美洲大蠊小肽对鸡小黄卵泡外膜细胞增殖和血管生成的影响

Effect of Periplaneta americana small peptides on proliferation and angiogenesis of chicken small yellow follicle adventitial cells

为了探讨美洲大蠊小肽对鸡小黄卵泡外膜细胞增殖和血管生成的影响,试验用不同浓度的美洲大蠊小肽(400,800,1200μg/mL)处理体外培养的鸡小黄卵泡外膜细胞,以正常细胞作为空白对照,采用CCK-8法分别在0,12,24,48小时检测细胞活力,筛选最佳药物浓度和作用时间;用EdU法检测细胞增殖能力;用实时荧光定量PCR法和Western-blot法分别检测各组细胞中ANXA2、VEGF的mRNA及其蛋白质的表达量;通过鸡胚绒毛尿囊膜(CAM)试验检测不同浓度美洲大蠊小肽对血管生成的作用。结果表明:与对照相比,美洲大蠊小肽能够提高鸡小黄卵泡外膜细胞的细胞活力,显著促进细胞增殖(P<0.05),最佳药物浓度及作用时间为800μg/mL、24 h。800μg/mL美洲大蠊小肽能够显著上调鸡小黄卵泡外膜细胞中ANXA2和VEGF的mRNA和蛋白质表达量(P<0.05)。不同浓度美洲大蠊小肽均具有显著的促血管生成作用,800μg/mL组效果最明显(P<0.05)。说明美洲大蠊小肽能显著促进鸡小黄卵泡外膜细胞增殖和血管生成,其可能是通过ANXA2和VEGF发挥调控作用的。

In order to discuss the effect of Periplaneta americana small peptides on proliferation and angiogenesis of chicken small yellow follicle adventitial cells, in this experiment, different concentrations of Periplaneta americana small peptides(400, 800, 1 200 μg/mL) were used to treat in vitro cultured chicken small yellow follicle adventitial cells, using normal cells as blank control. Cell viability was detected at 0, 12, 24, and 48 h by CCK-8 method, and the optimal drug concentration and action time were screened. Cell proliferation ability was detected by EdU method. The expression levels of ANXA2, VEGF mRNA and its protein in the cells of each group were detected by real-time PCR and Western-blot methods, respectively. The effect of different concentrations of Periplaneta americana small peptides on angiogenesis were detected by chick chorioallantoic membrane(CAM) assay. The results showed that Periplaneta americana small peptides could significantly increase the cell viability of chicken small yellow follicle adventitial cells(P<0.05) and promote cell proliferation(P<0.05). The optimal drug concentration and action time were 800 μg/mL and 24 h, respectively. 800 μg/mL Periplaneta americana small peptides could significantly up-regulate the mRNA and protein expressions of ANXA2 and VEGF in chicken small yellow follicle adventitial cells(P<0.05). Different concentrations of Periplaneta americana small peptides had significant effect on promoting angiogenesis, and the 800 μg/mL group had the most obvious effect(P<0.05). The results indicated that Periplaneta americana small peptides could significantly promote the proliferation and angiogenesis of chicken small yellow follicle adventitial cells, which may be regulated by ANXA2 and VEGF.