人羊膜间充质干细胞膜片的构建及其成软骨诱导的实验研究

Experimental Study on Construction of Human Amniotic Mesenchymal Stem Cell Sheet and Its Chondrogenic Induction

目的:探讨应用一种简单的方法构建人羊膜间充质干细胞(human amniotic mesenchymal stem cells,HAMSCs)膜片,并研究其向成软骨细胞分化的潜能,探索利用HAMSCs膜片构建组织工程软骨的可行性。方法:取产妇胎盘通过酶消化法获得HAMSCs,通过流式细胞术鉴定其表型特征;通过免疫荧光检测细胞波形蛋白和CK-19的表达。取第3代HAMSCs通过CCK-8检测细胞增殖活性;取第3代HAMSCs加入成膜片培养基培养以构建HAMSCs膜片;取第3代HAMSCs加入成膜片培养基培养7天,再换用成软骨诱导培养基培养以构建成软骨诱导的HAMSCs膜片。通过流式细胞术检测成膜片诱导后HAMSCs表型分子的表达;扫描电镜(SEM)观察细胞形态和细胞外基质的分泌。RT-PCR定量分析成软骨分化相关基因(SOX9、ACAN、COLⅡ)的表达;HE染色观察细胞形态、分布;甲苯胺蓝和番红染色检测蛋白聚糖的分泌,Ⅱ型胶原免疫组化检测Ⅱ型胶原的表达。结果:流式细胞术结果表明HAMSCs表达间充质干细胞(MSCs)表型。免疫荧光检测结果示:波形蛋白阳性表达,CK-19阴性表达。CCK-8检测结果示:细胞生长曲线呈S型,第3天进入对数生长期,第7天达到顶峰。HAMSCs成膜片诱导后流式结果表明干细胞特性分子CD44、CD73、CD90、CD105仍高表达。SEM结果示:HAMSCs膜片呈复层结构,梭形的细胞分泌大量细胞外基质,细胞被细胞外基质所包埋并逐渐融合。RT-PCR结果显示:与HAMSCs膜片组相比,成软骨诱导的膜片组Ⅱ型胶原、蛋白聚糖、SOX9 mRNA的表达量显著增高,差异有统计学意义(P<0.05)。HE染色结果示:成软骨诱导的HAMSCs膜片可见分布均匀的类圆形细胞并被大量细胞外基质包围;甲苯胺蓝染色结果显示:椭圆形细胞分泌大量细胞胞外基质,成铺路石样排列;番红染色结果示:成软骨诱导的膜片有大量蛋白聚糖分泌;Ⅱ型胶原免疫组化结果示:成软骨诱导的膜片高表达Ⅱ型胶原。结论:本实验应用一种简单的方法在普通培养皿上成功构建了HAMSCs膜片,体外研究证实HAMSCs膜片具有良好的成软骨分化潜能。因此,HAMSCs膜片可以作为软骨组织工程的种子细胞之一,HAMSCs膜片的应用将为软骨缺损修复提供一种新思路。

Objective To construct a human amniotic mesenchymal stem cell(HAMSC) sheet using a simple method and to explore its potential to differentiate into chondrocytes,so as to find out the feasibility of constructing tissue engineered cartilage with the HAMSC sheet. Methods HAMSCs were separated using the enzyme digestion,and their phenotypic characteristics were tested using the flow cytometry. The expression of the cellular vimentin and cytokeratin 19(CK-19) was detected using the immunofluorescence assay. The proliferation activity of the passage-3 HAMSCs was measured using the cholecystokinin-octopeptide(CCK-8). The membrane-forming medium was added to the third-generation HAMSCs for 7 days to construct the HAMSCs membrane,and then replaced with chondrogenic induction medium to construct a chondrogenic-induced HAMSCs membrane. The phenotypic characteristics of HAMSCs after the sheet induction was detected using the flow cytometry. The cell morphology and secretion of extracellular matrix were observed using the scanning electronic microscopy(SEM). The expression of genes involved in the cartilage differentiation of SOX9,ACAN and COLⅡ was analyzed using the reverse transcription-polymerase(RT-PCR). Hematoxylin and eosin(HE) staining was used to observe the morphology and distribution of cells. The secretion of the aggrecan was detected using the toluidine blue staining and safranin staining. The expression of type Ⅱ collagen was evaluated using the immunohistochemistry. Results The flow cytometry results indicated that HAMSCs expressed the MSCs phenotype. The immunofluorescence assay showed that the vimentin expression was positive,while that of CK-19 was negative. The CCK-8 test showed that the cell growth curve was of Sshape,entering the logarithmic-growth phase on the 3 rd day and reaching the peak on the 7 th day.The flow cytometry results of HAMSCs induced by sheet culture medium showed that stem cell characteristic molecules of CD44, CD73, CD90 and CD105 were still highly expressed. According to the SEM results,the HAMSCs sheet was of a multi-layered structure,and the fusiform cells secreted a large amount of extracellular matrix,and they were embedded by the extracellular matrix and integrated gradually. RT-PCR results showed that compared with HAMSCS sheet,the expression of SOX9,ACAN and COLⅡ mRNA increased significantly in the cell sheet group induced using the new method(P<0.05). HE staining results showed that the cartilage-induced HAMSCs sheet showed a uniform distribution of round-like cells and was surrounded by a large number of extracellular matrices. The results of toluidine blue staining showed that the elliptical cells secreted a large number of extracellular matrix arranged into paving stones. The safranin staining showed that the cartilage-induced sheet had a large amount of aggrecan secretion. Moreover,The results of immunohistochemistry showed that the collagen type Ⅱ was highly expressed in the cartilage-induced sheet. Con-clusion In this experiment,an HAMSCs sheet is successfully constructed on a common culture dish using a simple method. The sheet can be operated easily and is proved of good chondrogenic differentiaton potential in vitro research. Therefore,HAMSCs sheet can be used as one of the seed cells of cartilage tissue engineering.Such HAMSCs sheet will provide a new idea for cartilage defect repair.