骨髓间质干细胞抑制肝星状细胞增殖与活化的体外研究

Inhibition of hepatic stellate cell proliferation and activation by bone marrow mesenchymal stem cells in vitro

目的:探讨骨髓间质干细胞(MSCs)对活化态肝星状细胞(HSCs)增殖的影响。方法:分别从骨髓和肝脏分离纯化培养大鼠MSCs及HSCs,塑料板传代培养激活HSCs;在半透膜(transwellinsert)上接种MSCs,在6孔塑料培养板上接种HSCs,建立上下双层细胞共培养体系;大鼠正常肝细胞系(BRLs)及HSCs培养分别作为对照。免疫细胞化学检测平滑肌激动蛋白(α-SMA)与结蛋白的表达,IBAS2.5软件分析阳性染色表达量。结果:HSCs与MSCs共培养24h,HSCs表现轻度增殖抑制,随着培养时间延长,HSCs增殖活性受抑制更明显,在48h和72h的抑制率分别达15.7%与30.3%,与BRLs共培养体系比较有显著差异;与MSCs共培养72h,HSCs表达α-SMA量明显低于两个对照组BRLs及HSCs培养体系(50.2%vs90.2%、95.6%,P<0.01);而结蛋白表达的量在3组共培养体系中均无显著差异。结论:MSCs具有分泌细胞因子抑制HSCs增殖活性的潜能,在治疗肝纤维化中可能发挥作用。

AIM: To investigate the effect of bone marrow (BM)-derived mesenchymal stem cells (MSCs) on the proliferation of hepatic stellate cells (HSCs) in vitro. METHODS: MSCs and HSCs were isolated from BM and liver respectively in Fischer 344 rats. HSCs were re-cultured on plastic and appeared activated morphologically. The coculture system was set up by transwell insert with pore size 0.4 μm and 6-well plates. The 5th passage HSCs were cultured on the 6-well plastic plate and MSCs or buffalo rat liver cells (BRLs) or HSCs seeded on the transwell insert. Smooth muscle α-actin (α-SMA) and desmin expression of HSCs were tested by immunocytochemistry and quantified by IBAS 2.5 software. RESULTS: After coculture for 24 h, the proliferation of HSCs was inhibited to a moderate extent by MSCs. With the coculture time lasted, the significant growth suppression of HSCs was affected by MSCs (15.7% and 30.3%) but not BRL cells in 48 h and in 72 h. After coculture for 72 h, the expression of α-SMA in HSCs was lower in MSCs coculture system than that with BRLs or HSCs alone system (50.2% vs 90.2%, 95.6%, P<0.01), while the expression of desmin in HSCs had no difference in three coculture system. CONCLUSION: MSCs can inhibit the proliferation and activation of hepatic stellate cells by secreting some cytokines, which might play a protective role (against) liver fibrosis.