摘要
目的探讨人脐带间充质干细胞(hucMSCs)对人真皮成纤维细胞(HDFib)增殖与细胞外基质相关基因表达的影响及其可能机制。方法采用Ⅱ型胶原酶消化法从新生儿脐带中分离制备hucMSCs,用流式细胞术鉴定P3代hucMSCs表面标记抗原,用油红O染色、茜素红S染色和Masson染色进行间充质干细胞三向分化能力鉴定;随后与P3代HDFib进行transwell共培养,对照组transwell小室上添加等量培养基,观察24、48和72h时两组HDFib的细胞形态,CCK8法进行细胞增殖检测,qRT-PCR法测定培养24和48h时HDFib主要细胞外基质蛋白collagenⅠ、elastin、fibronectin、MMP-1和细胞信号转导相关基因PI3K、Akt、p38和caspase3 mRNA表达水平。结果制备获得的hucMSCs呈典型成纤维细胞样并贴壁生长,其表面间充质干细胞标志性抗原CD105、CD90、CD73阳性表达率分别为96.97%、99.75%和99.81%,诱导培养16 d后出现明显的成脂、成骨和成软骨细胞特性。hucMSCs共培养处理24、48和72 h,HDFib细胞存活率较对照组分别显著增加了7.29%(P<0.01)、25.82%(P<0.05)和11.74%(P<0.05),与形态学观察结果一致。qRT-PCR结果显示,hucMSCs共培养处理24 h,HDFib elastin和fibronectin基因表达分别上调为对照组的1.45倍(P<0.01)和1.30倍(P<0.05),PI3K mRNA水平上调为对照组的1.19倍(P<0.05);共培养48 h时,collagen I、elastin、fibronectin和PI3K、Akt分别上调2.60倍(P<0.001)、3.66倍(P<0.05)、3.45倍(P<0.01)和1.43倍(P<0.05)、1.65倍(P<0.05),MMP-1、p38和caspase3转录水平显著降低至对照组的0.70倍(P<0.01)、0.60倍(P<0.01)和0.86倍(P<0.05)。结论hucMSCs能够显著促进正常HDFib增殖,同时提高HDFib I型胶原、弹性蛋白和纤维粘连蛋白转录水平,是理想的皮肤组织工程学种子细胞。hucMSCs对HDFib细胞外基质的调节作用可能与激活HDFib皮肤创伤修复信号通路PI3K/Akt、下调细胞外基质调节基因p38、凋亡基因caspase3的mRNA水平有关。
Objective To investigate the effects of human umbilical cord mesenchymal stem cells(hucMSCs)on the cell proliferation and extracellular matrix genes expression of normal human dermal fibroblast(HDFib)and potential mechanisms.Methods The hucMSCs were derived from healthy full-term newborn umbilical cord through a collagenase digestion method.Flow cytometry was applied to determine the characteristic mesenchymal stem cell antigenic markers of P3-generation hucMSCs,and oil red O,alizarin red S and Masson staining were used for testing its adipogenic,osteogenic and chondrogenic differentiation capacities,respectively.Then,P3-generation HDFib were treated with hucMSCs by transwell experiment(co-culture group),while equal volume of medium was added to the control group.After a period of 24,48 and 72 h co-culture,the proliferation of HDFib was measured by CCK8 assay and cell morphology were also recorded.The mainly extracellular matrix proteins,collagen I,elastin,fibronectin,matrix metalloproteinase-1(MMP-1)and cellular signal transduction pathways PI3K,Akt,p38 and caspase3 mRNA expression after 24 and 48 h culture were further characterized by qRT-PCR.Results The adherent hucMSCs showed the shape of fibroblast,which could differentiate into osteoblasts,adipocytes and chondrocytes after 16 d induction culture period in vitro.The expression of CD105,CD90 and CD73 on hucMSCs were 96.97%,99.75%and 99.81%,respectively.Consistent with the morphologic results,the viability of HDFib treated with hucMSCs was significantly increased for 7.29%(P<0.01),25.82%(P<0.05)and 11.74%(P<0.05)after 24,48 and 72 h co-culture,respectively.Moreover,elastin,fibronectin and PI3K gene expression of HDFib were up-regulated by 1.45(P<0.01),1.30(P<0.05)and 1.19(P<0.05)folds after 24 h co-culture with hucMSCs compared to the control group,and collagen I,elastin,fibronectin,PI3K and Akt mRNA level were up-regulated by 2.60(P<0.001),3.66(P<0.05),3.45(P<0.01),1.43(P<0.05)and 1.65(P<0.05)folds after 48 h co-culture,while MMP-1,p38 and caspase3 were down-regulated by 0.70(P<0.01),0.60(P<0.01)and 0.86(P<0.05)folds of control.Conclusion Our results show that hucMSCs can significantly promote the proliferation of HDFib and increase the transcription levels of collagen I,elastin and fibronectin,which indicates that hucMSCs are potential seed cells for skin tissue engineering.Besides,our results indicate that the regulation of hucMSCs on HDFib extracellular matrix may be related to the activation of skin wound repair pathway PI3K/Akt and the decrease of p38 and caspase3 expression.
作者
章毅
陈侃俊
伍婷
祁成
金魏名
孔凡瑞
陈亮
ZHANG Yi;CHEN Kan-jun;WU Ting;QI Cheng;JIN Wei-ming;KONG Fan-rui;CHEN Liang(Shanghai Cord Blood Bank/Shanghai Stem Cell Technology Co.,Ltd./China Stem Cell Group Co.,Ltd.,Shanghai 200051,China)
出处
《中国医药生物技术》
2019年第3期215-223,共9页
Chinese Medicinal Biotechnology
基金
上海市自然科学基金(16ZR1429200)
上海市科委研发平台专项(16DZ2293000)
上海市科委工程技术研究中心建设专项(18DZ2250800)
上海张江国家自主创新示范区专项发展资金重大项目(ZJ2017-ZD-010)
关键词
人脐带间充质干细胞
人真皮成纤维细胞
细胞外基质
胶原
Human umbilical cord mesenchymal stem cells
Human dermal fibroblast
Extracellular matrix
Collagen
