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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 culture hair follicle neural stem cells and study its effect on the dedifferentiation of damaged optic nerve. Methods Totally 90g male SD rat hair follicles of the tentorium hair follicles were attached to the buccal mucosa, cultured in serum-free medium or induced by 15% fetal bovine serum. The cells were identified by immunofluorescence cytochemistry and PCR. The cells were stably transfected with the protein adeno-associated virus (rAAV2-EGFP). 54 adult male Sprague-Dawley rats were randomly divided into normal control group, injured group, transplantation group and transplantation group. The neural stem cells were transplanted after optic nerve injury. The optic nerve was observed under fluorescent microscope 7 days, 14 days and 30 days after transplantation. The injured optic nerve and the untransfected cell transplantation group were examined by Affymetrix gene chip and real-time fluorescence quantitative PCR 7 days after operation. The lateral optic nerve was removed 14 days after operation and examined by immunohistochemistry. Results The hair follicle-derived neural stem cells were successfully cultured and labeled with neural progenitor cells. After induced, the cells could express mature neural cell marker molecules. After the transplantation of hair follicle-derived neural stem cells to the damaged optic nerve for 30 days, they can still survive and migrate. Compared with the injury group, there were 240 differentially expressed genes in the transplantation group, some of which were associated with dedifferentiation such as stem cell apoptosis, proliferation, signal transduction, transcription, differentiation and development, cell adhesion and other related genes, real-time fluorescence quantitative PCR Gene chip results are basically consistent. The number of cells in the distal optic nerve of the transplantation group was significantly increased compared with the injury group. The expressions of nestin, MBP, ERK1 / 2 and glial fibrillary acidic protein (GFAP) Decreased expression, and optic nerve proximal segment of nerve filaments (NF) expression increased. Conclusion The hair follicle-derived neural stem cells promote the dedifferentiation of their large glial cells by adjusting the expression of some genes in the damaged optic nerve, which is conducive to the change of nerve regeneration.