中国血液净化

›› 2005, Vol. 4 ›› Issue (6): 317-321.

• 基础研究 • 上一篇    下一篇

TGFβ1反义寡核苷酸对人腹膜间皮细胞增殖和纤维连接蛋白的影响

刘映红 刘伏友 彭佑铭 袁 芳 段绍斌 刘 虹   

  1. 410011 长沙， 中南大学肾病研究所 中南大学湘雅二院肾内科 
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2005-06-12 发布日期:2005-06-12
  • 通讯作者: 刘伏友

  • Received:1900-01-01 Revised:1900-01-01 Online:2005-06-12 Published:2005-06-12

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摘要：

目的 探讨阳离子脂质体介导的TGFβ1反义寡核苷酸 （antisense oligonucleotides，ASON）对人腹膜间 皮细胞(HPMC)体外增殖及其分泌纤维连接蛋白（FN）的影响，初步探索腹膜间皮细胞对腹膜纤维化影响和TGFβ1反义寡核苷酸对其的干预作用，为临床防治腹膜纤维化寻找有效途径。方法 ①人腹膜间皮细胞原代培养，第三代用于实验；②合成特异性针对TGFβ1mRNA转译起始区ASON及正义、错义寡核苷酸，并和阳离子脂质体形成复合物；③将合成的寡核苷酸与第三代腹膜间皮细胞共同孵育24、48h，分为对照组（F12培养基组）、TGFβ1反义寡核苷酸组、TGFβ1正义寡核苷酸组和TGFβ1错义寡核苷酸组，采用MMT法观察细胞增殖，ELISA法测定细胞上清液内FN蛋白含量以及RT－PCR测定FNmRNA表达。结果 ①TGFβ1反义寡核苷酸作用人腹膜间皮细胞24、48h后，其细胞增殖率明显低于其它各组（ P均<0.05）；② TGFβ1反义寡核苷酸作用人腹膜间皮细胞24、48h后其上清液中FN蛋白质的含量明显降低；TGFβ1正义寡核苷酸组明显升高（ P<0.05）；③正义、反义和错义各组寡核苷酸作用人腹膜间皮细胞24h时 FNmRNA表达与对照组比较，正义组上调22％，而反义组和错义组分别下调22％和11％。结论 TGFβ1反义寡核苷酸能够抑制人腹膜间皮细胞增殖和FN的过度表达。

关键词: TGFβ1反义寡核苷酸, 人腹膜间皮细胞, 细胞增殖, 纤维连接蛋白

Abstract:

Objective To investigate whether the TGFβ1 antisense oligonucleotides(ASON) inhibit proliferation of human peritoneal mesothelial cell and expression of fibronectin (FN). Methods ①Isolated human peritoneal mesothelial cells (HPMC) were cultured,the tertiary cultured HPMC were treated with F12 without serum (control group)，F12 with TGF-beta1 sense oligodeoxynucleotides（sense group）, F12 with TGF-beta1 antisense oligodeoxynucleotides（antisense group）, F12 with TGF-beta1missense oligonucleotides（missense group）, ② synthesized phosphorothioate antisense oligonucleotides and their sense or missense oligonucleotides, and then treated HPMC with these oligonucleotides, cationic liposorne lipofect AMINE 2000 complexes at different time. The protein expression of FN was measured by ELISA methods, mRNA of FN was measured by RT-PCR methods. Results ①the TGF-β1 ASON could inhibit the HPMC proliferation at 24h or 48 h; ②FN protein expression was lower in antisense group than other groups（P<0.05）；③FN mRNA expression in antisense group was downregulated compared with other groups. Conclusion TGF-β1 ASON can inhibit；HPMC proliferation and overexpression of FN

Key words: Human peritoneal mesothelial cells, Proliferation, Fibronectin

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