转染SMDF基因的小鼠肌源性干细胞分化为施万样细胞的初步研究

Preliminary study of muscle-derived stem cells in mice with SMDF gene transfected into Schwann-like cells

目的将构建的Neu基因调节素-1(NRG-1)Ⅲ型感觉运动衍生因子(SMDF)真核表达载体转染肌源性干细胞(MDSC),通过与施万细胞(Schwann cell,SC)共培养液的方法,诱导转染后的MDSC分化为类SC,证实SMDF基因可促进类SC的增殖、分化,进一步验证SMDF蛋白的功能,为研究周围神经损伤后SC髓鞘化过程及分子机制奠定了实验基础。方法 (1)通过蛋白质印迹法分析小鼠脑、脊髓、背根神经节(DRG)、骨骼肌、肝脏、脾脏等组织中SMDF蛋白的表达情况,同时通过免疫组织化学染色法鉴定SMDF在DRG中的表达;(2)PCR获得小鼠SMDF基因编码区全序列,构建重组真核表达载体p EGFP-N2-SMDF;(3)应用脂质体介导的方法将真核表达载体p EGFP-N2-SMDF和空质粒p EGFP-N2转染体外培养液的MDSC,分为重组质粒组、质粒组和未转染组,检测转染前后SMDF基因和蛋白的表达情况;(4)采用细胞条件培液诱导转染SMDF基因的MDSC分化为施万样细胞,并采用免疫细胞化学方法进行鉴定。数据比较采用one-way ANOVA和t检验。结果 (1)p EGFP-N2-SMDF真核表达载体转染MDSC后,荧光显微镜下可见绿色荧光蛋白的表达。(2)PCR和蛋白质印迹法结果显示,重组载体p EGFP-N2-SMDF转染后的细胞中SMDF mRNA水平和蛋白水平的表达明显增高。重组质粒组SMDF/GAPDH值为1.7321±0.1346,分别与空质粒组(0.5975±0.0084)和未转染质粒组(0.4816±0.0092)比较,差异均有统计学意义(t=4.1258、4.3314,P值均小于0.01)。(3)转染SMDF基因的MDSC,经SC条件培液诱导后S-100β的阳性率明显增高。结论实验证明SMDF蛋白可在MDSC中表达,p EGFP-N2-SMDF转染后的细胞中SMDF mRNA水平和蛋白水平的表达明显提高,SMDF基因可促进施万样细胞的增殖、分化。

Objective The constructed Neuregulin-1（NRG-1） type Ⅲ sensory and motor neuronderived factor（ SMDF） eukaryotic expression vector was transfected into muscle-derived stem cell（ MDSC）and the MDSC were induced to differentiate into Schwann-like cells by co-culture method. It was confirmed that SMDF gene can promote the proliferation and differentiation of Schwann cells,and further verify the function of SMDF protein,which lays an experimental foundation for studying the process and molecular mechanism of SC myelination after peripheral nerve injury. Methods（ 1） Western blotting method was used to analyze the SMDF protein expression in brain,spinal cord,dorsal root ganglia（ DRG）,skeletal muscle,liver,spleen and other tissues,and identification of SMDF in DRG by immunohistochemical staining expression.（ 2） The complete sequence of the mouse SMDF gene coding region was obtained by PCR,and the recombinant eukaryotic expression vector p EGFP-N2-SMDF was constructed.（ 3） The eukaryotic expression vector p EGFP-N2-SMDF and empty plasmid p EGFP-N2 were transfected into MDSC cultured in vitro by liposome-mediated method,and they were divided into recombinant plasmid group,plasmid group and non-transformed group to detect of SMDF gene and protein expression before and after transfection.（4） MDSC transfected with SMDF gene were induced to differentiate into Schwann-like cells by cell culture medium,and identified by immunocytochemistry. Data were compared with one-way ANOVA and t test. Results After p EGFP-N2-SMDF eukaryotic expression vector was transfected into MDSC,the expression of green fluorescent protein was observed under fluorescence microscope.（2） PCR and Westernblotting results showed that the expression of SMDF mRNA and protein in the cells transfected with recombinant vector p EGFP-N2-SMDF was significantly increased. The SMDF/GAPDH values of the recombinant plasmid group were 1. 7321 ± 0. 1346,compared with the empty plasmid group（ 0. 5975 ±0. 0084） and the untransfected plasmid group（0. 4816 ± 0. 0092）,the difference was statistically significant（t = 4. 1258,4. 3314,with P values below 0. 01）.（3） The positive rate of S-100β was significantly increased in MDSC transfected with SMDF gene after induction by SC conditioned medium. Conclusions The results showed that SMDF protein could be expressed in MDSC,and the expression of SMDF mRNA and protein in p EGFP-N2-SMDF cells was significantly increased. SMDF gene could promote the proliferation and differentiation of Schwann-like cells.