温石棉对内皮细胞Wnt5a、p16和p21表达的影响

doi:10.11958/20240060

摘要/Abstract

摘要：

目的探讨温石棉对人脐静脉内皮细胞（HUVECs）的影响。方法实验组以50、100、200 mg/L温石棉纤维液刺激HUVECs 24、48、72 h，对照组仅加RPMI 1640培养基培养细胞，观察细胞形态变化，β-半乳糖苷酶法分析细胞衰老情况，四甲基偶氮唑蓝法检测细胞存活率。采用实时荧光定量PCR法检测细胞中Wnt5a、p16和p21 mRNA的表达情况。结果实验组HUVECs多呈梭形，部分呈圆形或不规则形，出现裸核及空泡现象，可见死亡细胞。随温石棉质量浓度及暴露时间的增加，细胞活性逐渐降低，衰老细胞逐渐增多。100 mg/L温石棉处理HUVECs 24 h时，细胞生长较活跃。与对照组相比，实验组Wnt5a、p16和p21 mRNA表达水平均增高（P<0.05）。结论温石棉可促进HUVECs衰老，Wnt5a、p16和p21参与此过程。

关键词: 石棉，蛇纹石, 人脐静脉内皮细胞, 肺纤维化, Wnt-5a蛋白, 基因，p16, 基因，p21

Abstract:

Objective To investigate the influence of chrysotile on human umbilical vein endothelial cells (HUVECs). Methods HUVECs in the experimental group were treated with 50, 100 and 200 mg/L chrysotile for 24, 48 and 72 h. The control group was cultured with RPMI 1640 medium only. Morphological changes of HUVECs were observed. Cell viability was detected by MTT assay. β-Galactosidase staining was used to analyze the cell senescence. The cell survival rate was determined by tetramethylazolium blue assay. Wnt5a, p16 and p21 expression were investigated by qRT-PCR. Results Cells of the experimental group were oval and irregular, and some of them were round or irregular, with naked nuclei and vacuolar, and dead cells were present. With the increase of chrysotile concentration and exposure time, the level of cell viability decreased and the number of aged cells increased gradually (P<0.05). The cell growth of HUVECs was more active when 100 mg/L chrysotile treated for 24 h. Compared with the control group, the mRNA expression levels of Wnt5a, p16 and p21 were increased in the experimental group (P<0.05). Conclusion Chrysotile can promote HUVECs senescence, and Wnt5a, p16 and p21 are involved in this mechanism.

Key words: asbestos, serpentine, human umbilical vein endothelial cells, pulmonary fibrosis, Wnt-5a protein, genes, p16, genes, p21

中图分类号:

R563.9

图/表 6

表1 qRT-PCR引物序列

Tab.1 Primer sequences of qRT-PCR

基因名称	引物序列（5’→3’）	产物大小/bp
Wnt5a	上游：CTAACTTAGCTGTGTGGGACATG	235
	下游：AAATGCAGAAAGCAAGCTAGCAG	235
p16	上游：CGGGGACATCAAGACATCGT	203
	下游：GCCGGATTTAGCTCTGCTCT	203
p21	上游：ACATCTCAGGGCCGAAAACG	173
	下游：AAGACACACAGAGTGAGGGC	173
β-actin	上游：CCACTGGCATCGTGATGGAC	171
	下游：GCGGATGTCCACGTCACACT	171

图1 温石棉刺激后HUVECs形态学表现（×400）

Fig.1 Morphological changes of HUVECs after treatment with chrysotile (×400)

图2 不同质量浓度温石棉作用24 h情况下HUVECs衰老情况（×200）

Fig.2 Cell senescence of HUVECs after treatment with different levels of chrysotile for 24 hours (×200)

表2 3组在温石棉刺激下的细胞衰老情况

Tab.2 The aged cell rate of HUVECs after treatment with chrysotile in three groups （n=5，%，$\bar{x}±s$）

组别	24 h	48 h	72 h
对照组	4.20±0.84	9.20±1.48	11.80±1.92
50 mg/L实验组	8.20±1.92	13.20±2.39	17.40±3.05
100 mg/L实验组	11.40±1.52	17.20±2.86	28.60±3.91
200 mg/L实验组	22.00±4.36	29.60±3.58	37.60±6.42

图3 不同时间及温石棉质量浓度作用下细胞存活率变化情况

Fig.3 The level of cell viability of HUVECs treated by different concentrations of chrysotile for different times

表3 对照组和试验组的Wnt5a、p16和p21 mRNA表达水平

Tab.3 The levels of Wnt5a, p16 and p21 mRNA expression in two groups （n=6，×10-3，$\bar{x}±s$）

组别	Wnt5a	p16	p21
对照组	5.44±3.74	6.56±1.74	2.23±0.91
实验组	34.18±6.08	20.63±0.86	7.80±0.73
t	5.167^＊	9.881^＊	6.091^＊

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