兰州大学机构库 >学院待认领
CTGF特异性小分子干扰RNA对大鼠肝纤维化逆转的影响
Alternative TitleSmall interfering RNA targeting connective tissue growth factor interference in the reverse impact of rats liver fibrosis
薛苗
Thesis Advisor陈红
2011-05-19
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword结缔组织生长因子 小分子干扰RNA 肝纤维化
Abstract目的 本研究通过设计和合成针对抗肝纤维化关键基因的结缔组织生长因子(connective tissue growth factor,CTGF)特异性小分子干扰RNA(small interfering RNA,siRNA),并通过细胞转染筛选出高效的CTGFsiRNA抗肝纤维化序列,探讨通过尾静脉注射CTGF siRNA观察对大鼠肝纤维化肝脏功能及肝纤维化指标的影响及是否能抑制大鼠体内肝脏CTGF基因表达防治大鼠肝纤维化。方法 1.参照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,分别转染正常肝细胞株(L-02),以转染非特异性siRNA(与CTGF mRNA无同源性)作为对照,应用Western Blot检测L-02细胞CTGF蛋白质表达,根据蛋白表达结果筛选出高效的CTGF干扰序列。2. 雄性SD大鼠30只,随机分成5组,分别为模型组:腹腔注射40%CCl4 (3ml/kg)及尾静脉注射生理盐水,1次/3d,连续8周;预防组:腹腔注射40%CCl4 及尾静脉注射抗CTGF siRNA(0.1mg/kg),1次/3d,连续8周;治疗组:腹腔注射40%CCl4 2周,后给予抗CTGF siRNA及CCl4 6周;晚期治疗组:腹腔注射40%CCl4 4周,后给予抗CTGF siRNA及CCl4 4周;空白对照组:尾静脉注射生理盐水8周。于最后一次CCl4注射3天后取血及组织标本,检测血清转氨酶(ALT、AST)、白蛋白(ALB)、总胆红素(TBIL)及肝纤维化(透明质酸 HA、层黏连蛋白 LN、人Ⅲ型前胶原 hpcⅢ、Ⅳ型胶原 Ⅳ. C)指标,应用Real-Time PCR及western-blot印迹法检测CTGF mRNA及蛋白质在大鼠肝组织表达,应用H-E染色检测肝组织炎症及纤维化,参照慢性肝炎分级分期标准对大鼠肝脏炎症活动度及肝纤维化程度进行病理分级。
Other AbstractObjectives: This research by design and the synthesis of the liver fibrosis key gene layer of small interfering RNA targeting connective tissue growth factor,and through cell transfection bloting high-performance CTGF siRNA of liver fibrosis sequence. To explore effect of small interfering RNA targeting connective tissue growth factor on liver function and fibre index of rats with liver fibrosis and whether inhibit CTGF expression in rats liver in vivo and prevent rats hepatic fibrosis.Methods: 1.Reference siRNA design principles, the application of RNA design software online, the design of RNA interference three candidates, and to transfection their normal cell(L-02),transfection non-specificity siRNA(non-homology of CTGF mRNA) in comparison,with western blot detection L-02 cell CTGF protein, expressed by the albumen bloting the interference sequence of CTGF.2. Thirty male rats were randomly divided into five groups,Rats received intraperitoneally injection of 40 % CCl 4(3 ml/ kg) together with tail vein injection of saline every three days for 8 consecutive weeks were served as model group;CCl4 together with tail vein delivery of siRNA (0. 1 mg/ kg) as preventive group;CCl4 for 2 weeks followed by CCl4 and CTGF siRNA for more than 6 weeks as curative group;CCl4 for 4 weeks followed by CCl4 and CTGF siRNA for more than 4 weeks as advanced curative group and only tail vein injection of saline as control group. Tail vein pressure in all rats at 3 days af ter the last CCl4 injection were blood and hepatic tissue from rats were harvested. Serum transaminase(ALT,AST), albumin(ALB), total bilirubin(TBIL) and hepatic fibrosis indices were measured. Expression of CTGF mRNA and protein in rats liver was evaluated by RT-PCR and Western blot , respectively. Inflammation and fibrosis in rats liver was analyzed by H-E.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/224179
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
薛苗. CTGF特异性小分子干扰RNA对大鼠肝纤维化逆转的影响[D]. 兰州. 兰州大学,2011.
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