间充质干细胞移植后抑制肝星状细胞活化减轻肝纤维化
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南京市科技局项目(201402003);江苏省医学创新团队与领军人才资助项目(LJ201121)


Autologous mesenchymal stem cells transplantation protect experimental liver fibrosis rats from inhibition of hepatic stellate cell activation
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    目的:研究间充质干细胞(mesenchymal stem cell,MSC)移植后对肝纤维化及肝星状细胞(hepatic stellate cells,HSC)活化的抑制作用-方法:Ficoll-Hypaque梯度密度离心法及细胞贴壁培养法分离纯化4周龄SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cell,BM-MSC)-取18只SD大鼠,通过腹腔小剂量注射四氯化碳(CCl4)8周,进行肝纤维化造模,在造模4周时,取其中9只作为治疗组,每周经尾静脉移植给予该组每只大鼠6×106个MSC;另外9只作为模型对照组,经尾静脉给予不含MSCs的等量生理盐水;另取9只大鼠,作为正常对照组,仅给予尾静脉等量生理盐水注射,持续4周-自实验开始,每周用全自动生化分析仪测定血清丙氨酸氨基转移酶(ALT)-天门冬氨酸氨基转移酶(AST)-总胆红素(TBIL)-白蛋白(ALB)水平,至结束共测定8次-实验终点时通过免疫组化对α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)-转化生长因子β1(transforming growth factor β1,TGF-β1)和Ⅰ型胶原蛋白(collagenⅠ,COLⅠ)在肝组织中的定位和表达进行研究;原位灌注和梯度密度离心法分离HSC,326 nm紫外光激发和α-SMA免疫荧光染色法鉴定HSC;qRT-PCR和Western blot检测HSC中α-SMA-TGF-β1基因和蛋白的表达水平-结果:与模型组相比,治疗组经MSC移植后,血清ALT-AST-TBIL水平,肝组织纤维化和炎症程度,肝组织α-SMA-TGF-β1和COLⅠ表达水平,HSC中α-SMA-TGF-β1基因和蛋白表达水平均明显降低-结论:肝纤维化SD大鼠经BM-MSC尾静脉移植治疗后肝功能显著改善-肝纤维化程度减轻,可能与抑制HSC活化有关-

    Abstract:

    Objective:To investigate the inhibitory effects of mesenchymal stem cells (MSC) on hepatic fibrosis and activation degree of hepatic stellate cells (HSC) after transplantation. Methods: Ficoll-Hypaque density gradient centrifugation and adherent culture were performed to separate and purify bone marrow mesenchymal stem cells (BM-MSCs) from 4 months old SD rats. Liver fibrosis model of SD rats was induced by long-term intraperitoneal injection of low-dose of CCl4 for 8 weeks. A total of 18 rats were performed to make model, since 4 weeks after the modeling process, 9 of them were taken out to receive treatment of 6×106 MSCs for 4 weeks by tail vein injection, the remains were injected the same volume of saline without MSCs. The normal control group containing 9 rats were only given the same volume of saline injection for 4 weeks. ALT, AST, TBIL,and ALB levels of serum were determined by automatic biochemical analyzer every week from the start to the end of research in a total of 8. The localization and expression of alpha-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β1), and collagen type Ⅰ (COL-Ⅰ) in liver tissues were analyzed by the immunohistochemical method. Density gradient centrifugation after situ perfusion and 326 nm of ultraviolet excitation together with α-SMA immunofluorescence staining were performed to isolate and identify HSC, respectively. The mRNA and protein expression levels of α-SMA and TGF-β1 in HSC were detected by qRT-PCR and Western blot, respectively. All above experiments were done at the end of the experiment at 8 weeks. Results: Compared with the model group, ALT, AST,and TBIL levels of serum; fibrosis and inflammation degree; α-SMA, TGF-β1,and COLⅠ expression levels in liver; the mRNA and protein expression levels of α-SMA and TGF-β1 in HSC were significantly reduced in the treatment group after MSCs transplantation. Conclusion: Liver fibrosis degree and liver function of SD rat were significantly reduced and improved after BM-MSC transplantation, respectively, which may be related to its inhibition of the activation level of HSC.

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施启鹏,郭圆圆,周 晗,蔡 洁,陈 念,李 军,章莉莉.间充质干细胞移植后抑制肝星状细胞活化减轻肝纤维化[J].南京医科大学学报(自然科学版),2015,(7):981-987

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  • 收稿日期:2015-03-12
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  • 在线发布日期: 2015-07-12
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