瞬时受体电位香草酸亚型4特异性激活剂对人血管内皮细胞功能和大鼠穿支皮瓣血供的影响及其机制

邝依敏; 黄昕; 蒙旭昌; 顾舒晨; 张泽伟; 刘云菡; 骆申英; 昝涛

doi:10.3760/cma.j.cn501120-20210419-00138

瞬时受体电位香草酸亚型4特异性激活剂对人血管内皮细胞功能和大鼠穿支皮瓣血供的影响及其机制

doi: 10.3760/cma.j.cn501120-20210419-00138

1.
上海交通大学医学院附属第九人民医院整复外科，上海　　200011
2.
广西医科大学第一附属医院整形美容外科，南宁　　530021
3.
郑州大学第一附属医院整形外科，郑州　　　　450052

基金项目:

国家自然科学基金面上项目 81772086, 82072177

上海市“医苑新星”青年医学人才培养资助计划

上海交通大学“晨星-优秀青年学者奖励计划”副教授支持计划A类

上海交通大学医学院“双百人”计划

详细信息

通讯作者:
昝涛，Email：zantaodoctor@yahoo.com

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出版历程
- 收稿日期: 2021-04-19
- 网络出版日期: 2022-07-20
- 刊出日期: 2022-05-20

Effects of transient receptor potential vanilloid type 4-specific activator on human vascular endothelial cell functions and blood supply of rat perforator flap and its mechanism

1.
Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
2.
Department of Plastic and Cosmetic Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
3.
Department of Plastic and Reconstructive Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

Funds:

General Program of National Natural Science Foundation of China 81772086, 82072177

"Outstanding Youth Medical Talents" of Shanghai "Rising Stars of Medical Talent" Youth Development Program

Associate Professor Type A Category of Shanghai Jiao Tong University's "Chenxing" Youth Development Program

