GCaMP基因工程化的C17.2神经干细胞的钙离子成像研究  

Calcium imaging features of GCa MP-engineered C17. 2 neural stem cells

作　　者：宋蔚林 赵从健[1] 阴正勤[1] 王一[1] 

机构地区：[1]第三军医大学西南医院全军眼科中心视觉损伤与再生修复重庆市重点实验室,重庆400038

出　　处：《第三军医大学学报》2015年第11期1064-1069,共6页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划;2013CB967002)~~

摘　　要：目的研究GCa MP(属于基因编码钙指示剂/蛋白钙指示剂)基因工程化的C17.2神经干细胞的钙离子成像。方法用慢病毒转染C17.2细胞,获得稳定表达GCa MP基因的C17.2-GCa MP细胞。Real-time PCR定量检测C17.2细胞及C17.2-GCa MP细胞中神经干细胞标志物巢蛋白(nestin)的表达情况。将C17.2细胞(Fluo-4 AM染色后)和C17.2-GCa MP细胞置于定制数字荧光显微镜下进行钙离子荧光检测。结果 Real-time PCR检测结果提示C17.2细胞及C17.2-GCa MP细胞中nestin表达差异无统计学意义(P>0.05)。C17.2-GCa MP细胞较Fluo-4 AM染色C17.2细胞钙离子荧光锐波(spike)波形持续时间长且相对荧光强度强(P<0.05),在Fluo-4 AM染色C17.2细胞中能观察到钙离子荧光慢波(wave)波形,而C17.2-GCa MP wave波形不典型。结论 C17.2-GCa MP细胞在保持神经干细胞干性的同时能够监测细胞内钙离子变化,能够运用于神经干细胞移植治疗视网膜色素变性功能整合的研究。Objectiv indicator） engineered C17.2 e To investigate the function of GCaMP （a genetically encoded calcium neural stem cells in calcium imaging. Methods C17.2-GCaMP cells were generated from C17.2 cells by infection of the lentivirus carrying GCaMP gene. Real-time PCR was applied to test the expression of nestin （the marker of neural stem cells） between C17.2 cells and C17.2-GCaMP cells. The calcium imaging of C17.2 cells loaded with Fluo-4 AM and C17.2-GCaMP cells was recorded under customerized digital fluorescence microscope. Results Real-time PCR results revealed that there was no significant difference in the expression of nestin between C17.2 cells and C17.2-GCaMP cells （ P 〉 0.05 ）. The calcium spikes in the C17.2-GCaMP cells had longer duration and higher intensity than those in C17.2 cells loaded with Fluo-4 AM （ P 〈 0.05 ）. In addition, typical calcium waves were observed in Fluo-4 AM-loaded C17.2 cells but not in C17.2-GCaMP cells. Conclusion C17.2-GCaMP cells maintain their sternness of neural stem cells after gene modification, and are able to instantly monitor the intracellular calcium dynamics, which could be applied to investigate the functional integration of transplanted neural stem ceils in vivo.

关 键 词：C17.2神经干细胞 GCaMP GECI Fluo-4 AM 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学] R774.13[医药卫生—基础医学]