The predictive effect of overexpressed miR-34a on good survival of cancer patients: a systematic review and meta-analysis.

BACKGROUND: MicroRNA-34a (miR-34a) is a potential prognostic factor for survival in patients with several types of cancer according to previous clinical researches. We conducted a systematic review and meta-analysis to summarize the significance of increased miR-34a expression in the prognosis of patients' overall survival. MATERIALS AND METHODS: The present systematic review and meta-analysis of 15 researches included 2,597 patients. Overexpression of miR-34a may predict good overall survival ([OS], HR =0.76, 95% confidence interval: 0.55-1.06, P=0.105), but the effect was not significant enough. Subgroup analysis results showed miR-34a was an ideal predictor for digestive system cancer (OS, HR =0.50, 95% confidence interval: 0.25-0.99, P=0.048). The predictive effects of elevated expression of miR-34a on the OS of untreated and treated patients were not of obvious differences. CONCLUSION: This systematic review and meta-analysis showed that miR-34a has a predictive effect on overall survival of patients with digestive system cancer.

Clinical analysis of 156 cases of multiple organ failure caused by fish bile.

OBJECTIVE: To analyze the clinical features and prognosis of patients suffering from fish bile poisoning. METHODS: A total of 156 multiple organ failure (MOF) patients caused by fish bile poisoning were hospitalized in our department over the past 28 years. The patients' symptoms, examination results, treatment and outcomes were collected and analyzed. RESULTS: All patients' first symptom was gastrointestinal discomfort, including unbearable nausea and intractable vomiting. The symptoms that followed were oliguria or anuria, edema, headache, fatigue, jaundice, palpitation, alimentary tract hemorrhage, gross hematuria, dyspnea, shock, tachycardia, bradycardia, arrhythmia, coma, and cardiac arrest. The symptom severity and cohort were different among different patients. Twenty-one cases received gastroscopy, which exhibited diffuse gastric mucosal bleeding. Twelve patients received renal biopsy, which exhibited focal necrosis of tubular epithelial cells. One patient received a liver biopsy, which exhibited extensive hepatocyte necrosis. All patients received blood purification therapy. Of the four patients who died, 4 out of 5 organs had failed. The general mortality rate was 2.6%. CONCLUSIONS: Compared with the MOF caused by trauma and sepsis, the fish bile poisoning MOF has a much lower morality rate. However, patients with higher age, more underlying diseases, and more organ failure tended to have a worse prognosis.

Elevated expression of miR-210 predicts poor survival of cancer patients: a systematic review and meta-analysis.

BACKGROUND: MiRNAs are important regulators of different biological processes, including tumorigenesis. MiR-210 is a potential prognostic factor for survival in patients with cancer according to previous clinical researches. We conducted a systematic review and meta-analysis to summarize the significance of increased miR-210 expression in the prognosis of indicated cancers. METHODOLOGY/PRINCIPAL FINDINGS: The present systematic review and meta-analysis of 16 researches included 1809 patients with 7 different types of cancers from 7 countries, and aimed to explore the association between miR-210 expression and the survival of cancer patients. Over-expression of miR-210 may predict poor overall survival (OS, HR = 1.33, 95% CI: 0.85-2.09, P = 0.210), but the effect was not significant. While the predictive effect on disease-free survival (DFS, HR = 1.89, 95% CI: 1.30-2.74, P = 0.001), progression-free survival (PFS, HR = 1.20, 95% CI: 1.05-1.38, P = 0.007) and relapse-free survival(RFS, HR = 4.42, 95% CI: 2.14-9.15, P = 0.000) for patients with breast cancer, primary head and neck squamous cell carcinoma (HNSCC), renal cancer, soft-tissue sarcoma, pediatric osteosarcoma, bladder cancer or glioblastoma was certain. Subgroup analysis showed the limited predictive effect of over-expressed miR-210 on breast cancer OS (HR = 1.63, 95% CI: 0.47-5.67, P = 0.443), breast cancer DFS (HR = 2.03, 95% CI: 0.90-4.57, P = 0.088), sarcoma OS (HR = 1.24, 95% CI: 0.20-7.89, P = 0.818) and renal cancer OS (HR = 1.16, 95% CI: 0.27-4.94, P = 0.842). CONCLUSIONS/SIGNIFICANCE: This systematic review and meta-analysis suggests that miR-210 has a predictive effect on survival of patients with studied cancer types as indexed by disease-free survival, progression-free survival and relapse-free survival. While the predictive effect on overall survival, breast cancer overall survival, breast cancer disease-free survival, sarcoma overall survival and renal cancer overall survival was not statistically significant.

