天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2015年
2期
142-146
,共5页
脐带%间质干细胞%胶原酶类%传代效应%沃顿胶
臍帶%間質榦細胞%膠原酶類%傳代效應%沃頓膠
제대%간질간세포%효원매류%전대효응%옥돈효
umbilical cord%mesenchymal stem cells%collagenases%passage effect%Wharton’s jelly
目的：观察不同胶原酶消化方法对人脐带沃顿胶间充质干细胞（mesenchymal stem cell，MSC）分离和培养结果的影响，并鉴定其分化潜能；探讨传代效应对其免疫表型的影响。方法将制备好的脐带标本分别加入Ⅰ型、Ⅱ型及Ⅳ型胶原酶，持续消化4～18 h，筛网过滤，离心收集细胞，用DMEM/F12培养基重悬细胞，调整细胞密度4.8×103～1×104/cm2，接种培养，比较不同消化法分离人脐带沃顿胶MSC的效果。Von kossa钙结节染色、四环素荧光标记鉴定人脐带沃顿胶MSC向成骨方向分化的能力，RT-PCR鉴定其向心肌细胞分化的能力。应用流式细胞仪检测连续传代后MSC的免疫表型变化。结果Ⅰ型胶原酶消化法能够从人脐带沃顿胶获取了数量较多、活力较高的MSC，而且细胞出现伸展的时间及原代培养时间均短于Ⅱ型胶原酶及Ⅳ型胶原酶消化法。表面标记分析显示：随着传代次数的增加，阳性标记CD29、CD44、CD73、CD90、CD105的表达百分率没有变化，而阴性标记CD31、CD34和HLA-DR的表达率增加明显。体外诱导实验表明：来源于人脐带沃顿胶的MSC具有体外成骨和成心肌样细胞分化的能力。结论Ⅰ型胶原酶消化法简单易行，对细胞损伤小，能稳定、高效地从人脐带沃顿胶中分离出MSC。
目的：觀察不同膠原酶消化方法對人臍帶沃頓膠間充質榦細胞（mesenchymal stem cell，MSC）分離和培養結果的影響，併鑒定其分化潛能；探討傳代效應對其免疫錶型的影響。方法將製備好的臍帶標本分彆加入Ⅰ型、Ⅱ型及Ⅳ型膠原酶，持續消化4～18 h，篩網過濾，離心收集細胞，用DMEM/F12培養基重懸細胞，調整細胞密度4.8×103～1×104/cm2，接種培養，比較不同消化法分離人臍帶沃頓膠MSC的效果。Von kossa鈣結節染色、四環素熒光標記鑒定人臍帶沃頓膠MSC嚮成骨方嚮分化的能力，RT-PCR鑒定其嚮心肌細胞分化的能力。應用流式細胞儀檢測連續傳代後MSC的免疫錶型變化。結果Ⅰ型膠原酶消化法能夠從人臍帶沃頓膠穫取瞭數量較多、活力較高的MSC，而且細胞齣現伸展的時間及原代培養時間均短于Ⅱ型膠原酶及Ⅳ型膠原酶消化法。錶麵標記分析顯示：隨著傳代次數的增加，暘性標記CD29、CD44、CD73、CD90、CD105的錶達百分率沒有變化，而陰性標記CD31、CD34和HLA-DR的錶達率增加明顯。體外誘導實驗錶明：來源于人臍帶沃頓膠的MSC具有體外成骨和成心肌樣細胞分化的能力。結論Ⅰ型膠原酶消化法簡單易行，對細胞損傷小，能穩定、高效地從人臍帶沃頓膠中分離齣MSC。
목적：관찰불동효원매소화방법대인제대옥돈효간충질간세포（mesenchymal stem cell，MSC）분리화배양결과적영향，병감정기분화잠능；탐토전대효응대기면역표형적영향。방법장제비호적제대표본분별가입Ⅰ형、Ⅱ형급Ⅳ형효원매，지속소화4～18 h，사망과려，리심수집세포，용DMEM/F12배양기중현세포，조정세포밀도4.8×103～1×104/cm2，접충배양，비교불동소화법분리인제대옥돈효MSC적효과。Von kossa개결절염색、사배소형광표기감정인제대옥돈효MSC향성골방향분화적능력，RT-PCR감정기향심기세포분화적능력。응용류식세포의검측련속전대후MSC적면역표형변화。결과Ⅰ형효원매소화법능구종인제대옥돈효획취료수량교다、활력교고적MSC，이차세포출현신전적시간급원대배양시간균단우Ⅱ형효원매급Ⅳ형효원매소화법。표면표기분석현시：수착전대차수적증가，양성표기CD29、CD44、CD73、CD90、CD105적표체백분솔몰유변화，이음성표기CD31、CD34화HLA-DR적표체솔증가명현。체외유도실험표명：래원우인제대옥돈효적MSC구유체외성골화성심기양세포분화적능력。결론Ⅰ형효원매소화법간단역행，대세포손상소，능은정、고효지종인제대옥돈효중분리출MSC。
Objective To observe the effects of different collagenase digestions on isolating human umbilical cord mesenchymal stromal cells (MSC) from Wharton’s jelly, to exam their differentiation ability and to investigate their passage effect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠorⅡorⅣfor 4-18 hours then were passed through sieves . Cells were collected by centrifugation then inoculated in DMEM/F12 medium at concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa staining and tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employed to identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected by flow cytometry after subculture. Results Using collagenaseⅠdigestion, the number of MSCs isolated from human umbilical cord in Wharton’s jelly and their vitality were much higher while the period to show cell extension and primary culture time were shorter than those using collagenaseⅡorⅣdigestions. The analysis of surface marker revealed that the expression of positive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markers such as CD31, CD34 and HLA-DR increased significantly with passages;Differential experiments induced in vitro show that human umbilical cord MSC in wharton’s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Con?clusion The human umbilical cord MSC in Wharton’s jelly was successfully isolated by collagenaseⅠdigestion. This meth?od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possi? ble. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.
