人羊膜间充质干细胞旁分泌对成骨细胞的影响

Effect of human amniotic mesenchymal stem cells on osteoblast by paracrine

目的探究人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)旁分泌对人成骨细胞生物活性的影响。方法酶消化法提取原代hAMSCs,并检测细胞表面标记物表达。收取hAMSCs 24 h培养上清与新鲜培养液1∶1配比作为条件培养基(condition medium,hAMSC-CM),以新鲜培养液作为阴性对照,作用于人成骨样细胞系MG63,观察对细胞生物活性的影响。MTT法检测连续1~4 d细胞增殖情况;Transwell小室测定细胞2.5 h迁移情况,苏木素-伊红染色后拍照计数;对硝基苯磷酸二钠(pNPP)法检测培养5 d后细胞内ALP表达。结果hAMSCs呈典型的成纤维样细胞形态,细胞表面阳性表达CD29、CD105、CD49d、CD44;阴性表达CD34、CD45、CD31和HLADR。hAMSC-CM在第1~3天均可显著促进MG63增殖(P<0.05);hAMSC-CM显著增强MG63迁移能力(P<0.05);hAMSC-CM培养组细胞中ALP表达显著强于对照组(P<0.05)。结论hAMSCs可通过CM介导的旁分泌作用增强成骨细胞生物活性。

Objective To investigate the effect of human amniotic mesenchymal stem cells(hAMSCs)on osteoblast by paracrine manner.Methods Primary hAMSCs were extracted by enzymatic digestion,and surface antigen markers were identified.Culture supernatant of hAMSCs after 24 h was collected and mixed with fresh medium by 1∶1,and was defined as condition medium(hAMSC-CM),while fresh medium was set as negative control.Their effects on the activities of MG63 were then detected.Cell proliferation was detected by MTT continuously for 4 days.The migrated cell number was estimated and counted after hematoxylin eosin staining by means of transwell system 2.5 h later.Alkaline phosphatase activity was measured after 5 days with p-Nitrophenyl Phosphate Disodium(pNPP).Results hAMSCs showed the shape of fibroblast.Surface antigen markers were positive for CD29,CD105,CD49 d,CD44,while negative for CD34,CD45,CD31,HLADR.hAMSC-CM promoted MG63 proliferation significantly during the first 3 days(P<0.05).Migration ability in hAMSC-CM group was enhanced prominently(P<0.05).ALP activity in cells cultured with hAMSC-CM exceeded control group markedly(P<0.05).Conclusion hAMSCs are able to enhance the biological activity of osteoblast through CM mediated paracrine.