人胚胎神经干细胞体外条件下对胶质瘤细胞的趋向性特点  

Tropism of fetal neural stem cells for glioma patch in vitro

作　　者：姬西团[1] 章翔[1] 费舟[1] 张剑宁[1] 刘卫平[1] 蒋晓帆[1] 宋少军[1] 宋蕾[1] 梁景文[1] 

机构地区：[1]解放军第四军医大学西京医院全军神经外科研究所,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2007年第24期4690-4692,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军医药卫生重点基金(04LX037)~~

摘　　要：目的:以Hs683、3T3成纤维细胞作为对照,探讨人胚胎神经干细胞在体外条件下对SHG44、C6胶质瘤细胞的趋向性。方法:①来源于怀孕子宫肌瘤切除术或流产的10～14周胚胎(西安市中心医院妇产科、西安交大二院妇产科、北方医院妇产科提供),产妇及其家属均签署知情同意书。Hs683成纤维细胞系由田晓峰博士馈赠,SHG44胶质瘤细胞系、C6胶质瘤细胞系、3T3成纤维细胞系均为本研究所冻藏。②将原代培养8d的人胚胎神经干细胞悬液离心、收集备用,最终神经球浓度达8×104 L-1。③将预先消毒的盖玻片(24mm×24mm)放置于培养皿内,将SHG44胶质瘤细胞系、C6胶质瘤细胞系、Hs683成纤维细胞系、3T3成纤维细胞系的4组细胞复苏后,加入含体积分数为0.1胎牛血清的DMEM培养基。当各组细胞爬片上的细胞贴壁达75%～85%时,取出盖玻片,与上述制备的人胚神经球悬浮液共同培养。④将预先制备好的SHG44胶质瘤细胞、C6胶质瘤细胞、Hs683成纤维细胞、3T3成纤维细胞爬片(各10张)从培养瓶取出,置于新的培养皿,分别与神经干细胞克隆球在含体积分数为0.01～0.02胎牛血清的DMEM培养液中共培养72h,观察并统计各细胞系每张爬片的克隆球数。上述4种细胞爬片周边3mm以外相同面积、无胶质瘤/成纤维细胞区域作为其各自空白对照。⑤分别将神经干细胞克隆球+C6胶质瘤细胞+3T3成纤维细胞、神经干细胞克隆球+SHG44胶质瘤细胞+Hs683成纤维细胞同法共培养72h,观察人胚神经干细胞在SHG44、Hs683、C6、3T3细胞爬片上的分布情况。结果:①人胚神经干细胞对SHG44、C6胶质瘤细胞的趋向作用:SHG44/C6胶质瘤细胞+人胚神经干细胞克隆球共培养72h后,胶质瘤细胞爬片上的神经克隆球体积均有所增大,且数量均明显高于外周无胶质瘤细胞的空白对照区(P均<0.05)。②人胚神经干细胞对Hs683、3T3成纤维细胞的趋向作用:Hs683/3T3成�AIM： The experiment is designed to explore the tropism of fetal neural stem calls （NSCs） for glioma cells （SHG44, C6） cultured in vitro, taking Hs683 and 3T3 as controls. METHODS： ①With the informed consents of the puerpera and their relatives, the embryos aging 10-14 weeks were recruited from the puerpera after myomectomy or abortion （Department of Gynaecology and Obstetrics at Xi＇an Central Hospital, Department of Gynaecology and Obstetrics at Second Hospital of Xi＇an Jiaotong University, and Department of Gynaecology and Obstetrics at North Hospital）. Hs683 fibreblast was presented by Professor Tian as a gift. SHG44 glioma cell line, C6 glioma cell line, and 3T3 fibroblast cell line were preserved by this institute.②The NSC suspension primarily cultured for 8 days in vitrowere centrifuged to reserve at the neuresphere concentration of 8×10^4 L^-1. ③SHG44 calls, C6 calls, Hs683 flbroblasts and 3T3 flbroblasts were resuscitated and cultured in DMEM containing 10% fetal bovine serum （FBS） on the 24 mm ×24 mm culture dish, which was disinfected previously. The cover slip was removed when the calls reached 75%-85% adhesion on the patch, and the cells were co-cultured with NSCs. ④Ten patches of cells in four groups were placed in a new culture dish and co-cultured with NSCs in DMEM containing 1%-2% FBS for 72 hours. The number of clone sphere on each patch was recorded. The equal area of over 3 mm surrounding the patch was taken as controls, no glioma/fibreblast was found.⑤The neurosphere＋C6 cells＋3T3 fibreblasts, neurosphere＋SHG44 calls＋Hs683 fibreblasts were co-cultured for 72 hours. The distributions of NSCs on SHG44 calls, C6 calls, Hs683 fibroblasts and 3T3 flbreblasts patches were analyzed. RESULTS：①A lot of neurespheres expanded on glioma call patch after they were co-cultured with SHG44 cells and C6 cells for 72 hours, and the number of neurespheres was significantly higher than that of control group （P 〈 0.05）.②After Hs683/3T3 flbreblast and NSCs were

关 键 词：趋向性 SHG44细胞 C6细胞 神经干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]