基因芯片研究尿道下裂的基因表达差异

目的筛选出与尿道下裂发病相关的基因。方法尿道下裂病人的尿道或包皮组织,年龄配对的包皮过长患儿组织作为对照,提取组织mRNA,合成双股cDNA并纯化,再逆转录成cRNA,生物素标记的cRNA打成碎片后与人类基因芯片进行杂交,计算机工作站自动扫描并记录芯片上的信号强度,转化为EXCEL数据库,总共分析了22000个基因的表达差异。结果发现一组表达有显著差异的基因,包括CYR61,CTGF,ATF3和GADD45。结论这些表达差异的基因可能参与尿道下裂的发生和发展,为进一步研究关键基因提供了依据。     

腹膜间皮细胞的培养及TGF-β1 CTGF蛋白含量测定

目的建立简便有效的腹膜间皮细胞体外培养鉴定及TGF-β1、CTGF蛋白含量测定方法。方法胰蛋白酶-EDTANa2消化网膜组织培养腹膜间皮细胞;用形态学、免疫组化鉴定细胞。结果分离培养的细胞符合间皮细胞特征;ELISA法测定细胞培养液中TGF-β1、CTGF蛋白含量简便可行。结论胰蛋白酶消化法是一种简单实用的分离腹膜间皮细胞的方法;ELISA法测定细胞培养液中TGF-β1、CTGF蛋白含量,可能成为衡量预防术     

Nephroblastoma overexpressed gene (NOV) expression in rat hepatic stellate cells

Using the expression-profiling method, we identified nephroblastoma overexpressed gene (NOV) mRNA as one member of the mRNA population that was upregulated in cultured activated hepatic stellate cell (HSC). Northern analysis showed that NOV mRNA was increasingly expressed during progressive activation of cultured rat HSCs, and a significant increase was observed in both the carbon tetrachloride-induced and bile duct ligation/scission rat models of liver fibrosis. RT-PCR showed human NOV mRNA was increased in most fibrotic livers compared with normal livers. The expression of NOV protein in fibrotic rat and human livers was predominantly located in areas of ductular proliferation and HSC of the fibrous septa. HSCs stimulated with transforming growth factor beta1 showed increased expression of NOV protein without changing its mRNA levels. Dexamethasone stimulated the expression of NOV mRNA and protein. Furthermore, we demonstrated that bile acids have a modulating effect on the induction of NOV mRNA expression. In conclusion, this study suggests that NOV is expressed during liver fibrogenesis and HSCs may be an important source of hepatic NOV.     

甘草甜素对大鼠肾小球硬化早期的防护作用

目的探讨甘草甜素对大鼠肾小球硬化早期的保护作用。方法采用两次经尾iv阿霉素方法建立大鼠肾小球硬化模型。实验随机分为3组,模型组、甘草甜素治疗组和对照组。检测各组大鼠第4、6、8周各项指标的变化,包括24 h尿蛋白定量、血清尿素氮(BUN)、肌酐(SCr)、胆固醇(TC)和白蛋白(Alb)。各组取肾皮质进行光镜等组织病理学检测。应用免疫组织化学方法检测肾组织内转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)蛋白的表达。采用标准曲线法进行荧光定量PCR检测肾组织内TGF-β1、CTGF和金属蛋白酶组织抑制物-1(TIMP-1)mRNA的表达。结果甘草甜素治疗组大鼠24 h尿蛋白定量、BUN、SCr、TC和Alb等指标与模型组相比较均有不同程度改善(P0.05)。肾小球硬化程度治疗组明显轻于模型组。治疗组与模型组比较肾组织内TGF-β1和CTGF蛋白表达均有不同程度降低,表达峰值下降(P0.05)。治疗组与模型组比较TGF-β1、CTGF和TIMP-1 mRNA表达均有不同程度降低,表达峰值下降(P0.05)。结论从蛋白和mRNA水平同时证实甘草甜素对TGF-β1、CTGF和TIMP-1的表达有明显的抑制作用,证实甘草甜素对大鼠肾小球硬化有早期保护作用。     

抗结缔组织生长因子特异性小干扰RNA的设计、合成及筛选

目的 设计和合成针对抗肝纤维化关键基因抗结缔组织生长因子(connective tissue growth factor,CTGF)的特异性小干扰RNA(siRNA),并筛选高效的CTGF siRNA抗肝纤维化序列.方法 参照siRNA设计原则,应用RNA在线设计软件,设计3段RNA干扰候选序列,分别转染正常干细胞株(...     

Pulmonary hypoplasia in the connective tissue growth factor (Ctgf) null mouse.

Connective tissue growth factor (CTGF) is a mediator of growth factor activity, and Ctgf knockouts die at birth from respiratory failure due to skeletal dysplasia. Previous microarray analysis revealed Ctgf down-regulation in the hypoplastic lungs of amyogenic mouse embryos. This study, therefore, examined pulmonary development in Ctgf-/- mouse fetuses to investigate if respiration could also have been impaired by lung abnormalities. The Ctgf-/- lungs were hypoplastic, with reduced cell proliferation and increased apoptosis. PDGF-B, its receptor and IGF-I, were markedly attenuated and the TTF-1 gradient lost. Type II pneumocyte differentiation was perturbed, the cells depicting excessive glycogen retention and diminished lamellar body and nuclear size, though able to synthesize surfactant-associated protein. However, type I pneumocyte differentiation was not affected by Ctgf deletion. Our findings indicate that the absence of Ctgf and/or its protein product, CTGF, may induce pulmonary hypoplasia by both disrupting basic lung developmental processes and restricting thoracic expansion.     