"Two-hundred Talent" Program of Shanghai Jiao Tong University School of Medicine

More Information

Corresponding author: Zan Tao, Email: zantaodoctor@yahoo.com

摘要

摘要:
目的分析瞬时受体电位香草酸亚型4（TRPV4）激活对人脐静脉内皮细胞（HUVEC）功能及内皮-间质转化（EndMT）的作用，并探讨TRPV4激活对大鼠穿支皮瓣血流灌注和成活的影响及其机制。
方法采用实验研究方法。取第3~6代HUVEC进行实验，分为0.5 μmol/L 4α-佛波醇12，13-二癸酸酯（4αPDD）组、1.0 μmol/L 4αPDD组、3.0 μmol/L 4αPDD组、10.0 μmol/L 4αPDD组、磷酸盐缓冲液（PBS）组，分别采用相应终物质的量浓度的4αPDD及PBS培养，采用细胞计数试剂盒8（CCK-8）法检测培养6、12 h细胞增殖活性。另取细胞分为PBS组、1 μmol/L 4αPDD组、3 μmol/L 4αPDD组，同前处理并检测培养6、12、24、48 h的细胞增殖活性；采用划痕试验检测划痕后12、24、48 h剩余划痕面积，并计算剩余划痕面积百分比；采用Transwell实验检测培养24、48 h细胞迁移数量；采用成管实验检测培养4、8 h管状结构数量；采用蛋白质印迹法检测培养24 h细胞上皮钙黏素、神经钙黏素、Slug、Snail蛋白表达。体外实验每组各时间点样本数均为3。取36只8~10周龄雄性SD大鼠，按随机数字表法分为皮瓣延迟组、4αPDD组和生理盐水组，每组12只，均建立背部髂腰动脉穿支皮瓣模型。皮瓣延迟组大鼠仅于皮瓣移植术前1周行皮瓣延迟。4αPDD组和生理盐水组大鼠不行皮瓣延迟，在术前10 min及术后24、48 h，分别经腹腔注射4αPDD和等量的生理盐水。分别于术后0（即刻）、1、4、7 d，行大体观察并计算术后7 d的皮瓣成活率；于术后1、4、7 d，采用激光散斑对比成像技术检测皮瓣血流灌注；术后7 d，采用免疫组织化学染色法检测皮瓣闭塞血管区域微血管密度。体内实验每组各时间点样本数均为12。对数据行析因设计方差分析、重复测量方差分析、单因素方差分析、LSD-t检验、Bonferroni校正。
结果培养6、12 h，PBS组、0.5 μmol/L 4αPDD组、1.0 μmol/L 4αPDD组、3.0 μmol/L 4αPDD组、10.0 μmol/L 4αPDD组细胞增殖活性组间总体比较，差异均无统计学意义（P>0.05）。培养6、12、24、48 h，PBS组、1 μmol/L 4αPDD组、3 μmol/L 4αPDD组细胞增殖活性组间总体比较，差异均无统计学意义（P>0.05）。划痕后12 h，1 μmol/L 4αPDD组、3 μmol/L 4αPDD组细胞剩余划痕面积百分比均与PBS组相近（P>0.05）；划痕后24、48 h，与PBS组比较，3 μmol/L 4αPDD组细胞剩余划痕面积百分比均明显降低（t值分别为2.83、2.79，P<0.05），1 μmol/L 4αPDD组细胞剩余划痕面积百分比均无明显变化（P>0.05）。培养24 h，1 μmol/L 4αPDD组、3 μmol/L 4αPDD组细胞迁移数量均与PBS组相近（P>0.05）；培养48 h，1 μmol/L 4αPDD组、3 μmol/L 4αPDD组细胞迁移数量均明显多于PBS组（t值分别为6.20、9.59，P<0.01）。培养4 h，1 μmol/L 4αPDD组、3 μmol/L 4αPDD组细胞管状结构数量均明显多于PBS组（t值分别为4.68、4.95，P<0.05或P<0.01）；培养8 h，1 μmol/L 4αPDD组、3 μmol/L 4αPDD组细胞管状结构数量均与PBS组相近（P>0.05）。培养24 h，与PBS组比较，3 μmol/L 4αPDD组细胞上皮钙黏素的蛋白表达明显降低（t=5.13，P<0.01），1 μmol/L 4αPDD组细胞上皮钙黏素的蛋白表达无明显变化（P>0.05）；3 μmol/L 4αPDD组细胞神经钙黏素蛋白表达明显升高（t=4.93，P<0.01），1 μmol/L 4αPDD组细胞神经钙黏素的蛋白表达无明显变化（P>0.05）；1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞Slug的蛋白表达均明显升高（t值分别为3.85、6.52，P<0.05或P<0.01）；3 μmol/L 4αPDD组细胞Snail的蛋白表达明显升高（t=4.08，P<0.05），1 μmol/L 4αPDD组细胞Snail的蛋白表达无明显变化（P>0.05）。1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞上皮钙黏素、神经钙黏素、Slug、Snail的蛋白表达组间两两比较，差异均无统计学意义（P>0.05）。术后0 d，3组大鼠皮瓣一般情况均良好；术后1 d，3组大鼠皮瓣远端均出现青紫肿胀；术后4 d，3组大鼠皮瓣肿胀消退、远端变成暗褐色、发生坏死，其中生理盐水组大鼠皮瓣坏死面积比4αPDD组和皮瓣延迟组大；术后7 d，3组大鼠皮瓣坏死面积基本稳定，其中皮瓣延迟组大鼠皮瓣远端坏死面积最小。术后7 d，4αPDD组［（80±13）%］和皮瓣延迟组［（87±9）%］大鼠的皮瓣成活率相近（P>0.05）且均明显高于生理盐水组［（70±11）%，t值分别为2.24、3.65，P<0.05或P<0.01］。术后1 d，3组大鼠皮瓣整体血流信号基本一致，闭塞血管区域的血流信号均相对较强，远端均存在一定程度的灌注不足。术后4 d，3组大鼠皮瓣存活区与坏死区分界渐清晰，闭塞血管区域的血流信号增强，其中生理盐水组大鼠皮瓣远端低灌注区范围大于皮瓣延迟组和4αPDD组。术后7 d，3组大鼠皮瓣整体的血流信号基本稳定，其中皮瓣延迟组和4αPDD组大鼠皮瓣闭塞血管区域及整体血流信号强度均明显大于生理盐水组。术后7 d，4αPDD组和皮瓣延迟组大鼠皮瓣闭塞血管区域中的微血管密度相近（P>0.05）且均明显高于生理盐水组（t值分别为4.11、5.38，P<0.01）。
结论 TRPV4激活后可能通过EndMT机制促进人血管内皮细胞的迁移和成管，从而增加穿支皮瓣的血流灌注、闭塞血管区域微血管密度，提高皮瓣成活率。
- TRPV阳离子通道 /
- 内皮细胞 /
- 穿支皮瓣 /
- 新生血管化，病理性 /
- 内皮-间质转化
Abstract:
Objective To analyze the effects of transient receptor potential vanilloid type 4 (TRPV4) activation on the function and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), as well as to explore the effects of TRPV4 activation on blood perfusion and survival of rat perforator flap and the mechanism.
Methods The experimental research methods were used. The 3^rd to 6^th passages of HUVECs were used for experiments and divided into 0.5 μmol/L 4α-phorbol 12, 13-didecanoate (4αPDD) group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, 10.0 μmol/L 4αPDD group, and phosphate buffer solution (PBS) group, which were cultivated in corresponding final molarity of 4αPDD and PBS, respectively. The cell proliferation activity at 6 and 12 h of culture was detected using cell counting kit-8 (CCK-8). Another batch of cells was acquired and divided into PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group, which were treated similarly as described before and then detected for cell proliferation activity at 6, 12, 24, and 48 h of culture. The residual scratch area of cells at post scratch hour (PSH) 12, 24, and 48 was detected by scratch test, and the percentage of the residual scratch area was calculated. The number of migrated cells at 24 and 48 h of culture was detected by Transwell experiment. The tube-formation assay was used to measure the number of tubular structures at 4 and 8 h of culture. The protein expressions of E-cadherin, N-cadherin, Slug, and Snail at 24 h of culture were detected by Western blotting. All the sample numbers in each group at each time point in vitro experiments were 3. A total of 36 male Sprague-Dawley rats aged 8 to 10 weeks were divided into delayed flap group, 4αPDD group, and normal saline group according to the random number table, with 12 rats in each group, and iliolumbar artery perforator flap models on the back were constructed. The flap surgical delay procedure was only performed in the rats in delayed flap group one week before the flap transfer surgery. Neither rats in 4αPDD group nor normal saline group had flap surgical delay; instead, they were intraperitoneally injected with 4αPDD and an equivalent mass of normal saline, respectively, at 10 min before, 24 h after, and 48 h after the surgery. The general state of flap was observed on post surgery day (PSD) 0 (immediately), 1, 4, and 7. The flap survival rates were assessed on PSD 7. The flap blood perfusion was detected by laser speckle contrast imaging technique on PSD 1, 4, and 7. The microvascular density in the flap's choke vessel zone was detected by immunohistochemical staining. All the sample numbers in each group at each time point in vivo experiments were 12. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction.
