CAJ | 학술논문

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200 nmol/L bpv(pic).分别于接种后24h、48 h、72 h、5d行实时定量PCR(RT-PCR)检测及免疫荧光染色,观察各组PTEN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(24 h、48 h、72 h),正常对照组细胞轴突最长;到后期(5 d),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PTEN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PTEN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.
목적 관찰억암분자린산매급장력단백동원물기인(PTEN)억제제bpv(pic)대억제성미배경중신경원생장적영향,병탐토기가능적궤제.방법 진행태서피층신경원적원대배양,이성년대서뇌수초막단백작위세포배양적억제성저물.취배양후세포분위3조:(1)정상대조조:상규배양,불주특수처리;(2)억제대조조:배양공파편용수초막단백포피;(3)bpv(pic)처리조:수초막단백포피배양공,배양액중함200 nmol/L bpv(pic).분별우접충후24h、48 h、72 h、5d행실시정량PCR(RT-PCR)검측급면역형광염색,관찰각조PTEN화포유동물뢰파매소파단백(mTOR)기인표체정황급각조신경원돌기생장정황.결과 면역형광결과현시,4개시간점각조간세포축돌장도차이균유통계학의의(P＜0.05);재배양조기(24 h、48 h、72 h),정상대조조세포축돌최장;도후기(5 d),bpv(pic)처리조세포축돌최장.PT-PCR결과현시:3조세포mTOR급PTEN재4개시간점지간표체량기본은정,차이균무통계학의의(P＞0.05).동일시간점불동조별세포간mTOR급PTEN표체량차이유통계학의의(P＜0.05),기중bpv(pic)처리조mTOR급PTEN표체량균명현고우정상대조조여억제대조조,차이유통계학의의(P＜0.05).결론 억암기인PTEN적작용수도조억후,억제성미배경중신경원적생장상황득도개선,가능여린지선기순-3,4,5-삼린산(PIP3)/사-소안산단백격매(Akt)/mTOR통로적활화유관.
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 (mTOR) genes,and immunofluorescence staining was used to observe the growth ofneurites 24,48 and 72 h,and 5 d after inoculation.Results Immunofluorescence staining revealed that significant differences of the growth of neurites 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±35.4 μm),those in the bpv treatment group enjoyed the second prize (28.8±10.2,75.4±22.5 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 ±29.8 μm).But the difference on the 5th 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 mTOR genes of the three groups between each two time points (P＞0.05); while at the same time point,significantly increased expressions ofmTOR 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 improved on condition that the tumor suppressor gene PTEN is inhibited,which is probably related to the activation of PI3K/AKT/MTOR pathway.