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过氧化物酶体增殖物激活受体激动剂罗格列酮对人子宫肌瘤细胞的影响
【作者】 秦忠芳；
【导师】 李美蓉；
【作者基本信息】 山西医科大学 ， 妇产科学， 2010， 硕士
【摘要】 目的：通过不同浓度过氧化物酶体增殖物激活受体激动剂罗格列酮作用于人体外子宫肌瘤细胞后,观察对人子宫肌瘤细胞的增值抑制及对过氧化物酶体增殖物激活受体(PPARy)和凋亡基因Bax、Bcl-2表达的影响,来探讨子宫肌瘤可能的发生机制及能否抑制子宫肌瘤的生长,并提示临床应用PPARγ激动剂治疗子宫肌瘤的潜在可能。方法：应用胶原酶消化法分离、获得子宫肌瘤细胞,用鼠抗人平滑肌肌动蛋白(a-actin)进行免疫细胞化学法鉴定。给予原代培养的子宫肌瘤不同浓度的罗格列酮处理,以四甲基偶氮唑盐微量酶反应比色(MTT)法测定细胞生长抑制率；以半定量逆转录聚合酶链反应(RT-PCR)法检测PPARymRNA以及凋亡基因Bax、Bcl-2在子宫肌瘤细胞中的表达及罗格列酮对子宫肌瘤细胞增殖活化的影响。结果：(1)应用消化法获得了大量原代子宫肌瘤细胞,并通过免疫化学细胞证实。(2)MTT法结果显示：不同浓度罗格列酮作用24h,对子宫肌瘤细胞的增值无明显抑制,差异无统计学意义(P>0.05)；不同剂量的罗格列酮作用48h、72h后对各实验组子宫肌瘤细胞的增值有明显的抑制,与相同条件下作用24h的子宫肌瘤细胞的增值抑制率相比具有明显的差异(P<0.05)；而且各组之间细胞增殖抑制率差异有统计学意义(P<0.05)。5×10-8mol／L罗格列酮作用子宫肌瘤各个时间点对子宫肌瘤的增值无明显抑制,差别无统计学意义P>0.05);而相同剂量(5×10-6,5×10-7mol／L)的罗格列酮作用子宫肌瘤,随作用时间的延长对各实验组子宫肌瘤细胞的增值有明显的抑制(P<0.05)；而且各组之间细胞增殖抑制率差异有统计学意义(P<0.05)。这种抑制作用存在剂量和时间依赖性。(3)半定量RT-PCR结果显示：罗格列酮(5×10-6、5×10-7、5×10-8mol／L)作用于子宫肌瘤细胞72h后,可明显升高加药组细胞PPARγ, Bax的表达水平,下调Bcl-2的表达水平,呈剂量依赖性。表现出明显活化抑制。结论：(1)罗格列酮可显著抑制体外培养的人子宫肌瘤细胞增殖,随着药物剂量的增加和时间的延长,子宫肌瘤细胞的增殖和活化明显抑制,呈剂量和时间依赖性。(2)罗格列酮通过激活PPARy抑制子宫肌瘤细胞的增值可能是通过上调Bax,下调Bcl-2的表达实现的。(3)罗格列酮用于子宫肌瘤的治疗可能具有潜在价值。
【Abstract】 Objective:To explore the leiomyoma cells growth inhibition induced by a peroxisome proliferator activated receptor-Y activator rosiglitazone and its molecular mechanism.and to explore the expression of PPARγ、Bax、Bcl-2 mRNA, and the suppressive effect of rosilitazone on the proliferation and apoptosis of uterine leiomyoma cells in vitro. Our purpose was to explore the possible mechanism of uterine leiomyoma, and the possible to inhibit the growth of uterine leiomyoma, and prompt clinical application of PPAR Y agonist treatment of uterine leiomyma potential.Methods:Uterine leiomyoma cells were cultered in vitro,after the action of rosigliatzone with different concentration,MTT was used to examne the inhibition activity of uterine leiomyoma cells.RT-PCR was used to determine the expression of the PPARγand of the apoptosi genes Bax、Bcl-2 in uterine leiomyoma.Result:(1)The uterine leiomyma cells were got by using collagenase dissection and identified by immunocytochemistry.(2)MTT method showed that:The treatment after2、48、72h(P< 0.05) resulted in an inhibition of the cell growths with (5 X 10-6、5×10-7 mol/L) rosiglitazone(P< 0.05),and there were no significant difference between eath other with 5 X 10-8mol/Lrosiglitazone (P> 0.05). The treatment with rosiglitazone(5 X 10-6、5 X 10-7、5 X 10-8mol/L) resulted in an inhibition of the cell growths after 72、48 house (P< 0.05),and there were no significant difference between each other in the treatment of 24h(P> 0.05).(3)Semi-quantitative RT-PCR results showed that:Rosiglitazone(5 X 10-6、5 X 10-7、5 X 10-8mol/L) affect uterine leiomyma cells 72h,will increase the expression of PPARγ,Bax,reduce the expression of Bcl-2,and its role is adose dependent manner., showing apparent activation inhibition.Conclusion:(1)Rosiglitazone significantly inhibited the in vitro cultured human uterine leiomyoma cell proliferation, with the increase in drug dose and the extension of the time, uterine leiomyoma cell proliferation and activation are inhibited, show a dose-and time-dependent.(2) Rosiglitazone can suppress the cell proliferation partly through the regulations of PPAR-γ、Bax and Bcl-2 expressions. The cross-talk between the signal pathways of PPAR-y and Bax、Bcl-2 may be involved in the process. (3)PPARγligands may be of potential use for uterine leiomyoma treantment.