Results At 6 and 12 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 0.5 μmol/L 4αPDD group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, and 10.0 μmol/L 4αPDD group (P>0.05). At 6, 12, 24, and 48 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group (P>0.05). At PSH 12, the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At PSH 24 and 48, compared with those in PBS group, the percentages of the residual scratch area of cells in 3 μmol/L 4αPDD group were significantly decreased (with t values of 2.83 and 2.79, respectively, P<0.05), while the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group showed no significant differences (P>0.05). At 24 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At 48 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD groups were significantly greater than that in PBS group (with t values of 6.20 and 9.59, respectively, P<0.01). At 4 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly greater than that in PBS group (with t values of 4.68 and 4.95, respectively, P<0.05 or <0.01). At 8 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD and 3 μmol/L 4αPDD groups were similar to that in PBS group (P>0.05). At 24 h of culture, compared with those in PBS group, the protein expression level of E-cadherin of cells in 3 μmol/L 4αPDD group was significantly decreased (t=5.13, P<0.01), whereas there was no statistically significant difference in the protein expression level of E-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression level of N-cadherin of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.93, P<0.01), whereas there was no statistically significant difference in the protein expression level of N-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression levels of Slug of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly increased (with t values of 3.85 and 6.52, respectively, P<0.05 or P<0.01); and the protein expression level of Snail of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.08, P<0.05), whereas there was no statistically significant difference in the protein expression level of Snail of cells in 1 μmol/L 4αPDD group (P>0.05). There were no statistically significant differences in the protein expression levels of E-cadherin, N-cadherin, Slug, or Snail of cells between 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group (P>0.05). The general condition of flaps of rats in the three groups was good on PSD 0. On PSD 1, the flaps of rats in the three groups were basically similar, with bruising and swelling at the distal end. On PSD 4, the swelling of flaps of rats in the three groups subsided, and the distal end turned dark brown and necrosis occurred, with the area of necrosis in flaps of rats in normal saline group being larger than the areas in 4αPDD group and delayed flap group. On PSD 7, the necrotic areas of flaps of rats in the 3 groups were fairly stable, with the area of necrosis at the distal end of flap of rats in delayed flap group being the smallest. On PSD 7, the flap survival rates of rats in 4αPDD group ((80±13)%) and delayed flap group ((87±9)%) were similar (P>0.05), and both were significantly higher than (70±11)% in normal saline group (with t values of 2.24 and 3.65, respectively, P<0.05 or P<0.01). On PSD 1, the overall blood perfusion signals of rats in the 3 groups were basically the same, and the blood perfusion signals in the choke vessel zone were relatively strong, with a certain degree of underperfusion at the distal end. On PSD 4, the boundary between the surviving and necrotic areas of flaps of rats in the 3 groups became evident, and the blood perfusion signals in the choke vessel zone were improved, with the normal saline group's distal hypoperfused area of flap being larger than the areas in delayed flap group and 4αPDD group. On PSD 7, the blood perfusion signals of overall flap of rats had generally stabilized in the 3 groups, with the intensity of blood perfusion signal in the choke vessel zone and overall flap of rats in delayed flap group and 4αPDD group being significantly greater than that in normal saline group. On PSD 7, the microvascular density in the choke vessel zone of flap of rats in 4αPDD group and delayed flap group were similar (P>0.05), and both were significantly higher than that in normal saline group (with t values of 4.11 and 5.38, respectively, P<0.01).
Conclusions After activation, TRPV4 may promote the migration and tubular formation of human vascular endothelial cells via the EndMT pathway, leading to the enhanced blood perfusion of perforator flap and microvascular density in the choke vessel zone, and therefore increase the flap survival rate.
- TRPV cation channels /
- Endothelial cells /
- Perforator flap /
- Neovascularization, pathologic /
- Endothelial-to-mesenchymal transition
邝依敏（马来西亚籍）、黄昕、蒙旭昌：酝酿和设计实验、实施研究、采集数据、分析/解释数据、起草文章、统计分析；顾舒晨、张泽伟、刘云菡、骆申英：对文章的知识性内容作批评性审阅；昝涛：研究指导、行政支持、技术支持、材料支持

