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

Effect of TGFβ1 antisense oligodeoxynucleotides (ASON) proliferation and fibronectin expression on human peritoneal mesothelial cell

目的探讨阳离子脂质体介导的TGFβ1反义寡核苷酸(antisenseoligonucleotides,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);②TGFβ1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。

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 lipofectAMINE 2 000 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);③ FN mRNA expression in antisense group was downregulated compared with other groups. Conclusion TGF-β1 ASON can inhibit; HPMC proliferation and overexpression of FN