中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2014年
6期
1141-1147
,共7页
辛毅%李娜%张颖%黄益民%刘飒%许秀芳%张兆光
辛毅%李娜%張穎%黃益民%劉颯%許秀芳%張兆光
신의%리나%장영%황익민%류삽%허수방%장조광
脐带%骨髓%间充质干细胞%生物学特性%绿色荧光蛋白%转基因小鼠
臍帶%骨髓%間充質榦細胞%生物學特性%綠色熒光蛋白%轉基因小鼠
제대%골수%간충질간세포%생물학특성%록색형광단백%전기인소서
Umbilical cord%Bone marrow%Mesenchymal stem cells%Biological characteristics%Green fluo-rescent protein%Transgenic mice
目的：探讨绿色荧光蛋白转基因小鼠脐带和骨髓源性间充质干细胞（ MSCs ）的体外分离培养方法、生物学特性、表面标志及多向分化潜能。方法：应用Ⅱ型胶原酶消化培养法分离脐带MSCs及密度梯度离心法分离骨髓MSCs进行体外培养。在倒置显微镜下观察2种细胞的生长特点，运用生长曲线和MTT法检测其原代细胞增殖能力，台盼蓝法测定细胞传代成活率，采用流式细胞术测定2种第3代（ P3）细胞DNA周期及表面标志物的表达，并比较其向成脂细胞和成骨细胞的分化潜能。结果：酶消化法分离培养的脐带MSCs 1 d后，细胞贴壁呈成纤维形，2 d后呈漩涡状生长且增殖明显，3 d后达80％融合即可传代；应用密度梯度离心法分离骨髓MSCs，体外培养4 d后，细胞贴壁呈圆形、梭形和多角形生长，5 d后呈克隆样生长且增殖明显，7 d后达80％融合即可传代。原代培养的脐带MSCs生长曲线近似“S”形，骨髓MSCs 生长曲线较平缓；MTT法显示脐带MSCs在3～5 d增殖较明显，骨髓MSCs 7 d后细胞增殖较明显。2种P3细胞传代成活率均为96％以上，G0／G1期细胞均为85％以上，无明显差异（P＞0．05）；2种P3细胞CD44、CD90和CD105阳性率均为（60．7±2．3）％以上高表达，CD45、CD19、CD14和CD79a均为（25．6±4．8）％低表达，两者无明显差异（ P＞0．05）；2种MSCs在体外均具有向成骨细胞和成脂细胞分化的潜能，脐带MSCs向成骨及成脂细胞分化率均为90％以上，与骨髓MSCs的分化潜能比较有显著差异（ P＜0.05）。结论：脐带MSCs较骨髓MSCs具有较强的增殖能力及分化潜能。绿色荧光蛋白转基因小鼠的脐带MSCs可作为较好干细胞示踪的细胞源。
目的：探討綠色熒光蛋白轉基因小鼠臍帶和骨髓源性間充質榦細胞（ MSCs ）的體外分離培養方法、生物學特性、錶麵標誌及多嚮分化潛能。方法：應用Ⅱ型膠原酶消化培養法分離臍帶MSCs及密度梯度離心法分離骨髓MSCs進行體外培養。在倒置顯微鏡下觀察2種細胞的生長特點，運用生長麯線和MTT法檢測其原代細胞增殖能力，檯盼藍法測定細胞傳代成活率，採用流式細胞術測定2種第3代（ P3）細胞DNA週期及錶麵標誌物的錶達，併比較其嚮成脂細胞和成骨細胞的分化潛能。結果：酶消化法分離培養的臍帶MSCs 1 d後，細胞貼壁呈成纖維形，2 d後呈漩渦狀生長且增殖明顯，3 d後達80％融閤即可傳代；應用密度梯度離心法分離骨髓MSCs，體外培養4 d後，細胞貼壁呈圓形、梭形和多角形生長，5 d後呈剋隆樣生長且增殖明顯，7 d後達80％融閤即可傳代。原代培養的臍帶MSCs生長麯線近似“S”形，骨髓MSCs 生長麯線較平緩；MTT法顯示臍帶MSCs在3～5 d增殖較明顯，骨髓MSCs 7 d後細胞增殖較明顯。2種P3細胞傳代成活率均為96％以上，G0／G1期細胞均為85％以上，無明顯差異（P＞0．05）；2種P3細胞CD44、CD90和CD105暘性率均為（60．7±2．3）％以上高錶達，CD45、CD19、CD14和CD79a均為（25．6±4．8）％低錶達，兩者無明顯差異（ P＞0．05）；2種MSCs在體外均具有嚮成骨細胞和成脂細胞分化的潛能，臍帶MSCs嚮成骨及成脂細胞分化率均為90％以上，與骨髓MSCs的分化潛能比較有顯著差異（ P＜0.05）。結論：臍帶MSCs較骨髓MSCs具有較彊的增殖能力及分化潛能。綠色熒光蛋白轉基因小鼠的臍帶MSCs可作為較好榦細胞示蹤的細胞源。
목적：탐토록색형광단백전기인소서제대화골수원성간충질간세포（ MSCs ）적체외분리배양방법、생물학특성、표면표지급다향분화잠능。방법：응용Ⅱ형효원매소화배양법분리제대MSCs급밀도제도리심법분리골수MSCs진행체외배양。재도치현미경하관찰2충세포적생장특점，운용생장곡선화MTT법검측기원대세포증식능력，태반람법측정세포전대성활솔，채용류식세포술측정2충제3대（ P3）세포DNA주기급표면표지물적표체，병비교기향성지세포화성골세포적분화잠능。결과：매소화법분리배양적제대MSCs 1 d후，세포첩벽정성섬유형，2 d후정선와상생장차증식명현，3 d후체80％융합즉가전대；응용밀도제도리심법분리골수MSCs，체외배양4 d후，세포첩벽정원형、사형화다각형생장，5 d후정극륭양생장차증식명현，7 d후체80％융합즉가전대。원대배양적제대MSCs생장곡선근사“S”형，골수MSCs 생장곡선교평완；MTT법현시제대MSCs재3～5 d증식교명현，골수MSCs 7 d후세포증식교명현。2충P3세포전대성활솔균위96％이상，G0／G1기세포균위85％이상，무명현차이（P＞0．05）；2충P3세포CD44、CD90화CD105양성솔균위（60．7±2．3）％이상고표체，CD45、CD19、CD14화CD79a균위（25．6±4．8）％저표체，량자무명현차이（ P＞0．05）；2충MSCs재체외균구유향성골세포화성지세포분화적잠능，제대MSCs향성골급성지세포분화솔균위90％이상，여골수MSCs적분화잠능비교유현저차이（ P＜0.05）。결론：제대MSCs교골수MSCs구유교강적증식능력급분화잠능。록색형광단백전기인소서적제대MSCs가작위교호간세포시종적세포원。