Efficacy and safety of Abelmoschus manihot for primary glomerular disease: a prospective, multicenter randomized controlled clinical trial.

BACKGROUND: Abelmoschus manihot, a single medicament of traditional Chinese medicine, has been widely used to treat kidney disease. This is the first randomized controlled clinical trial to assess its efficacy and safety in patients with primary glomerular disease. STUDY DESIGN: Prospective, open-label, multicenter, randomized, controlled, clinical trial. SETTING & PARTICIPANTS: From May 2010 to October 2011, a total of 417 patients with biopsy-proven primary glomerular disease from 26 hospitals participated in the study. INTERVENTIONS: A manihot in the form of a huangkui capsule, 2.5 g, 3 times per day; losartan potassium, 50mg/d; or combined treatment, a huangkui capsule at 2.5 g 3 times per day, was combined with losartan potassium, 50mg/d. The duration of intervention was 24 weeks. OUTCOMES & MEASUREMENTS: The primary outcome was change in 24-hour proteinuria from baseline after treatment. Change in estimated glomerular filtration rate (eGFR) from baseline after treatment was a secondary outcome. The 24-hour proteinuria was measured every 4 weeks and eGFR was measured at 0, 4, 12, and 24 weeks. RESULTS: Mean baseline urine protein excretion was 1,045, 1,084, and 1,073 mg/d in the A manihot, losartan, and combined groups, respectively, and mean eGFR was 108, 106, and 106 mL/min/1.73 m2, respectively. After 24 weeks of treatment, mean changes in proteinuria were protein excretion of -508, -376, and -545 mg/d, respectively (P=0.003 for A manihot vs losartan and P<0.001 for the combined treatment vs losartan). Mean eGFR did not change significantly. The incidence of adverse reactions was not different among the 3 groups (P>0.05), and there were no severe adverse events in any group. LIMITATIONS: Results cannot be generalized to those with nephrotic syndrome or reduced eGFR. CONCLUSIONS: A manihot is a promising therapy for patients with primary kidney disease (chronic kidney disease stages 1-2) with moderate proteinuria.

Costimulatory molecule VSIG4 exclusively expressed on macrophages alleviates renal tubulointerstitial injury in VSIG4 KO mice.

BACKGROUND: Activation and infiltration of T cells and macrophages are key features of renal tubulointerstitial injury. The costimulatory molecule V-set and immunoglobulin domain-containing protein-4 (VSIG4), which is exclusively expressed on macrophages, is capable of inhibiting the T cell response. However, it is unclear whether VSIG4 is involved in renal tubulointerstitial injury. This study was designed to investigate the role of VSIG4 in renal tubulointerstitial injury and the related T cell infiltration. METHODS: The unilateral ureteric obstruction (UUO) model of renal inflammation and tubulointerstitial fibrosis was established in VSIG4 transgenic knock-out C57BL/6 mice (VSIG4(-/-)) and wild-type C57BL/6 mice (VSIG4(+/+)). Comparative analysis of renal biological indices were assessed by quantitative real-time PCR and immunofluorescence staining. RESULTS: Both the VSIG4(-/-) and VSIG4(+/+) mice showed UUO-related temporal changes in renal expression of CD3, CD4 and CD8 T cell markers, with the protein levels being significantly lower in the VSIG4(+/+) UUO mice. Moreover, at each time point examined the UUO VSIG4(+/+) mice showed significantly lower renal mRNA levels of the cytokines interleukin (IL)-2, interferon- and tumor necrosis factor-, but significantly higher IL-10, than the UUO VSIG4(-/-) mice. CONCLUSIONS: The macrophage-expressed VSIG4 may act to alleviate renal tubulointerstitial injury via inhibition of T cell infiltration and secretion of inflammation related factors.

The influence of cathelicidin LL37 in human anti-neutrophils cytoplasmic antibody (ANCA)-associated vasculitis.

