV), F-wave and sympathetic skin response (SSR) were detected in 27 patients with diabetes mellitus but without symptoms and signs of lesions of nerve system. Our results showed that 48.1%, 44.4%, 51.9% of the patients were found to have abnormal NCV, F-wave and SSR respectively. The abnormalities were mainly characterized by prolonged latency, reduced velocity and absence of wave-form. There were significant differences between the controls and the DM group (P<0.05). Both the distal and proximal segments of nerves were affected and the distal lesions took place earlier than proximal ones and the changes in low extremities were more severe than those of upper extremities. F-wave can be used as a sensitive indicator for the early diagnosis of peripheral neuropathy and it can help to detect the subclincial lesions. SSR can be used for the evaluation of functional status of autonomic nerves in DM patients.

To investigate the effect of preceding naloxone injection into the third cerebroventricle or acute subdiaphragmatic vagotomy on the gastric acid secretion inhibited by the somatostatin analogue octreotide given by intracerebroventricular (icv) injection. The third ventricles were cannulated in male Wistar rats anesthetized with sodium pentobarbital. One week later, acute gastric lumen perfusion was carried out. The gastric perfusion samples were collected every 10 min and were titrated by 0.01 mol/L NaOH to neuter. On the basis of subcutaneous injection of pentagastrin (G-5, 160 μ g/kg), icv injection of physiological saline (group A, n=20), icv injection of octreotide (0.05 μ g) (group B, n=20), icv injection of naloxone (2.5 μ g)+octreotide (0.05 μ g) (group C, n=20), acute subdiaphragmatic vagotomy+ icv injection of physiological saline (group D, n=20), or acute subdiaphragmatic vagotomy+icv injection of octreotide (0.05 μ g) (group E, n=20) were conducted. Before and after icv injection, 1-h total acid output (TAO) was determined and compared. The experimental data were expressed in change rate (%) of TAO. The change rates (%) of TAO were 4.60% in group A, −20.35% in group B, −18.06% in group C, 5.01% in group D and −21.59% in group E, respectively. Comparison of group B or C versus group A showed that P<0.01 and comparison between the group E versus group D showed that P<0.01. Whereas the differences between group C and group B, group E and group B were not statistically significant (P>0.05 for all). The results indicate that the central inhibition of gastric acid secretion by octreotide may not be mediated by the endogenous opiate substance or its receptor and the peripheral pathway for icv injection of octreotide to suppress gastric acid secretion is via extra-vagus route.

To systematically evaluate the importance of protein synthesis in ischemic preconditioning (PC)-induced ischemic tolerance (IT), temporary middle cerebral artery occlusion (MCAO) by Longa (20 min) was used for PC (ischemic precondioning). Twenty-four hours of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance (IT) to compare with non-PC (sham-operated) rats (n=5 for each group). Infarct size and neurological deficits were measured 24 h after PMCAO. Samples of brain were taken for the determination of HSP70 expression by Western blot analysis. The effects of the protein synthesis inhibitor cycloheximide administered just before PC or administered long after PC but just before PMCAO on IT were also determined (n=5 for each group). Our results showed that hemispheric infarct was significantly reduced (P<0.01) only if PC was performed after 24 h, and PC significantly (P<0.05) reduced neurological deficits (similar to reductions in infarct size). Cycloheximide eliminated ischemic PC-induced IT effects on both brain injury and neurological deficits if administered before PC but not if administered long after PC but before PMCAO. PC produced no brain injury but did increase HSP70 protein 24 h after PC. Cycloheximide eliminated that effect. The results suggest that PC is a powerful inducer of ischemic brain tolerance as reflected by the preservation of brain tissue and motor function. PC induces IT that is dependent on de novo protein synthesis.

To examine the changes in erythropoietin (Epo) protein and its mRNA expression in rat brain subjected to focal ischemia and possible mechanism of the preconditioning of mitochondrial toxin 3-nitropropionic acid (3-NPA), rats were administrated either vehicle or 3-NPA at a dose of 20 mg/kg, intraperitoneally (ip), 3 days prior to a 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Infarct volumes were measured by using 2, 3, 5 triphenylte trazolinm chloride (TTC) staining, and Epo protein and its mRNA levels were assessed by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Our results showed that after reperfusion, Epo was found to be expressed extensively in the rat brain. It was most apparent in the basal nuclei and hippocampus, and was, to some extent, present in cortex. Preconditioning with 3-NPA caused a reduction in infarct volume. The expression of both Epo protein and mRNA increased significantly in the different brain areas in the 3-NPA pretreated group as compared with the non-pretreated ischemia model group. These results suggested that preconditioning with low dose 3-NPA could induce ischemic tolerance and neuro-protective effects by increasing the Epo expression in the ischemic and ischemia-related areas.

