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目的为了研究MMP-13蛋白在瘢痕形成的作用,我们构建MMP-13基因真核重组表达质粒,检测并鉴定了MMP-13基因在转染了重组质粒的皮肤角质形成细胞HaCat中的表达。方法通过RT-PCR方法将MMP-13基因克隆到真核表达载体pcDNA3.1(+)中,构建其真核表达重组质粒pcDNA3.1(+)/MMP-13,基因测序鉴定。重组质粒以阳离子脂质体2000介导转染HaCat细胞,RT-PCR检测外源基因的表达,间接免疫荧光和Western blot检测外源蛋白的表达。以明胶酶谱法和MTT法分别检测MMP-13的活性以及其对HaCat细胞增殖的影响。结果通过RT-PCR检测到MMP-13基因在重组质粒转染后的HaCat细胞中大量表达,以间接免疫荧光反应和Western blot可检测到MMP-13蛋白在在重组质粒转染后的HaCat细胞中呈阳性,明胶酶谱法和MTT法检测显示真核表达质粒pcDNA3.1(+)/MMP-13表达的MMP-13蛋白具有活性,并且可以抑制HaCat细胞的增殖。结论构建的重组质粒pcDNA3.1(+)/MMP-13能在Hacat细胞中表达活性蛋白MMP-13,并可以抑制HaCat细胞的增殖,这为研究MMP-13在瘢痕形成中的作用奠定了基础,从而为瘢痕治疗提供有效靶标。
Objective To investigate the role of MMP-13 in scar formation, we constructed eukaryotic recombinant expression plasmid of MMP-13 gene and detected and identified the expression of MMP-13 gene in HaCaT cells transfected with the recombinant plasmids. Methods The gene of MMP-13 was cloned into eukaryotic expression vector pcDNA3.1 (+) by RT-PCR. The recombinant plasmid pcDNA3.1 (+) / MMP-13 was constructed and identified by sequencing. The recombinant plasmid was transfected into HaCat cells with cationic liposome 2000, the expression of foreign gene was detected by RT-PCR, and the expression of foreign protein was detected by indirect immunofluorescence and Western blot. The activity of MMP-13 and its effect on the proliferation of HaCat cells were detected by gelatin zymography and MTT respectively. Results The expression of MMP-13 gene in HaCat cells transfected with the recombinant plasmids was detected by RT-PCR. The expression of MMP-13 protein in HaCat cells transfected with recombinant plasmids was detected by indirect immunofluorescence and Western blot Positive, gelatin zymography and MTT assay showed that the expression of eukaryotic expression plasmid pcDNA3.1 (+) / MMP-13 MMP-13 protein activity, and can inhibit HaCat cell proliferation. Conclusion The constructed recombinant plasmid pcDNA3.1 (+) / MMP-13 can express active protein MMP-13 in Hacat cells and inhibit the proliferation of HaCat cells, which lays the foundation for studying the role of MMP-13 in scar formation , Thus providing an effective target for scar treatment.