INTRODUCTION: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterised by the autoinflammation and necrosis of blood vessel walls. The renal involvement is commonly characterised by a pauci-immune crescentic glomerulonephritis (PiCGN) with a very rapid decline in renal function. Cathelicidin LL37, an endogenous antimicrobial peptide, has recently been implicated in the pathogenesis of autoimmune diseases. To assess whether serum LL37 reflects renal crescentic formation, we measured the serum levels of LL37 in AAV patients with and without crescentic glomerulonephritis (crescentic GN) as compared to healthy controls (HCs). We also analysed the correlation of the serum levels of LL37 and interferon-α (IFN-α) with the clinical characteristics of the patients. METHODS: The study population consisted of 85 AAV patients and 51 HCs. In 40 ANCA-positive patients, a parallel analysis was performed, including the assessment of LL37 and IFN-α levels in the serum and renal biopsies. Of those studied, 15 AAV patients had biopsy-proven crescentic GN, and 25 AAV patients lacked crescent formation. The serum levels of cathelicidin LL37 and IFN-α were both measured by ELISA, and the clinical and serological parameters were assessed according to routine procedures. Immunofluorescence staining was performed on frozen sections of kidney needle biopsies from AAV patients with crescentic GN. RESULTS: The serum levels of LL37 and IFN-α were significantly increased in AAV patients with crescentic GN compared to AAV patients without crescentic formation and HCs, and patients with high LL37 and IFN-α levels were more likely to be in the crescentic GN group. The LL37 levels were positively correlated with the IFN-α levels, and both LL37 and IFN-α levels showed a positive correlation with serum creatinine and no correlation with complement C3. The renal tissue of crescentic GN patients showed expression of LL37 and IFN-α at the Bowman's capsule and extracellular sites, suggesting the active release of LL37 and IFN-α. CONCLUSIONS: Significantly higher levels of LL-37 and IFN-α were observed in AAV patients, particularly those with crescentic formation, and LL37 and IFN-α were expressed in the renal tissue of patients with crescentic GN. These data suggest that serum levels of LL37 and IFN-α may reflect both local renal inflammation and systemic inflammation.

Efficacy and safety of traditional chinese medicine (Shenqi particle) for patients with idiopathic membranous nephropathy: a multicenter randomized controlled clinical trial.

BACKGROUND: To compare the safety and efficacy of the traditional Chinese medicine Shenqi particle and standard therapy with prednisone and cyclophosphamide (control) in adult patients with idiopathic membranous nephropathy (IMN). STUDY DESIGN: Open-label, multicenter, parallel, randomized, controlled clinical trial. SETTING & PARTICIPANTS: From April 2008 to February 2011, a total of 190 patients with biopsy-proven IMN from 7 hospitals in China participated in the study. All patients had nephrotic syndrome with estimated glomerular filtration rate (eGFR) >30 mL/min/1.73 m(2). INTERVENTION: Shenqi particle (9.6 g 3 times per day) or prednisone (1 mg/kg/d tapering to 0.17 mg/kg/d) and cyclophosphamide (total dose of 9-12 g per square meter of body surface area) for 48 weeks. OUTCOMES: Primary outcomes included complete remission, defined as proteinuria (24-hour urine protein excretion) ≤0.3 g/d, or partial remission, defined as proteinuria with protein excretion >0.3-<3.5 g/d and a 50% reduction from its peak value at 48 weeks. Secondary outcomes included serum albumin level, eGFR, doubling of serum creatinine level, end-stage renal disease, and death. RESULTS: Baseline values for proteinuria and eGFR were 5.34 ± 2.74 g/d and 84.0 ± 27.4 mL/min/1.73 m(2) for the Shenqi particle group and 5.33 ± 2.47 g/d and 83.8 ± 24.9 mL/min/1.73 m(2) for the control group, respectively. 132 patients (63 Shenqi particle group, 69 control group) completed the study. Change in urinary protein excretion in the Shenqi particle group was -3.01 (95% CI, -3.68 to -2.34) g/d, and in the control group, -3.28 (95% CI, -3.98 to -2.58) g/d; the mean difference between groups was 0.27 (95% CI, -0.70 to 1.23) g/d (P = 0.6). Changes in eGFR were 12.3 (95% CI, 4.99 to 19.6) mL/min/1.73 m(2) in the Shenqi particle group and -2.8 (95% CI, -10.32 to 4.77) mL/min/1.73 m(2) in the control group; the mean difference between groups was 15.1 (95% CI, 4.56 to 25.55) mL/min/1.73 m(2) (P = 0.005). Severe adverse events occurred in only the control group (14.5%) and included lung infection, liver injury, and pneumonia. LIMITATIONS: High rate of loss to follow-up and lack of observation period prior to the study. CONCLUSIONS: Shenqi particle may be a promising alternative therapy for adults with IMN and nephrotic syndrome.

