3种组织来源的间充质干细胞体外成骨能力的比较

摘要

目的 比较成人骨髓间充质干细胞(BMSCs)、人脐带间充质干细胞(UC-MSCs)和人胎盘间充质干细胞(P-MSCs)的成骨能力.方法 用含10%胎牛血清的DMEM/Ham's F-12培养液培养3种MSCs,CCK8法检测增殖能力,流式细胞仪鉴定3种细胞.碱性磷酸酶(ALP)和茜素红染色观察细胞经成骨诱导后成骨分化蛋白- ALP的分泌和矿化钙结节的沉积.实时荧光定量PCR(RT-qPCR)法检测MSCs骨再生相关基因的表达.Western blot方法检测MSCs成骨再生相关基因的蛋白表达.结果 MSCs在第3天进入对数增殖期.3种细胞的表面标志物阳性率:CD44、CD90和CD105均高于98%.3种MSCs成骨诱导9 d时,3种MSCs的实验组均表达大量成骨分化蛋白-ALP,成骨诱导18 d时3种MSCs均呈现较好的矿化能力;3种MSCs成骨诱导9 d时,实验组RUNX2和ALP基因显著性高表达(P<0.05),成骨诱导18 d时,实验组RUNX2和骨钙素(OCN)亦显著性高表达(P<0.05);3种MSCs成骨诱导9 d时,实验组均检测到RUNX2和ALP的蛋白表达;成骨诱导18 d时,实验组细胞亦检测到RUNX2和OCN的蛋白表达.结论 UC-MSCs和P-MSCs具有良好的成骨分化能力,有望作为骨组织工程的种子细胞用于治疗骨缺损.%Objective To find the seed cells for replacing bone marrow mesenchymal stem cells(BMSCs) to treat bone defects by compared the capacity of osteogenesis from the BMSCs, human umbilical cord mesenchymal stem cells(UC-MSCs) and human placental mesenchymal stem cells(P-MSCs). Methods Three MSCs were cultured in DMEM/Ham's F-12 medium containing 10% fetal bovine serum,cell proliferation curve was drawn by CCK8 detec-ted,and the cell surface antigens were measured using flow cytometry. Osteogenic ability was confirmed by the al-kaline phosphatase(ALP) staining,alizarin red staining. To further explore the difference in organic components, their underlying genotypes and proteins, including RUNX2, ALP and osteocalcin(OCN), were analyzed by RT-qPCR and Western blot. Results Cell growth curve analysis indicated that three MSCs were in the exponential stage at 3 days following incubation. Flow cytometry analysis showed that more than 98% cells were positive for CD44, CD90 and CD105. ALP and Alizarin red staining displayed that three MSCs presented good mineralizationg ability by osteogenesis induced medium. RT-qPCR and Western blot showed that the three MSCs experiment group higher expression levels of osteogenic markers including RUNX2, ALP and OCN during the initial 9 and 18 days when compared with control(P<0.05). Conclusions UC-MSCs and P-MSCs have good osteogenic ability, which may function as a potential bone tissue engineering seed cells for the treatment of bone defects.