康柏西普可逆转TGF-β2诱导的晶状体上皮细胞发生上皮间质转化：基于调节TGF-β/Smad信号通路

doi:10.12122/j.issn.1673-4254.2024.08.04

摘要/Abstract

摘要：

目的探讨康柏西普（conbercept）对转化生长因子-β₂（TGF-β₂）诱导人晶状体上皮细胞（HLECs）发生上皮间质转化（EMT）的逆转机制。方法体外培养HLEC-SRA01/04细胞并分为对照组、TGF-β₂组、conbercept组、TGF-β₂+conbercept组。采用MTT法、流式细胞术、划痕实验、Transwell检测细胞增殖、凋亡与迁移，Western blotting 和qRT-PCR 检测EMT相关上皮细胞标记物E-Cadherin、α-SMA、snail、细胞外基质和TGF-β/Smad信号通路相关基因的表达。结果 TGF-β₂+conbercept组EMT程度明显减轻，细胞间充质及细胞外基质标志物α-SMA、snail、collagen Ⅰ、collagen Ⅳ、FN1的表达量均明显减少，与TGF-β₂组的差异有统计学意义（P<0.05）。上皮细胞相关标志物E-Cadherin的蛋白及mRNA的表达上调（P<0.05）。Transwell结果显示，与TGF-β₂组相比，TGF-β₂+conbercept组细胞迁移能力减弱（P<0.05）。此外，TGF-β₂诱导HLEC-SRA01/04细胞发生EMT过程中发生Smad2/3磷酸化水平升高现象可被康柏西普抑制（P<0.01）。结论康柏西普可能通过TGF-β/Smad信号通路抑制HLEC-SRA01/04细胞EMT过程，具有预防及治疗PCO的能力。

关键词: 后发性白内障, 康柏西普, 上皮间质转化, 转化生长因子-β₂

Abstract:

Objective To investigate the mechanism by which conbercept reverses transforming growth factor-β₂ (TGF‑β₂)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). Methods Cultured HLEC SRA01/04 cells were treated with TGF‑β₂, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α‑SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway. Results Conbercept significantly reduced TGF-β₂-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P<0.05). Transwell assay showed significantly lower cell migration ability in TGF-β₂+conbercept group than in TGF-β₂ group (P<0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β₂-induced EMT (P<0.01). Conclusion Conbercept inhibits TGF‑β₂ induced EMT by downregulating the expression of pSmad2/3 in TGF‑β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

Key words: posterior capsule opacification, conbercept, epithelial-to-mesenchymal transition, transforming growth factor-β₂

朱梦云, 王剑锋. 康柏西普可逆转TGF-β₂诱导的晶状体上皮细胞发生上皮间质转化：基于调节TGF-β/Smad信号通路[J]. 南方医科大学学报, 2024, 44(8): 1459-1466.

Mengyun ZHU, Jianfeng WANG. Conbercept reverses TGF‑β₂-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway[J]. Journal of Southern Medical University, 2024, 44(8): 1459-1466.

图/表 11

表1 引物序列

Tab.1 The sequences of primer

Gene	Forward primer	Reverse primer
E-cadherin	GAAGTGTCCGAGGACTTTGG	CAGTGTCTCTCCAAATCCGATA
Snail	CGGAAGCCTAACTACAGCGA	GGACAGAGTCCCAGATGAGC
GAPDH	CAGCCTCAAGATCATCAGCA	TGTGGTCATGAGTCCTTCCA
α-SMA	ATAGACATGCCGCCCTTCTT	GGCATCAAGGTACCCACAGA
FN1	AAGACCAGCAGAGGCATAAGG	TGTAGGGGTCAAAGCACGAG
Collagen Ⅰ	TTGAGTTGTATCGTGTGGTG	AGAAGATGAAAATGAGACTG
Collagen Ⅳ	TAGAGAGGAGCGAGATGTTC	GTGACATTAGCTGAGTCAGG

图1 康柏西普对HLEC-SRA01/04细胞存活率的影响

Fig.1 Effects of conbercept on survival rate of lens epithelial cell line HLEC-SRA01/04. *P<0.05 vs 0 mg/mL at the same time point; #P<0.05 vs same concentration for 24 h; &P<0.05 vs the same concentration for 48 h.