AIM:To compare the biological characteristics , surface markers and multi-differentiation potential of the mesenchymal stem cells (MSCs) derived from the umbilical cord and bone marrow in the green fluorescent protein (GFP) transgenic mice.METHODS:Umbilical cord MSCs (UCMSCs) were isolated by collagen type II enzymatic diges-tion and bone marrow MSCs ( BMSCs) were isolated by density gradient centrifugation .The growth of the 2 types of MSCs was observed under inverted microscope .The cell proliferation was detected by determining the growth curve and MTT as-say.The Trypan blue method was performed to analyze the cell viability rate .The cell cycle and cell surface markers were measured by flow cytometry .The differentiation potentials of the 2 types of MSCs were tested by the differentiation kits to-ward adipocytes and osteoblasts .RESULTS:The UCMSCs attached to the culture surface 1 d after the isolation , and the cells showed spiral shape with notable growth and proliferation after 2 d of culture.After 3 d, the cell arrived sub-confluent and was ready for passage .BMSCs still showed circular shape and started to attach to the surface 4 d after culture .They formed the small colony shape only after 5 d with obvious proliferative potential .The cells became confluent 7 d after the culture.The original generation of cultivating UCMSCs growth curve was shown typically an “S” shape.But the BMSCs growth was slower than the UCMSCs .The cell proliferation was obvious for UC-MSCs in 3~5 d.BMSCs proliferated signif-icantly only after 7 d.The viability rate arrived more than 96%for both types of MSCs .The cell cycle of both MSCs did not show significant difference (G0/G1 phases were above 85%, P>0.05).Both MSCs positively expressed CD44, CD90 and CD105 (60.7%±2.3%) but the expression of CD45, CD19, CD14 and CD79 was negative (less than 25.6%±4.8%, P>0.05).More than 90%of the MSCs from the umbilical cord and bone marrow differentiated towards the adipocytes and osteoblasts without significant difference (P<0.05).CONCLUSION:UCMSCs have stronger ability of proliferation and multi-directional differentiation potentials .UCMSCs in GFP transgenic mice as a high-quality tracer can serve for tracking the stem cells in vivo.
