中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2010年
5期
457-460
,共4页
人骨髓间充质干细胞%膜片钳%电压依赖性钙通道%钙离子电流%细胞周期
人骨髓間充質榦細胞%膜片鉗%電壓依賴性鈣通道%鈣離子電流%細胞週期
인골수간충질간세포%막편겸%전압의뢰성개통도%개리자전류%세포주기
Human mesenchymal stem cells%Patch clamp%Voltage-operated Ca2+ channels%Calcium ion current%Cell cycle
目的 分离培养人骨髓间充质干细胞(hMSC),用膜片钳技术观察其膜表面电压依赖型钙通道.方法 取人骨髓血,用Percoll(1.073 g/ml)密度梯度离心及贴壁筛选结合的方法,体外培养扩增hMSC.台盼蓝拒染法计数活细胞数.流式细胞仪分析其免疫表型及细胞周期.用膜片钳技术观察hMSC膜表面电压依赖型钙通道的表达情况.结果 hMSC传代后形态上均为紧密排列的成纤维细胞样结构,细胞活力检测达99%.流式细胞检测表明,hMSC表达CD44、CD105,不表达CD31、CD34和CD45.细胞周期检测显示传代后的hMSC处于G1/G0期的细胞为81.52%,处于G2、M和S期的细胞约占18.48%,G2/G1为1.67.膜片钳检测显示,在20个记录中,有6个细胞可记录到对nifedipine敏感的内向钙电流,当激活电压大约在-40 mv时,最大内向峰值电流(Ica-Peak);在-0 mV为(96.67±13.50)pA,当加入5 μmol/L nifedipine,峰值钙电流为(47.75±11.24)pA,显著受到抑制(P＜0.05).结论 体外分离培养扩增的hMSC细胞活力强,主要为静止期细胞,其细胞膜表面存在电压依赖性钙通道.
目的 分離培養人骨髓間充質榦細胞(hMSC),用膜片鉗技術觀察其膜錶麵電壓依賴型鈣通道.方法 取人骨髓血,用Percoll(1.073 g/ml)密度梯度離心及貼壁篩選結閤的方法,體外培養擴增hMSC.檯盼藍拒染法計數活細胞數.流式細胞儀分析其免疫錶型及細胞週期.用膜片鉗技術觀察hMSC膜錶麵電壓依賴型鈣通道的錶達情況.結果 hMSC傳代後形態上均為緊密排列的成纖維細胞樣結構,細胞活力檢測達99%.流式細胞檢測錶明,hMSC錶達CD44、CD105,不錶達CD31、CD34和CD45.細胞週期檢測顯示傳代後的hMSC處于G1/G0期的細胞為81.52%,處于G2、M和S期的細胞約佔18.48%,G2/G1為1.67.膜片鉗檢測顯示,在20箇記錄中,有6箇細胞可記錄到對nifedipine敏感的內嚮鈣電流,噹激活電壓大約在-40 mv時,最大內嚮峰值電流(Ica-Peak);在-0 mV為(96.67±13.50)pA,噹加入5 μmol/L nifedipine,峰值鈣電流為(47.75±11.24)pA,顯著受到抑製(P＜0.05).結論 體外分離培養擴增的hMSC細胞活力彊,主要為靜止期細胞,其細胞膜錶麵存在電壓依賴性鈣通道.
목적 분리배양인골수간충질간세포(hMSC),용막편겸기술관찰기막표면전압의뢰형개통도.방법 취인골수혈,용Percoll(1.073 g/ml)밀도제도리심급첩벽사선결합적방법,체외배양확증hMSC.태반람거염법계수활세포수.류식세포의분석기면역표형급세포주기.용막편겸기술관찰hMSC막표면전압의뢰형개통도적표체정황.결과 hMSC전대후형태상균위긴밀배렬적성섬유세포양결구,세포활력검측체99%.류식세포검측표명,hMSC표체CD44、CD105,불표체CD31、CD34화CD45.세포주기검측현시전대후적hMSC처우G1/G0기적세포위81.52%,처우G2、M화S기적세포약점18.48%,G2/G1위1.67.막편겸검측현시,재20개기록중,유6개세포가기록도대nifedipine민감적내향개전류,당격활전압대약재-40 mv시,최대내향봉치전류(Ica-Peak);재-0 mV위(96.67±13.50)pA,당가입5 μmol/L nifedipine,봉치개전류위(47.75±11.24)pA,현저수도억제(P＜0.05).결론 체외분리배양확증적hMSC세포활력강,주요위정지기세포,기세포막표면존재전압의뢰성개통도.
Objective To isolate and culture human marrow mesenchymal stem cells (hMSCs),and to detect the voltage-operated Ca2+ channels (VOCCs) on the membrane surface of hMSCs using patch clamp technique. Methods Human bone marrow mononuclear cells were collected by gradient centrifugation on Percoll at a density of 1.073 g/ml combined with wall adherence method. The hMSCs were further cultured and expanded in vitro. Trypan blue exclusion staining method was used to count the number of living cells. The cellular phenotypes and cell cycle of hMSCs were identified by fluorescent activated cell sorting (FACS), and the expression of VOCCs on the membrane surface of hMSCs was observed with patch clamp technique. Results The typical morphology of passaged hMSCs featured a dense array of fibroblastlike structure figure. Up to 99% of hMSCs was viable. Flow cytometry demonstrated that hMSCs expressed CD44 and CD105 but not CD34, CD45 and CD31. Cell cycles of hMSCs showed that 81.52 % of hMSCs was in G1 and G0 stages, and 18.48 % in G2, M and S stages, with a G2/G1 ratio of 1.67. Patch clamp examination displayed nifedipine-sensitive calcium influx in 6 out of 20 hMSCs. The peak calcium influx (ICa-Peak) was (96.67±13.50) pA at -0 mV when activated voltage was about -40 mV. However, the Ica-Peak was restrained to (47.75± 11.24) pA with 5 μ mol/L nifedipine (P＜0.05). Conclusion hMSCs isolated and cultured in vitro were highly viable and mostly were at G0 or G1 stages. VOCCs were shown to be present on the membrane surface of hMSCs.