Computational prediction and experimental assessment of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from neutral endopeptidase.

Neutral endopeptidase (NEP) is the first target antigen identified on podocytes in human membranous nephropathy (MN). Cytotoxic T lymphocytes (CTLs) are considered essential for glomerular destruction in MN model. The aim of this study was to show that the CTL epitopes of NEP could be used to design more effective and better tolerated therapies. The CTL epitopes of NEP were screened using the long-distance prediction system SYFPEITHI and the Bioinformatics and Molecular Analysis Section of the MHC Peptide Binding Predictions program. Peptides were synthesized and immunoreactivity was assessed by peptide-MHC-binding affinity assay, cytotoxicity assay and HLA-A2.1/Kb transgenic mice immunization. Five candidates were identified according to the high scores generated by the computer predicting system. Peptide NEP(375-383) (FIMDLVSSL), which up-regulated HLA-A2.1 molecular expression, showed a high affinity to HLA-A2.1, whereas NEP(268-276), NEP(297--305) and NEP(492-500) (QLALEMNKV, MLLYNKMRL and KLNNEYLEL) showed a moderate affinity and NEP(559-567) (ILQPPFFSA) only had a low affinity. Cytotoxicity assay further showed that NEP(268-276) and NEP(375-383) could induce NEP-specific CTL responses in vitro. Unexpectedly, we found that a single CTL epitope, NEP(375-383), could induce proteinuria and glomerular injury in HLA-A2.1/K(b) transgenic mice in vivo. HLA-A*0201-restricted CTL epitope NEP(375-383) can serve as a potential candidate for designing MN vaccine.

Uraemic serum induces dysfunction of vascular endothelial cells: role of ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway (UPP) has been indicated to contribute to dysfunction of endothelial cells (ECs). Nevertheless, the relationship between UPP and vascular complications of uraemia remains unknown. We aimed to determine whether the UPP is activated in vascular ECs when cultured with uraemic serum, and to examine the role of the UPP on dysfunction of ECs in uraemia. Rabbit aortic endothelial cells (RAECs) were cultured with normal serum or different concentrations of uraemic serum. The expression of the ubiquitin-activating enzyme (E1), an indicator of the UPP, was detected by real-time RT-PCR and Western blot; proteasome activity was determined by fluorescence spectrophotometry; and nuclear factor-κB (NF-κB) activity and expression, as well as tumour necrosis factor-α (TNF-α) expression, were also detected. We found that the expression of E1 and the activities of three kinds of proteasomes were increased significantly in RAECs after incubation with uraemic serum. Proliferation of RAECs was increased significantly by incubation with 3-15% uraemic serum but decreased markedly when incubated with uraemic serum above 15% (increased apoptosis). Incubation of RAECs with uraemic serum induced increased NF-B DNA-binding activity and nuclear translocation of NF-κB, decreased nitric oxide production and increased expression of TNF-α, which is the final effector of inflammatory activation of cells. All of these responses in RAECs were suppressed by the specific proteasome inhibitor, MG132. The inhibition of inflammatory responses by MG132 was further supported by a parallel experiment with pyrrolidine dithiocarbamate, a specific inhibitor of κNF-B. These findings suggest that the UPP was activated in RAECs by administration of uraemic serum, and played a pivotal role in the dysfunction of vascular ECs, such as inflammatory activation.

Conditioned medium from renal tubular epithelial cells stimulated by hypoxia influences rat bone marrow-derived endothelial progenitor cells.

BACKGROUND AND AIMS: It has been well documented that endothelial progenitor cells (EPCs) participate in neovascularization in adults and that rarefaction of peritubular capillaries (PTCs) is closely associated with progressive kidney disease. Therefore, we investigated whether EPCs were influenced by conditioned medium (CM) of renal tubular epithelial cells (RTECs) stimulated by hypoxia, to provide evidence for EPCs transplantation in vivo in the future. METHODS: Mononuclear cells of rat bone marrow were isolated by density gradient centrifugation and were cultured according to previously described techniques. RTECs were cultured primarily with routine tissue block adhering wall method. In addition, CM was harvested from RTECs cultivated for 48 h in 5% O(2). EPCs proliferation and migration were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and transwell. The protein and mRNA expression of stromal cell-derived factor (SDF-1), vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1), and C-X-C chemokine receptor 4 (CXCR4) was separately assessed by Western blot, enzyme-linked immunosorbent assay (ELISA), and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. RESULTS: We showed that hypoxia increased the expression of SDF-1 and VEGF in RTEC. In addition, hypoxic CM improved proliferation, migration, and expression of VEGF, Ang-1, and CXCR4 of EPCs. CONCLUSIONS: These results suggest that hypoxic CM improves neovascularization of EPCs and may also be considered as therapeutic agents to supply the potent origin of reconstituion of PTCs of progressive kidney disease.

