肝细胞损伤条件培养基对大鼠骨髓间充质干细胞肝向分化的促进作用

doi:10.13481/j.1671-587x.20160613

摘要/Abstract

摘要：

目的：制备肝细胞损伤条件培养基（CM），体外模拟损伤组织微环境，探讨其对大鼠骨髓间充质干细胞（BMMSCs）肝向分化的作用。方法：取1周龄乳鼠，经差时贴壁和差速梯度法结合优化梯度离心和贴壁换液时间分离培养BMMSCs。取6~8周龄大鼠，腹腔注射60%四氯化碳溶液（0.75 mL·kg^-1）制备肝细胞损伤组织匀浆上清（CM1）和肝细胞损伤血清（CM2）。将第3代（P3）BMMSCs随机分为0% CM组（对照组，采用10% FBS L-DMEM培养）、10% CM1组、20% CM2组和10% CM1+10% CM2组。倒置显微镜下观察细胞形态表现，免疫组织化学法检测各组BMMSCs表面标志物CD105和各组细胞中白蛋白（ALB）的阳性表达率，油红O染色法检测BMMSCs成脂分化的脂滴形成阳性率；RT-PCR法检测各组细胞中甲胎蛋白（AFP）和ALB的表达水平，糖原染色法检测细胞中糖原合成阳性率，鉴定并比较各组BMMSCs肝向分化能力。结果：骨髓细胞原代培养72 h，贴壁细胞呈圆形或梭形，铺满瓶底时呈均匀有序的成纤维梭形状，P3 BMMSCs表面标志物CD105呈阳性表达。P3 BMMSCs经地塞米松、胰岛素、3-异丁基-1-甲基黄嘌呤和消炎痛（DX+IBMX+IS+ID）联合诱导后4 d，镜下可见脂滴形成。P3 BMMSCs经CM诱导后细胞中AFP和ALB呈阳性表达，细胞糖原染色呈阳性。诱导后7 d，含CM成分的3组BMMSCs中均可见细胞胞浆内容物变得丰富，细胞呈圆形、三角形或肝板样排列；诱导后7、14和21 d，与对照组比较，含CM成分的3组BMMSCs中ALB阳性表达率均明显增加（P<0.01）；10% CM1+10% CM2组BMMSCs中ALB阳性表达率高于10% CM1组和20% CM2组（P<0.05）。诱导后7和14 d，与对照组比较，含CM成分的3组BMMSCs中糖原合成阳性率均明显增加（P<0.01）；10% CM1+10% CM2组BMMSCs中糖原合成阳性率高于10% CM1组和20% CM2组（P<0.05）。结论：CM1和CM2可诱导大鼠BMMSCs向肝细胞分化，在达到相同诱导作用效果时所需的CM1和CM2浓度更低；CM1和CM2提供的微环境更有利于BMMSCs向肝细胞分化，其诱导细胞分化效率更高。

关键词: 条件培养基, 肝向分化, 骨髓间充质干细胞, 细胞增殖

Abstract:

Objective: To prepare the hepatocellular injury-conditioned medium (CM), and to simulate the microenvironment of injured tissue in vitro, and to explore the effect of CM on hepatic differentiation of bone marrow mesenchymal stem cells (BMMSCs) in the rats.Methods: The BMMSCs from 1-week-old rats were isolated and cultured by differential adherence and differential gradient method combined with optimized gradient centrifugation and culture time for adherent fluid exchange.The rats aged 6-8 weeks were injected intraperitoneally with 60% carbon tetrachloride solution (0.75 mL·kg^-1) to prepare the hepatocyte injured tissue homogenate supernatant (CM1) and hepatocyte injured serum (CM2). The third passage (P3) BMMSCs were randomly divided into 0%CM group (control group, cultured with 10%FBS L-DMEM), 10%CM1 group, 20%CM2 group, and 10%CM1+10%CM2 group. The morphology of cells was observed under inverted microscope;the expressions of cell-surface marker CD105 in BMMSCs and the positive expression rates of albumin (ALB) in cells in various groups were detected by immunohistochemistry method;the positive rate of lipid droplet formation in the pocess of adipogenic differentiation was detected by oil red O staining; the expression levels of alpha-fetoprotein (AFP) and ALB in the cells in various groups were detected by RT-PCR method;the positive rate of glycogen synthesis was detected by periodic acid-shiff staining; the hepatic differentiation ability of BMMSCs in various groups were identified and compared. Results: After primary culture of bone marrow cells for 72 h, the adherent cells showed round or spindle-shaped, covered with the bottom of the fibrous shuttle,and showed uniform and orderly shape, and the P3 BMMSCs surface markers CD105 showed positive expression.The P3 BMMSCs were induced f or 4 d by dexamethasone, insulin, 3-isobutyl-1-methylxanthine and indomethacin (DX+IBMX+IS+ID), the formation of lipid droplets was observed under microscope. After P3 BMMSCs were induced by CM, the expressions of AFP and ALB were positive,and the staining of cell glycogen was positive. After induced for 7 d, the cytoplasmic contents of P3 BMMSCs were abundant and the cells were round or triangular or liver board-like arrangement in CM groups. After induced for 7, 14 and 21 d, compared with control group, the positive expression rates of ALB in BMMSCs in CM groups were significantly increased (P<0.01); the positive expression rates of ALB in BMMSCs in 10% CM1+10% CM2 group were higher than those in 10%CM1 group and 20%CM2 group (P<0.05). After induced for 7 and 14 d by CM, compared with control group, the positive rates of glycogen synthesis in BMMSCs in CM groups were significantly increased (P<0.01);the positive rates of glycogen in 10%CM1+10%CM2 group were higher than those in 10% CM1 group and 20% CM2 group (P<0.05).Conclusion: CM1 and CM2 could induce the differentiation of BMMSCs into hepatocyte-like cells,and the lower concentrations of CM1 and CM2 can achieve the same effect.The microenvironment provided by CM1 and CM2 is more beneficial to the differentiation of BMMSCs into hepatocytes, which could induce cell differentiation more efficiently.

Key words: proliferation, conditioned medium, hepatic differentiation, bone marrow mesenchymal stem cells

中图分类号:

R657

赵美子, 王丹丹, 虞振静, 段颜莉, 马丽娜. 肝细胞损伤条件培养基对大鼠骨髓间充质干细胞肝向分化的促进作用[J]. 吉林大学学报(医学版), 2016, 42(06): 1108-1115.

ZHAO Meizi, WANG Dandan, YU Zhenjing, DUAN Yanli, MA Lina. Promotive effect of hepatocellular injury-conditioned medium on hepatic differentiation of bone marrow mesenchymal stem cells in rats[J]. Journal of Jilin University Medicine Edition, 2016, 42(06): 1108-1115.

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