毛囊神经干细胞促进受损大鼠视神经大胶质细胞去分化

Hair follicle neural stem cells promote dedifferentiation of macroglial cells in injured optic nerve of rats

目的培养毛囊神经干细胞,研究其对受损视神经大胶质细胞去分化的影响。方法取约90g雄性SD大鼠触须垫毛囊隆突区贴块,无血清培养或经15%胎牛血清诱导培养毛囊隆突区细胞,用免疫荧光细胞化学和PCR鉴定;以含增强型绿色荧光蛋白的腺相关病毒（rAAV2-EGFP）稳定转染该细胞。成年雄性SD大鼠54只,分为正常对照组、损伤组、移植组,移植组为视神经损伤后移植上述毛囊神经干细胞。转染细胞移植组术后7d、14d、30d,取视神经荧光显微镜下观察;损伤组和未转染细胞移植组术后7d取术侧视神经行Affymetrix基因芯片及实时荧光定量PCR检测,术后7d、14d取术侧视神经行HE、免疫组织化学检测。结果成功培养出神经前体细胞标记分子阳性的毛囊神经干细胞,诱导后该细胞可表达成熟神经细胞标记分子。毛囊神经干细胞移植到受损视神经30d后,仍能存活和迁移。与损伤组相比,移植组差异表达基因有240条,其中一些与去分化相关,如干细胞、凋亡、增殖、信号转导、转录、分化发育、细胞黏附等相关基因,实时荧光定量PCR与基因芯片结果基本相符。移植组视神经远侧段细胞数较损伤组明显增多,巢蛋白、髓鞘碱性蛋白（MBP）、细胞外信号调节激酶1/2（ERK1/2）表达增加,胶质纤维酸性蛋白（GFAP）表达减少,且视神经近侧段神经丝（NF）表达增加。结论毛囊神经干细胞通过调节受损视神经中的一些基因表达,促进了其大胶质细胞去分化并向有利于神经再生方向变化。

Objective To investigate the effect of cultured hair follicle neural stem cells （HFNSCs） on dedifferentiation of macroglial cells in injured optic nerve of rats. Methods Follicle bulge cells isolated from vibrissa pad of male SD rats weighing about 90g were cultured in DMEM/F12 without serum or containing 15% FBS, identified as HFNSCs by immunofluorescenee cytochemistry and PCR. The cells were stably transfected with rAAV2-EGFP. Adult male SD rats were randomly divided into 3 groups as normal control group, injury group and transplantation group in which HFNSCs were transplanted into injured optic nerves. At 7 days, 14 days and 30 days after EGFP-HFNSCs transplantation, optic nerves were observed under fluorescence microscope. At 7 days post operation, optic nerves from injury group and transplantation group were detected by gene chip of Affymetrix and real-time PCR. At 7 days, 14 days post operation, optic nerves were harvested and detected by HE staining and immunohistochemistry. Results Bulge cell in primary culture were labeled by neural progenitor cell markers, however the ceils induced by 15 % FBS expressed some makers of mature neural cells. EGFP-HFNSCs could survive and migrate in the injured optic nerve 30 days after transplantation. With the injury group, there were 240 differentially expressed genes including genes related stem cell, apoptosis, proliferation, transcription, differentiation and development, cell adhesion, signal transduction and so on in the transplantation group. The result of real time PCR was consistent with that of gene chip. There were more cells, more immunoreactivity of Nestin, MBP, Erk1/2, and less immunoreactivity of GFAP in the distal optic nerves and more immunoreactivity of NF in the proximal optic nerves in the transplantation group than that in the injured group. Conclusion HFNSCs regulate some genes expression of glial cells in the injured optic nerve to promote macroglial cells dedifferentiation and nerve regeneration.