所有作者均声明不存在利益冲突

该文旨在探讨吸入性损伤和ARDS对炎症新型生物标志物胰石蛋白和现有生物标志物的影响。此外，该文还着重研讨了吸入性损伤对感染性/脓毒性血症炎症因子的干扰。胰石蛋白是由胰腺腺泡细胞分泌的，是全身感染和脓毒症早期激活中性粒细胞的急性期蛋白，常作为预测脓毒症发生的标志物。作者收集了瑞士苏黎世烧伤中心2015年5月—2018年10月收治的90例烧伤总面积≥15%TBSA的烧伤患者入院后14 d的观察期内的血液标本，检测了传统炎症生物标志物C反应蛋白(CRP)、白细胞计数（WBC）、降钙素原和胰石蛋白，结果显示90例患者中有25例(27%)出现吸入性损伤(中位年龄52岁，中位烧伤总面积31.5%TBSA，平均简明烧伤严重指数评分为73分）；吸入性损伤组患者入院时仅WBC显著高于对照组(P=0.011)；32%的患者出现ARDS，但与吸入性损伤的严重程度无关(P=0.11)；大部分时间点的WBC、CRP和降钙素原的结果说明吸入性损伤相关的炎症与脓毒症进展无关；胰石蛋白水平较高且随着时间的推移急剧升高(P<0.001)，是诊断脓毒症的最佳生物标志物。因此，该文得出结论：胰石蛋白是识别吸入性损伤继发脓毒症的可靠标志物。

李江锋，编译自《Burns》，2021，47(2) :338-348；郇京宁，审校

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1 2种分组后人脐静脉内皮细胞培养各时间点的增殖活性（样本数均为3， $\bar{x} \pm s$ ）。1A.5组细胞增殖活性；1B.3组细胞增殖活性

