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

王淞; 李海胜; 钱卫; 张小容; 贺伟峰; 罗高兴

doi:10.3760/cma.j.cn501120-20211210-00410

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

doi: 10.3760/cma.j.cn501120-20211210-00410

1.
陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所，创伤、烧伤与复合伤国家重点实验室，重庆市创面修复与组织再生重点实验室，重庆　400038
2.
解放军中部战区总医院烧伤整形科，武汉　　430064

基金项目:

国家自然科学基金重点项目 81630055

国家自然科学基金青年科学基金项目 82102340

湖北省自然科学基金 2020CFB210

详细信息

通讯作者:
罗高兴，Email：logxw@yahoo.com

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出版历程
- 收稿日期: 2021-12-10

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

1.
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing Key Laboratory for Wound Repair and Regeneration, Chongqing 400038, China
2.
Department of Burns and Plastic Surgery, General Hospital of Central Theater Command of People's Liberation Army, Wuhan 430064, China

Funds:

Key Program of National Natural Science Foundation of China 81630055

Youth Science Foundation Project of National Natural Science Foundation of China 82102340

Hubei Provincial Natural Science Foundation 2020CFB210

More Information

Corresponding author: Luo Gaoxing, Email: logxw@yahoo.com

摘要

摘要: 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（21 241±1 139）μm，明显长于空载腺病毒+阴性对照siRNA组的（17 005±1 156）μm、P311腺病毒+siRNA-VEGFR2组的（13 494±2 465）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（11 600±2 776）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（20 318±1 433）μm，明显长于空载腺病毒+DMSO组的（16 846±1 464）μm、P311腺病毒+ERK1/2抑制剂组的（15 114±1 950）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（13 188±2 306）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。
- 伤口愈合 /
- 血管内皮生长因子受体2 /
- 血管新生 /
- 人微血管内皮细胞1 /
- P311 /
- 胞外信号调节激酶1/2
Abstract: 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 (21 241±1 139) μm, which was significantly longer than (17 005±1 156) μm in empty adenovirus+siRNA negative control group and (13 494±2 465) 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 (11 600±2 776) 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 (20 318±1 433) μm, which was significantly longer than (16 846±1 464) μm in empty adenovirus+DMSO group and (15 114±1 950) 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 (13 188±2 306) 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.
- Wound healing /
- Vascular endothelial growth factor receptor-2 /
- Angiogenesis /
- Human microvascular endothelial cells 1 /
- P311 /
- Extracellular signal-regulated kinase 1/2
王淞：实施研究、起草文章；李海胜：实施研究和分析数据；钱卫、张小容：采集数据和统计分析；贺伟峰：酝酿和设计实验；罗高兴：获取研究经费和对文章的知识性内容作批评性审阅

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严重烫伤后肠道缺血缺氧会引起肠上皮屏障受损，发生肠道细菌移位（EBT），从而导致严重的并发症，如SIRS、脓毒症和MOF的发生。囊性纤维化穿膜传导调节蛋白（CFTR）可因肠上皮细胞缺氧而表达下调，进而影响紧密连接蛋白的正常结构与功能，而紧密连接蛋白正是维持肠道屏障的关键结构。陆军军医大学（第三军医大学）第一附属医院全军烧伤研究所陈婧老师团队联合上海交通大学第九人民医院整复外科章一新教授团队近期在《Burns & Trauma》发文《Molecular mechanism mediating enteric bacterial translocation after severe burn: the role of cystic fibrosis transmembrane conductance regulator》，使用人Caco-2细胞系建立体外缺氧损伤模型，并建立C57小鼠严重烫伤模型，研究严重烫伤对小鼠肠黏膜屏障、CFTR和紧密连接蛋白表达的影响，同时选取DF 508小鼠（F508del CFTR基因突变小鼠）为体内模型，进一步证明CFTR在维持小鼠正常肠道屏障功能中的作用。研究结果显示，在缺氧条件下，人Caco-2细胞中CFTR的表达显著降低；胞外信号调节激酶（ERK）和核因子κB信号被激活；炎症因子（TNF-α、IL-1β和IL-8）分泌增加；带状闭合蛋白1（ZO-1）、闭合蛋白和E-钙黏合素的表达下调；跨上皮电阻值降低；并导致ZO-1在细胞膜上分布不连续，排列不规则。同样地，敲除CFTR导致了相似的改变，且敲除CFTR引起的炎症因子的上调和紧密连接蛋白（ZO-1和闭合蛋白）的下调，可以通过抑制特定的ERK或核因子κB而逆转。与此同时，在严重烫伤小鼠的肠道中观察到大量的促炎症介质分泌和EBT，进一步支持了体外实验结果。与野生型小鼠相比，DF 508小鼠回肠中的TNF-α、IL-1β和IL-8浓度升高。此外，维生素D3被证明可以保护肠道上皮屏障免受缺氧损伤，机制可能与其上调CFTR表达，从而抑制ERK通路激活，减少促炎性细胞因子释放有关。此项研究结果表明，严重烫伤后，肠上皮细胞通过CFTR/ERK/促炎性细胞因子途径调节细胞间紧密连接蛋白的表达，进而调节肠道菌群移位，维生素D3可对严重烫伤后的肠黏膜屏障起到保护作用。

