γ-分泌酶抑制剂在肺纤维化上皮间质转化中的作用

doi:10.11958/20220307

天津医药 ›› 2022, Vol. 50 ›› Issue (9): 917-920.doi: 10.11958/20220307

γ-分泌酶抑制剂在肺纤维化上皮间质转化中的作用

韩姣(), 王华兵, 徐玲文, 董芳()

武汉市第三医院（武汉大学附属同仁医院）重症医学科（邮编430200）

收稿日期:2022-02-28 修回日期:2022-04-19 出版日期:2022-09-15 发布日期:2022-09-05
通讯作者: 董芳 E-mail:308605312@qq.com;weiyuyan1981@163.com
作者简介:韩姣（1989）,女,主治医师,主要从事呼吸与危重症医学相关研究。E-mail： 308605312@qq.com
基金资助:
武汉市卫健委科研项目(WX21Z19)

The role of γ-secretase inhibitor in pulmonary fibrosis epithelial-mesenchymal transition

HAN Jiao(), WANG Huabing, XU Lingwen, DONG Fang()

Department of Critical Care Medicine, Wuhan Third Hospital (Tongren Hospital of Wuhan University), Wuhan 430200, China

Received:2022-02-28 Revised:2022-04-19 Published:2022-09-15 Online:2022-09-05
Contact: DONG Fang E-mail:308605312@qq.com;weiyuyan1981@163.com

韩姣, 王华兵, 徐玲文, 董芳. γ-分泌酶抑制剂在肺纤维化上皮间质转化中的作用[J]. 天津医药, 2022, 50(9): 917-920.

HAN Jiao, WANG Huabing, XU Lingwen, DONG Fang. The role of γ-secretase inhibitor in pulmonary fibrosis epithelial-mesenchymal transition[J]. Tianjin Medical Journal, 2022, 50(9): 917-920.

摘要/Abstract

摘要：

目的检测体外转化生长因子（TGF）-β1诱导肺纤维化上皮间质转化（EMT）的形成情况并检测Notch信号特异性抑制剂γ-分泌酶抑制剂（DAPT）对其影响及可能机制。方法将A549细胞分成对照组（RPMI 1640完全培养基培养）、TGF-β1组（在含有10 μg/L TGF-β1的RPMI 1640培养基中培养）、TGF-β1+DAPT组（在含有10 μg/L TGF-β1和2 μmol DAPT的RPMI 1640培养基中培养）、DAPT组（在含有2 μmol DAPT的RPMI 1640培养基中培养）。通过倒置显微镜观察细胞形态,实时荧光定量逆转录聚合酶链反应检测肺泡上皮细胞特异性蛋白E-钙黏合素以及间质细胞特异性蛋白α-肌动蛋白（α-SMA）mRNA相对表达水平,Western blot检测E-钙黏合素以及α-SMA蛋白表达水平。结果倒置显微镜检查示,对照组细胞呈多边形,铺路石样,细胞间连接紧密;TGF-β1组细胞呈梭行,纺锤状,细胞间连接减少;TGF-β1+DAPT组仅有少量细胞呈梭形,多数细胞形态与对照组相似;DAPT组细胞形态大致同对照组。与对照组相比,TGF-β1组α-SMA蛋白及mRNA表达增加,而E-钙黏合素蛋白及mRNA表达减弱（P<0.05）;与TGF-β1组比较,TGF-β1+DAPT组α-SMA蛋白及mRNA表达水平降低,而E-钙黏合素蛋白及mRNA表达水平增加（P<0.05）;DAPT组与对照组2指标的蛋白与mRNA相对表达水平差异无统计学意义（P>0.05）。结论 TGF-β1可诱导肺上皮间质转化,而Notch信号抑制剂DAPT能够阻断、部分或全部逆转这一过程。

关键词: 转化生长因子β1, 肺纤维化, 上皮-间充质转化, γ-分泌酶抑制剂

Abstract:

Objective To investigate the formation of pulmonary fibrosis epithelial-interstitial transformation (EMT) induced by transforming growth factor TGF-β1 in vitro and to detect the effect of Notch signal specific inhibitor DAPT on this transformation and its possible mechanism. Methods A549 cells were divided into the control group (cultured in RPMI 1640 complete medium), the TGF-β1 group (containing 10 μg/L TGF-β1 in RPMI 1640 medium), the TGF-β1+DAPT group (containing 10 μg/L TGF-β1 and 2 μmol DAPT in RPMI 1640 medium) and the DAPT group (only 2 μmol DAPT was cultured in RPMI 1640 medium). The cell morphology was observed by inverted microscope. The mRNA expression of alveolar epithelial cell specific protein levels of E-cadherin and interstitial cell specific protein α-actin (α-SMA) were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the expression levels of E-cadherin and α-SMA were detected by Western blot assay. Results The cells in the control group were polygonal, irregular and closely linked, while those in the TGF-β1 group were spindle-shaped with reduced intercellular connections. In the TGF-β1 + DAPT group, only a few cells showed spindle shape, and most cell morphology was similar to that of the control group. The cell morphology of the DAPT group was similar to that of the control group. Compared with the control group, expression levels of α-SMA protein and mRNA increased significantly in the TGF-β1 group (P<0.05), while the expression of E-cadherin and its mRNA decreased significantly (P<0.05). Compared with the TGF-β1, the expression of α-SMA and mRNA decreased significantly in the TGF-β1 and DAPT group, while the expression of E-cadherin and mRNA increased significantly (P<0.05). There were no significant differences in expression levels of α-SMA and E-cadherin protein and mRNA between the DAPT group and the control group (P>0.05). Conclusion TGF-β1 can induce lung epithelial interstitial transformation, while Notch signal inhibitor DAPT can block, partially or completely reverse this transformation process.

Key words: transforming growth factor-β1, pulmonary fibrosis, epithelial-mesenchymal transition, γ-secretase inhibitor

中图分类号:

R563.9

图/表 5

表1 qRT-PCR引物序列

Tab.1 Primer sequence for qRT-PCR

基因名称	引物序列（5′→3′）	产物大小（bp）
β-actin	上游：AGCGAGCATCCCCCAAAGTT	285
β-actin	下游：GGGCACGAAGGCTCATCATT	285
α-SMA	上游：TCATGGTCGGTATGGGTCAG	150
α-SMA	下游：CGTTGTAGAAGGTGTGGTGC	150
E-cadherin	上游：CGTAGCAGTGACGAATGTGG	175
E-cadherin	下游：CTGGGCAGTGTAGGATGT	175

图1 倒置显微镜下各组细胞形态的比较（Bar=20 µm）

Fig.1 Comparison of cell morphology between the four groups (Bar=20 µm)

表2 各组细胞E-钙黏合素和α-SMA mRNA相对表达水平比较（n=3,$\bar{x}±s$）

Tab.2 Comparison of relative expression levels of E-cadherin and α-SMA mRNA between the four groups

组别	E-钙黏合素	α-SMA
对照组	0.997±0.149	1.045±0.174
TGF-β1组	0.044±0.036^a	1.863±0.095^a
TGF-β1+DAPT组	0.802±0.093^b	1.369±0.149^b
DAPT组	1.026±0.168^b	1.075±0.065^b
F	14.254^＊	26.142^＊

图2 E-钙黏合素和α-SMA蛋白的Western blot图 A：对照组;B：TGF-β1组;C：TGF-β1+DAPT组;D：DAPT组。

Fig.2 Western blot assay of E-cadherin and α-SMA

表3 各组细胞E-钙黏合素和α-SMA蛋白相对表达水平比较（n=3,$\bar{x}±s$）

Tab.3 Comparison of proteins of E-cadherin and α-SMA between the four groups

组别	E-钙黏合素	α-SMA
对照组	0.551±0.052	0.357±0.037
TGF-β1组	0.286±0.023^a	0.647±0.042^a
TGF-β1+DAPT组	0.497±0.057^b	0.484±0.049^ab
DAPT组	0.590±0.059^b	0.380±0.040^b
F	22.121^＊	29.753^＊

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γ-分泌酶抑制剂在肺纤维化上皮间质转化中的作用

The role of γ-secretase inhibitor in pulmonary fibrosis epithelial-mesenchymal transition

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