左炔诺孕酮对人子宫肌瘤细胞体外增殖与凋亡的影响及机理研究
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摘要
子宫肌瘤是女性生殖器官的常见肿瘤,其发生与细胞增殖及细胞凋亡的失衡有关。近年来,随着对子宫肌瘤病因学研究的进展,局部应用孕激素类药物的治疗方法备受关注。左炔诺孕酮宫内缓释系统(LNG-IUS)是当前国际公认性能优良的宫内抗生育系统。国外一项前瞻性自身对照研究发现使用LNG-IUS能使肌瘤患者的子宫肌瘤变小,经量减少,其对子宫肌瘤的治疗作用可能与抑制内膜生长有关。然而LNG-IUS所形成的局部高效孕激素环境,是否还存在其它治疗作用靶点和机制尚不清楚。本研究首先构建纯度高、稳定性好的人子宫平滑肌瘤原代培养细胞,并在此基础上,直接以其为作用靶标,从增殖与凋亡的角度探讨左炔诺孕酮对子宫肌瘤的作用及其相关机制。研究共分三部分:
一、人子宫平滑肌瘤细胞的原代培养与鉴定
采用贴块法和酶消化法均成功获得肌瘤细胞。采用贴块法的第7～10 d始见单个细胞从组织块边缘游出,半月左右细胞长满并可传代;应用酶消化法时,不同肌瘤所需的消化时间稍有差异,消化所得的细胞经台盼蓝染色,活率可达90%。光镜下肌瘤细胞呈梭形,核椭圆形,可呈典型“峰-谷”样生长。原代与第3、9代细胞的倍增时间之间无统计学差异。免疫组化检测α-SMA阳性细胞达95%以上。以上结果表明,贴块法和酶消化法均能获得大量高纯度,功能良好,性状稳定的肌瘤细胞。
二、左炔诺孕酮对人子宫肌瘤细胞体外增殖与凋亡的影响
MTT结果显示,10μg/mL LNG开始抑制肌瘤细胞的生长,随浓度增加及时间延长,抑制作用加强;电镜观察20μg/mL LNG组细胞,胞质空泡化、内质网扩张、线粒体固缩、染色质浓缩边聚;流式细胞术分析,加药组细胞凋亡率随LNG浓度的增加而逐渐升高;AO/EB双染在荧光显微镜下观察,对照组无明显凋亡细胞,10μg/mL LNG组开始出现早期凋亡细胞,15μg/mL、20μg/mL、25μg/mL LNG组晚期凋亡细胞逐渐增多,核浓聚、偏位、被染成桔红色,可见明显的凋亡小体。这说明,10μg/mL LNG开始于体外发挥抑制肌瘤细胞增殖并诱导其凋亡的效应。
三、左炔诺孕酮抑制体外培养人子宫肌瘤细胞增殖及诱导凋亡的机制研究
RT-PCR结果显示,10μg/mL以上LNG作用后,肌瘤细胞中IGF-Ⅰ、Survivin mRNA表达显著下降(P＜0.05,);Western blot证实,10μg/mL和20μg/mL LNG作用后,Survivin蛋白表达量显著下降,p38磷酸化水平升高,Caspase 3被明显激活。由此可以推测,高浓度LNG对子宫肌瘤的治疗机制可能与降调IGF-Ⅰ表达,抑制Survivin抗凋亡活性,并直接激活p38 MAPK信号通路,诱导Caspase 3活化有关。
综上所述,本文以原代培养的人子宫肌瘤细胞为研究对象,通过多种实验手段,证明高浓度LNG能够借助于IGF-Ⅰ表达的下调有效抑制肌瘤细胞增殖,并通过激活p38 MAPK信号通路、活化Caspase3及抑制Survivin、IGF-Ⅰ表达而诱导肌瘤细胞的凋亡。
Uterine leiomyoma is benign smooth muscle cell tumor of the myometrium,occurring in as many as 30%adult women.Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death;too much growth can come from too little death as well as from too much proliferation.Actually,apoptosis is known to occur in tumors either spontaneously or in response to treatment.Recently,it has been found that the use of LNG-IUS is effective in reduction of uterine myoma volumes.To explore the mechanisms of inhibitory effect of high level levonorgestrel on uterine leiomyomas growth,our researches obtained primarily cultured uterine leiomyoma cells,and then observed the effects of levonorgestrel on proliferation and apoptosis in it.