杨云稀，编译自《Burns & Trauma》，2021，8:tkaa042；孙炳伟，审校

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1 2组人微血管内皮细胞1划痕后各时间点剩余划痕面积倒置相差显微镜×200，图中标尺为100 μm。1A、1B、1C.分别为P311腺病毒组划痕后0（即刻）、6、11 h，随着划痕后时间的延长，剩余划痕面积逐渐减少，至划痕后11 h划痕基本愈合；1D、1E、1F.分别为空载腺病毒组划痕后0、6、11 h，图1E、1F剩余划痕面积分别较图1B、1C明显增大

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2 2组人微血管内皮细胞1培养8 h血管形成情况倒置相差显微镜×200，图中标尺为100 μm。2A. P311腺病毒组管状结构节点数较多，管状结构总长度较长；2B.空载腺病毒组管状结构节点数少于图2A，管状结构总长度短于图2A

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3 蛋白质印迹法检测2组人微血管内皮细胞1转染48 h VEGFR2/ERK1/2信号通路相关蛋白的表达量。3A.条带图；3B.条图（样本数为6，x¯±s）

注：VEGFR2为血管内皮生长因子受体2，ERK1/2为胞外信号调节激酶1/2，GAPDH为3-磷酸甘油醛脱氢酶；条带图上方1、2分别为P311腺病毒组和空载腺病毒组；与空载腺病毒组比较，^aP<0.01

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4 蛋白质印迹法检测4组人微血管内皮细胞1转染24 h VEGFR2/ERK1/2信号通路相关蛋白的表达量。4A.条带图；4B.条图（样本数为6，x¯±s）

注：VEGFR2为血管内皮生长因子受体2，ERK1/2为胞外信号调节激酶1/2，GAPDH为3-磷酸甘油醛脱氢酶；条带图上方1、2、3、4分别为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；与P311腺病毒+阴性对照siRNA组比较，^aP<0.01；与空载腺病毒+阴性对照siRNA组比较，^bP<0.01，^cP<0.05

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5 4组人微血管内皮细胞1转染24 h血管形成情况倒置相差显微镜×200，图中标尺为100 μm。5A.P311腺病毒+阴性对照小干扰RNA（siRNA）组管状结构节点数较多，管状结构总长度较长；5B.空载腺病毒+阴性对照siRNA组管状结构节点数明显少于图5A，管状结构总长度明显短于图5A；5C.P311腺病毒+siRNA-血管内皮生长因子受体2（VEGFR2）组管状结构节点数明显少于图5A，管状结构总长度明显短于图5A；5D.空载腺病毒+siRNA-VEGFR2组管状结构节点数明显少于图5B，管状结构总长度明显短于图5B，且均与图5C相近

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6 蛋白质印迹法检测4组人微血管内皮细胞1处理2 h ERK1/2信号通路相关蛋白的表达量。6A.条带图；6B.条图（样本数为6，x¯±s）

注：F值、P值为4组间各指标总体比较所得；ERK1/2为胞外信号调节激酶1/2，GAPDH为3-磷酸甘油醛脱氢酶；条带图上方1、2、3、4分别为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；与P311腺病毒+DMSO组比较，^aP<0.01；与空载腺病毒+DMSO组比较，^bP<0.05

