磷脂转运蛋白在烟草诱导RLE-6TN细胞株发生上皮间质转化中的作用

The effect of phospholipid transfer protein on cigarette smoke extract induced epithelial-mesenchymal transition of rat alveolar type Ⅱ cells

目的探讨磷脂转运蛋白（PLTP）在烟草提取物（CSE）诱导大鼠Ⅱ型肺泡上皮细胞株RLE-6TN发生上皮间质转化（EMT）中的作用。方法体外培养RLE-6TN细胞株24h，分为4组，每组3孔，分别加入0％、0，25％、0．5％和1％CSE培养2d，检测E-钙黏蛋白和波形蛋白mRNA表达以及细胞和CSE共培养3d检测PLTP、EMT相关蛋白（E-钙黏蛋白、N-钙黏蛋白和波形蛋白）的表达量。将siRNA．PLTP转染入RLE-6TN中，Westernblot法检测CSE对转染后PLTP和EMT相关蛋白的影响。结果不同浓度的CSE作用后，E-钙黏蛋白mRNA分别为1．01±0．05、0．74±0．05、0．65±0．03、0．30±0．08，呈浓度依赖下降；波形蛋白mRNA在1％CSE组（1．88±0．49）表达与对照组（1．01±0．20）相比增加。PLTP蛋白含量分别为0．42±0．02、0．89±0．25、1．08±0．18、1．61±0．06；E-钙黏蛋白蛋白含量分别为1．61±0．04、1．08±0．10、0．63±0．08、0．68±0．17；N-钙黏蛋白蛋白含量分别为0．60±0．15、0．57±0．26、0．88±0．30、1．94±0．54；波形蛋白蛋白含量分别为0．61±0．05、0．98±0．16、1．07±0．14、1．34±0。19。1％组与对照组比较差异有统计学意义（均P〈0．05）。siRNA．PLTP和CSE处理细胞后，E-钙黏蛋白蛋白表达量较对照组明显增加（P〈0．05），N-钙黏蛋白和波形蛋白蛋白表达量较对照组明显降低（P〈0．05），siRNA—PLTP可部分逆转CSE诱导RLE-6TN细胞发生上皮间质转化。结论PLTP能促进烟草诱导RLE-6TN发生上皮间质转化。

Objective To investigate the effect of phospholipid transfer protein（PLTP） on cigarette smoke extract（CSE） induced epithelial-mesenchymal transition（EMT） in rat alveolar Type Ⅱ cells （RLE- 6TN）. Methods CSE of different concentrations （0% ,0. 25 % ,0. 5 % and 1% ） was co-cultured for 2 or 3 days with RLE-6TN, either pre-treated or not pre-treated with siRNA-PLTP for 6 h. Expression levels of E-cadherin mRNA and Vimentin mRNA were examined by RT-PCR, while expression levels of PLTP, E-cadherin, N-cadherin and Vimentin were examined by Western blot. Results Our results showed that the expression of E-eadherin mRNA decreased in CSE-treated groups： 1.01 ± 0. 05,0. 74 ± 0. 05,0. 65 ±0. 03, 0. 30±0. 08 respectively at different concentrations of CSE （0 % , 0. 25% , 0. 5 % , and 1.0% ） ; while the level of Vimentin mRNA increased significantly in 1% CSE treated cells （ 1.88 ±0.49） , compared with control cells （ 1.01 ±0. 20）. Treatment with CSE at different concentrations （0% ,0. 25% ,0. 5% and 1% ） showed that the protein levels of PLTP were 0.42 ± 0.02,0.89 ±0.25, 1.08 ±0.18, 1.61 ±0.06 respectively; those of E-cadherin were 1.61± 0. 04,1.08 ± 0. 10,0. 62 ± 0.08,0. 68 ± 0. 17, respectively ; those of N-cadherin were 0. 60±0. 14,0. 57 ± 0. 26,0. 88 ± 0. 30,1.94 ± 0. 54, respectively; and those of Vimentin were 0. 61± 0. 05,0. 98 ± 0. 16,1.07 ± 0. 14,1.34±0. 19, respectively; all P 〈 0.05 when the 1% CSE group was compared with the control group. EMT induced by CSE was significantly inhibited by siRNA-PLTP. Conclusion PLTP may be involved in CSE induced EMT of rat alveolar cells.