白细胞介素6对人脐静脉血管内皮细胞-间质细胞转化的作用

郭玲; 何静; 崔莉; 米俊伟; 张蜀; 孙剑会; 杜娟; 文大林; 张华才; 蒋建新; 王建民; 黄宏

doi:10.3760/cma.j.cn.501120-20201215-00530

白细胞介素6对人脐静脉血管内皮细胞-间质细胞转化的作用

doi: 10.3760/cma.j.cn.501120-20201215-00530

1. 陆军特色医学中心野战外科研究部,创伤、烧伤与复合伤国家重点实验室,重庆 400042;
2. 陆军特色医学中心干细胞再生医学科,重庆 400042

基金项目:

重庆市院士牵头科技创新引导专项（cstc2017zdcy-yszxX0002）

国家自然科学基金面上项目（81571912，30871025）

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出版历程
- 收稿日期: 2020-12-15
- 网络出版日期: 2021-10-28
- 刊出日期: 2021-05-20

Role of interleukin-6 in human umbilical vein endothelial cell to mesenchymal cell transformation

1. State Key Laboratory of Trauma, Burns and Combined Injury, Department of Surgical Research, the Army Medical Center, Chongqing 400042, China;
2. Department of Stem Cell & Regenerative Medicine, the Army Medical Center, Chongqing 400042, China

