层粘连蛋白促脐带间充质干细胞向成骨细胞分化与黏着斑激酶表达

背景：目前对脐带间充质干细胞的研究集中在分离纯化及药物作用下诱导分化。分化过程中细胞外基质成分的作用及信号通路的关系的研究报道很少。目的：研究层粘连蛋白在脐带间充质干细胞向成骨细胞分化过程中的作用与黏着斑激酶表达之间的关系。方法：取对数生长脐带间充质干细胞分为4组：层粘连蛋白诱导组：层粘连蛋白质量浓度分别为50,25,5,1mg/L;成骨诱导组：用成骨诱导液处理;复合诱导组：用层粘连蛋白和成骨诱导液处理。对照组：未用层粘连蛋白和成骨诱导液处理。连续培养4周。观察细胞形态、茜素红染色分析钙盐沉积,检测碱性磷酸酶活性,组织化学组化与Westenblot检测ERK1/2、P-ERK、黏着斑激酶的表达。结果与结论：①倒置显微镜下对照组比诱导组细胞胞体呈矮柱状、有短突起彼此相连,核圆形。②茜素红染色细胞形成红色,表现为阳性。复合诱导组比其他组要高、不同时间点比21d钙盐沉积明显增多（P〈0.05）。③层粘连蛋白组碱性磷酸酶活性比对照组高、其诱导第7天后作用显著（P〈0.05）。层粘连蛋白不同质量浓度组之间碱性磷酸酶活性,差异无显著性意义（P〉0.05）。④组织化学组化鉴定ERK、P-ERK、黏着斑激酶表达均与对照比层粘连蛋白组表达增强（P〈0.05）。⑤WestenBlot检测成骨诱导组与复合诱导组有ERK、P-ERK、黏着斑激酶蛋白表达,层粘连蛋白组与对照组无表达。结果提示,脐带间充质干细胞向成骨细胞分化过程中层粘连蛋白有促分化作用,层粘连蛋白上调信号转导蛋白黏着斑激酶、ERK、P-ERK表达。

Promoting effects of laminin on osteogenetic differentiation of umbilical cord mesenchymal stem cells and focal adhesion kinase expression

BACKGROUND：Recent studies on umbilical cord mesenchymal stem cells（UCMSCs） are focused on the isolation,purification and induction and differentiation of UCMSCs,and there are a few reports about the studies of the effects of extracellular matrix components and correlations to signaling pathway.OBJECTIVE：To study the effects of laminin on the osteogenesis differentiation of UCMSCs and expression of focal adhesion kinase（FAK）.METHODS：The logarithmic cells were divided in to four groups.In laminin induction group,laminin concentrations were at 50,25,5,1 mg/L.In osteogenic induction group,osteogenic solution was used.In co-induction group,laminin and osteogenic solution were used.In control group,no Laminin or osteogenic solution.Observation of cell morphology,Alizarin red staining analysis of calcium salt deposits,detection of alkaline phosphatase（ALP） activity,histochemical and western blotting analysis of ERK1/2,P-ERK and FAK expression were performed after 4 weeks of culture.RESULTS AND CONCLUSION：①The cells of induction groups were formed to short column and linked together with short neurites,and nucleus appeared roundness under inverted microscopes.② Alizarin red staining analysis indicated that the cells were positive with red color.Co-induction group appeared more calcium salt deposits than other groups.Calcium salt deposits were higher in 21-day cells than other time points cells（P 0.05）.③ ALP activity analysis indicated that laminin induction group had higher ALP activity than control group,and 7-day induction cells had statistical significant differences（P 0.05）.There were no differences among the cells treated with different laminin concentrations（P 0.05）.④ Histochemical analysis indicated that the expressions of ERK1/2,P-ERK and FAK were improved in laminin induction group compared with control group（P 0.05）.⑤ Western blotting analysis indicated that there were ERK1/2,P-ERK and FAK proteins in induction and co-induction groups,but no above-mentioned expression in laminin induction and control groups.Results indicated that laminin promoted the differentiation of UCMSCs into osteoblasts and upregulated ERK1/2,P-ERK and FAK protein expression.