硫酸吲哚酚通过TGF-β1诱导腹膜间皮细胞转分化

doi:10.3877/cma.j.issn.2095-1221.2022.06.002

目的

探讨转化生长因子β1 （TGF-β1）在硫酸吲哚酚诱导腹膜间皮细胞转分化中的作用。

方法

对人腹膜间皮细胞株HMrSV5进行体外培养，先用含10%胎牛血清的RPMI1640培养液培养，分别在培养液中加入1 000 μmol/L的硫酸吲哚酚，4.25%腹膜透析液（PDF），2 μmol/L TGF-β1受体抑制剂LY364947 （TRI）+PDF，TRI +1 000 μmol/L的硫酸吲哚酚共培养。用免疫荧光法观察细胞在0、4、24、48、72 h的形态改变，用qRT-PCR方法测得各组各时间点TGF-β1、Twist1和E-钙粘蛋白（E-cadherin）基因相对表达量。多组间比较采用单因素方差分析，组间两两比较采用SNK-q检验。

结果

硫酸吲哚酚剌激细胞后，细胞由铺路石样变细变长，呈长梭形改变，而TRI +1 000 μmol/L硫酸吲哚酚干预的细胞变化不明显。免疫荧光染色结果显示，硫酸吲哚酚干预细胞后，从24 h开始α-平滑肌肌动蛋白（α-SMA）的表达量增加，48 h表达最明显，而同时段硫酸吲哚酚+TRI干预细胞后，α-SMA表达相对减少。qRT-PCR结果显示，与对照比较，加入硫酸吲哚酚或PDF干预48 h后TGF-β1、Twist基因相对表达量（1.03±0.01，1.07±0.01比0.28±0.01）升高，E-cadherin基因相对表达量（0.86±0.02，0.88±0.02比3.34±0.02）下降（P < 0.05）；与硫酸吲哚酚培养液比较，加入硫酸吲哚酚+TRI培养48 h后细胞TGF-β1、Twist1基因相对表达量（0.14±0.00比1.03±0.01）降低，E-cadherin基因相对表达量（1.69±0.03比0.86±0.02）升高，差异有统计学意义（P < 0.05）。与PDF培养比较，加入PDF+TRI培养48 h后细胞中TGF-β1、Twist1基因相对表达量（0.14±0.00比1.07±0.01）降低，E-cadherin基因相对表达量（1.80±0.03比0.88±0.02）升高，差异有统计学意义（P < 0.05）。

结论

硫酸吲哚酚可通过上调TGF-β1基因表达来促进腹膜间皮细胞EMT，Twist1参与其中。

关键词: 上皮细胞-间充质转分化, 硫酸吲哚酚, 转化生长因子β1, Twist1

Objective

To investigate the role of transforming growth factor (TGF-β1) in the induction of indoxyl sulfate in the transdifferentiation of peritoneal mesenchymal cells.

Methods

Human peritoneal mesenchymal cell line HMrSV5 was cultured in vitro using RPMI1640 medium containing 10% fetal bovine serum. 1 000 μmol/L indoxyl sulfate and 4.25% peritoneal dialysate (PDF) were added into the medium, respectively. 2 μmol/L TGF-β1 receptor inhibitor LY364947 (TRI) +PDF, TRI+1 000 μmol/ L indoxyl sulfate co-culture. The morphologic changes of cells at 0, 4, 24, 48 and 72 h were observed by immunofluorescence method. The relative expression levels of TGF-β1, Twist1 and E-cadherin at each time point were measured by qRT-PCR method. One-way analysis of variance was used for comparison between multiple groups, and SNK-q test was used for pairwise comparison between different groups.

Results

After the stimulation of indoxyl sulfate, the cells became thin and long like paving stones, but the changes were not obvious after the intervention of TRI +1 000 μmol/L indoxyl sulfate. Immunofluorescence staining showed that the expression of alpha-smooth muscle actin (α-SMA) was increased from 24 h after indoxyl sulfate intervention, and the expression was most obvious at 48 h, while the expression of α-SMA was decreased after indoxyl sulfate+TRI intervention at the same time. The results of qRT-PCR showed that compared with the control, the relative expression of TGF-β1 and Twist (1.03±0.01, 1.07±0.01 vs 0.28±0.01) gene was increased after 48 h intervention with indoxyl sulfate or PDF. E-cadherin (0.86± 0.02, 0.88± 0.02 vs 3.34±0.02) gene expression was decreased (P < 0.05) ; Compared with indoxyl sulfate culture medium, the relative gene expression of TGF-β1 and Twist1 (0.14±0.00 vs 1.03±0.01) was decreased after being added with indoxyl sulfate+TRI culture for 48 h. The relative expression level of E-cadherin (1.69±0.03 vs 0.86±0.02) was increased, and the difference was statistically significant (P < 0.05) . Compared with PDF culture, TGF-β1 and Twist1 48 h (0.14±0.00 vs 1.07±0.01) gene relative expression levels were decreased after adding PDF+TRI culture for 48 h. The relative expression level of E-cadherin (1.80±0.03 vs 0.88±0.02) was increased, and the difference was statistically significant (P < 0.05) .

