人胎盘不同组织分离间充质干细胞的生物学特性

Biological characters of mesenchymal stem cells separated from different components of human placenta

背景:胎盘间充质干细胞来源稳定,正逐渐成为广受关注的再生医学种子细胞来源。目的:比较从胎盘组织绒毛膜与绒毛滋养层分离获取人胎盘间充质干细胞生物学特性的差异。方法:手术剪剥离人胎盘表面羊膜,分别剪取胎儿侧的绒毛膜与绒毛滋养层组织机械破碎后,分别加含Ⅱ型胶原酶的PBS消化液消化,分离为单个核细胞,用含有体积分数10%胎牛血清的DMEM培养基在37℃、体积分数5%CO2、95%饱和湿度下培养,48 h后全量换液,去除非贴壁细胞,添加新鲜培养基,当细胞融合90%左右时,胰酶传代。通过观察原代细胞总数,细胞生长形态,以及间充质干细胞表面标记CD90、CD73、CD105的流式细胞测定结果比较从不同胎盘组织分离获得胎盘间充质干细胞的差异。结果与结论:流式细胞仪测定结果显示,从绒毛膜与绒毛滋养层中分离得到细胞间充质干细胞表面标记CD90、CD73以及CD105的阳性率都在90%以上,两种来源的细胞生长都呈现典型的成纤维细胞形态,这表明绒毛膜与绒毛滋养层中分离得到细胞都具有间充质干细胞的特性。提示消化时间相同,酶浓度相同,以及摇床转速相同的情况下,可从绒毛膜中可以得到更多的细胞,对于后续培养更易获得较多的细胞。

BACKGROUND：Human placenta is a stable source for mesenchymal stem cels, which is becoming a cellsource in the regenerative medicine that attracts widespread attentions. OBJECTIVE： To compare the biological characters of mesenchymal stem cels that separated from different components of human placenta （human chorion and vilous trophoblast）. METHODS：The amniotic membrane of placenta surface was detached surgicaly. Human chorion and vilous trophoblast in the fetal side was cut into pieces. After digestion with PBS containing colagenase II, mononuclear cels were separated and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum in 37℃ and 5% CO2, 95% saturated humidity, after 48 hours the ful amount in liquid, dislodge suspension cels. Forty-eight hours later, the medium was changed completed, and non-adherent cels were removed. When cellfusion reached about 90%, trypsin digestion was employed for cellpassage. Biological characters of mesenchymal stem cels separated from different components of human placenta were compared through observation of total number of primary cels, cellmorphology, and surface markers expression （CD90, CD73 and CD105）. RESULTS AND CONCLUSION：The flow cytometric analysis revealed that the cels separated from the human chorion and vilous trophoblast were over 90% strongly positive for CD90, CD73, CD105. These two sources of cels showed typical fibroblast morphology, suggesting that the cels have the characteristics of mesenchymal stem cels. Under the same enzyme digestion time, enzyme concentration, and shaking speed, more cels are visible from the chorion, and the subsequent culture is easier to harvest cels.