培养Schwann细胞用于构建毛囊神经嵴干细胞定向分化饲养层  

Primary Culture of Schwann cells as a Feed Layer of Hair Follicle Neural Crest Stem Cells

作　　者：周红利[1,2] 张艳[3] 任应博[3] 林森[1,2] 

机构地区：[1]成都医学院人体解剖与组胚教研室,四川成都610083 [2]成都医学院"发育与再生"四川省重点实验室,四川成都610083 [3]四川大学华西医院临床医学院毕业后继续教育部,四川成都610041

出　　处：《成都医学院学报》2011年第4期309-315,共7页Journal of Chengdu Medical College

基　　金：中华医学会-欧莱雅中国人健康皮肤研究项目(H2008020407);四川省教育厅自然科学基金资助项目(07ZB016);成都医学院校管基金资助项目(209Z029)

摘　　要：目的培养原代Schwann细胞,用于构建毛囊神经嵴干细胞饲养层构建。探索Schwann细胞(Schwann cells)对胎牛血清浓度的依耐性;检测Schwann细胞在不同血清浓度下的活性及分泌状态,并应用此饲养层定向诱导毛囊神经嵴干细胞分化。方法原代培养及纯化Schwann细胞;用不同血清浓度的胎牛血清(FBS)培养基培养Schwann细胞,测定并用四氮唑盐(MTT)比色法检测Schwann细胞生长及增殖情况;使用S100特异性标识抗体间接免疫荧光法与荧光染料Hoechst 33342对Schwann细胞进行鉴定。用ELISA方法检测Schwann细胞的分泌因子。利用丝裂霉素C处理Schwann细胞建立饲养层,培养Schwann细胞和毛囊神经嵴干细胞。结果体外成功培养Schwann细胞,用S100间接免疫荧光法与核染料Hoechst33342鉴定Schwann细胞呈阳性表达,纯度约95%,成功培养毛囊神经嵴干细胞。无血清的培养基可使Schwann细胞生长状态良好。饲养层细胞能够稳定分泌神经生长因子(nerve growth factor,NGF)和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF),可诱导毛囊神经嵴干细胞定向分化。结论无血清的培养基更利于毛囊神经嵴干细胞饲养层的构建。饲养层Schwann细胞可持续分泌NGF和BDNF等神经营养因子,可成为使毛囊神经嵴干细胞定向分化的稳定饲养层细胞。Objective To construct hair follicle neural crest stem celI（NCSC）feed layerby Primary cultured Schwann cells. Schwann cells were cultured with different concentration of fetal bovin serum（FBS）, whose activity was measured by ELISA and MTT assay. The feed layer was used to feed and induce NCSC differentiation. Methods The original generations of purification Sehwann cells were cultured with different concentration of FBS medium. The growth and proliferated ability of the cells were measured by MTT assay. Immunofluorescence was performed on Schwann cell with S100 and GFAP, and the figures were scanned under a confocal microscope. Mytomycin C was used to make the feed layer to eeaze the proliferation of Schwann cells. ELISA was perform to measure the release of nerve growth factor（NGF）and brain-derived neurotrophic factor（BDNF） by feed layer. NCSC was cocuhured with this constructed feed layer. Results Schwann cells were successful cultured in vitro. S100 was used to identify the cells by indirect immunofluorescence. It was found that FBS-free medium may provide a compatible condition to the growth of Schwann cell in the first 7 days. NGF and BDNF may also be released by the feed layer cells. The NCSC could be induced into neuro-glia cells by the feed layer. Conclusion FBS-free medium could provide a steady condition of Schwann cell growth. The feed layer can release NGF and BDNF steadily and may induce the NCSC into neuro-glia cells.

关 键 词：SCHWANN细胞 饲养层 毛囊神经嵴干细胞 定向分化 

分 类 号：R651.3[医药卫生—外科学]