过表达音猬因子的毛囊干细胞在毛囊再生中的作用研究

Effect study of Sonic hedgehog overexpressed hair follicle stem cells in hair follicle regeneration

目的 明确音猬因子(Sonic hedgehog,Shh)在毛囊干细胞(hair follicle stem cells,HFSCs)传代过程中的表达水平,分析过表达Shh对HFSCs增殖活性的影响,探究过表达Shh后HFSCs的存活情况及其对毛囊再生的影响。方法 取20例40~50岁女性脱发患者捐赠的头皮正常区(H1组)和脱发区(H2组)毛发,显微镜下剪取毛囊中间部分培养,获取原代HFSCs并传代;流式细胞术鉴定第4代HFSCs表面阳性标志物(CD29、CD71)和阴性标志物(CD34)。过表达Shh慢病毒转染两组HFSCs,并分别用流式细胞术和细胞计数试剂盒8法测定转染前后的细胞周期和增殖活性。然后将Shh慢病毒转染后的HFSCs移植至裸鼠背部皮下作为实验组,以注射等量生理盐水作为对照组,5周后采用Western blot法检测裸鼠背部皮肤组织内Shh蛋白表达情况,HE染色和免疫荧光染色分别比较各组毛囊数量及HFSCs存活情况。结果 分离培养的细胞呈长梭形,贴壁较牢;流式细胞术检测示CD29和CD71在细胞表面呈高表达,CD34呈低表达;提示培养细胞为HFSCs。实时荧光定量PCR和Western blot检测示,H1组和H2组第4、7、10代细胞中Shh蛋白和基因表达水平随体外培养时间延长而逐渐下降。过表达Shh后两组HFSCs增殖活性均显著高于空白组(未转染慢病毒)和阴性对照组(转染阴性对照慢病毒),且H1组HFSCs转染前后增殖活性均显著高于H2组,差异均有统计学意义(P<0.05)。裸鼠细胞移植5周后,各实验组背部皮肤内Shh蛋白稳定表达;各实验组毛囊数量和HFSCs标记物(CD71、细胞角蛋白15)表达水平均显著多于对照组,且实验组中的H1组毛囊数量和HFSCs标记物表达水平均显著多于H2组,差异均有统计学意义(P<0.05)。结论 慢病毒介导的Shh可成功转染人HFSCs,过表达Shh后HFSCs增殖活性显著升高,能持续稳定地分泌和表达Shh,并结合干细胞和Shh的优势共同促进毛囊再生。

Objective To determine the expression level of Sonic hedgehog(Shh)in the passage of hair follicle stem cells(HFSCs),analyze the effect of Shh overexpression on the proliferation activity of HFSCs,and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration.Methods Hair follicles from the normal area(H1 group)and alopecia area(H2 group)of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken,and the middle part of the hair follicle was cut under the microscope to culture,and the primary HFSCs were obtained and passaged;the positive markers(CD29,CD71)and negative marker(CD34)on the surface of the fourth generation HFSCs were identified by flow cytometry.The two groups of HFSCs were transfected with Shh-overexpressed lentivirus.Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection,respectively.Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group,and the same amount of saline was injected as the control group.At 5 weeks after cell transplantation,the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot.HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups.Results The isolated and cultured cells were fusiform and firmly attached to the wall flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells,while CD34 was lowly expressed,suggesting that the cultured cells were HFSCs.The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th,7th,and 1oth passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro.After overexpression of Shh,the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group(not transfected with lentivirus)and the negative control group(transfected with negative control lentivirus),and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection,showing significant differences(P<0.05).At 5 weeks after cell transplantation,Shh protein was stably expressed in the dorsal skin of each experimental group;the number of hair follicles and the expression levels of HFSCs markers(CD71,cytokeratin 15)in each experimental group were significantly higher than those in the control group,and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group,and the differences were significant(P<0.05).Conclusion Lentivirus-mediated Shh can be successfully transfected into HFSCs,the proliferation activity of HFSCs significantly increase after overexpression of Shh,which can secrete and express Shh continuously and stably,and promote hair follicle regeneration by combining the advantages of stem cells and Shh.