In order to investigate the correlation between protein expression of PTEN and the proliferation, infiltration, metastasis and prognosis in pancreatic cancer, immunohistochemical SP method was used to examine the protein expression of PTEN, PCNA, MVD, MMP-2, MMP-9 and TUNEL method to detect the levels of apoptosis of pancreatic cells in 41 pancreatic head cancers from regional pancreatectomy (RP) and 10 normal pancreatic tissues. The results showed that among 41 cases of pancreatic cancers, the positive staining of PTNE (39.02 %) was significantly weaker than that in normal pancreatic tissues (P<0.05). The levels of PCNA labeling index (LI), apoptotic index (AI), microvessel density (MVD), MMP-2 LI and MMP-9 LI were decreased gradually with the increase of the expression intensity of PTEN, and there was a significant difference in the above parameters among the patients having different expression levels of PTEN (P<0.01 or P<0.05). There was a negative correlation between the expression of PTEN and PCNA LI, MVD, MMP-2 LI, MMP-9 LI, and a positive correlation between AI and the expression of PTEN. The expression intensity of PTEN was correlated with the postoperative survival of the patients with pancreatic cancer (x²=22.3400, P<0.0001, RR=2.030). It was suggested that the expression levels of PTEN protein were closely related with proliferation, infiltration and metastasis in human pancreatic cancer, and the expression of PTEN protein was one of the prognostic factors for pancreatic cancer following RP.

To study the role and mechanisms of hypoxia-inducible factor-1alpha (HIF-1α) on the growth and tumorigenicity of lung cancer cells A549, the antisense oligonucleotide of HIF-1α was transfected to A549 cells. The effect of the antisense oligonucleotide on tumor growth in vitro and in vivo was evaluated by the growth rate suppression of A549 cells and subcutaneous implanted tumor in nude mice, and the effect on tumorigenicity was evaluated by the expression inhibition of angiogenic factors, the microvessel density (MVD) and vascular endothelial growth factor (VEGF) protein expression which were detected by immohistochemistry and western blot respectively. This study revealed that in vitro the growth rate of antisense oligonucleotide group was significantly decreased as compared with that of control group, sense oligonucleotide group and false-sense oligonucleotide group; in vivo the weight of implanted tumors in nude mice of antisense oligonucleotide group was 1.51±0.40 g, which was significantly lower than that of control group (2.79±0.33 g), sense oligonucleotide group (2.81±0.45g) and false-sense oligonucleotide group (2.89±0.39 g) and the inhibitory rate was 47 %. Both MVD and VEGF protein expression were significantly inhibited in antisense oligonucleotide group compared with those in other groups. These results indicated that antisense oligonucleotide of HIF-1α could inhibit lung cancer cells A549 growth in vitro and in vivo, and the mechanism may be due to the inhibition of vascular growth and VEGF protein expression.

To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to dtect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

Phrenic nerve injury after cardiac surgery increases postoperative pulmonary complications. The purpose of this study was to analyze the causes and effects of phrenic nerve injury after cardiac surgery. Prospectively collected data on 2084 consecutive patients who underwent cardiac surgery from Jan. 1995 to Feb. 2002 were analyzed. Twenty-eight preoperative and operation related variables were subjected to logistic analysis with the end point being phrenic nerve injury. Then phrenic nerve injury and 6 perioperative morbidities were included in the analysis as variables to determine their independent predictive value for perioperative pulmonary morbidity. An identical approach was used to identify the independent risk factors for perioperative mortality. There were 53 phrenic nerve injuries (2.5 %). There was no phrenic nerve injury in non-coronary surgery or coronary surgery using conduits other than the internal mammary artery. The independent risk factors for phrenic nerve injury were the use of internal mammary artery (Odds ratio (OR)=14.5) and the presence of chronic obstructive pulmonary disease (OR=2.9). Phrenic nerve injury was an independent risk factor (OR=8.1) for perioperative pulmonary morbidities but not for perioperative mortality. Use of semi-skeletonized internal mammary artery harvesting technique and drawing attention to possible vascular or mechanical causes of phrenic nerve injury may reduce its occurrence. Unilateral phrenic nerve injury, although rarely life-threatening, is an independent risk factor for postoperative respiratory complications. When harvesting internal mammary arteries, it should be kept in mind avoiding stretching, compromising, or inadvertently dissecting phrenic nerve is as important as avoiding damage of internal mammary artery itself.