图3 康柏西普对HLEC-SRA01/04迁移能力的影响

Fig.3 Effects of conbercept on migration ability of lens epithelial cells (Scale bar=200 μm). **P<0.01 vs control.

图4 倒置显微镜观察TGF-β2诱导HLECs SRA01/04细胞形态变化

Fig.4 Inverted microscopy of morphological changes of human lens epithelial cells SRA01/04 induced by TGF-β2 (Scale bar=200 μm).

图5 TGF-β2诱导HLEC-SRA01/04发生EMT过程

Fig.5 TGF-β2 at 10 ng/mL for 24 h induces EMT of human lens epithelial cells SRA01/04 cells. A: Expression level of E-cadherin after TGF-β2 treatment detected by qRT-PCR. B: Expression level of α-SMA after TGF-β2 treatment detected by qRT-PCR. C: Expression level of Snail after TGF-β2 treatment detected by qRT-PCR. *P<0.05, **P<0.01 vs control.

图6 Western blotting检测TGF-β2诱导HLEC-SRA01/04细胞成EMT模型

Fig.6 Western blotting for detecting TGF-β2-induced EMT in HLEC-SRA01/04 cells treated with TGF-β2 for 24 h by detecting epithelial cell markers E-cadherin and mesenchymal cell markers α‑SMA and Snail. *P<0.05, **P<0.01 vs control.

图7 Western blotting检测康柏西普对TGF-β2诱导HLEC-SRA01/04的E-cadherin、α-SMA与snail的蛋白表达

Fig.7 Western blotting for detecting expressiona of E-cadherin, α-SMA and Snail in TGF-β2-induced HLEC-SRA01/04 cells after conbercept treatment. At least 3 replicates were performed in each experiment. **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01, ###P<0.001 vs TGF-β2.

图8 qRT-PCR检测康柏西普对TGF-β2诱导HLEC-SRA01/04的E-Cadherin、α-SMA与snail的mRNA表达

图9 康柏西普对TGF-β2诱导HLEC-SRA01/04的ECM基因表达的影响

Fig.9 Effect of conbercept on TGF-β2-induced expression of ECM genes in HLEC-SRA01/04 cells. A: Collagen I mRNA levels in cells treated with TGF-β2 and conbercept for 24 h detected by real-time PCR. B: FN1 mRNA levels in cells treated with TGF-β2 and conbercept for 24 h The mRNA levels. C: Collagen IV mRNA levels in cells treated with TGF-β2 and conbercept for 24 h. At least 3 replicates were performed in each experiment. **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01 vs TGF-β2.

图10 康柏西普对TGF-β2诱导HLEC-SRA01/04的细胞迁移能力的影响

Fig.10 Effects of conbercept on migration of SRA01/04 cells induced by TGF-β2. At least 3 replicates were performed in each experiment. Giemsa staining, Scale bar: 50 μm. ***P<0.001 vs control; ##P<0.01 vs TGF-β2.

图11 康柏西普抑制TGF-β2诱导HLEC-SRA01/04细胞的TGF-β/Smad信号通路

Fig.11 Conbercept inhibits TGF-β/Smad signaling pathway in SRA01/04 cells induced by TGF-β2. The expressions of Smad2/3 and p-Smad2/3 were detected by Western blotting. At least 3 replicates were performed in each experiment. **P<0.01 vs control; ##P<0.01 vs TGF-β2.

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康柏西普可逆转TGF-β₂诱导的晶状体上皮细胞发生上皮间质转化：基于调节TGF-β/Smad信号通路

Conbercept reverses TGF‑β₂-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway

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