摘要
目的观察Hedgehog信号通路在肝星状细胞（HSC）中的表达情况及Hedgehog信号通路对HSC激活和增殖的调控作用。方法采用逆转录一聚合酶链反应（RT—PCR）的方法检测大鼠HSC细胞株rHSC-99中Hedgehog信号通路各成分的表达。构建含Ihh、Smo、Gli2的干扰片段的质粒，分别转染HSC，用SYBRGreen荧光定量PCR的方法检测转染后Ihh、Smo、Gli2的表达，West—cm blot方法检测HSC中α-SMA表达，酶联免疫吸附试验（ELISA）方法检测转染后HSC细胞培养上清中I型胶原含量的变化，用噻唑蓝（M1Tr）比色法检测转染后HSC的增殖情况。结果Hedgehog信号通路中Ihh、Smo、Ptc、Gli2、Gli3在HSC中表达；荧光定量PCR结果显示转染含Ihh、Smo、Gli2的siRNA质粒后的HSC中相应基因Ihh、Smo、Gh2的mRNA表达明显下降（0．254±0．130、0．221±0．150、0．235±0．110比1，P〈0．01）；Westernblot结果显示HSC中α-SMA表达明显下降（0．191±0．014、0．357±0．021、0．086±0．016比1．143±0．017，P〈0．01）；ELISA结果显示转染后的HSC中Ⅰ型胶原分泌明显减少（22．9±2．0、16．4±1．4、17．6±1．8比40．7±4．3，P〈0．01）；MTT结果显示细胞增殖受到抑制（0．204±0．019、0．226±0．014、0．228±0．015比0．412±0．016，P〈0．05）。结论HSC表达Hedgehog信号通路中的Ihh、Smo、Ptc、Gli2、Gh3，下调Hedgehog信号通路可以抑制HSC激活和增殖。
Objective To study the expression of Hedgehog signal pathway in rat hepatic stellate cell （HSC） line rHSC-99. The method of RNAi was adopted to inhibit Hedgehog signal pathway,and estimate the regulation role of Hedgehog signal pathway in activation and proliferation of HSC. Methods RTPCR was used to detect the expression of Hedgehog signal pathway in rat HSC hne rHSC-99. Transcripts of siRNA sequence of the genes Ihh, Smo, and ,Gli2 were designed, and transfected into HSC respectively. Then the expression of these mRNAs were detected by SYBR green flurogenic quantitative PCR. The expression of α-SMA was detected by Western blot. The variation of type Ⅰ collagen in culture supematant of HSC was detected by ELISA. The proliferation of HSC was measured by MTT assay. Results HSC expressed mRNAs of Ihh,Smo, Ptc, Gli2, Gli3. The expression of these mRNAs could be reduced by transfecting plasmids encoded siRNA of Ihh, Smo or Gli2 （0.254 ± 0. 130,0. 221 ± 0. 150,0. 235 ± 0.110 vs 1 ,P 〈0.01）. Transfection experiment demonstrated the reduction of the expression of α-SMA （0. 191± 0.014,0. 357 ± 0. 021,0. 086 ± 0. 016 vs 1. 143 ± 0. 017, P 〈 0. 01 ） and secretion of collagenⅠ （22.9 ± 2.0,16.4 ± 1.4,17.6 ± 1.8 vs 40.7 ± 4.3, P 〈 0.01 ） in HSC, and HSC proliferation was decreased （0.204 ± 0. 019,0. 226 ± 0. 014, 0. 228 ± 0. 015 vs 0. 412 ± 0. 016, P 〈 0.05 ）. Conclusion This study showed the expression of Hedgehog signal pathway in HSC. Down-regulation of Hedgehog signal pathway may inhibit HSC activation and proliferation.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第12期1642-1644,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30672064）
北京市自然科学基金资助项目（7083116）
关键词
肝星状细胞
信号通路
增殖
Hepatic stellate cell
Signal pathway
Proliferation
