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目的:探讨甘油三酯(TG)和极低密度脂蛋白(VLDL)及其刺激的Kupffer细胞(KC)对大鼠肝星状细胞(HSC)增殖的影响。方法:用链霉蛋白酶和胶原酶原位灌流,Nvcodenz密度梯度离心分离大鼠HSC和KC 应用免疫组织化学、吞噬功能试验、电镜等方法进行鉴定:制备TG和VLDL刺激的KC培养的上清液(KCCM).并以MTT比色去观察TG和VLDL及KCCM耐肝星状细胞增殖效应。结果:本法能成功地获得高纯度的HSC和KC:TG和VLDL浓度分别为12.5μg/ml和25～100μg/ml时.其对HSC增殖有促进作用(P<0.05或P<0.01):与正常和KCCM组相比,KCCM+VLDL组和KCCM+TG组对HSC增殖有促进作用(P<0.01).KCCM-VLDL组和KCCM+TG组之间无差异(P>0.05):KCCM组高于正常组,但无统计学差异(P>0.05) 结论:本实验所用的HSC和KC分离培养方法简单、易行、可靠,细胞纯度高:体外细胞培养表明.TG和VLDL及其刺激的KCCM可促进HSC增殖.其与脂肪肝肝纤维化的发生可能有关。
Objective: To investigate the effects of triglyceride (TG) and very low density lipoprotein (VLDL) and its stimulated Kupffer cells (KC) on the proliferation of rat hepatic stellate cells (HSCs). Methods: HSC and KC were isolated by in situ perfusion with pronase and collagenase, and were isolated by Nvcodenz density gradient centrifugation. Immunohistochemistry, phagocytosis test and electron microscopy were used to identify HSCs and KCs. The supernatants of KC culture stimulated by TG and VLDL (KCCM). MTT colorimetric assay was used to observe the effects of TG and VLDL and KCCM on hepatic stellate cell proliferation. Results: The method could successfully obtain high purity HSC and KC: TG and VLDL concentrations of 12.5μg / ml and 25 ~ 100μg / ml respectively, which could promote the proliferation of HSC (P <0.05 or P <0.01) There was no difference between KCCM-VLDL group and KCCM + TG group (P> 0.05): KCCM + VLDL group and KCCM + TG group could promote the proliferation of HSC compared with normal and KCCM group (P <0.01) (P> 0.05) Conclusion: The method of HSC and KC used in this experiment is simple, easy and reliable, and has high cell purity. In vitro cell culture showed that TG and VLDL and their stimulated KCCM can promote the proliferation of HSC, which may be related to the occurrence of hepatic fibrosis in fatty liver.