摘要

摘要:
目的观察白细胞介素6（IL-6）对人脐静脉内皮细胞（HUVEC）表型和功能的影响，探索IL-6在内皮-间充质转化（EndMT）过程中的作用。方法采用实验研究方法。取产妇行分娩手术后弃用的新鲜正常胎儿脐带，分离培养原代细胞的第2天在倒置相差显微镜下观察细胞形态；免疫荧光法鉴定第4代细胞是否为HUVEC后，取2批第3～5代HUVEC，用于后续实验。将第1批细胞按随机数字表法（下同）分为6组：空白对照组、5 ng/mL IL-6组、10 ng/mL IL-6组、25 ng/mL IL-6组、50 ng/mL IL-6组、100 ng/mL IL-6组，将第2批细胞分为4组：空白对照组、10 ng/mL IL-6组、25 ng/mL IL-6组、50 ng/mL IL-6组；空白对照组仅加入完全培养液，其余各组细胞还加入相应终质量浓度的IL-6。第1批细胞，分组培养后72 h，倒置相差显微镜下观察6组HUVEC形态；免疫荧光法检测6组HUVEC凝血因子Ⅷ和α平滑肌肌动蛋白（α-SMA）的阳性表达，并计算双阳性细胞数占凝血因子Ⅷ阳性细胞数的比值（以下简称双阳性细胞数的比值），样本数为6；实时荧光定量反转录PCR法检测6组HUVEC 血管内皮钙黏蛋白和α-SMA蛋白的mRNA表达量，样本数为3。第2批细胞，分组培养后72 h，蛋白质印迹法检测4组HUVEC 血管内皮钙黏蛋白、α-SMA和Ⅰ型胶原的蛋白表达量，样本数为3。对数据行单因素方差分析、Bonferroni校正。结果原代分离培养的第2天细胞呈短梭形或多边形，免疫荧光法鉴定第4代细胞为HUVEC。第1批细胞，分组培养后72 h，6组细胞随IL-6浓度的逐渐增加，其形态向长梭形变化，细胞间连接减少或消失、间隙变大。分组培养后72 h，与空白对照组双阳性细胞数的比值相比，25 ng/mL IL-6组、50 ng/mL IL-6组、100 ng/mL IL-6组显著增高（
P
<0.01）；与5 ng/mL IL-6组双阳性细胞数的比值相比，25 ng/mL IL-6组、50 ng/mL IL-6组和100 ng/mL IL-6组显著升高（
P<
0.01）；与10 ng/mL IL-6组双阳性细胞数的比值相比，50 ng/mL IL-6组和100 ng/mL IL-6组显著升高（
P
<0.01）；100 ng/mL IL-6组双阳性细胞数的比值均显著高于25 ng/mL IL-6组、50 ng/mL IL-6组（
P
<0.01）。分组培养后72 h，与空白对照组细胞血管内皮钙黏蛋白的mRNA表达量比，25 ng/mL IL-6组、50 ng/mL IL-6组和100 ng/mL IL-6组明显下降（
P
<0.05或
P
<0.01）；与5 ng/mL IL-6组细胞血管内皮钙黏蛋白的mRNA表达量比较，50 ng/mL IL-6组和100 ng/mL IL-6组均明显下降（
P
<0.01）；与10 ng/mL IL-6组细胞血管内皮钙黏蛋白的mRNA表达量比较，50 ng/mL IL-6组和100 ng/mL IL-6组明显下降（
P
<0.01）；与25 ng/mL IL-6组细胞血管内皮钙黏蛋白mRNA的表达量比较，50 ng/mL IL-6组和100 ng/mL IL-6组明显下降（
P
<0.01）。分组培养后72 h，5 ng/mL IL-6组、10 ng/mL IL-6组、25 ng/mL IL-6组、50 ng/mL IL-6组、100 ng/mL IL-6组细胞 α-SMA的 mRNA表达水平显著高于空白对照组（
P
<0.05或
P
<0.01）。第2批细胞分组培养后72 h，与空白对照组细胞血管内皮钙黏蛋白的蛋白表达量1.391±0.026比较，10 ng/mL IL-6组（1.185±0.063）、25 ng/mL IL-6组（0.717±0.078）、50 ng/mL IL-6组（0.293±0.064）显著降低（
P
<0.05）；与10 ng/mL IL-6组细胞血管内皮钙黏蛋白的蛋白表达量比较，25 ng/mL IL-6组、50 ng/mL IL-6组显著降低（
P
<0.01）；与25 ng/mL IL-6组细胞血管内皮钙黏蛋白的蛋白表达量比较，50 ng/mL IL-6组显著降低（
P
<0.01）。分组培养后72 h，与空白对照组细胞α-SMA的蛋白表达量比较，10 ng/mL IL-6组、25 ng/mL IL-6组、50 ng/mL IL-6组显著升高（
P
<0.01）；与10 ng/mL IL-6组细胞α-SMA的蛋白表达量比较，25 ng/mL IL-6组、50 ng/mL IL-6组显著增加（
P
<0.01）。分组培养后72 h，与空白对照组细胞Ⅰ型胶原的蛋白表达量比较，25 ng/mL IL-6组、50 ng/mL IL-6组显著增加（
P
<0.05）。结论 IL-6作用于HUVEC后，细胞的表型和功能呈浓度依赖性表现为间质细胞的特征。炎症因子可促进EndMT进程，成为调控组织纤维化机制的重要因子之一。
- 白细胞介素6 /
- 内皮细胞 /
- 纤维化 /
- 内皮-间质转化
Abstract:
Objective To observe the effect of interleukin-6 (IL-6) on the phenotype and function of human umbilical vein endothelial cells (HUVECs) and explore the role of IL-6 in the process of endothelial-to-mesenchymal transition (EndMT). Methods The experimental research method was used. Fresh umbilical cord discarded after normal maternal delivery was collected. On the second day of the primary cell isolation and cultivation, the cell morphology was observed under inverted phase contrast microscope. HUVECs of the 4th passage were identified by immunofluorescence method, and 2 batches of HUVECs ofthe 3rd to 5th passages were used for the subsequent experiments. The first batch of cells were divided into 6 groups according to the random number table (the same below): blank control group, 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group. The second batch of cells were divided into 4 groups: blank control group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group,and 50 ng/mL IL-6 group; the cells in blank control group was cultured with complete culture medium only, while the cells in the other groups were added with IL-6 of the corresponding final mass concentrations.Cells from the 1st batch were cultured for 72 hours after grouping, the morphology of HUVECS in the 6 groups was observed under inverted phase contrast microscope. At 72 h after grouping culture, the positive expressions of coagulation factor Ⅷ and α vascular smooth muscle actin (α-SMA) in HUVECs in the 6 groups were detected by immunofluorescence method, and the ratio of the number of double positive cells to the number of coagulation factor Ⅷ positive cells (the ratio of double positive cells for short) was calculated, with 6 samples per group; mRNA expression levels of vascular endothelial cadherin and α-SMA of HUVECs in 6 groups were detected by reverse transcription-polymerase chain reaction, with 3 samples per group.Cells from the 2nd batch were cultured 72 hours after grouping, the protein expression levels of vascular endothelial cadherin, α-SMA, and type Ⅰ collagen in the 4 groups were detected by Western blotting, with 3 samples per group. Data were statistically analyzed with one-way analysis of variance and Bonferroni correction. Results On the 2nd day after isolation and cultivation, the primary cells were in short spindle shape or polygon, cells of the 4th passage were identified as HUVECs by immunofluorescence method. At 72 hours of culture after grouping, the cells from the 1st batch in the 6 groups changed to long spindle shape morphologically along with the increase of IL-6 concentration, the intercellular connections decreased or disappeared with the gap between cells becoming larger. At 72 h after grouping culture, compared with that inblank control group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (
P
<0.01); compared with that in 5 ng/mL IL-6 group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (
P
<0.01); compared with that in 10 ng/mL IL-6 group, the ratio of double positive cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly increased (
P
<0.01); the ratio of double positive cells in 100 ng/mL IL-6 group was significantly increased compared with those in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group (
P
<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly decreased (
P
<0.01 or
P
<0.05); compared with that in 5 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (
P
<0.01); compared with that in 10 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (
P
<0.01); compared with that in 25 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (
P
<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of α-SMA of cells in 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6, group, and 100 ng/mL IL-6 group were significantly increased (
P
<0.05 or
P
<0.01). Cells from the 2nd batch were cultured for 72 hours after grouping. Compared with 1.391±0.026 in blank control group, the protein expressions of vascular endothelial cadherin of cells in 10 ng/mL IL-6 group (1.185±0.063), in 25 ng/mL IL-6 group (0.717±0.078), and in 50 ng/mL IL-6 group (0.239±0.064) were significantly decreased (
P
<0.05); compared with that in 10 ng/mL IL-6 group, the protein expressions of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly decreased (
P
<0.01); compared with that in 25 ng/mL IL-6 group, the protein expression of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group was significantly decreased (
P
<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expression levels of α-SMA of cells in 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, and 50 ng/mL IL-6 group were significantly increased (
P
<0.01); compared with that in 10 ng/mL IL-6 group, the protein expression levels of α-SMA of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (
P
<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expressions of type Ⅰ collagen of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (
P
<0.05). Conclusions After IL-6 treatment, the phenotype and function of HUVECS showed the characteristics of mesenchymal cells in a concentration-dependent manner. The inflammatory factor can promote the process of EndMT, and become one of the important factors regulating the mechanism of tissue fibrosis.
- Interleukin-6 /
- Endothelial cells /
- Fibrosis /
- Endothelial-to-mesenchymal transition