反义I型内皮素受体抑制人子宫肌瘤细胞增殖

Antisense endothelin A receptor oligonucleotide inhibits human uterine leiomyoma cell proliferation

目的 应用反义技术研究 I型内皮素受体反义寡核苷酸 (ETA- ODN)对人类子宫肌瘤增殖的抑制作用 .方法 取人子宫肌瘤细胞培养传代 ,经鉴定后加入不同浓度的人工合成的 ETA- ODN片段 ,观察反义片段的膜透性及细胞生长计数和细胞增殖情况 ,并应用放射免疫方法检测 ETA蛋白合成 .结果 1培养后 12 h即可见地高辛标记的反义片段进入细胞并逐渐增多 ;2 15μmol· L- 1 浓度的 ETA- ODN对子宫肌瘤细胞生长有明显的抑制作用 (P<0 .0 5 ) ,细胞生长计数有明显的相关性 ;315 μmol· L- 1浓度的 ETA- ODN对 ETA蛋白合成亦有明显抑制作用 .结论 ETA- ODN以浓度和时间依赖性方式抑制子宫肌瘤细胞增殖以及 ETA蛋白合成 ;ETA介导子宫肌瘤平滑肌细胞生长增殖过程 ;ODN通过反义机制实现对

AIM To evaluate the effect of antisense endothelin A receptor oligonucleotides (ETA ODN) on human uterine leiomyoma cell growth. METHODS ① SMC culture: The human uterine leiomyomas cell were harvested, digested and cultured. The cells of passage 4-6 were routinely used for study after the identification with α actin antibody. ② ODN synthesis: The antisense sequences were designed and screened by genebank scanning. After these procedures, eighteen phosphorothioate antisenses complementary to ETA cDNA were synthesized, and eventually labeled with digoxin, by which the sequence stability augmented and membranous permeability was tested. ③ MTT examination: MTT, as efficient as 3HTdR in evaluating DNA synthesis, was used in ODN treated SMC. ④ 125 I ET 1 radio banding assay: 125 I ET 1 radio banding assay to ETA and immunohistochemistry were utilized to explore protein expression of the receptors and to analyze the roles of ODNs. RESULTS ① ODN could be found in cultured cells 12 h after culture and gradually increased. ② ETA ODN with the concentration of 15 μmol·L -1 suppressed uterine leiomyoma cell proliferation, indicating the inhibitory action peaked 24 h after culture, whereas no inhibitory action was found in sense. ③ ETA protein expression was reduced, showing that CPM progressively decreased 24 h after culture. CONCLUSION ETA ODN suppresses ETA expression and uterine leiomyoma cell proliferation in a time and concentration dependent manner. ETA plays an important role in mediating cell proliferation. ODN exerts its inhibitory function through antisense mechanism.