一、Primary culture and identification of human uterine leiomyoma cells
By explant culture,cells could be seen emigrating from the edge of tissue pieces after 7-10 days,and subcultured after about half of a month;Whereas cells obtained by digestive method having an initial viability of 90%by dye exclusion.Cells morphologically appeared spindle-shaped with a big oviform nuclei and grew in the "hill-valley" mode.There weren't significant differences in doubling time of cells at the first,third and ninth passages.Immunohistochemical study revealed strong expression ofα-SMA,showing that the purity of primary cells was above 95%.In conclusion,We have successfully developed a good in vitro model of primary leiomyoma cells with high purity and stability either by explant or digestive method.
二、Effects of levonorgestrel on proliferation and apoptosis in human uterine leiomyoma cells in vitro
MTT assay demonstrated that the inhibitory effect of levonorgestrel on UtLMCs growth developed when the concentration was up to 10μg/mL and was in a dose and time dependent manner.The resultes of FCM analysis showed that levonorgestrel treatment could induce a substantial apoptotic response in UtLMCs,which was enhanced with the raise of levonorgestrel.The earlier apoptotic changes,such as endoplasmic vacuolization and condensed chromatin,were observed under TEM in LNG treated cells.AO/EB double staining disclosed that there were more viable apoptotic cells in LNG treated groups than control group and the number of non-viable apoptotic cells and dead cells increased with dose increasing.Thus,certain concentration of levonorgestrel resulted in a decrease in proliferation and an increase in apoptosis in UtLMCs.
三、The mechanism of anti-proliferative and apoptotic effects of levonorgestrel on human uterine leiomyoma cells in vitro
Survivin expression in LNG treated cells was decreased both at mRNA and protein levels according to RT-PCR and Western blot results. It had been observed by RT-PCR that the IGF-Ⅰexpression decreased with LNG addition.Western blot analyses showed that phosphorylated p38 and Caspase 3 expressions were up-regulated by 10μg/mL and 20 μg/mL LNG treatment.So,the molecular mechanism by which levonorgestrel participate in the inhibition of human uterine leiomyoma growth might be due to the marked down-regulation of IGF-Ⅰand Survivin,as well as the direct activation of p38 MAPK and Caspase 3.
In summary,based on the model of primary cultured human uterine leiomyoma cells,we demonstrated that LNG could efficiently inhibit the proliferation of leiomyoma cells and induce cell apoptosis,either by down-regulating IGF-Ⅰ,Survivin expressions,or activating p38 MAPK and Caspase 3.
一、人子宫平滑肌瘤细胞的原代培养与鉴定
采用贴块法和酶消化法均成功获得肌瘤细胞。采用贴块法的第7～10 d始见单个细胞从组织块边缘游出,半月左右细胞长满并可传代;应用酶消化法时,不同肌瘤所需的消化时间稍有差异,消化所得的细胞经台盼蓝染色,活率可达90%。光镜下肌瘤细胞呈梭形,核椭圆形,可呈典型“峰-谷”样生长。原代与第3、9代细胞的倍增时间之间无统计学差异。免疫组化检测α-SMA阳性细胞达95%以上。以上结果表明,贴块法和酶消化法均能获得大量高纯度,功能良好,性状稳定的肌瘤细胞。
二、左炔诺孕酮对人子宫肌瘤细胞体外增殖与凋亡的影响
MTT结果显示,10μg/mL LNG开始抑制肌瘤细胞的生长,随浓度增加及时间延长,抑制作用加强;电镜观察20μg/mL LNG组细胞,胞质空泡化、内质网扩张、线粒体固缩、染色质浓缩边聚;流式细胞术分析,加药组细胞凋亡率随LNG浓度的增加而逐渐升高;AO/EB双染在荧光显微镜下观察,对照组无明显凋亡细胞,10μg/mL LNG组开始出现早期凋亡细胞,15μg/mL、20μg/mL、25μg/mL LNG组晚期凋亡细胞逐渐增多,核浓聚、偏位、被染成桔红色,可见明显的凋亡小体。这说明,10μg/mL LNG开始于体外发挥抑制肌瘤细胞增殖并诱导其凋亡的效应。
三、左炔诺孕酮抑制体外培养人子宫肌瘤细胞增殖及诱导凋亡的机制研究
RT-PCR结果显示,10μg/mL以上LNG作用后,肌瘤细胞中IGF-Ⅰ、Survivin mRNA表达显著下降(P＜0.05,);Western blot证实,10μg/mL和20μg/mL LNG作用后,Survivin蛋白表达量显著下降,p38磷酸化水平升高,Caspase 3被明显激活。由此可以推测,高浓度LNG对子宫肌瘤的治疗机制可能与降调IGF-Ⅰ表达,抑制Survivin抗凋亡活性,并直接激活p38 MAPK信号通路,诱导Caspase 3活化有关。
