构建肿瘤坏死因子α刺激基因6慢病毒表达及干扰载体及对人瘢痕疙瘩成纤维细胞增殖与凋亡的影响

Construction of recombinant lentiviral vector and interfering carrier for tumor necrosis factor alpha stimulated gene 6 and its effect on proliferation and apoptosis of human keloid fibroblasts

背景:研究表明,肿瘤坏死因子α刺激基因6(tumor necrosis factorαstimulated gene 6,TSG-6)具有抗炎作用,早期创面局部注射TSG-6蛋白可抑制瘢痕形成,但其参与瘢痕形成的机制尚不明确。目的:构建人TSG-6重组慢病毒表达载体与干扰载体,建立稳定转染的过表达及干扰细胞株,观察TSG-6对瘢痕疙瘩成纤维细胞株增殖与凋亡的影响。方法:采用酶消化法分离纯化得到人瘢痕疙瘩成纤维细胞,再以免疫组织化学法鉴定。构建p LVX-puro-TSG-6及pL VX-shR NA1-TSG-6重组慢病毒载体,感染人瘢痕疙瘩成纤维细胞,并行RT-PCR和Western blot检验各组细胞中TSG-6的表达。用MTT法和流式细胞术检测转染后不同时间段内各组细胞增殖和凋亡情况。用Western blot检测各组细胞中Bcl-2、P53及Active-caspase-3的表达。结果与结论:(1)成功分离原代人瘢痕疙瘩成纤维细胞,光学显微镜下细胞多呈梭形,传至5代后行波形蛋白的免疫组织化学染色,阳性率为100%,细胞角蛋白染色阴性;(2)重组慢病毒载体及稳定转染细胞株构建成功,细胞中TSG-6的表达发生明显变化。与对照组相比,TSG-6过表达组细胞增殖减缓,凋亡率显著升高,TSG-6干扰组细胞增殖加快,细胞凋亡率降低(P<0.05);(3)Western blot结果显示,TSG-6过表达组Bcl-2表达显著降低,P53及Active-caspase-3明显上升(P<0.05);(4)结果表明,TSG-6对瘢痕疙瘩成纤维细胞具有抑制增殖和诱导凋亡的作用,且其机制可能与下调Bcl-2蛋白表达、上调P53蛋白表达、升高Caspase-3活性有关。

BACKGROUND：Current research has shown that tumor necrosis factorαstimulated gene 6 （TSG-6） has anti-inflammatory effect, and the scar formation can be inhibited by local injection of TSG-6 protein at the early stage of trauma. However, the mechanism of this effect is stil unclear. OBJECTIVE：To construct the lentiviral expression vector and shRNA vector for human TSG-6, with stable overexpression, transfection and interference, and to explore the effect of TSG-6 on proliferation and apoptosis of keloid fibroblast cel lines. METHODS：Human keloid fibroblast cel s were isolated from the keloid’s tissue by enzyme digestion and identified by immunocytochemistry assay. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed and transfected into human keloid fibroblast, exclusively. Expression levels of TSG-6 mRNA and protein were detected by RT-PCR and western blot assay. MTT assay and flow cytometry were used to estimate the cel proliferation and apoptosis in each group after transfection. In addition, expression of Bcl-2, p53 and active-caspase-3 were detected by western blot assay in each group. RESULTS AND CONCLUSION：（1） Human keloid fibroblasts were separated successful y. Under the light microscope, cel s were spindle. Immunohistochemical staining for vimentin was performed in the fifth passage of cel s, with the positive rate of 100%. Cel s were negative for cytokeratin. （2） Recombinant lentiviral vectors and stably transfected cel lines were successful y established. TSG-6 gene expression was altered apparently. Compared with the control group, cel proliferation was delayed and apoptotic rate was noticeably increased in TSG-6 gene overexpression group. Cel proliferation increased and apoptotic rate decreased in the TSG-6 gene intervention group （P〈0.05）. （3） Western blot assay results demonstrated that Bcl-2 expression reduced, P53 and Active-caspase-3 expression significantly increased in the TSG-6 gene overexpression group （P〈0.05）. （4） These finding showed that TSG-6 could inhibit proliferation and induce apoptosis in keloid fibroblasts. Its mechanism may be associated with the down-regulation of Bcl-2 protein expression, up-regulation of P53 protein expression and increased Caspase-3 activity.