To further investigate the mechanisms of action of icariin (ICA), we assessed the effects of ICA on the in vitro formation of cGMP and cAMP in isolated rabbit corpus cavernosum. Isolated segments of rabbit corpus cavernosum were exposed to increasing concentrations of ICA and the dose-dependent accumulation of cGMP and cAMP was determined in the tissues samples by means of ¹²⁵I radioimmunoassay. Responses of the isolated tissues preparations to ICA were compared with those obtained with the reference compounds sildenafil (Sild). Furthermore, the effects of ICA on the mRNA expression of specific cGMP-binding phosphodiesterase type V (PDE5) in rat penis were also observed. After incubation with ICA for 6 h or 14 h respectively, the levels of PDE5 mRNA were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that ICA increased cGMP concentrations directly (P<0.05), but there was no significant effect on cAMP concentrations (P<0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both ICA and Sild increased cGMP concentrations with increasing dose (P<0.01). Their EC₅₀ was 4.62 (ICA) and 0.42 (Sild) μ mol/L respectively. Under the same condition, ICA and Sild unaltered cAMP level significantly (P>0.05). There were PDE5A1 and PDE5A2 mRNA expressions in rat corpus cavernosum with PDE5A2 being the dominant isoform. ICA could obviously inhibit these two isoforms mRNA expression in rat penis, and decrease PDE5A1 more pronouncedly (P<0.01). The present study indicated that the aphrodisiac mechanisms of icariin involved the NO-cGMP signal transduction pathway, with increasing cGMP levels in the corpus cavernosum smooth muscle. The inhibitory effect of icariin on PDE5 mRNA expression, especially on PDE5A1, might account for its molecular mechanisms for its long-term activity.

In order to explore the effect of high glucose concentration and high glucose concentration with hypoxia on the production of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), human RPE cells were cultured in 5.56 mmol/L glucose (control group), 5.56 mmol/L glucose with 150 μ mol/L CoCl₂ (hypoxic group), 25 mmol/L glucose (high glucose group) and 25 mmol/L glucose with 150 μ mol/L CoCl₂ (combination group). RT-PCR was used to detect the expression of HIF-1α and VEGF mRNAs. Western blot analysis was used to measure the levels of HIF-1α and VEGF proteins. Although the small amount of HIF-1α protein was able to be detected in high glucose group but not in control group, there was no significant difference between the expression of HIF-1α mRNA of RPE cells in high glucose group and that of RPE cells in control group. As compared with RPE cells in control group, the mRNA expression and the protein synthesis of VEGF in high glucose group were up-regulated. As compared with RPE cells in hypoxic group, the expression of HIF-1α mRNA of RPE cells in combination group was not different, but the protein synthesis of HIF-1α, the mRNA expression and the protein synthesis of VEGF were more obviously up-regulated. In conclusion, high concentration glucose mainly influence the protein synthesis of HIF-1α of RPE cell, and HIF-1α protein is able to be accumulated in high concentration glucose. Under hypoxia, the HIF-1α protein induced by high concentration glucose is more stable, and the expression of VEGF is obviously increased. It is suggested that high concentration glucose may play a role in retinal neovascularization, especially at ischemia stage of diabetic retinopathy.

In this study, we investigated the expression of CXCL12 (SDF-1)/CXCR4 in trophoblasts and the role they play in the gestation. Immunochemistry was used to detect the expression of CXCR4 and CXCL12 in human villi and placenta. Highly purified extra-villous trophoblasts (EVTs) ere detected for CXCR4 and CXCL12 in vitro by immunocytochemistry. The chemotaxis of CXCL12 was tested in transwell and the chemotactic activity was quantitatively examined. It was suggested that both CXCR4 and CXCL12 were expressed in trophoblasts and were decreased with the gestation time P<0.05). In a certain coverage, CXCL12 exhibited chemotactic activity which was positively correlated with its concentration [(r)=0.68, P<0.01], the maximum chemotactic index (CI) was 1.62±0.12. Our results suggest that interaction between CXCR4 and CXCL12 is involved in materno-fetal immunological tolerance in all three trimesters of gestation and contributes to the invasion of EVTs during pregnancy.