综上所述,本文以原代培养的人子宫肌瘤细胞为研究对象,通过多种实验手段,证明高浓度LNG能够借助于IGF-Ⅰ表达的下调有效抑制肌瘤细胞增殖,并通过激活p38 MAPK信号通路、活化Caspase3及抑制Survivin、IGF-Ⅰ表达而诱导肌瘤细胞的凋亡。
Uterine leiomyoma is benign smooth muscle cell tumor of the myometrium,occurring in as many as 30%adult women.Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death;too much growth can come from too little death as well as from too much proliferation.Actually,apoptosis is known to occur in tumors either spontaneously or in response to treatment.Recently,it has been found that the use of LNG-IUS is effective in reduction of uterine myoma volumes.To explore the mechanisms of inhibitory effect of high level levonorgestrel on uterine leiomyomas growth,our researches obtained primarily cultured uterine leiomyoma cells,and then observed the effects of levonorgestrel on proliferation and apoptosis in it.
一、Primary culture and identification of human uterine leiomyoma cells
By explant culture,cells could be seen emigrating from the edge of tissue pieces after 7-10 days,and subcultured after about half of a month;Whereas cells obtained by digestive method having an initial viability of 90%by dye exclusion.Cells morphologically appeared spindle-shaped with a big oviform nuclei and grew in the "hill-valley" mode.There weren't significant differences in doubling time of cells at the first,third and ninth passages.Immunohistochemical study revealed strong expression ofα-SMA,showing that the purity of primary cells was above 95%.In conclusion,We have successfully developed a good in vitro model of primary leiomyoma cells with high purity and stability either by explant or digestive method.
二、Effects of levonorgestrel on proliferation and apoptosis in human uterine leiomyoma cells in vitro
MTT assay demonstrated that the inhibitory effect of levonorgestrel on UtLMCs growth developed when the concentration was up to 10μg/mL and was in a dose and time dependent manner.The resultes of FCM analysis showed that levonorgestrel treatment could induce a substantial apoptotic response in UtLMCs,which was enhanced with the raise of levonorgestrel.The earlier apoptotic changes,such as endoplasmic vacuolization and condensed chromatin,were observed under TEM in LNG treated cells.AO/EB double staining disclosed that there were more viable apoptotic cells in LNG treated groups than control group and the number of non-viable apoptotic cells and dead cells increased with dose increasing.Thus,certain concentration of levonorgestrel resulted in a decrease in proliferation and an increase in apoptosis in UtLMCs.
三、The mechanism of anti-proliferative and apoptotic effects of levonorgestrel on human uterine leiomyoma cells in vitro
Survivin expression in LNG treated cells was decreased both at mRNA and protein levels according to RT-PCR and Western blot results. It had been observed by RT-PCR that the IGF-Ⅰexpression decreased with LNG addition.Western blot analyses showed that phosphorylated p38 and Caspase 3 expressions were up-regulated by 10μg/mL and 20 μg/mL LNG treatment.So,the molecular mechanism by which levonorgestrel participate in the inhibition of human uterine leiomyoma growth might be due to the marked down-regulation of IGF-Ⅰand Survivin,as well as the direct activation of p38 MAPK and Caspase 3.
In summary,based on the model of primary cultured human uterine leiomyoma cells,we demonstrated that LNG could efficiently inhibit the proliferation of leiomyoma cells and induce cell apoptosis,either by down-regulating IGF-Ⅰ,Survivin expressions,or activating p38 MAPK and Caspase 3.
引文
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