Indoxyl Sulfate诱导腹膜间皮细胞转分化的实验研究

doi:10.3877/cma.j.issn.2095-1221.2017.03.006

目的

硫酸吲哚酚（IS）刺激永生化人腹膜间皮细胞株（HMrSV5）后，研究细胞是否发生上皮细胞-间充质转分化（EMT），同时探索转化生长因子β1（TGF-β1）可能在其中的作用。

方法

体外培养HMrSV5，传代后，随机将人腹膜间皮细胞株分成以3组：对照组：加入4.25﹪腹膜透析液与培养液共培养；IS处理组：分别用含250μmol/LIS、500μmol/LIS、1 000μmol/LIS浓度的培养液与4.25﹪腹膜透析液共培养；TGF-β1受体抑制剂组：用含2μmol/LLY364947+IS的培养液与4.25﹪腹膜透析液共培养。分别在培养0、4、12、24、48和72 h时间下，采用细胞免疫荧光法观察细胞形态变化，Western Blot法研究细胞α平滑肌肌动蛋白（α-SMA）的表达规律，48 h时用ELISA法测定纤维连接蛋白（FN），层粘连蛋白（LN5）两种基质蛋白的分泌情况。

结果

（1）免疫荧光法显示：与正常细胞比较，IS处理组细胞由典型的铺路样鹅卵石状，逐渐变细变长，呈梭形改变；而TGF-β1受体抑制剂组细胞形态变化不明显，不如IS处理组；（2）Western Blot法检测结果显示：与对照组比较，IS处理组α-SMA的表达水平明显增加，48 h时1 000μmol/LIS表达最明显（39.929±2.610vs0.996±0.001；P < 0.01），差异有统计学意义；与IS处理组比较，TGF-β1受体抑制剂组的表达则明显减少，其中48 h时抑制明显（7.418±3.075vs39.929±2.610，P < 0.01），差异有统计学意义；（3）ELISA法结果显示：与对照组比较，IS处理组基质蛋白FN（1.368±0.040vs1.094±0.036，P < 0.01）、LN5（1.031±0.085vs0.860±0.063，P < 0.01）的表达均上调，差异有统计学意义；与IS处理组比较，TGF-β1受体抑制剂组FN（0.925±0.15vs1.368±0.040，P < 0.01）、LN5（0.795±0.426vs1.031±0.085，P < 0.01）分泌均减少，差异有统计学意义。

结论

IS可诱导腹膜间皮细胞发生EMT，TGF-β1的参与可能起重要作用，这为治疗腹膜纤维化可能提供新的靶点。

关键词: 腹膜间皮细胞, 硫酸吲哚酚, 细胞上皮-间充皮转分化, 转化生长因子β

Objective

To induce epithelial mesenchymal transition (EMT) of immortalized human peritoneal mesothelial cell line (HMrSV5) with indoxyl sulfate (IS) and investigate the role of transforming growth factor (TGF-β1) .

Methods

HMrSV5 cells were randomly divided into 3 groups: the control group: with 4.25﹪peritoneal dialysate fluid in culture medium; IS groups: with IS at final concentrations oft 250μmol/L, 500μmol/L, and 1 000μmol/L and 4.25﹪peritoneal dialysate fluid; TGF-β1 inhibitor group: with 2μmol/L LY364947, IS and 4.25﹪peritoneal dialysate fluid. At 0 h, 4 h, 12 h, 24 h, 48 h, and 72 h, the morphological changes were observed by immunofluorescence method, and alpha smooth muscle actin (α-SMA) expression was evaluated with Western Blot. At 48 h, supernatant was collected to determinate fibronectin and laminin5 by ELISA.

Results

After IS induction, cells change from typical cobblestone-like shape to long fusiform shape, which was prevented but TGF-β1 inhibitor. Western Blot showed that: compared with control group the expression level ofα-SMA increased significantly in the IS treatment group. The level ofα-SMA was highest at 48 h with 1 000μmol/L IS (39.929±2.610vs0.996±0.001;P< 0.05) , which was decrease to 7.418±3.075 in the presence of TGF-β1 inhibitor (P< 0.05) .Compared with control group, IS increased the secretion of FN (1.368±0.040vs1.094±0.036, P< 0.05) and laminin 5 (1.031±0.085vs0.860±0.063,P< 0.05) , which were decreased by TGF-βinhibitor (0.925±0.15 and 0.795±0.426 respectively,P< 0.05) .