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7 4组人微血管内皮细胞1处理2 h血管形成情况倒置相差显微镜×200，图中标尺为100 μm。7A.P311腺病毒+二甲基亚砜（DMSO）组管状结构节点数较多，管状结构总长度较长；7B.空载腺病毒+DMSO组管状结构节点数少于图7A，管状结构总长度短于图7A；7C.P311腺病毒+胞外信号调节激酶1/2（ERK1/2）抑制剂组管状结构节点数少于图7A，管状结构总长度短于图7A；7D.空载腺病毒+ERK1/2抑制剂组管状结构节点数和总长度与图7C相近

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表1 2组人微血管内皮细胞1培养各时间点增殖活性比较（x¯±s）

组别	样本数	1 d	3 d	5 d
P311腺病毒组	6	0.188±0.030	0.415±0.032	0.713±0.030
空载腺病毒组	6	0.185±0.019	0.392±0.030	0.687±0.031
t值		-0.23	-1.30	-1.52
P值		0.822	0.222	0.160
注：处理因素主效应，F=5.10，P=0.074；时间因素主效应，F=720.12，P<0.001；两者交互作用，F=0.81，P=0.473

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表2 2组人微血管内皮细胞1划痕后各时间点剩余划痕面积百分比比较（%，x¯±s）

组别	样本数	6 h	11 h
P311腺病毒组	6	47±7	9±5
空载腺病毒组	6	55±5	20±10
t值		-2.47	-2.62
P值		0.033	0.025
注：初始剩余划痕面积百分比均为100%；处理因素主效应，F=18.42，P=0.008；时间因素主效应，F=443.86，P<0.001；两者交互作用，F=7.89，P=0.041

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表3 4组人微血管内皮细胞1转染24 h后血管形成情况（x¯±s）

组别	样本数	管状结构节点数（个）	管状结构总长度（μm）
P311腺病毒+阴性对照siRNA组	6	720±62	21 241±1 139
空载腺病毒+阴性对照siRNA组	6	428±38	17 005±1 156
P311腺病毒+siRNA- VEGFR2组	6	364±57	13 494±2 465
空载腺病毒+siRNA- VEGFR2组	6	310±75	11 600±2 776
F值		57.07	26.32
P值		<0.001	<0.001
P₁值		<0.001	0.002
P₂值		<0.001	<0.001
P₃值		0.002	<0.001
P₄值		0.127	0.121
注：F值、P值为4组间各指标总体比较所得；P₁值、P₂值分别为P311腺病毒+阴性对照小干扰RNA（siRNA）组与空载腺病毒+阴性对照siRNA组、P311腺病毒+siRNA-血管内皮生长因子受体2（VEGFR2）组比较所得，P₃值、P₄值分别为空载腺病毒+siRNA-VEGFR2组与空载腺病毒+阴性对照siRNA组、P311腺病毒+siRNA-VEGFR2组比较所得

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表4 4组人微血管内皮细胞1处理2 h血管形成情况（x¯±s）

组别	样本数	管状结构节点数（个）	管状结构总长度（μm）
P311腺病毒+DMSO组	6	726±72	20 318±1 433
空载腺病毒+DMSO组	6	421±39	16 846±1 464
P311腺病毒+ERK1/2抑制剂组	6	365±41	15 114±1 950
空载腺病毒+ERK1/2抑制剂组	6	317±67	13 188±2 306
F值		63.49	16.53
P值		<0.001	<0.001
P₁值		<0.001	0.004
P₂值		<0.001	<0.001
P₃值		0.005	0.002
P₄值		0.162	0.083
注：F值、P值为4组间各指标总体比较所得；DMSO为二甲基亚砜，ERK1/2为胞外信号调节激酶1/2；P₁值、P₂值分别为P311腺病毒+DMSO组与空载腺病毒+DMSO组、P311腺病毒+ERK1/2抑制剂组比较所得，P₃值、P₄值分别为空载腺病毒+ERK1/2抑制剂组与空载腺病毒+DMSO组、P311腺病毒+ERK1/2抑制剂组比较所得

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