中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2013年
32期
2592-2596
,共5页
刘玲英%柴家科%段红杰%侯玉森%尹会男%郁永辉%胡泉%郝岱峰%冯光
劉玲英%柴傢科%段紅傑%侯玉森%尹會男%鬱永輝%鬍泉%郝岱峰%馮光
류령영%시가과%단홍걸%후옥삼%윤회남%욱영휘%호천%학대봉%풍광
间质干细胞%脐带%胶原酶类%胰蛋白酶%细胞分离
間質榦細胞%臍帶%膠原酶類%胰蛋白酶%細胞分離
간질간세포%제대%효원매류%이단백매%세포분리
Mesenchymal stem cells%Umbilical cord%Collagenases%Trypsin%Cell separation
目的 对比分析分离人脐带间充质干细胞(MSCs)常用方法的效果,筛选比较高效的分离方法.方法 采集足月剖宫产手术胎儿的脐带组织(n=15),充分洗涤后去除3条脐血管及脐带外膜,将其剪碎至1 mm3的组织块,洗涤离心确定组织块的总体积,平均分为4组,分别采用组织块法和单纯胶原酶Ⅱ消化法、胶原酶Ⅱ+胰蛋白酶消化法、胶原酶Ⅱ+透明质酸酶消化法,分离获得人脐带MSCs,行锥虫蓝染色计数细胞;倒置相差显微镜观察细胞形态;使用免疫荧光染色检测细胞表面标志物CD105、CD90、CD73、CD31、CD44、CD45、人类白细胞抗原I(HLA-I)和人类白细胞抗原Ⅱ类分子(HLA-DR)的表达情况,进行细胞鉴定;噻唑蓝(MTT)法检测细胞的增殖曲线.结果 组织块法和单纯胶原酶Ⅱ消化法、胶原酶Ⅱ+胰蛋白酶消化法、胶原酶Ⅱ+透明质酸酶消化法4种方法均可分离获得干细胞,但获得细胞的数量和增殖能力不同.4种方法获得的细胞数量分别为(5.44±0.21)×105、(4.03 ±0.24)×105、(4.91 ±0.33) ×l05和(5.94 ±0.40)×105,胶原酶Ⅱ+透明质酸酶消化法显著多于其他3种方法,差异均有统计学意义(均P ＜0.05).组织块法种植后第9～11天观察到细胞在组织块周围生长,而其他3种方法种植后第2天即可见贴壁生长的细胞.4种方法细胞达到90％ ～95％融合的时间分别为(18.5±3.5)、(8.0±1.0)、(7.5±1.5)、(3.5 ±±0.5)d,胶原酶Ⅱ+透明质酸酶消化法显著短于其他3组,差异均有统计学意义(均P ＜0.05).组织块法原代细胞的形态多为多角形,不规则,细胞的体积较大,单纯胶原酶Ⅱ消化法和胶原酶Ⅱ+胰蛋白酶消化法原代细胞的形态均较短小,而胶原酶Ⅱ+透明质酸酶消化法细胞形态呈细长的梭形.4种方法得到的细胞均阳性表达CD105、CD90和CD73,弱表达HLA-I,不表达CD31、CD45和HLA-DR.胶原酶Ⅱ+透明质酸酶消化法细胞的增殖速率显著快于其他3种方法.结论 胶原酶Ⅱ+透明质酸酶消化法是一种高效的提取人脐带MSCs的方法.
目的 對比分析分離人臍帶間充質榦細胞(MSCs)常用方法的效果,篩選比較高效的分離方法.方法 採集足月剖宮產手術胎兒的臍帶組織(n=15),充分洗滌後去除3條臍血管及臍帶外膜,將其剪碎至1 mm3的組織塊,洗滌離心確定組織塊的總體積,平均分為4組,分彆採用組織塊法和單純膠原酶Ⅱ消化法、膠原酶Ⅱ+胰蛋白酶消化法、膠原酶Ⅱ+透明質痠酶消化法,分離穫得人臍帶MSCs,行錐蟲藍染色計數細胞;倒置相差顯微鏡觀察細胞形態;使用免疫熒光染色檢測細胞錶麵標誌物CD105、CD90、CD73、CD31、CD44、CD45、人類白細胞抗原I(HLA-I)和人類白細胞抗原Ⅱ類分子(HLA-DR)的錶達情況,進行細胞鑒定;噻唑藍(MTT)法檢測細胞的增殖麯線.結果 組織塊法和單純膠原酶Ⅱ消化法、膠原酶Ⅱ+胰蛋白酶消化法、膠原酶Ⅱ+透明質痠酶消化法4種方法均可分離穫得榦細胞,但穫得細胞的數量和增殖能力不同.4種方法穫得的細胞數量分彆為(5.44±0.21)×105、(4.03 ±0.24)×105、(4.91 ±0.33) ×l05和(5.94 ±0.40)×105,膠原酶Ⅱ+透明質痠酶消化法顯著多于其他3種方法,差異均有統計學意義(均P ＜0.05).組織塊法種植後第9～11天觀察到細胞在組織塊週圍生長,而其他3種方法種植後第2天即可見貼壁生長的細胞.4種方法細胞達到90％ ～95％融閤的時間分彆為(18.5±3.5)、(8.0±1.0)、(7.5±1.5)、(3.5 ±±0.5)d,膠原酶Ⅱ+透明質痠酶消化法顯著短于其他3組,差異均有統計學意義(均P ＜0.05).組織塊法原代細胞的形態多為多角形,不規則,細胞的體積較大,單純膠原酶Ⅱ消化法和膠原酶Ⅱ+胰蛋白酶消化法原代細胞的形態均較短小,而膠原酶Ⅱ+透明質痠酶消化法細胞形態呈細長的梭形.4種方法得到的細胞均暘性錶達CD105、CD90和CD73,弱錶達HLA-I,不錶達CD31、CD45和HLA-DR.膠原酶Ⅱ+透明質痠酶消化法細胞的增殖速率顯著快于其他3種方法.結論 膠原酶Ⅱ+透明質痠酶消化法是一種高效的提取人臍帶MSCs的方法.
