明胶-胶原复合凝胶联合诱导因子调控大鼠骨髓间充质干细胞的肝向分化

Gelatin collagen composite hydrogel and inducible factor regulate differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells

背景:体外人工肝系统已成为临床上非常有效且实用的肝衰竭治疗手段,但仍存在细胞来源有限和获得细胞的功能有限等问题。目的:优化和筛选符合细胞生长需要的明胶-胶原复合凝胶,以复合凝胶为细胞生长的基底材料,探究诱导骨髓间充质干细胞肝向分化的方案。方法:将明胶溶液与胶原溶液按照不同的体积比(1∶1、2∶1、4∶1)混合,分别制备明胶质量浓度为50,100,150,200 g/L的明胶-胶原复合凝胶,通过熔点、微观形貌、吸水率和孔隙率筛选溶液的体积比与明胶的质量浓度,进行后续实验。将大鼠骨髓间充质干细胞接种于复合水凝胶中(实验组),以单独培养的细胞为对照,采用CCK-8法检测细胞增殖;采用两阶段分步诱导方案来诱导两组大鼠骨髓间充质干细胞的肝向分化,于诱导培养的3,7,14,21 d检测上清中白蛋白与尿素的水平。结果与结论:(1)对比熔点、微观形貌、吸水率和孔隙率检测结果,当明胶溶液与胶原溶液按照4∶1体积比混合、明胶质量浓度为150 g/L时制备的复合凝胶最适宜用于细胞培养。(2)CCK-8检测显示,实验组培养1,3,5 d的细胞增殖活性低于对照组,此后两组间的差距逐渐减小,至10 d时已无差异。(3)肝向诱导分化实验显示,随着诱导时间的延长,两组白蛋白合成量逐渐增加,其中实验组14,21 d的白蛋白合成量高于对照组(P<0.05);两组诱导3-7 d内的尿素分泌量增幅较大,7 d后生成量趋于稳定,实验组14,21 d的尿素分泌量稍高于对照组,但差异无显著性意义(P>0.05)。(4)结果表明,以明胶-胶原复合凝胶为细胞生长的基底材料联合诱导因子可诱导大鼠骨髓间充质干细胞分化为肝样细胞。

BACKGROUND:In vitro artificial liver system has become a very effective and practical treatment for liver failure,but there are still some problems such as limited source of cells and limited function of cells.OBJECTIVE:To optimize and screen gelatin collagen composite hydrogel that meet the needs of cell growth,the hydrogel was used as the basal cell growth material to explore the scheme of inducing hepatic differentiation of bone marrow mesenchymal stem cells.METHODS:Gelatin collagen composite hydrogel with gelatin concentration of 50,100,150,200 g/L was prepared by mixing gelatin solution and collagen solution at different volume ratios(1:1,2:1,4:1).The volume ratio of the solution and the gelatin concentration were selected for subsequent experiments by melting point,microstructure,water absorption and porosity.Rat bone marrow mesenchymal stem cells seeded in composite hydrogel were used as experimental group;rat bone marrow mesenchymal stem cells cultured alone were used as controls.Cell proliferation was detected by CCK-8 assay.The twostep induction regimen was used to induce the hepatic differentiation of two groups of rat bone marrow mesenchymal stem cells.The levels of albumin production and urea secretion in the supernatant were tested at 3,7,14,and 21 days after induced culture.RESULTS AND CONCLUSION:(1)After testing the melting point,microscopic morphology,water absorption and porosity of the experimental groups with different ratios,the composite gel was most suitable for cell culture when the 150 g/L gelatin solution was mixed with collagen solution at 4:1 volume ratio.(2)CCK-8 assay showed that the proliferation activity of the experimental group was lower than that of the control group at 1,3 and 5 days,and the difference between the two groups gradually decreased,and there was no difference at 10 days.(3)Experiment of hepatic differentiation showed that with the extension of induction time,the amount of albumin secretion in the two groups increased gradually,and the amount of albumin in the experimental group was higher than that in the control group at 14 and 21 days(P<0.05).The amount of urea secretion in the two groups increased significantly within 3-7 days of induction,and stabilized after 7 days.The amount of urea secretion in the experimental group was slightly higher than that in the control group at 14 and 21 days,but the difference was not significant(P>0.05).(4)The results showed that gelatin collagen composite hydrogel could induce rat bone marrow mesenchymal stem cells to differentiate into hepatocyte-like cells.