[Effects of costimulatory molecule VSIG4 exclusively expression in macrophage on renal interstitial fibrosis].

AIM: To investigate the relationship between costimulatory molecule VSIG4 expression in macrophage and mice renal interstitial fibrosis. METHODS: A total of 40 healthy male C57B6 mice (VSIG4(-/-); and VSIG4(+/+);) were divided into two groups, VSIG4(-/-); tubulointerstitial Nephritis (n=20) and VSIG4(+/+); tubulointerstitial Nephritis (n=20), mice received left urethral obstruction operation(UUO) then were killed at 7 d before operation, 3 d, 7 d and 14 d post operation for both groups.Blood SCr and BUN were detected. Renal interstitial fibrosis was measured by HE stain. The expression of TGF-beta1, MMP-2, in UUO mice(VSIG4(-/-); and VSIG4(+/+);) were detected by immunohistochemistry. RESULTS: SCr and BUN levels in the two groups have no significantly different. TGF-beta1 was significantly increased in the VSIG4(-/-); UUO mice group in comparison with the other group, while MMP-2 was reduced. CONCLUSION: The interstitial inflammatory cell infiltration and tubular lesion of VSIG4(-/-); UUO mice were increased, with the expression of TGF-beta1 increased and MMP-2 reduced. VSIG4 may play a role in inhibiting interstitial fibrosis.

Preventive effect of a proteasome inhibitor on the formation of accelerated atherosclerosis in rabbits with uremia.

Inflammation plays a central role in the pathogenesis of atherosclerosis. This study investigated whether the proteasome inhibitor has the same preventive effect on the formation of accelerated atherosclerosis in rabbits with uremia compared with a NF-kappaB inhibitor. New Zealand white rabbits were subjected to five-sixths nephrectomy (chronic renal failure [CRF]) or to a sham operation. Rats in each group were randomly assigned into three subgroups (n = 24 in each group) and treated with repeated intramuscular injections of proteasome inhibitor MG132 or NF-kappaB inhibitor PDTC for a specified period. Compared with sham rabbits, CRF rabbits displayed typical atherosclerotic changes (endothelial cell damage, intimal thickens, and appearance of foam cells). CRF rabbits had significantly higher levels of proteasome activity, NF-kappaB mRNA, protein, and DNA binding activity as well as tumor necrosis factor-a and proliferative cell nuclear antigen protein expression in aortic wall cells. CRF rabbits also showed lower levels of IkappaBalpha. Compared with CRF rabbits, CRF rabbits treatment with proteasome inhibitor MG132 showed restoration of IkappaBalpha mRNA and protein expression and decreased NF-kappaB DNA binding activity and tumor necrosis factor-a expression. Treatment with either proteasome inhibitor MG132 or NF-kappaB inhibitor PDTC could reverse these pathologic changes in the aortic wall cells of CRF rabbits. A comparison between the inhibitory effects of the two treatments revealed no statistical difference. These results suggest that ubiquitin-proteasome activation play a pivotal role in the pathogenesis of uremia-accelerated atherosclerosis. The ubiquitin-proteasome signaling pathway in aortic cells may therefore be an important target for preventing uremia-accelerated atherosclerosis.

Inhibition of indoleamine 2, 3-dioxygenase-mediated tryptophan catabolism accelerates crescentic glomerulonephritis.

Immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. Via catalysing tryptophan degradation, IDO suppresses adaptive T cell-mediated immunity and plays an important role in various forms of immune tolerance. Its role in T helper type 1 (Th1)-directed, cell-mediated crescentic glomerulonephritis (GN) is still unclear. Therefore, we investigated the activity and role of IDO in crescentic GN using a model of nephrotoxic serum nephritis (NTN), and IDO activity was inhibited by 1-methyl-tryptophan (1-MT) in vivo. Our results showed that activity of IDO, as determined by high performance liquid chromatography analysis of the kynurenine/tryptophan ratio, was increased markedly in the serum and renal tissue of NTN mice, and immunohistochemistry revealed that expression of IDO was up-regulated significantly in glomeruli and renal tubular epithelial cells during NTN. Treatment with 1-MT resulted in significantly exacerbated kidney disease with increased glomerular crescent formation, accumulation of CD4(+)T cells and macrophages in renal tissue, and augmented renal injury compared with phosphate-buffered saline-treated NTN mice, which was associated with enhanced Th1 responses and intrarenal cellular proliferation. These findings suggest that the development of NTN was regulated negatively by increased IDO activity, and IDO might play an important role in the pathogenesis of crescentic GN.

[Hypoxia responsive element regulated herpes simplex virus-thymidine kinase system enhances killing effect of gancyclovir on Ewing's sarcoma cell line under hypoxic condition].

OBJECTIVE: To find out a possible approach to improve the effectiveness of radiotherapy and chemotherapy for Ewing's sarcoma by constructing a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia and to evaluate the effects of this HRE regulated HSV-TK system on killing effect of gancyclovir (GCV) on Ewing's sarcoma cell line SK-ES under hypoxic condition. METHODS: The HRE was synthesized according to the literature and cloned into the enhancer site of pIRES(2)-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the multiple clone site of pIRES(2)-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing's sarcoma cell line SK-ES was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxic (21% oxygen) or hypoxic (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing's sarcoma cell line SK-ES transfected with pTK or pHRE-TK recombinant plasmid to the anti-tumour drug GCV was determined with the method of tetrazolium (MTT) after treating with GCV for five days. RESULTS: (1) The result of sequencing showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction. (2) Comparison of fluorescent optical density (FOD) showed that (1) the EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia (P < 0.01); (2) when the cells were exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group (P < 0.01); (3) there was no significant difference among the four groups of cells when they were exposed to normoxia (P > 0.05). (3) Comparison of the sensitivity of four groups of cells to GCV showed that (1) the cells in pHRE-TK and pTK groups were much more sensitive to GCV than the cells in pHRE group under hypoxia condition (P < 0.01), the higher the GCV concentration, the greater the difference; (2) the cells of pHRE-TK group were more sensitive to GCV than those in pTK group under hypoxic condition (P < 0.01), but was almost equally sensitive under normoxic condition (P > 0.05); (3) the pHRE-TK group cells had higher sensitivity to GCV under hypoxia than normoxia (P < 0.01) while the pTK group cells had almost the same sensitivity to GCV under hypoxia and normoxia (P > 0.05). CONCLUSION: (1) The eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed successfully. (2) HRE could up-regulate expression of EGFP by SK-ES cells under hypoxia condition. (3) HRE could enhance the killing effect of HSV-TK/GCV system on human Ewing's sarcoma cell line SK-ES under hypoxic condition.

[Reconstruction of urothelium tissue using issue-engineering technique].

OBJECTIVE: To evaluate the feasibility of reconstruction of urothelium tissue in vivo using tissue-engineering technique. METHODS: The urothelium cells were obtained from young rabbit, bladder by mechanical and enzyme digested methods. After expanded in vitro, the 4th to 5th generation urothelium cells were seeded onto the surface of 8 Polylatical/glycolic acid copolymer polymer, the polymer matrix without seeding cells served as control group. A total of 8 cell-polymer scaffolds and 4 simply scaffolds were separately implanted into subcutaneous pockets of athymic mice. The experiment groups included cell-polymer scaffolds 4 weeks and cell-polymer scaffolds 8 weeks. The control group included simply scaffold 4 weeks and simply scaffold 8 weeks. After 4 and 8 weeks, the specimens were obtained and examined by gross inspection, histologically and immunohistochemically. RESULTS: The results of HE and Masson staining showed that the polymer were covered by urothelium cells layers and cells layers increased markley in experimental group. Immunocytochemical studies revealed that the cells were stained positively for anti-cytokeratins (AE1/AE3) in experimental group. Fiber tissue deposition were found on the surface of polymers in control group by HE and Masson staining. Immunocytochemical staining of implants showed the negative result for cytokeratins in control group. CONCLUSION: It is feasibility that reconstruction of urothelium tissue using tissue-engineering technique,which provides basic understandings for further development of the bladder and ureteral tissue engineered research.