Conclusion

Indoxyl sulfate can promote EMT through upregulation of TGF-β1 gene expression, and Twist1 was involved in the process induced by indoxyl sulfate in peritoneal mesenchymal cells.

Key words: Epithelial-mesenchymal transition, Indoxol sulfate, Transforming growth factor-β1, Twist1

表1 引物序列信息

基因	序列（5'→3'）	预期扩增产物长度（bp）	退火温度（℃）
Twist1	上游GTCCGCGTCCCACTAGC	90	59.72
	下游TCCATTTTCTCCTTCTCTGGAA		52.79
E-cadherin	上游GCCGAGAGCTACACGTTCAC	117	58.91
	下游GTCGAGGGAAAAATAGGCTG		53.27
TGF-β1	上游CCCTGGACACCAACTATTGC	92	56.27
	下游CTTCCAGCCGAGGTCCTT		57.57
hGAPDH	上游GGCTGAGAACGGGAAGCTTGTCAT	110	62.05
	下游CAGCCTTCTCCATGGTGGTGAAGA		61.51

表1 引物序列信息

基因	序列（5'→3'）	预期扩增产物长度（bp）	退火温度（℃）
Twist1	上游GTCCGCGTCCCACTAGC	90	59.72
	下游TCCATTTTCTCCTTCTCTGGAA		52.79
E-cadherin	上游GCCGAGAGCTACACGTTCAC	117	58.91
	下游GTCGAGGGAAAAATAGGCTG		53.27
TGF-β1	上游CCCTGGACACCAACTATTGC	92	56.27
	下游CTTCCAGCCGAGGTCCTT		57.57
hGAPDH	上游GGCTGAGAACGGGAAGCTTGTCAT	110	62.05
	下游CAGCCTTCTCCATGGTGGTGAAGA		61.51

图1 倒置显微镜下观察腹膜间皮细胞株形态（×100）注：a图为对照组，细胞呈鹅卵石铺路样；b图为硫酸吲哚酚，细胞呈梭形；c图为硫酸吲哚酚+TRI组，细胞呈长梭形改变

图2 共聚焦显微镜下观察HMrSV5细胞形态（IF染色，×400）注：蓝色信号为细胞核；红色信号为α-SMA

表2 不同时间TGF-β1基因相对表达量情况（ ± s，n = 3）

分组	0 h	4 h	24 h	48 h	72 h
对照	0.10±0.00	0.22±0.01	0.32±0.00	0.34±0.00	0.48±0.01
硫酸吲哚酚	0.10±0.01	0.42±0.01^a	0.51±0.01^a	1.42±0.02^a	1.26±0.02^a
PDF	0.11±0.00	0.42±0.01^a	0.51±0.01^a	1.60±0.03^a	1.54±0.02^a
硫酸吲哚酚+TRI	0.10±0.00	0.21±0.00^bc	0.22±0.00^abc	0.51±0.01^abc	0.50±0.01^b
PDF+TRI	0.10±0.00	0.22±0.01^bc	0.23±0.00^abc	0.48±0.01^abc	0.46±0.01^bc
F值	3.094	1081.438	3023.632	5010.075	4881.937
P值	0.067	< 0.001	< 0.001	< 0.001	< 0.001

表2 不同时间TGF-β1基因相对表达量情况（ ± s，n = 3）

分组	0 h	4 h	24 h	48 h	72 h
对照	0.10±0.00	0.22±0.01	0.32±0.00	0.34±0.00	0.48±0.01
硫酸吲哚酚	0.10±0.01	0.42±0.01^a	0.51±0.01^a	1.42±0.02^a	1.26±0.02^a
PDF	0.11±0.00	0.42±0.01^a	0.51±0.01^a	1.60±0.03^a	1.54±0.02^a
硫酸吲哚酚+TRI	0.10±0.00	0.21±0.00^bc	0.22±0.00^abc	0.51±0.01^abc	0.50±0.01^b
PDF+TRI	0.10±0.00	0.22±0.01^bc	0.23±0.00^abc	0.48±0.01^abc	0.46±0.01^bc
F值	3.094	1081.438	3023.632	5010.075	4881.937
P值	0.067	< 0.001	< 0.001	< 0.001	< 0.001

