牛羊水间充质干细胞对小鼠酒精性肝病的影响

试验旨在分离培养牛羊水间充质干细胞（AF-MSCs）,并研究其对酒精性肝病（ALD）小鼠治疗效果以及对肝组织中缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）及Toll样受体4（TLR4）mRNA表达的影响。从4~5月龄雌性胎牛羊水中获取AF-MSCs,进行RT-PCR、免疫荧光及诱导成脂鉴定。将48只ICR小鼠（雄性）随机分为4组：空白对照组（BC组）、模型对照组（MC组）、AF-MSCs组（MC+AF-MSCs组）和水飞蓟宾胶囊（SC）组（MC+SC组）,每组12只。除BC组灌胃0.2 mL生理盐水,其余各组均灌胃56度红星二锅头（5 g· kg ^-1,2次· d ^-1）,连续4周;第29天MC+AF-MSCs组进行肝外注射Dil标记的AF-MSCs（5×10⁶个·mL ^-1）,MC+SC组分2次灌胃SC 2.4 mg（溶于56度红星二锅头）;试验结束后,测定小鼠血清谷丙转氨酶（ALT）、谷草转氨酶（AST）和甘油三酯（TG）活性。HE染色、免疫组化法观察造模情况,Dil示踪AF-MSCs在肝内定植分布;qRT-PCR法检测HIF-1α、VEGF及TLR4基因表达情况。RT-PCR、免疫荧光结果表明,分离得到牛AF-MSCs,且有诱导成脂分化能力。血清学结果显示,MC组ALT、AST和TG活性极显著高于BC组（P<0.01）。组织病理切片显示,MC组肝组织中出现脂肪性变、炎性细胞浸润及肝血窦充血等病理性变化,说明56度红星二锅头成功复制ALD小鼠模型;与MC+SC组相比,MC+AF-MSCs组ALT、AST活性显著降低（P<0.01）,肝组织中脂肪性变减轻,肝血窦未见充血,说明AF-MSCs对肝功能的改善比SC效果显著;qRT-PCR结果显示,MC+AF-MSCs组下调HIF-1α、VEGF、TLR4基因的表达量较MC+SC组显著（P<0.01）,说明AF-MSCs参与HIF-1α/VEGF信号通路的调控,抑制TLR4表达的效果优于SC;细胞示踪术证明,AF-MSCs定植在病变部位并趋化更多AF-MSCs向炎症部位迁移参与肝组织的修复。综上,本研究成功分离得到牛AF-MSCs,其能够参与调节氧化应激、血管生成及抑制炎性因子的释放,且改善ALD的效果优于SC。

The Effect of Bovine Amniotic Fluid Mesenchymal Stem Cells on Alcoholic Liver Disease in Mice

The purpose of the experiment is to isolate and culture bovine amniotic fluid mesenchymal stem cells (AF-MSCs), and to study its therapeutic effect on alcoholic liver disease (ALD) mice and its effect on the mRNA expression of hypoxia inducible factor-1 α(HIF-1α), vascular endothelial growth factor (VEGF) and Toll-like receptor 4 (TLR4).AF-MSCs were isolated from amniotic fluid mesenchymal stem cells obtained from 4-to 5-month-old bovine embryos, and were identified by RT-PCR, immunofluorescence and induction of adipogenesis. Forty-eight ICR mice (male) were randomly divided into 4 groups:Blank control group (BC group), Model control group (MC group), AF-MSCs group (MC+AF-MSCs group) and silybin capsule (SC) group (MC+SC group), with 12 mice in each group.Except for the mice in BC group were intragastric administrated with 0.2 mL of normal saline, the rest of the groups were intragastrically administered with 56-degree Hongxing Erguotou (5 g·kg ^-1, 2 times·d ^-1) for 4 consecutive weeks; on the 29th day, the MC+AF-MSCs group was given extrahepatic injection Dil-labeled AF-MSCs (5×10⁶ cells·mL^-1), the MC+SC group was given intragastric SC twice a day for one day, total amount of SC was 2.4 mg (dissolve in 56-degree Hongxing Erguotou); after the experiment, the activities of serum glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and triglyceride (TG) were measured. HE staining and immunohistochemical methods were used to observe the modeling. Dil traced the colonization and distribution of AF-MSCs in the liver; qRT-PCR was used to detect the expression of HIF-1α, VEGF and TLR4 genes. RT-PCR and immunofluorescence results showed that bovine AF-MSCs were successfully isolated and had the ability to induce adipogenic differentiation. Serological results showed that the activities of ALT, AST and TG in the MC group were significantly higher than those in the BC group (P<0.01). Histopathological section showed that there were pathological changes such as steatosis, inflammatory cell infiltration and hepatic sinusoidal congestion in the liver tissue of the MC group, indicating that the 56-degree Hongxing Erguotou successfully replicated the ALD mouse model; Compared with the MC+SC group, the ALT and AST activities of the MC+AF-MSCs group were significantly reduced (P<0.01), the fatty change in the liver tissue was mitigated, and the liver sinusoids were not congested, indicating that AF-MSCs improved liver function more effective than SC; The qRT-PCR results showed that the expression of HIF-1α, VEGF and TLR4 genes in the MC+AF-MSCs group was significantly lower than that in the MC+SC group (P<0.01), indicating that AF-MSCs are involved in the regulation of HIF-1α/VEGF signaling pathway, and the effect of inhibiting TLR4 expression is better than SC; Cell tracing proved that AF-MSCs colonized the diseased site and chemotaxis more AF-MSCs migrated to the inflammation site and participated in the repair of liver tissue. In summary, this study successfully isolated bovine AF-MSCs. It can participate in the regulation of oxidative stress, angiogenesis, and inhibit the release of inflammatory factors. And the effect of improving ALD is better than SC.