抑制PTEN蛋白对抑制性微环境中神经元生长的影响  

Phosphatase and tensin homology deleted on chromosome ten inhibition in improving growth of neurons cultured in inhibitive microenvironment

作　　者：范建民[1] 沈剑虹[2] 杨甫[2] 周非[2] 姚麒[2] 顾志恺[2] 

机构地区：[1]南通大学附属妇幼保健院神经内科,南通226001 [2]南通大学附属医院神经外科,南通226001

出　　处：《中华神经医学杂志》2014年第10期994-998,共5页Chinese Journal of Neuromedicine

基　　金：基金项目：南通市社会事业科技创新与示范计划项目（HS2011024）

摘　　要：目的 观察抑癌分子磷酸酶及张力蛋白同源物基因（PTEN）抑制剂bpv（pic）对抑制性微环境中神经元生长的影响，并探讨其可能的机制。方法 进行胎鼠皮层神经元的原代培养，以成年大鼠脑髓鞘膜蛋白作为细胞培养的抑制性底物。取培养后细胞分为3组：（1）正常对照组：常规培养，不做特殊处理；（2）抑制对照组：培养孔玻片用髓鞘膜蛋白包被；（3）bpv（pic）处理组：髓鞘膜蛋白包被培养孔，培养液中含200nmol／Lbpv（pic）。分别于接种后24h、48h、72h、5d行实时定量PCR（RT—PCR）检测及免疫荧光染色，观察各组册Ⅳ和哺乳动物雷帕霉素靶蛋白（mTOR）基因表达情况及各组神经元突起生长情况。结果 免疫荧光结果显示，4个时间点各组间细胞轴突长度差异均有统计学意义（P〈0．05）；在培养早期（24h、48h、72h），正常对照组细胞轴突最长；到后期（5d），bpv（pic）处理组细胞轴突最长。PT-PCR结果显示：3组细胞mTOR及PTEN在4个时间点之间表达量基本稳定，差异均无统计学意义（P〉0．05）。同一时间点不同组别细胞间mTOR及PTEN表达量差异有统计学意义（P〈0．05），其中bpv（pic）处理组mTOR及删表达量均明显高于正常对照组与抑制对照组，差异有统计学意义（P〈0．05）。结论 抑癌基因删的作用受到阻抑后，抑制性微环境中神经元的生长状况得到改善，可能与磷脂酰肌醇-3，4，5-三磷酸（PIP3）／丝-苏氨酸蛋白激酶（Akt）／mTOR通路的活化有关。Objective To investigate the effect of inhibiting tumor suppressor gene phosphatase and tensin homology deleted on chromosome ten （PTEN） on growth of neurons cultured in inhibitive microenvironment. Methods Primary cortical neurons were separated from fetal rats and cultured with adult rat brain-derived myelin membrane proteins as inhibitory substrate. Cells were divided into 3 groups： normal control group, inhibitory control group （myelin membrane proteins as inhibitory substrate） and bpv treatment group （200 nmol/L bpv＋myelin membrane proteins）. Real-time quantitative-PCR was performed to detect the expressions of PTEN and mammalian target ofrapamycin （roTOR） genes, and immunofluorescence staining was used to observe the growth ofneurites 24, 48 and 72 h, and 5 datter inoculation. Results Immunofluorescence staining revealed that significant differences of the growth ofneurites at the four observation time points existed among the 3 groups （P〈0.05）; during the early days of culture （24, 48 and 72 h）, cells in normal control group had the longest neurite （43.5±12.2, 94.9±23.6 and 154.3±35.4μm）, those in the bpv treatment group enjoyed the second prize （28.8±10.2, 75.4±2.5 and 136.8±40.8 μm） and those in the inhibitory control group was obviously inhibited （17.4±5.8, 46.3± 13.7 and 78.4±29.8μm）. But the difference on the 5^th d of cultivation changed into bpv treatment group〉normal control group〉inhibitory control group with the neurite length of（225.9＋50.6）, （218.4±58.1） and （173.6±48.7） μm, respectively （P〈0.05）. PCR revealed no significant difference in expressions of PTEN and raTOR genes of the three groups between each two time points （P〉0.05）; while at the same time point, significantly increased expressions of roTOR and PTEN in the bpv treatment group were noted as compared with those in the normal control group and inhibitory control group （P〈0.05）. Conclusions Growth of neurons in inhibitive microenvironment is i

关 键 词：磷酸酶及张力蛋白同源物基因 抑癌基因 神经再生 原代细胞培养 

分 类 号：Q421[生物学—神经生物学]