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
10.3969/j.issn.2095-4344.2016.29.009
- VernacularTitle: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
- Author:
Zhao CHEN
;
Xiaojing LI
;
Hui WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2016;20(29):4319-4327
- CountryChina
- Language:Chinese
-
Abstract:
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.
