摘要
背景：目前绝大多数研究者将目标指向功能细胞，为改善细胞的生存环境而给予一些生长因子。睾丸支持细胞能分泌大量营养物质及生长因子，且其所具有的Fas配体可降低移植入体内后宿主的免疫排斥。目的：探讨睾丸支持细胞对体外培养鼠胚胎大脑皮质细胞的影响。设计、时间及地点：细胞学体外观察实验，于2005-10／2007-09在首都医科大学生殖医学研究中心实验室完成。材料：健康SPF级10～15d龄雄性ICR小鼠40只，用于提取睾九支持细胞；15d龄胎鼠40只，用于提取大脑皮质细胞。方法：将分离的大脑皮质细胞密度调整为2×10^9L^-1，分为2组：实验组接种于预铺有70％汇合传代的睾丸支持细胞培养瓶中，加入含体积分数为0．1的FBS、1×10^5U／L青毒素、0．01g／L链霉素的DMEM／F12完会培养基，置于37℃、体积分数为0．05的CO2饱和湿度环境下培养17d；对照组接种于预铺有0．05％多聚右旋赖氨酸的培养瓶中单独培养。主要观察指标：采用免疫组织化学法、免疫细胞化学法、免疫细胞荧光法进行细胞鉴定，榆测神经元、神经球数量及酪氨酸羟化酶阳性率。结果：两组细胞神经元特异性烯醇化酶、巢蛋白、酪氨酸羟化酶均呈阳性表达。与培养2h比较，随培养时间的延长两组贴壁神经元数均明显减少（P〈0．01），但实验组各时间点贴壁神经元数均明显多于对照组（P〈0．01）。实验组于培养3d出现神经球，数量多且变化较快，7d后开始呈现明显的下降趋势，至17d神经球接近消失；对照组仅可见散在巢蛋白阳性表达细胞，未出现神经球。实验组酪氨酸羟化酶阳性细胞率随培养时间的延长而逐渐升高，至11d达高峰，且各培养时间点酪氨酸羟化酶阳性细胞率均高于对照组（P〈0．01）。结论：睾丸支持细胞可有效促进鼠胎脑皮质细胞的贴壁及生长，在促进神经干细胞增殖的同时诱导其向多巴胺能神经元分化。
BACKGROUND： Functioning cell is focus of current studies to prove some growth factors for improving survival environment of cells. Sertoli cells can secrete plenty of nutritive substances and growth factors, and its Fas ligand can reduce host immunologic rejection following implantation. OBJECTIVE： To investigate the effect of Sertoli cells on mouse embryonic cerebral cortical cells in vitro. DESIGN, TIME AND SETTING： In vitro observation of cytology was performed at the laboratory of Reproductive Medical Center, Capital Medical University between October 2005 and September 2007. MATERIALS： Forty SPF male ICR mice, aged 10- 15 days, were selected to harvest Sertoli cells; forty 15-day-old fetal mice were selected to harvest cerebral cortical cells. METHODS： The density of isolated cerebral cortical cells was adjusted to 2×10^9 L^-1 and divided into two groups. The cells in the experimental group were seeded on a culture flask coated with 70% passaged Sertoli cells and added DMEM/F12 culture medium containing 0.1 volume fraction of fetal bovine serum, 1×10^5 U/L penicillin, and 0.01 g/L streptomycin and incubated in 5% CO2 at 37 ℃ for 17 days. The cells in control group were cultured in a flask coated with 0.05% polyadenylic acid. MAIN OUTCOME MEASURES： Cells were identified using immunohistechemistry, immunocytochemistry and immunocytological fluorescent methods. The number of neurons and neurospheres were counted. Tyrosine hydroxylase （TH） positive rate was detected. RESULTS： All cells in two groups were neuron specific enolase, nestin, and TH positive. Compared with 2 hours of culture, the number Of attached neurons significantly decreased with time （P 〈 0.01）, but the number of attached neurons in the experimental group was still significantly higher than the control group at each time point （P 〈 0.01）. On the 3rd day, there was formation of neurospheres in the experimental group. The number of the neurospheres decreased from the 7th day, and nearly disappeared to the 17th day. Only scattered nestin-positive cells were found in the control group. TH-positive neurons were increased with culture time, and reached the peak level on the 11th day. At any stage, the number of TH-positive cells was higher in the experimental group than in the control group （P 〈 0.01）. CONCLUSION： Sertoli cells can significantly promote cerebral cortex cell attachment and growth. The neural stem cells can be directly induced to differentiate into dopaminergic progenitors by cocultured with Sertoli cells
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第43期8490-8494,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
北京市教委科技发展计划(KM2003100250898)
北京市自然科学基金(7083105)~~
