慢病毒载体介导VEGF_(165)基因修饰大鼠毛囊干细胞的实验研究

EXPERIMENTAL STUDY ON VASCULAR ENDOTHELIAL GROWTH FACTOR_(165) GENE-MODIFIED RAT HAIR FOLLICLE STEM CELLS MEDIATED BY LENTIVIRAL VECTOR

目的为获得持续高表达VEGF165的大鼠毛囊干细胞（rathairfolliclestemcells，rHFSCs），观察慢病毒载体介导VEGF165基因对rHFSCs的转染及其表达情况。方法切取1周龄SD大鼠触须部皮肤，Dispase酶和Ⅳ型胶原酶混合消化，显微镜下分离毛囊隆突部，组织块培养rHFSCs；经Ⅳ型胶原差速贴壁纯化后，分别取不同代次rHFSCs绘制细胞生长曲线；联合应用免疫荧光染色及实时荧光定量PCR（realtimequantitativePCR，RT-qPCR）检测相关基因鉴定细胞。钙转法包装pLV-内部核糖体进入位点（internalribosomeentrysite，IRES）-VEGF165-增强型绿色荧光蛋白（enhancedgreenfluorescentprotein，EGFP）（实验组）和pLV-IRES．EGFP空载体（对照组）慢病毒，用两组慢病毒转染rHFSCs。倒置荧光显微镜下观察实验组绿色荧光表达情况，RT-PCR和Westemblot法分别检测VEGF165mRNA和蛋白表达情况。结果分离、培养、纯化的rHFSCs呈典型“铺路石”状，贴壁较牢，克隆形成能力强；纯化后细胞培养1～2d处于生长潜伏期，5～6d处于对数生长期；细胞免疫荧光染色可见毛囊干细胞标志物角蛋白15（cytokeratin15，CK15）、整合素α6和整合素β1表达呈阳性；RT-qPCR检测可见干细胞标志物CK15、CK19、整合素α6和整合素β1表达量高，而表皮干细胞标志物CD34和角质形成细胞标志物CK10表达量均较低。倒置荧光显微镜观察示，实验组慢病毒介导VEGF165基因转染14d后转染效率可达85．76％±1．91％；RT-PCR和Westernblot检测示，实验组VEGF165mRNA和蛋白表达均呈阳性，对照组均呈阴性。结论经显微镜联合组织块分离培养、Ⅳ型胶原差速贴壁筛选后，可得到高纯度rHFSCs，细胞增殖能力强。慢病毒介导的VEGF165，基因修饰方法可成功转染并得到高表达VEGF165mRNA和蛋白的rHFSCs，可为组织工程构建人工毛囊、血管及皮肤等提供良好的种子细胞。

Objective To obtain rat hair follicle stem cells （rHFSCs） which can constantly and highly express vascular endothelial growth factor 165 （VEGF165）, and to observe the expression of VEGF165 gene in rat HFSCs. Methods The cirri skin of 1-week-old Sprague Dawley rat was harvested and digested by using combination of Dispase and type IV collagenases. The bulge was isolated under microscope. The rHFSCs were cultured by tissue block method. After purified by rapid adhering on collagen type IV, the growth curve of different generations rHFSCs was drawn. The cells were identified by immunofluorescence staining and real time quantitative PCR （RT-qPCR） analysis that tested the expression level of correlated genes. Lentivirus of pLV-internal ribosome entry site （IRES）-VEGF165-enhanced green fluorescent protein （EGFP） （experimental group） and pLV- IRES-EGFP empty vector （control group） was packaged by calcium transfected method and the rHFSCs were transfected. The green fluorescent protein expression was observed by inverted fluorescence microscope, and VEGF165 mRNA and protein expressions were detected using RT-PCR and Western blot. Results The rHFSCs which were isolated, cultured, and purified were like the ＂slabstone＂, and had strong adhesion ability and colony formation ability. The purified cells were in latent growth phase at 2-3 days; they were in exponential growth phase at 5-6 days. The expressions of cytokeration 15 （CK15）, integrin α6, and integrin β1 （markers of HFSCs） were positive by immunocytochemistry. The RT-qPCR analysis showed that CK15, CK19, integrin α6, and integrin β1 expressed highly, but CD34 （a marker of epidermal stem cells） and CK10 （a marker ofkeratinocyte） expressed lowly. After 14 days, the transfection efficiency was up to 85.76% ± 1.91%. RT-PCR analysis and Western blot showed that VEGF165mRNA and protein expressions were positive in experimental group, and were negative in controlgroup. Conclusion The rHFSCs with high purity and strong proliferation ability can be obtained by using microscope combined with tissue cultivation and rapid cell adhesion on collagen type IV. The rHFSCs with high expression of VEGF165 can be successfully obtained by lentiviral transfection. This method provides good seeding cells for tissue engineering to construct artificial hair follicles, blood vessels, and skins.