This study examined the inhibitory effect of topical cyclosporine (CsA) treatment on conjunctiva epithelial apoptosis in a murine model of xerophthalamia. Dry eye was induced in 3 groups of C57BL6 mice by subcutaneous injection of scopolamine (t.i.d) and exposure to an air draft and low-humidity environment for 16 h each day for 12 days. The dry eye control group received no topical treatment; another group received 1 μL of 0.05 % CsA topically (t.i.d, dry eye+CsA); and the third group received 1 μL of the castor oil vehicle of CsA topically (t.i.d, dry eye + vehicle). Normal mice were used as untreated controls. Twelve days later, the mice were killed, and their conjunctivas were excised. The number of the conjunctival goblet cells was counted in tissue sections stained with periodic acid Schiff (PAS) reagent. Their conjunctiva epithelium had been investigated by immuno-histochemical staining to detect the goblet cells and the expression of Caspase-3, Bax and bcl-2. Our results showed that compared with dry eye control and dry eye mice + vehicle groups, the number of conjunctival epithelial goblet cells was significantly greater in the untreated controls and dry eye mice receiving CsA (P < 0.01 for both groups). There was no significant difference in the number of conjunctival epithelial goblet cells between the dry eye control and dry eye+vehicle group. It was also true of the number of conjunctival epithelial goblet cells when comparison was made between the normal group and the dry eye+CsA group. Expressions of Caspas-3 and Bax were increased and ex-pression of bcl-2 was decreased in conjunctival epithelial cells in dry eye control and dry eye mice+vehicle groups. There was a significant positive correlation between goblet cell number and the number of cells that expressed bcl-2, and a negative correlation between goblet cells and Caspase-3 and Bax expression. It is concluded that the topical use of CsA could significantly reduce conjuncti-val epithelial apoptosis and protect goblet cell against the loss in experimental murine xerophathala-mia. Inhibition of apoptosis appears to be a key mechanism responsible for the therapeutic effect of CsA on xerophthalamia.

To describe and evaluate a refraction-derived method and a clinically derived method to calculate the correct corneal power for intraocular lens (IOL) power calculations after laser in situ keratomileusis (LASIK) and to compare the results to the commonly used history-derived method. Retrospective analysis were conducted in consecutive case from clinical practice. For each patient, we established the pre-LASIK and post-LASIK spectacle refraction, the pre-LASIK (K_pre) and post-LASIK K readings (K_post). We then calculated the pre-and post-LASIK refraction at the corneal plane and the amount of correction obtained by the refraction surgery (ΔSEQco). The cases were divided into two groups. Group I was used to derive two formulas. The values obtained with the two methods were compared with the K by history-derived method (K_c.hd) in group II to validate the results. The K values calculated by using the refraction-derived method (K_c.rd) and the K values calculated using the clinically derived method (K_c.cd) correlated highly with K_c.hd. The correct corneal power for intraocular lens (IOL) power calculations after LASIK can use refraction-derived method and clinically derived method instead of history-derived method when some refractive parameters are not available.

To compare and evaluate two methodologies, entire-sampling and micro-sampling for the harvesting of vitreous humor, the vitreous humor of rabbits were sampled with the two methods respectively, and the concentrations of calcium, chlorine, potassium, sodium and phosphorus of the were measured. The results showed that the differences in the variance coefficient and two-eye concentrations of micro-sampled specimens were less than those of the entire-sampled specimens. In the micro-sampling group, the concentrations of repeated micro-sampling showed no differences among different groups (P>0.05) and the intra-ocular fluid dynamics did not have significant influence on post-mortem sampling. The sampling technique may affect the concentrations of specimen collected. Our study suggests that micro-sampling is less influenced by the human factor and is reliable, reproducible, and more suitable for forensic investigation.