注：PBS为磷酸盐缓冲液，4αPDD为4α-佛波醇12，13-二癸酸酯；图1A处理因素主效应，F=2.91，P=0.078；时间因素主效应，F=313.11，P<0.001；两者交互作用，F=1.15，P=0.388；图1B处理因素主效应，F=2.34，P=0.244；时间因素主效应，F=814.28，P<0.001；两者交互作用，F=5.75，P=0.010

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2 通过划痕试验观察3组人脐静脉内皮细胞划痕后各时间点剩余划痕面积光学显微镜×100，图中标尺为200 μm。2A、2B、2C、2D.分别为磷酸盐缓冲液组划痕后0（即刻）、12、24、48 h的剩余划痕面积，随划痕后时间的延长，剩余划痕面积逐渐减少；2E、2F、2G、2H及2I、2J、2K、2L.分别为1 μmol/L 4α-佛波醇12，13-二癸酸酯（4αPDD）组及3 μmol/L 4αPDD组划痕后0、12、24、48 h的剩余划痕面积，其中图2F、2G、2H剩余划痕面积均分别较图2B、2C、2D小，但均分别较图2J、2K、2I大

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3 Transwell实验观察3组人脐静脉内皮细胞培养各时间点迁移情况光学显微镜×200，图中标尺为20 μm。3A、3B、3C.分别为磷酸盐缓冲液（PBS）组、1 μmol/L 4α-佛波醇12，13-二癸酸酯（4αPDD）组、3 μmol/L 4αPDD组培养24 h迁移至Transwell小室下层的细胞情况，图3B、3C细胞数量均较图3A多；3D、3E、3F.分别为PBS组、1 μmol/L 4αPDD组、3 μmol/L 4αPDD组培养48 h迁移至Transwell小室下层的细胞情况，图3E、3F细胞数量均较图3D明显增多

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4 3组人脐静脉内皮细胞培养各时间点的血管形成情况倒置荧光显微镜×10，图中标尺为50 μm。4A、4B、4C.分别为磷酸盐缓冲液（PBS）组、1 μmol/L 4α-佛波醇12，13-二癸酸酯（4αPDD）组、3 μmol/L 4αPDD组培养4 h管状结构形成情况，图4B、4C管状结构数量均较图4A多；4D、4E、4F.分别为PBS组、1 μmol/L 4αPDD组、3 μmol/L 4αPDD组培养8 h管状结构形成情况，图4E、4F管状结构数量均较图4D多

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5 蛋白质印迹法检测3组人脐静脉内皮细胞培养24 h的上皮钙黏素、神经钙黏素、Slug和Snail的蛋白表达水平。5A.条带图；5B.条图（样本数为3， $\bar{x} \pm s$ ）

注：GAPDH为3-磷酸甘油醛脱氢酶，PBS为磷酸盐缓冲液，4αPDD为4α-佛波醇12，13-二癸酸酯；条带图上方和条图横坐标下1、2、3均分别为PBS组、1 μmol/L 4αPDD组、3 μmol/L 4αPDD组；与PBS组比较，^aP<0.05，^bP<0.01

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6 3组行背部髂腰动脉穿支皮瓣原位转移的大鼠术后各时间点大体情况。6A、6B、6C、6D.分别为皮瓣延迟组术后0（即刻）、1、4、7 d大体情况，0 d情况良好，1 d时皮瓣远端青紫肿胀，4 d时远端开始变成暗褐色，7 d时坏死范围稳定；6E、6F、6G、6H.分别为4α-佛波醇12，13-二癸酸酯组术后0、1、4、7 d大体情况，其中4 d远端明显变成暗褐色，开始结痂坏死；6I、6J、6K、6L.分别为生理盐水组术后0、1、4、7 d大体情况，其中图6K、6I皮瓣远端坏死分别较图6C、6D与6G、6H重，坏死范围更大

