摘要
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.
出处
《中国组织工程研究》
CAS
北大核心
2016年第29期4319-4327,共9页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金资助项目(81272107)~~