丁苯酞在成人脂肪基质细胞分化为神经元样细胞过程中的分化促进及自噬激动作用观察  

Effects of butyphthalide on differentiation and autophagy agonism in differentiation of adipose-derived stromal cells into neuronal-like cells

作　　者：陆艳卉 徐康 赵紫烨[1] 李斌[1] 郭佳 周桂娟[1] LU Yanhui;XU Kang;ZHAO Ziye;LI Bin;GUO Jia;ZHOU Guijuan(Neurology Department,Tangshan People's Hospital,Tangshan 063000,China;不详)

机构地区：[1]唐山市人民医院神经内科,河北唐山063000 [2]唐山市人民医院检验科

出　　处：《山东医药》2022年第21期14-19,共6页Shandong Medical Journal

摘　　要：目的观察丁苯酞在成人脂肪基质细胞(ADSC)分化为神经元样细胞过程中的分化促进及自噬激动作用。方法取成人ADSC,应用1 mmol/L的β-巯基乙醇进行预诱导24 h后,分为β-巯基乙醇组、β-巯基乙醇+丁苯酞组、β-巯基乙醇+雷帕霉素组。其中β-巯基乙醇组加入含5 mmol/L的β-巯基乙醇,β-巯基乙醇+丁苯酞组加入5 mmol/L的β-巯基乙醇和10μmol/L的丁苯酞,β-巯基乙醇+雷帕霉素组加入5 mmol/L的β-巯基乙醇和200μg/L的雷帕霉素。在预诱导、诱导1 h、诱导3 h、诱导5 h、诱导8 h时,采用倒置相差显微镜观察各组细胞形态学变化,计算细胞分化率。在诱导1 h、诱导5 h时,在透射电镜下观察细胞的超微结构,计数自噬小体和自噬溶酶体数量。在诱导1 h、诱导3 h、诱导5 h、诱导8 h时,采用MTT法检测细胞存活率。在诱导5 h时,采用免疫细胞化学法检测各组细胞中神经元特异性烯醇化酶(NSE)、神经丝蛋-200(NF-200)。在诱导1 h、诱导3 h、诱导5 h、诱导8 h时,采用免疫细胞化学法检测各组细胞中LC3蛋白。结果诱导后的三组细胞形态学变化相似,分化的细胞呈典型的神经元样细胞形态;细胞核体积大而圆,染色浅,其中常染色质多、异染色质较少,部分神经元样细胞的细胞浆可见大量内质网扩张,线粒体出现肿胀、嵴断裂,细胞内出现具有双层膜结构的自噬小体。随着诱导时间的延长,各组细胞分化率、LC3阳性细胞率显著升高(P均<0.05),在诱导5 h时达到峰值;与β-巯基乙醇组相比,β-巯基乙醇+雷帕霉素组、β-巯基乙醇+丁苯酞组细胞分化率、LC3阳性细胞率均显著升高(P均<0.05);与β-巯基乙醇+雷帕霉素组相比,β-巯基乙醇+丁苯酞组在诱导1 h、3 h时细胞分化率、LC3阳性细胞率均显著升高(P均<0.05),在诱导8 h时显著降低(P<0.05)。诱导5 h时各组细胞自噬小体及自噬溶酶体数量均高于诱导1 h时(P均<0.05),β-巯基乙醇+丁苯酞Objective To observe the effects of butyphthalide on the differentiation and autophagy agonism in the differentiation of adipose-derived stromal cells(ADSC)into neuronal-like cells.Methods ADSC was divided into theβ-mercaptoethanol group,β-mercaptoethanol+butylphthalide group,andβ-mercaptoethanol+rapamycin group after ADSC was pre-induced for 24 h by 1 mmol/Lβ-mercaptoethanol.Cells in theβ-mercaptoethanol group were added with 5 mmol/Lβ-mercaptoethanol,β-mercaptoethanol+butyphthalide group with 5 mmol/Lβ-mercaptoethanol and 10μmol/L butylphthalide,andβ-mercaptoethanol+rapamycin group with 5 mmol/Lβ-mercaptoethanol and 200μg/L rapamycin.In the pre-induction,and at 1,3,5,and 8 h of induction,cell morphology changes were observed using inverted phase contrast microscopy,and the cell differentiation rate was calculated.At 1 and 5 h of induction,the ultrastructure of cells was observed under transmission electron microscopy,the numbers of autophagosomes and autophagy lysosomes were counted.At 1,3,5,and 8 h of induction,the cell survival rate was detected by MTT.At 5 h of induction,neuron-specific enolase(NSE)and neuorfliamne-200(NF-200)were detected by immunocytochemistry in each group.At 1,3,5,and 8 h of induction,the LC3 protein in each group was detected by immunocytochemistry.Results The morphology of cells was similar in the three groups,the differentiated cells showed a typical neuron-like cell morphology.The nuclei became round and big,the staining was shallow,and they had a large amount of euchromatin but little heterochromatin.The cytoplasm of some neuronal-like cells had a large number of outstretched endoplasmic reticulum,mitochondrial edema,volume increasing and cristae rupture;and double-membrane autophagosome appeared.Over the course of induction,the differentiation rate and expression rate of LC3-positive cells in each group increased significantly(both P<0.05)and reached a peak at 5 h of induction.Compared with theβ-mercaptoethanol group,theβ-mercaptoethanol+rapamycin group and theβ-merc

关 键 词：丁苯酞 脂肪基质细胞 神经元样细胞 细胞分化 细胞自噬 雷帕霉素 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]