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

doi:10.3969/j.issn.2095-4344.2016.29.009

Abstract

Abstract:

BACKGROUND: Current research has shown that tumor necrosis factor α stimulated 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 still 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 cell lines.
METHODS: Human keloid fibroblast cells 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 cell 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 successfully. Under the light microscope, cells were spindle. Immunohistochemical staining for vimentin was performed in the fifth passage of cells, with the positive rate of 100%. Cells were negative for cytokeratin. (2) Recombinant lentiviral vectors and stably transfected cell lines were successfully established. TSG-6 gene expression was altered apparently. Compared with the control group, cell proliferation was delayed and apoptotic rate was noticeably increased in TSG-6 gene overexpression group. Cell 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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: RNA Interference, Cicatrix, Fibroblasts, Cell Proliferation, Apoptosis, Tissue Engineering

CLC Number:

R318

Chen Zhao, Li Xiao-jing, Wang Hui. 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[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(29): 4319-4327.

Figures/Tables 2

2.1 人瘢痕疙瘩成纤维细胞的鉴定结果倒置相差显微镜下观察可见：刚接种的成纤维细胞尚未贴壁时呈球形，贴壁后逐渐伸展，呈梭形、长条形或三角形，约3 d后呈排列紧密的汇合状态。细胞密度低时细胞之间排列疏松，有较大细胞间隙。细胞密度高时，细胞相互平行排列或呈放射状和漩涡状排列[13]，为典型成纤维细胞形态[14](图1)。波形蛋白抗体的免疫化学染色阳性，角蛋白19抗体的免疫化学染色阴性，可判断为成纤维细胞[15]。 2.2 PCR获得TSG-6全长基因结果利用人瘢痕疙瘩成纤维细胞cDNA，通过PCR方法扩增出TSG-6的全长基因，经1%琼脂糖凝胶电泳分离后得到大小约800 bp特异性单一条带(图2)。 2.3 RT-PCR及Western blot鉴定过表达及干扰效果RT-PCR的检测结果显示：转染TSG-6表达载体的人瘢痕疙瘩成纤维细胞，其TSG-6基因在mRNA水平的表达明显增高(图3A)；转染TSG-6干扰载体的人瘢痕疙瘩成纤维细胞，其TSG-6基因在mRNA水平的表达明显降低(图3B)；与此同时，Westernblot检测也得到了相同结果，证实TSG-6基因在蛋白水平表达量的变化与其在mRNA水平表达变化一致(图3C，D)。 2.4 MTT法检测细胞增殖结果对各组细胞培养24，48，72，96 h后观察，可见TSG-6基因表达上调对细胞增殖有明显的抑制作用。转染TSG-6表达载体的人瘢痕疙瘩成纤维细胞，其增殖效应较人瘢痕疙瘩成纤维细胞对照组明显减缓(P < 0.05)，而转染TSG-6干扰载体的人瘢痕疙瘩成纤维细胞，其增殖较人瘢痕疙瘩成纤维细胞对照组有所加快(P < 0.05)，人瘢痕疙瘩成纤维细胞对照组、过表达对照组、干扰对照组细胞的增殖趋势差异无统计学意义(图4)。 2.5 流式细胞仪检测细胞凋亡结果对各组细胞传代贴壁培养72 h后，进行流式细胞术分析，结果显示TSG-6基因表达上调对细胞凋亡有明显的促进作用。与人瘢痕疙瘩成纤维细胞对照组相比，转染TSG-6表达载体的人瘢痕疙瘩成纤维细胞，其细胞凋亡率明显增高(P < 0.05)；转染TSG-6干扰载体的人瘢痕疙瘩成纤维细胞，其细胞凋亡率明显降低(P < 0.05)；人瘢痕疙瘩成纤维细胞对照组、过表达对照组、干扰对照组间差异无统计学意义(图5)。 2.6 Western blot检测抗凋亡蛋白Bcl-2、凋亡蛋白P53及Active-caspase-3的表达 Western blot的检测结果显示：转染TSG-6表达载体的人瘢痕疙瘩成纤维细胞，其抗凋亡蛋白Bcl-2的表达显著降低，凋亡蛋白P53及Active-caspase-3的表达显著升高；转染TSG-6干扰载"

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