Conclusion

EMT of PMC could be induced by IS via TGF-β1 signaling pathway and is potential target for the treatment of peritoneal fibrosis.

Key words: Peritoneum, Stromal cells, Sulfuric acids, Indophenol, Epithelial-mesenchymal-transition, Transforming growth factor beta

图1 倒置显微镜下观察正常腹膜间皮细胞株形态（×100）

图2 共聚焦显微镜下观察IS处理组HMrSV5细胞发形态（IF染色，×400）

图3 共聚焦显微镜下观察TGF-β1受体抑制剂组HMrSV5细胞形态（IF染色，×400）

图4 不同时间各组刺激细胞表达α-SMA蛋白的条带变化

表1 对照组、IS处理组及TGF-β1受体抑制剂组培养不同时间后灰度值比较（ ± s）

分组	0 h	4 h	12 h	24 h	48 h	72 h
对照组	1.012±0.001	1.115±0.116	1.066±0.114	1.034±0.066	0.996±0.001	1.115±0.116
250 μmol/LIS	1.035±0.305	2.358±0.310^a	2.725±0.310^a	2.151±0.355	0.873±0.419	25.025±2.332^a
500 μmol/LIS	1.245±0.202	3.491±0.328^a	4.079±0.189^a	4.744±0.199^a	1.831±0.505	36.491±2.227^a
1 000 μmol/LIS	1.176±0.316	2.518±0.180^a	5.317±0.275^a	35.707±2.008^a	39.929±2.610^a	25.518±1.173^a
TGF-β1受体抑制剂组	0.821±0.265	1.871±0.535^b	4.305±0.240^b	14.738±2.364^b	7.418±3.075^b	1.871±0.535^b
F值	1.331	21.377	145.845	321.323	254.515	308.506
P值	0.324	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

表1 对照组、IS处理组及TGF-β1受体抑制剂组培养不同时间后灰度值比较（ ± s）

分组	0 h	4 h	12 h	24 h	48 h	72 h
对照组	1.012±0.001	1.115±0.116	1.066±0.114	1.034±0.066	0.996±0.001	1.115±0.116
250 μmol/LIS	1.035±0.305	2.358±0.310^a	2.725±0.310^a	2.151±0.355	0.873±0.419	25.025±2.332^a
500 μmol/LIS	1.245±0.202	3.491±0.328^a	4.079±0.189^a	4.744±0.199^a	1.831±0.505	36.491±2.227^a
1 000 μmol/LIS	1.176±0.316	2.518±0.180^a	5.317±0.275^a	35.707±2.008^a	39.929±2.610^a	25.518±1.173^a
TGF-β1受体抑制剂组	0.821±0.265	1.871±0.535^b	4.305±0.240^b	14.738±2.364^b	7.418±3.075^b	1.871±0.535^b
F值	1.331	21.377	145.845	321.323	254.515	308.506
P值	0.324	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

表2 对照组、IS处理组及TGF-β1受体抑制剂组培养48 h后LN5、FN浓度（μg /L）比较（ ± s）

分组	FN	LN-5
对照组	1.094±0.036	0.860±0.063
250 μmol/LIS	1.077±0.038	0.829±0.085
500 μmol/LIS	1.225±0.035	1.031±0.085^a
1 000 μmol/LIS	1.368±0.040^a	0.860±0.069
TGF-β1受体抑制剂组	0.925±0.154^c	0.795±0.426^b
F值	14.053	7.146
P值	0.000	0.003

表2 对照组、IS处理组及TGF-β1受体抑制剂组培养48 h后LN5、FN浓度（μg /L）比较（ ± s）

分组	FN	LN-5
对照组	1.094±0.036	0.860±0.063
250 μmol/LIS	1.077±0.038	0.829±0.085
500 μmol/LIS	1.225±0.035	1.031±0.085^a
1 000 μmol/LIS	1.368±0.040^a	0.860±0.069
TGF-β1受体抑制剂组	0.925±0.154^c	0.795±0.426^b
F值	14.053	7.146
P值	0.000	0.003

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Experimental study on epithelial-mesenchymal-transition of peritoneal mesothelial cells induced by indoxyl sulfate

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Experimental study on epithelial-mesenchymal-transition of peritoneal mesothelial cells induced by indoxyl sulfate