목적 대비분석분리인제대간충질간세포(MSCs)상용방법적효과,사선비교고효적분리방법.방법 채집족월부궁산수술태인적제대조직(n=15),충분세조후거제3조제혈관급제대외막,장기전쇄지1 mm3적조직괴,세조리심학정조직괴적총체적,평균분위4조,분별채용조직괴법화단순효원매Ⅱ소화법、효원매Ⅱ+이단백매소화법、효원매Ⅱ+투명질산매소화법,분리획득인제대MSCs,행추충람염색계수세포;도치상차현미경관찰세포형태;사용면역형광염색검측세포표면표지물CD105、CD90、CD73、CD31、CD44、CD45、인류백세포항원I(HLA-I)화인류백세포항원Ⅱ류분자(HLA-DR)적표체정황,진행세포감정;새서람(MTT)법검측세포적증식곡선.결과 조직괴법화단순효원매Ⅱ소화법、효원매Ⅱ+이단백매소화법、효원매Ⅱ+투명질산매소화법4충방법균가분리획득간세포,단획득세포적수량화증식능력불동.4충방법획득적세포수량분별위(5.44±0.21)×105、(4.03 ±0.24)×105、(4.91 ±0.33) ×l05화(5.94 ±0.40)×105,효원매Ⅱ+투명질산매소화법현저다우기타3충방법,차이균유통계학의의(균P ＜0.05).조직괴법충식후제9～11천관찰도세포재조직괴주위생장,이기타3충방법충식후제2천즉가견첩벽생장적세포.4충방법세포체도90％ ～95％융합적시간분별위(18.5±3.5)、(8.0±1.0)、(7.5±1.5)、(3.5 ±±0.5)d,효원매Ⅱ+투명질산매소화법현저단우기타3조,차이균유통계학의의(균P ＜0.05).조직괴법원대세포적형태다위다각형,불규칙,세포적체적교대,단순효원매Ⅱ소화법화효원매Ⅱ+이단백매소화법원대세포적형태균교단소,이효원매Ⅱ+투명질산매소화법세포형태정세장적사형.4충방법득도적세포균양성표체CD105、CD90화CD73,약표체HLA-I,불표체CD31、CD45화HLA-DR.효원매Ⅱ+투명질산매소화법세포적증식속솔현저쾌우기타3충방법.결론 효원매Ⅱ+투명질산매소화법시일충고효적제취인제대MSCs적방법.
Objective To explore the most appropriate method for the isolation of human umbilical cord mesenchyamal stem cells (MSCs) through a comparison of different methods.Methods Fifteen umbilical cord specimens from full-term healthy fetus with caesarean birth were completely rinsed with phosphate buffer saline (PBS) and sliced into 1 mm3 tissue blocks after removal of umbilical vessels and external membrane.These tissue blocks were averagely divided into 4 groups after washing and centrifuge.Then four methods for the isolation of human umbilical cord MSCs were compared:an explant culture and three enzymatic methods of collagenase Ⅱ,collagenase Ⅱ/trypsin and collagenase Ⅱ/hyaluronidase.The count of living cells was evaluated by trypan blue dye exclusion test.Cell morphology was observed under inverted microscope.The expressions of cell surface markers CD105,CD90,CD73,CD31,CD44,CD45,human leukocyte antigen-I (HLA-I) and human leukocyte antigen class Ⅱ molecules (HLA-DR) were detected by immunofluorescent staining.Cell proliferation was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).Results The human umbilical cord MSCs were successfully isolated by four isolated methods.However the isolation method used profoundly altered the cell number and proliferation capacity of isolated cells.Isolated cells using four methods were counted at (5.44 ± 0.21) × 105,(4.03 ± 0.24) × l05,(4.91 ± 0.33) × 105 and (5.94 ± 0.40) × l05 respectively.More cells were obtained with collagenase Ⅱ/hyaluronidase than other three methods (all P ＜ 0.05).Cells out of tissue blocks were observed at Day 9-11 and cells were observed at Day 2 with three types of enzyme digestion.The fusion time of cells were (18.5 ±3.5),(8.0 ± 1.0),(7.5 ± 1.5) and (3.5 ±0.5) days respectively.The fusion time of cells obtained with collagenase Ⅱ/hyaluronidase was lower than other methods (all P ＜ 0.05).Cell morphology:polygonal,irregular and of large volume for explant culture ; relatively short and small for collagenase Ⅱ and collagenase Ⅱ/trypsin methods ; thin spindle for collagenase Ⅱ/hyaluronidase method.Immunofluorescent staining revealed that CD105,CD73,CD90 and CD44 were expressed in all groups while there was no expression of CD31,CD45 or HLA-DR.And the cells obtained with collagenase Ⅱ/hyaluronidase method were in a higher cell proliferation rate and activity compared to other methods.Conclusion The collagenase Ⅱ/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods.