Cyr61、CTGF、NOV、WISP-1基因在肾癌中的表达

目的〓〖HTK〗探讨肾癌组织CCN蛋白中Cyr61、CTGF、NOV和WISP-1 基因的蛋白表达水平及意义。〖HTW〗方法〓〖HTK〗采用Western blot半定量方法检测31例肾癌组织和18例癌旁正常肾组织中Cyr61、CTGF、NOV、WISP-1基因的蛋白表达。 〖HTW〗结果〓〖HTK〗Cyr61、CTGF、NOV、WISP-1蛋白在肾癌组织中的表达水平均低于癌旁正常组织(P<0.05)，其中乳头状肾癌的NOV和WISP-1 表达水平高于透明细胞癌(P<0.05)，G1级肿瘤NOV表达水平高于G2，3肿瘤(P<0.05)。 〖HTW〗结论〓〖HTK〗Cyr61、CTGF、NOV和WISP-1在肾癌中低表达， CCN基因在肾癌的发生和发展中可能起抑制作用。     

肺癌组织中CTGF和WISP-1基因表达的研究

目的研究肺癌中CTGF和WISP-1基因表达与临床病理因素的关系。方法采用RT-PCR和免疫组化染色法对60名肺癌患者的癌组织和癌旁正常肺组织中CTGF和WISP-1表达水平进行测定。结果65%(39/60)的肺癌组织CTGF基因表达水平低于癌旁正常肺组织(t=-1.59,P=0.016);83%(50/60)的肺癌组织WISP-1基因表达水平高于癌旁正常肺组织(t=4.15,P=0.000),此结果得到免疫组化的证实。Pearson相关分析:WISP-1和CTGF基因的表达呈显著负相关(r=-0.299;P=0.020)。多元线性回归分析:肿瘤位置和性别对癌组织CTGF基因表达有重要影响;病理类型、年龄和家族史对WISP-1基因表达有影响。结论CTGF和WISP-1基因中单个基因或两个基因相互作用对肺癌的进展均起重要作用及其与临床病理因素的关系。     

Hepatitis C virus-replicating hepatocytes induce fibrogenic activation of hepatic stellate cells

BACKGROUND & AIMS: The mechanism by which hepatitis C virus induces liver fibrosis remains largely obscure. To characterize the profibrogenic potential of hepatitis C virus, we used the hepatitis C virus replicon cell line Huh-7 5-15, which stably expresses the nonstructural hepatitis C virus genes NS3 through NS5B, and hepatic stellate cells as fibrogenic effector cells. METHODS: Rat and human hepatic stellate cells were incubated with conditioned media from replicon cells, and expression of fibrosis-related genes was quantified by using real-time polymerase chain reaction, protein, and functional assays. Transforming growth factor beta1 activity was determined by bioassay. RESULTS: Hepatitis C virus replicon cells release factors that differentially modulate hepatic stellate cell expression of key genes involved in liver fibrosis in a clearly profibrogenic way, up-regulating procollagen alpha1(I) and procollagen alpha1(III) and down-regulating fibrolytic matrix metalloproteinases. Transforming growth factor beta1 expression and bioactivity were increased severalfold in hepatitis C virus-replicating vs mock-transfected hepatoma cells. However, transforming growth factor beta1 activity was responsible for only 50% of the profibrogenic activity. CONCLUSIONS: Hepatitis C virus nonstructural genes induce an increased expression of transforming growth factor beta1 and other profibrogenic factors in infected hepatocytes. The direct induction of profibrogenic mediators by hepatitis C virus in infected hepatocytes explains the frequent observation of progressive liver fibrosis despite a low level of inflammation and suggests novel targets for antifibrotic therapies in chronic hepatitis C.     

黄芪鸡血藤汤对实验性肝纤维化大鼠TGF-β_1、CTGF及PDGF-BB表达影响

目的:观察黄芪鸡血藤汤对大鼠肝纤维化的预防作用,并探讨该组方预防肝纤维化的作用机制。方法:选用SD大鼠58只,随机分为5组:正常对照组(10只):正常饮食饮水,皮下注射花生油。模型组(12只):按复合因素造模。中药治疗组:分为大、中、小剂量组(每组12只):在造模的同时每日分别给予中药每100g体重2g、1g、0.5g灌胃。8周末,处死大鼠后处理肝脏标本。肝组织用HE染色,光镜下观察各组大鼠组织学改变,并进行比较。免疫组织化学法观察黄芪鸡血藤汤对各组大鼠肝脏TGF-β1、CTGF、PDGF-BB表达的影响。结果:各组大鼠肝脏标本形态学的比较:8周后,中药组大鼠肝脏色泽、质地及表面光滑程度均明显优于模型组。免疫组织化学结果显示:中药各剂量组TGF-β1、CTGF、PDGF-BB在大鼠肝组织中的表达明显低于模型组(P<0.05)。其中大剂量组最低(P<0.01)。结论:黄芪鸡血藤汤能有效地预防实验性大鼠肝纤维化的形成。