摘要
目的:观察人骨髓间充质干细胞膜上钙离子通道的激活、失活峰值与离子通道选择性等电生理学特性,为研究干细胞在分化过程中离子通道变化奠定基础。方法:选择泸州医学院附属医院耳鼻咽喉科2004-03/2005-04获得正常发育的人捐献的骨髓标本10例。采用梯度离心法和单层贴附培养法分离,纯化,获取人骨髓间充质干细胞,选取传代第1～2代生长活性良好的人骨髓间充质干细胞进行全细胞膜片钳的记录与分析。实验采用PC-IIB型膜片钳放大器(华中理工大学研制),由计算机发出指令经数字量数据/模拟量数据转换经放大器输出到细胞,细胞信号由放大器放大再经模/数转换后记录到计算机,保存在数字硬盘上供分析调读,记录与分析软件为IBBClamp,采样间隔100μs。通过设定不同的实验程序,记录通道电流的幅度,激活和失活电流的峰值与时程,离子通道的选择性,I-V曲线。结果:①纳入10例骨髓标本,8例成功分离、培养出骨髓间充质干细胞,2例培养出现细菌污染。②原代培养的骨髓间充质干细胞形态较均一,主要为梭形,其生长特点为:24～48h内贴壁,3～9d进入对数生长期,9～12d进入生长平台期。③在8例成功培养细胞中,有7例骨髓间充质干细胞可成功记录到离子通道电流,并均可记录到对Nicardipine阻断剂敏感的钙离子通道电流,该钙离子通道电流表现特点为:在阻断骨髓间充质干细胞膜上钠、钾电流后,当钳制为-90mV,膜上钙离子电流可被连续记录,从-110到-10mV持续钳制用10mV阶跃去极化,在-60mV获得钙离子通道的反转电流。结论:人骨髓间充质干细胞膜上用全细胞电压膜片钳可记录到钙离子通道电流,其电生理学特性符合非兴奋细胞膜上的钙离子通道特点。
AIM： To observe the activation and deactivation peak value as well as the selectivity of calcium ions signaling pathways of whole cell membrane of human mesenchymal stem cells, so as to provide the basis for the change of calcium ions signaling pathways in the process of differentiation of stem cell. METHODS： Ten bone marrow samples donated by the persons who got normal development in the Department of Otolaryngology, Affiliated Hospital of Sichuan Luzhou Medical College from March 2004 to April 2005. With gradient centrifugation and monolayer attachement culture method, hMSCs were isolated from human bone marrow and were purified. The well-grown hMSCs of the first to second generation were chosen to perform the record and analysis with whole cell patch clamp technology, Type PC-IIB patch clamp amplifier （developed by Huazhong University of Technology was adopted in this experiment）. Signaling was given out by the computer, then transformed by digital data/ simulated data and output to the cells by amplifier. Cell signaling was amplified by amplifier, transformed through module/data, then recorded in the computer and stored at digital hard disc for analysis and reading with software IBBClamp. The samples were collected at interval of 100μs. Different experimental procedures were set. The current amplitude of pathway, peak value and time course of activation and deactivation, selectivity of ions pathway, I-V curve were recorded. RESULTS： （1）In the experiments, hMSCs of 8 cases were successfully isolated from bone marrow of 10 healthy persons and cultured successfully, but those of 2 cases presented contamination. （2） Most of hMSCs were spindle in shape and they were characterized by adherence within 24 to 48 hours, entering into logarithmic growth phase within 3 to 9 days and entering the growth platform phase within 9 to 12 days. （3） Among the cells cultured successfully in 8 cases, calcium ions pathway current could he detected in the MSCs of 7 cases. Calcium pathway current which was sensitive to Nicardipine could all be detected, these calcium ions pathway current was characterized by that after the sodium and potassium current of cell membrane of MSCs was blocked, calcium ions current on the membrane could be recorded continuously if the patch clamp was -90 mV. Fluctuation in amplitudes of inward calcium ions currents was recorded at between -110 and -10 mV, and the reverse currents were recorded at -60 mV in patch clamp experiment.CONCLUSION： Whole cell patch clamp can record the calcium ion pathway current on the hMSCs membrance, its electrophysiological characteristics are consistent with the characteristics of calcium ion pathway of non-excitable cell membrane.
出处
《中国临床康复》
CSCD
北大核心
2006年第25期7-9,共3页
Chinese Journal of Clinical Rehabilitation
基金
泸州市重点科研项目基金资助(20050101)~~
