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目的探讨缺氧缺血性脑损伤(HIBD)大鼠神经干细胞(NSCs)修复损伤的可能机制。方法传至2、3代的SD大鼠NSCs随机为空白对照组(未转染质粒者,CON),转染阴性对照质粒组(ncNSCs)和转染β-catenin siRNA真核表达质粒组(siNSCs),分别在不同脑组织匀浆上清液中培养,以模拟HIBD及正常脑内微环境。应用免疫荧光方法观察各组NSCs的分化情况;应用反转录PCR、Western blot法检测NSCs Ngn1,BMP4基因表达情况。结果与CON组比较,ncNSCs分化为神经元和少突胶质细胞的百分比明显增加(Pa<0.05),其中HIBD组增加较多;ncNSCs分化为星形胶质细胞的百分比显著减少(P<0.05)。与CON组相比,siN-SCs分化为神经元的百分比显著减少(P<0.01),分化为星形胶质细胞的百分比显著增加(P<0.01);少突胶质细胞的分化增加,但少于ncNSCs组(P<0.01),其中HIBD组分化为神经元和少突胶质细胞较多。与CON组比较,ncNSCs Ngn1 mRNA和Ngn1蛋白的表达显著增加(Pa<0.01),而BMP4 mRNA和BMP4蛋白的表达均显著减少(Pa<0.01);与CON组和ncNSCs组比较,siNSCs的Ngn1 mRNA和Ngn1蛋白表达显著减少(Pa<0.01),BMP4 mRNA和BMP4蛋白的表达显著增加(Pa<0.01),2个siNSCs组间Ngn1和BMP4的表达差异无统计学意义。结论 HIBD时受损的脑组织可进行自主修复,与β-catenin促进ncNSCs向神经元分化有关,BMP4和Ngn1在HIBD大鼠NSCs的增殖分化中起重要的协调作用。
Objective To investigate the possible mechanism of neural stem cells (NSCs) injury in hypoxic-ischemic brain damage (HIBD) rats. Methods The NSCs of passage 2 and 3 were randomly divided into blank control group (untransfected plasmid, CON), transfected negative control plasmid group (ncNSCs) and β-catenin siRNA eukaryotic expression plasmid group (siNSCs ) Were cultured in different brain tissue homogenates to simulate HIBD and normal brain microenvironment. Immunofluorescence was used to observe the differentiation of NSCs in each group. The expression of Ngn1 and BMP4 in NSCs was detected by reverse transcription PCR and Western blot. Results Compared with CON group, the percentages of ncNSCs differentiated into neurons and oligodendrocytes were significantly increased (P <0.05), especially in HIBD group. The percentage of ncNSCs differentiated into astrocytes was significantly decreased (P <0.05 ). Compared with CON group, the percentages of siN-SCs differentiated into neurons decreased significantly (P <0.01), the percentage of differentiated astrocytes increased significantly (P <0.01), and the differentiation of oligodendrocytes increased Less than ncNSCs group (P <0.01), of which HIBD group differentiated into neurons and oligodendrocytes more. Compared with CON group, the expression of Ngn1 mRNA and Ngn1 protein in ncNSCs was significantly increased (Pa <0.01), while the expressions of BMP4 mRNA and BMP4 protein were significantly decreased (P <0.01). Compared with CON group and ncNSCs group, Ngn1 mRNA (P <0.01). The expression of BMP4 mRNA and BMP4 protein was significantly increased (Pa <0.01). There was no significant difference in the expression of Ngn1 and BMP4 between the two groups of siNSCs. Conclusion The damaged brain tissue in HIBD can be repaired autonomously, which is related to the promotion of ncNSCs to neuron differentiation by β-catenin. BMP4 and Ngn1 play an important coordinating role in the proliferation and differentiation of NSCs in HIBD rats.