表3 不同时间Twist1基因相对表达量情况（ ± s，n = 3）

分组	0 h	4 h	24 h	48 h	72 h
对照	0.08±0.01	0.09±0.00	0.15±0.00	0.28±0.01	0.68±0.01
硫酸吲哚酚	0.07±0.00	0.16±0.00^a	0.20±0.00^a	1.03±0.01^a	0.98±0.01^a
PDF	0.08±0.00	0.17±0.00^a	0.19±0.00^a	1.07±0.01^a	1.00±0.04^a
硫酸吲哚酚+TRI	0.07±0.00	0.08±0.01^bc	0.15±0.00^bc	0.14±0.00^abc	0.14±0.00^abc
PDF+TRI	0.07±0.00	0.08±0.00^bc	0.14±0.00^bc	0.14±0.00^abc	0.14±0.00^abc
F值	2.860	274.909	617.471	12058.420	1886.263
P值	0.081	< 0.001	< 0.001	< 0.001	< 0.001

表3 不同时间Twist1基因相对表达量情况（ ± s，n = 3）

分组	0 h	4 h	24 h	48 h	72 h
对照	0.08±0.01	0.09±0.00	0.15±0.00	0.28±0.01	0.68±0.01
硫酸吲哚酚	0.07±0.00	0.16±0.00^a	0.20±0.00^a	1.03±0.01^a	0.98±0.01^a
PDF	0.08±0.00	0.17±0.00^a	0.19±0.00^a	1.07±0.01^a	1.00±0.04^a
硫酸吲哚酚+TRI	0.07±0.00	0.08±0.01^bc	0.15±0.00^bc	0.14±0.00^abc	0.14±0.00^abc
PDF+TRI	0.07±0.00	0.08±0.00^bc	0.14±0.00^bc	0.14±0.00^abc	0.14±0.00^abc
F值	2.860	274.909	617.471	12058.420	1886.263
P值	0.081	< 0.001	< 0.001	< 0.001	< 0.001

表4 不同时间E-cadherin基因相对表达量情况（ ± s，n = 3）

分组	0 h	4 h	24 h	48 h	72 h
对照	1.02±0.01	2.06±0.02	4.12±0.05	3.34±0.02	1.97±0.03
硫酸吲哚酚	1.04±0.03	1.34±0.02^a	2.31±0.06^a	0.86±0.02^a	0.57±0.02^a
PDF	1.00±0.01	1.45±0.03^ab	2.59±0.06^ab	0.88±0.02^a	0.62±0.03^a
硫酸吲哚酚+TRI	1.10±0.06	1.80±0.03^abc	2.51±0.05^abc	1.69±0.03^abc	0.73±0.01^abc
PDF+TRI	1.06±0.05	1.91±0.03^abc	3.14±0.10^abc	1.80±0.03^abc	0.78±0.02^abc
F值	2.988	422.539	324.526	5799.198	2005.336
P值	0.073	< 0.001	< 0.001	< 0.001	< 0.001

表4 不同时间E-cadherin基因相对表达量情况（ ± s，n = 3）

分组	0 h	4 h	24 h	48 h	72 h
对照	1.02±0.01	2.06±0.02	4.12±0.05	3.34±0.02	1.97±0.03
硫酸吲哚酚	1.04±0.03	1.34±0.02^a	2.31±0.06^a	0.86±0.02^a	0.57±0.02^a
PDF	1.00±0.01	1.45±0.03^ab	2.59±0.06^ab	0.88±0.02^a	0.62±0.03^a
硫酸吲哚酚+TRI	1.10±0.06	1.80±0.03^abc	2.51±0.05^abc	1.69±0.03^abc	0.73±0.01^abc
PDF+TRI	1.06±0.05	1.91±0.03^abc	3.14±0.10^abc	1.80±0.03^abc	0.78±0.02^abc
F值	2.988	422.539	324.526	5799.198	2005.336
P值	0.073	< 0.001	< 0.001	< 0.001	< 0.001

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Indoxyl sulfate induces transdifferentiation of peritoneal mesenchymal cells through TGF-β1

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Indoxyl sulfate induces transdifferentiation of peritoneal mesenchymal cells through TGF-β1