【摘要】 目的观察银杏酮酯(extract of ginkgo biloba leaves,EGb50)对体外培养的乳鼠SC增殖的影响,探讨其促进神经再生的作用机制。方法取20只3d龄SD乳鼠坐骨神经,以酶分步消化法分离SC。将纯化的第2代SC分为实验组和对照组,实验组采用终浓度为50μg/mL EGb50的FBS-DMEM培养液培养,对照组采用不含EGb50的FBS-DMEM培养液培养。于培养后1、3、5、7和9d,采用四唑氮衍生物[2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide,XTT]法检测吸光度(A)值并绘制生长曲线,流式细胞仪测定细胞周期。另在培养2d和3d用3H-胸腺嘧啶核甙(3H-thymine nucleoside,3H-TdR)测定法检测每分钟射线绝对衰变数(disintegration per minute,DPM)值,ELISA双夹心法测定培养液中神经生长因子(nerve growth factor,NGF)含量。结果培养的SC大多呈梭形及圆形,纯度达90%以上。XTT法检测对照组培养3dA值逐渐增加,5d达较高水平,此后缓慢增加。实验组变化规律与对照组一致,但不同时间点A值均高于对照组,差异有统计学意义(P<0.01)。3H-TdR掺入实验:实验组2、3dDPM值为1961.78±231.13及4601.51±605.08,对照组为1347.15±121.57及3740.42±158.73,差异均有统计学意义(P<0.01)。实验组和对照组细胞增殖指数分别为18.6%±3.2%及9.7%±2.9%,差异有统计学意义(P<0.01)。实验组和对照组SC培养液上清NGF含量分别(0.0656±0.0039)ng/mL和(0.0386±0.0036)ng/mL,差异有统计学意义(P<0.01)。结论EGb50有促进SC分裂增殖的作用,可能是其促进周围神经再生的重要机制之一。更多还原

【Abstract】 Objective To investigate the e ect of extract of ginkgo biloba leaves(EGb50) on the proliferation of SCs cultured in vitro.Methods The SCs were isolated from 3-day-old SD rats’ sciatic nerves by the method of enzyme gradation digestion(n=20) and the puri ed 2nd passage of SCs were divided into 2 groups:the experimental group,in which SCs were cultured in FBS-DMEM medium with EGb50(terminal concentration:50 μg/mL);the control group,in which SCs were cultured in the FBS-DMEM medium without EGb50.The absorbance(A) value was detected by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT) method 1,3,5,7 and 9 days after culture,then the growth curves was drawn.Cell cycle was detected by ow cytometry(FCM).Disintegration per minute(DPM) of SCs was detected by the method of 3H-thymine nucleoside(3H-TdR) 2 and 3 days after culture and nerve growth factor(NGF) synthesis in SCs culture media was detected by ELISA method.Results Most SCs were spindle-shaped with a purity above 90%.XTT detection showed that A value of SCs in the control group was gradually increased 3 days after culture,reached the peak 5 days after culture and gradually decreased from then;the A value in the experimental group experienced the similar changes,but it was higher than that in the control group at each time point(P < 0.01).3H-TdR showed that the DPM of the experimental group was 1 961.78 ± 231.13 and 4 601.51 ± 605.08 at 2 and 3 days after culture,while for the control group,the A value was 1 347.15 ± 121.57 and 3 740.42 ± 158.73 at the same time point,indicating a signi cant di erence between two groups(P < 0.01).FCM observation indicated that the SCs proliferation index of the experimental group and the control group was 18.6% ± 3.2% and 9.7% ± 2.9%,indicating a signi cant di erence between two groups(P < 0.01).ELISA observation showed that the NGF concentration in the experimental and the control group was(0.065 6 ± 0.003 9) ng/mL and(0.038 6 ± 0.003 6) ng/mL,indicating a signi cant di erence(P < 0.01).Conclusion EGb50 is capable of enhancing the proliferation of SCs cultured in vitro,which may be one of the important mechanisms to promote peripheral nerve regeneration.更多还原