P311对人微血管内皮细胞1体外血管形成能力的影响及其分子机制

Effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 in vitro and its molecular mechanism

目的探讨P311对人微血管内皮细胞1(HMEC-1)血管形成能力的影响及其可能的分子机制。方法采用实验研究方法。取HMEC-1,按随机数字表法(分组方法下同)分为P311腺病毒组和空载腺病毒组,分别进行48 h相应转染后,采用细胞计数试剂盒8法检测培养1、3、5 d细胞增殖活性;划痕试验检测细胞划痕后6、11 h剩余划痕面积,并计算剩余划痕面积百分比;体外血管形成实验观察细胞培养8 h血管形成情况,并测量管状结构节点数和总长度;蛋白质印迹法检测细胞中血管内皮生长因子受体2(VEGFR2)、磷酸化VEGFR2、胞外信号调节激酶1/2(ERK1/2)及磷酸化ERK1/2蛋白表达量。取HMEC-1,分为P311腺病毒+阴性对照小干扰RNA(siRNA)组、空载腺病毒+阴性对照siRNA组、P311腺病毒+siRNA-VEGFR2组和空载腺病毒+siRNA-VEGFR2组,分别进行相应的处理,蛋白质印迹法检测转染24 h细胞中VEGFR2、磷酸化VEGFR2、ERK1/2、磷酸化ERK1/2蛋白表达量;体外血管形成实验观察转染24 h细胞血管形成情况,并测量管状结构节点数和总长度。取HMEC-1,分为P311腺病毒+二甲基亚砜(DMSO)组、空载腺病毒+DMSO组、P311腺病毒+ERK1/2抑制剂组和空载腺病毒+ERK1/2抑制剂组,分别进行相应的处理。蛋白质印迹法检测处理2 h细胞中ERK1/2及磷酸化ERK1/2蛋白表达量;体外血管形成实验观察处理2 h细胞血管形成情况,并测量管状结构节点数和总长度。各组各时间点样本数均为6。对数据行独立样本t检验、重复测量方差分析、单因素方差分析、LSD检验。结果与空载腺病毒组比较,P311腺病毒组细胞培养1、3、5 d增殖活性均没有明显改变(t值分别为-0.23、-1.30、-1.52,P>0.05)。P311腺病毒组细胞划痕后6、11 h剩余划痕面积百分比均较空载腺病毒组明显降低(t值分别为-2.47、-2.62,P<0.05)。培养8 h,与空载腺病毒组比较,P311腺病毒组细胞管状结构节点数和总长度均明显增加(t值分别为4.49、4.78,P<0.01)。转染48 h,与空载腺病毒组比较,P311腺病毒组细胞VEGFR2、ERK1/2蛋白表达量均无明显变化(P>0.05),磷酸化VEGFR2、磷酸化ERK1/2蛋白表达量均明显升高(t值分别为17.27、16.08,P<0.01)。转染24 h,P311腺病毒+阴性对照siRNA组细胞磷酸化VEGFR2和磷酸化ERK1/2蛋白表达量均明显高于空载腺病毒+阴性对照siRNA组(P<0.01),P311腺病毒+阴性对照siRNA组细胞VEGFR2、磷酸化VEGFR2、磷酸化ERK1/2蛋白表达量均明显高于P311腺病毒+siRNA-VEGFR2组(P<0.01),空载腺病毒+阴性对照siRNA组细胞VEGFR2、磷酸化ERK1/2蛋白表达量均明显高于空载腺病毒+siRNA-VEGFR2组(P<0.05或P<0.01)。转染24 h,P311腺病毒+阴性对照siRNA组细胞管状结构节点数为(720±62)个,明显多于空载腺病毒+阴性对照siRNA组的(428±38)个、P311腺病毒+siRNA-VEGFR2组的(364±57)个(P值均<0.01);P311腺病毒+阴性对照siRNA组细胞管状结构总长度为(21241±1139)μm,明显长于空载腺病毒+阴性对照siRNA组的(17005±1156)μm、P311腺病毒+siRNA-VEGFR2组的(13494±2465)μm(P值均<0.01)。空载腺病毒+阴性对照siRNA组细胞管状结构节点数明显多于空载腺病毒+siRNA-VEGFR2组的(310±75)个(P<0.01),管状结构总长度明显长于空载腺病毒+siRNA-VEGFR2组的(11600±2776)μm(P<0.01)。处理2 h,P311腺病毒+DMSO组细胞磷酸化ERK1/2蛋白表达量明显高于空载腺病毒+DMSO组、P311腺病毒+ERK1/2抑制剂组(P值均<0.01),空载腺病毒+DMSO组细胞磷酸化ERK1/2蛋白表达量明显高于空载腺病毒+ERK1/2抑制剂组(P<0.05)。处理2 h,P311腺病毒+DMSO组细胞管状结构节点数为(726±72)个,明显多于空载腺病毒+DMSO组的(421±39)个、P311腺病毒+ERK1/2抑制剂组的(365±41)个(P值均<0.01);P311腺病毒+DMSO组细胞管状结构总长度为(20318±1433)μm,明显长于空载腺病毒+DMSO组的(16846±1464)μm、P311腺病毒+ERK1/2抑制剂组的(15114±1950)μm(P值均<0.01)。空载腺病毒+DMSO组管状结构节点数明显多于空载腺病毒+ERK1/2抑制剂组的(317±67)个(P<0.01),管状结构总长度明显长于空载腺病毒+ERK1/2抑制剂组的(13188±2306)μm(P<0.01)。结论P311能够通过激活VEGFR2/ERK1/2信号通路发挥促进HMEC-1血管形成的作用。