Morphine has been reported to suppress human immune response. We aimed to observe the effects of morphine, fentanyl and tramadol on NF-κB and IL-2 from both laboratory and clinical perspective. Jurkat cells were incubated with ten times clinically relevant concentrations of morphine, fentanyl and tramadol before being stimulated with PMA. NF-κB binding activity and IL-2 levels were measured. In the clinical study, 150 consenting patients were randomized into 3 groups according to the analgesics used in them, namely, group morphine (M), group fentanyl (F) and group tramadol (T). IL-2 was measured preoperatively and 1, 3 and 24 h after operation. Consequently, NF-κB activation was suppressed by morphine and fentanyl but not by tramadol. IL-2 was significantly decreased by morphine and fentanyl but not by tramadol in vitro. In the PCA patients, IL-2 was decreased in group M and increased in group F postoperatively. Whereas in group T, IL-2 was unchanged 1 h after operation but was significantly elevated 3 and 24 h after operation. Our results showed that the inhibition of morphine on IL-2 was most probably related to its suppression on NF-κB. Fentanyl had different effects on human immune response in vitro and in vivo. Tramadol may have immune enhancing effect.

The purpose of this study is to demonstrate if Gadolinium-enhanced MRI can detect early reversible ischemia of the femoral head epiphysis caused by hip hyper-abduction in piglets. Between 3 and 6 h consistent hyper-abduction, gadolinium-enhanced MRI was performed in 20 femoral heads of 10 piglets. After completion of MRI scan, the piglets were allowed to ambulate freely for 1 or 7 days and re-imaged. The enhanced-MRI results of epiphyseal and physeal cartilage and the secondary center of ossification were observed. MRI appearances and histological findings were compared. On Gadolinium-enhanced MRI, decreased or absent enhancement was seen in 14 cartilaginous epiphyses of all 20 femoral heads. Reperfusion was completed in 10 of 14 femoral heads after one day of ambulation and in the rest 4 after 7 days of ambulation. Gadolinium-enhanced MRI can identify early ischemia and its reversal of the capital femoral epiphysis induced by hip hyper-abduction.

To investigate the value of the guidance of three dimensional (3-D) reconstruction of multi-slice spiral CT (MSCT) for the placement of pedicle screws, the 3-D anatomical data of the thoracic pedicles were measured by MSCT in two embalmed human cadaveric thoracic pedicles spines (T₁−T₁₀) to guide the insertion of pedicle screws. After pulling the screws out, the pathways were filled with contrast media. The PW, PH, TSA and SSA of developed pathways were measured on the CT images and they were also measured on the real objects by caliper and goniometer. Analysis of variance demonstrated that the difference between the CT scans and real objects had no statistical significance (P>0.05). Moreover, the difference between pedicle axis and developed pathway also had no statistical significance (P>0.05). The data obtained from 3-D reconstruction of MSCT demonstrated that individualized standards, are not only accurate but also helpful for the successful placement of pedicle screws.

In this study we implanted magnetically labeled neural stem cells (NSCs) in PD rats and then monitored their survival and migration in the host brain by magnetic resonance imaging (MRI). The mesencephalic NSCs were obtained from the brain of SD rats. Superparamagnetic iron oxide (SPIO) was transferred to NSCs by Lipofectamine transfection. Eighteen PD lesioned rats were selected for transplantation by evaluation of their rotational behavior in response to amphetamine and randomly assigned to 3 groups, i.e., sham group, PBS group and NSCs transplanted group, with 6 rats in each group. MR scanning was performed at 1, 2, 4, 6, 8 and 10 week(s) following transplantation. At the meantime, rotational behavior was assessed in each group. Our results showed that SPIO particles were clearly visible with Prissian blue staining in neurospheres and cells derived from NSCs. The rotational behavior of the NSCs transplanted group was remarkably improved compared with that of sham group and PBS group (P<0.05). In vivo MR tracking of NSCs showed that SPIO labeling led to a strong susceptibility change of signal 1 week after transplantation on T₂ weighted images. And a large circular hypointense signal appeared in the transplanted area on T₂* gradient echo images. Ten weeks following transplantation, the hypointense signal on T₂ weighted and T₂* gradient echo images was still displayed. It is concluded that SPIO particles could label NSCs effectively, and MRI detection of SPIO labeled cells is a promising method and novel approach to analyzing the NSCs following transplantation in the treatment of PD.