注：白色箭头指示穿支皮瓣血管蒂

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7 激光散斑血流成像仪检测3组行背部髂腰动脉穿支皮瓣原位转移的大鼠术后各时间点的皮瓣血流灌注情况。7A、7B、7C.分别为皮瓣延迟组术后1、4、7 d皮瓣血流灌注情况，随时间延长，皮瓣整体和闭塞血管区域的血流信号增强明显；7D、7E、7F.分别为4α-佛波醇12，13-二癸酸酯组术后1、4、7 d皮瓣血流灌注情况，其中图7E皮瓣存活区与坏死区分界渐清晰，皮瓣远端血流信号弱，闭塞血管区域的血流信号增强；7G、7H、7I.分别为生理盐水组术后1、4、7 d皮瓣血流灌注情况，其中图7I皮瓣远端坏死面积较图7C、7F大，闭塞血管区域和整体存活区域的血流信号强度也较图7C、7F弱

注：白色箭头指示穿支皮瓣血管蒂；紫红色代表血流灌注多，蓝色代表血流灌注少，黑色代表无血流灌注

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8 免疫组织化学染色观察3组行背部髂腰动脉穿支皮瓣原位转移的大鼠术后7 d皮瓣闭塞血管区域CD31的表达二氨基联苯胺-苏木精×100，图中标尺为50 μm。8A、8B、8C.分别为皮瓣延迟组、4α-佛波醇12，13-二癸酸酯组和生理盐水组闭塞血管区域CD31染色阳性的微血管情况，图8C的微血管密度明显低于图8A、8B

注：CD31染色阳性（棕褐色）为微血管

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表1 划痕试验观测的3组人脐静脉内皮细胞划痕后各时间点剩余划痕面积百分比比较（%， $\bar{x} \pm s$ ）

组别	样本数	12 h	24 h	48 h
PBS组	3	76.5±13.8	56.2±16.0	48.2±16.0
1 μmol/L 4αPDD组	3	68.1±0.5	50.3±9.4	40.8±5.5
3 μmol/L 4αPDD组	3	58.6±0.8	36.4±3.1	28.6±7.4
t₁值		1.19	0.85	1.04
P₁值		0.733	1.000	0.920
t₂值		2.56	2.83	2.79
P₂值		0.052	0.028	0.030
注：PBS为磷酸盐缓冲液，4αPDD为4α-佛波醇12，13-二癸酸酯；处理因素主效应，F=8.53，P=0.002；时间因素主效应，F=89.06，P<0.001；两者交互作用，F=0.98，P=0.459；t₁值、P₁值，t₂值、P₂值分别为1 μmol/L 4αPDD组、3 μmol/L 4αPDD组与PBS组各时间点比较所得

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表2 Transwell实验观测的3组人脐静脉内皮细胞培养各时间点的迁移数量比较（个， $\bar{x} \pm s$ ）

组别	样本数	24 h	48 h
PBS组	3	95±14	131±8
1 μmol/L 4αPDD组	3	148±12	288±16
3 μmol/L 4αPDD组	3	157±28	374±66
t₁值		2.09	6.20
P₁值		0.176	<0.001
t₂值		2.44	9.59
P₂值		0.093	<0.001
注：PBS为磷酸盐缓冲液，4αPDD为4α-佛波醇12，13-二癸酸酯；处理因素主效应，F=37.89，P<0.001；时间因素主效应，F=79.36，P<0.001；两者交互作用，F=12.85，P=0.001；t₁值、P₁值，t₂值、P₂值分别为1 μmol/L 4αPDD组、3 μmol/L 4αPDD组与PBS组各时间点比较所得

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表3 3组人脐静脉内皮细胞培养各时间点管状结构数量比较（个， $\bar{x} \pm s$ ）

组别	样本数	4 h	8 h
PBS组	3	53.5±2.1	36.0±9.9
1 μmol/L 4αPDD组	3	79.5±2.1	44.0±1.4
3 μmol/L 4αPDD组	3	81.0±8.5	43.5±2.1
t₁值		4.68	1.44
P₁值		0.010	0.601
t₂值		4.95	1.35
P₂值		0.008	0.678
注：PBS为磷酸盐缓冲液，4αPDD为4α-佛波醇12，13-二癸酸酯；处理因素主效应，F=12.84，P=0.007；时间因素主效应，F=88.31，P<0.001；两者交互作用，F=3.93，P=0.081；t₁值、P₁值，t₂值、P₂值分别为1 μmol/L 4αPDD组、3 μmol/L 4αPDD组与PBS组各时间点比较所得

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