Objective To explore the effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1(HMEC-1)in vitro and the potential molecular mechanism.Methods The experimental research method was used.HMEC-1 was collected and divided into P311 adenovirus group and empty adenovirus group according to the random number table(the same grouping method below),which were transfected correspondingly for 48 h.The cell proliferation activity was detected using the cell counting kit 8 on 1,3,and 5 days of culture.The residual scratch area of cells at post scratch hour 6 and 11 was detected by scratch test,and the percentage of the residual scratch area was calculated.The blood vessel formation of cells at 8 h of culture was observed by angiogenesis experiment in vitro,and the number of nodes and total length of the tubular structure were measured.The protein expressions of vascular endothelial growth factor receptor 2(VEGFR2),phosphorylated VEGFR2(p-VEGFR2),extracellular signal-regulated kinase 1/2(ERK1/2),and phosphorylated ERK1/2(p-ERK1/2)in cells were detected by Western blotting.HMEC-1 was collected and divided into P311 adenovirus+small interfering RNA(siRNA)negative control group,empty adenovirus+siRNA negative control group,P311 adenovirus+siRNA-VEGFR2 group,and empty adenovirus+siRNA-VEGFG2 group,which were treated correspondingly.The protein expressions of VEGFR2,p-VEGFR2,ERK1/2,and p-ERK1/2 in cells were detected by Western blotting at 24 h of transfection.The blood vessel formation of cells at 24 h of transfection was observed by angiogenesis experiment in vitro,and the number of nodes and total length of the tubular structure were measured.HMEC-1 was collected and divided into P311 adenovirus+dimethylsulfoxide(DMSO)group,empty adenovirus+DMSO group,P311 adenovirus+ERK1/2 inhibitor group,and empty adenovirus+ERK1/2 inhibitor group,which were treated correspondingly.The protein expressions of ERK1/2 and p-ERK1/2 in cells were detected by Western blotting at 2 h of treatment.The blood vessel formation of cells at 2 h of treatment was observed by angiogenesis experiment in vitro,and the number of nodes and total length of the tubular structure were measured.The sample number at each time point in each group was 6.Data were statistically analyzed with independent sample t test,analysis of variance for repeated measurement,one-way analysis of variance,and least significant difference test.Results Compared with that of empty adenovirus group,the proliferation activity of cells in P311 adenovirus group did not show significant difference on 1,3,and 5 days of culture(with t values of-0.23,-1.30,and-1.52,respectively,P>0.05).The residual scratch area percentages of cells in P311 adenovirus group were significantly reduced at post scratch hour 6 and 11 compared with those of empty adenovirus group(with t values of-2.47 and-2.62,respectively,P<0.05).At 8 h of culture,compared with those of empty adenovirus group,the number of nodes and total length of the tubular structure of cells in P311 adenovirus group were significantly increased(with t values of 4.49 and 4.78,respectively,P<0.01).At 48 h of transfection,compared with those of empty adenovirus group,the protein expressions of VEGFR2 and ERK1/2 of cells in P311 adenovirus group showed no obvious changes(P>0.05),and the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus group were significantly increased(with t values of 17.27 and 16.08,P<0.01).At 24 h of transfection,the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA negative control group(P<0.01).The protein expressions of VEGFR2,p-VEGFR2,and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in P311 adenovirus+siRNA-VEGFR2 group(P<0.01).The protein expressions of VEGFR2 and p-ERK1/2 of cells in empty adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA-VEGFR2 group(P<0.05 or P<0.01).At 24 h of transfection,the number of nodes of the tubular structure in cells of P311 adenovirus+siRNA negative control group was 720±62,which was significantly more than 428±38 in empty adenovirus+siRNA negative control group and 364±57 in P311 adenovirus+siRNA-VEGFR2 group(with P values both<0.01).The total length of the tubular structure of cells in P311 adenovirus+siRNA negative control group was(21241±1139)μm,which was significantly longer than(17005±1156)μm in empty adenovirus+siRNA negative control group and(13494±2465)μm in P311 adenovirus+siRNA-VEGFR2 group(with P values both<0.01).The number of nodes of the tubular structure in cells of empty adenovirus+siRNA negative control group was significantly more than 310±75 in empty adenovirus+siRNA-VEGFR2 group(P<0.01),and the total length of the tubular structure of cells in empty adenovirus+siRNA negative control group was significantly longer than(11600±2776)μm in empty adenovirus+siRNA-VEGFR2 group(P<0.01).At 2 h of treatment,the protein expression of p-ERK1/2 of cells in P311 adenovirus+DMSO group was significantly higher than that in empty adenovirus+DMSO group and P311 adenovirus+ERK1/2 inhibitor group(with P values both<0.01),and the protein expression of p-ERK1/2 of cells in empty adenovirus+DMSO group was significantly higher than that in empty adenovirus+ERK1/2 inhibitor group(P<0.05).At 2 h of treatment,the number of nodes of the tubular structure in cells of P311 adenovirus+DMSO group was 726±72,which was significantly more than 421±39 in empty adenovirus+DMSO group and 365±41 in P311 adenovirus+ERK1/2 inhibitor group(with P values both<0.01).The total length of the tubular structure of cells in P311 adenovirus+DMSO group was(20318±1433)μm,which was significantly longer than(16846±1464)μm in empty adenovirus+DMSO group and(15114±1950)μm in P311 adenovirus+ERK1/2 inhibitor group(with P values both<0.01).The number of nodes of the tubular structure in cells of empty adenovirus+DMSO group was significantly more than 317±67 in empty adenovirus+ERK1/2 inhibitor group(P<0.01),and the total length of the tubular structure of cells in empty adenovirus+DMSO group was significantly longer than(13188±2306)μm in empty adenovirus+ERK1/2 inhibitor group(P<0.01).Conclusions P311 can enhance the angiogenesis ability of HMEC-1 by activating the VEGFR2/ERK1/2 signaling pathway.