摘要
目的探讨胆管结扎诱导的大鼠肝纤维化模型的制备和改良的Friedman方法分离和培养肝星状细胞（HSC），以及HSC蛋白酶活化受体（PARs）的表达与HSC活化的关系。方法采用结扎大鼠胆管诱导肝纤维化动物模型，应用改良的Friedman方法和Nycodenz一步密度梯度离心法分离肝纤维化大鼠HSC。病理学检测大鼠肝纤维化分级，免疫组化检测Desmin α-SMA和PARs在HSC的表达，实验同时设立阴性对照。结果模型组大鼠肝纤维化病理学分级第1周为0，第2～3周多为1～2级，第4、5周为2～3级，第6周肝组织结构破坏，肝细胞变性坏死，炎性细胞浸润，病理学分级多为4级。改良的Friedman方法大鼠肝脏可获得5×10^6～1×10^7个细胞，存活率在96％以上，纯度平均为95％～96％。免疫组化检测α-SMA和PARs在培养的HSC于7、14天其表达逐渐增多，二者表达呈一致关系。结论结扎大鼠胆管建立的肝纤维化模型形成时间较短，实验中无有毒物质接触，是肝纤维化的理想模型；改良的Friedman方法分离和培养HSC，在保证HSC纯度的同时，提高了产量和活率，是研究HSC的有效方法；PARs的表达与HSC活化相关。
Objective To develop an economic, simple and reliable methods for isolation and culture of hepatic stellate cell （HSC） in rats, and to study the the relationship expression of proteinase-activated receptors（PARs） and between activation of HSC. Methods To adopt experimental model of hepatic fibrosis induced by BDL, and rat HSC were isolated from BDL rats and control rats according to a modification of the technique of Friedmen. This entails liver perfusion ex vivo and digestion with solutions containing pronase E followed by collagenase la. The digested liver specimen was filtered and the resulting suspension was cleaned and resuspended in a discontinuous Nycodenz gradient. HSC were cultured on plastic plates in Dulbecco＇s modified minimal essential medium containing 20% FCS. Expression of α-smooth muscle actin and proteinase-activated receptor 1 and 2 （PAR1,PAR2） were examined with immunocytochemistry method. Results The process of pathological changes of rat experimental model of hepatic fibrosis induced by BDL in six weeks was 4 grade by hepatic fibrosis pathological standard. Isolated HSC were more than 96% viable as assessed by trypan blue exclusion, and purity of cell was 95% - 96%. In the culture of rat HSC, the expression of PAR1/ PAR2 and the expression of α- SMA increased gradually,the expression of PAR1/ PAR2 paralleled with the expression of α - SMA, and correlated with activation and proliferation of HSC. Conclusion The experimental model of hepatic fibrosis induced by BDL was an ideal method, modification of the technique of friedmen was also an ideal one for the isolation and culture of HSC in rats. The expression of PAR1 and PARs correlated with the activation of HSC.
出处
《胃肠病学和肝病学杂志》
CAS
2006年第5期474-477,共4页
Chinese Journal of Gastroenterology and Hepatology
基金
中国人民解放军第三军医大学中青年基金
No.200543
关键词
肝纤维化
肝星状细胞
细胞分离
细胞培养
蛋白酶活化受体
Hepatic fibrosis
Hepatic stellate cells
Isolation
Cuhure
Proteinase-activated receptors
