茶多酚对前列腺癌PC-3M系细胞作用的实验研究
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摘要
前列腺癌是男性中仅次于肺癌、直接导致死亡的第二大癌症,而在中国、日本等长期具有饮茶习惯的亚洲国家,前列腺癌的发病率要远远低于欧美等西方国家,这是否与这两区域不同的饮茶习惯有关呢?
另外,前列腺癌组织包含两种癌细胞类型:激素依赖性和激素非依赖性,这导致中晚期前列腺癌治疗的短期效果与中长期效果差距甚大。
茶叶中的主要成分:茶多酚(TPs)具有多种生物活性,因此尝试用TPs来抗治前列腺癌便在情理之中。
本实验选用人雄激素非依赖性前列腺癌PC-3M细胞,设立对照组与实验组,倒置显微镜观察TPs处理后细胞数量与形态变化;采用MTT比色法检测TPs对雄激素非依赖性前列腺肿瘤PC-3M细胞增殖的影响;吖啶橙/溴化乙锭细胞染色法观察诱导细胞凋亡情况;流式细胞术分析细胞周期;划痕实验观察细胞迁移力;细胞侵袭实验观察细胞侵袭能力;RT-PCR和Western blot检测cyclin D1、Caspase-3、survivin、MMP-2、TIMP-2基因的转录与表达。
结果表明,与对照组比较,加药组细胞增殖速度减慢,细胞状态变差。在镜下表现为数量减少,且细胞细瘦、皱缩,局部可见细丝状“伪足”形成,细胞透光性减弱,细胞质内灰黑色颗粒状物增多。MTT法示实验组对PC-3M细胞的增殖抑制作用增强;细胞染色法示实验组凋亡率增高;流式细胞术显示细胞阻滞于G1期,增殖指数降低;划痕实验示茶多酚使PC-3M细胞迁移力下降;细胞侵袭实验示PC-3M细胞侵袭力减弱;RT-PCR与Western blot示Caspase-3、TIMP-2基因转录与翻译上调,而cyclin D1、survivin、MMP-2基因转录与翻译下调,且上述结果在一定范围内呈时间、剂量依赖性。
由此我们得出结论:茶多酚对人雄激素非依赖性前列腺癌PC-3M细胞具有阻滞细胞周期、抑制增殖、促进凋亡,减弱PC-3M细胞侵袭力的作用,上述作用与上调Caspase-3、TIMP-2基因,下调cyclin D1、survivin、MMP-2基因的转录与翻译有关。
Objective:
Tea,as a traditional health drink in China, has lasted for over three thousand years,and it is one of the three world's favourest beverages for it's speciality on health care.Today it's still popular.It has became more and more scientists' concerns that the habit of drinking tea regularly has advantages of keeping human health.Prostate cancer is the second largest cancer that result of the death,only second to lung cancer in men in western countries.The prostate tumor incidence in China,Japan and other asiatic countries in which peoples have the long-term habits of drinking tea is far lower than the west.Whether this difference is relative with the habits of drinking tea in above-mentioned zones?Many findings of experiments in vivo and in vitro have indicated that the main ingredient of tea:tea polyphenols(TPs) is anticarcinogenic,so it is reasonable that more and more investigators try to cure the tumor with the TPs.Our experiment also try to investigate the regulatory effect of tea polyphenol on proliferation inhibition apoptosis and invasion force of human androgen-independent prostate cancer PC-3M cells.
Methods:
The androgen-independent prostate cancer cells PC-3M is cultured in Iscove's Modified Dulbecco's Medium(IMDM) containing 10% of fetal bovine serum,and is incubatored in incubation at 37℃, 5% CO2 and constant humidity.
When cell fusion reachs about 80%,intervention treatment is conducted.The PC-3M cells are divided into treatment group and control group.
At 24 hour and 48 hour after intervention,we observe the change about cell number,morphology and state by phase contrast microscope.Final concentrations of TPs are 0,20,40,60,80,100ug/ml.
The inhibitions of cell proliferation at 24 hour,48 hour and 72 hour after intervention are detected by MTT assay.Final concentrations of TPs are same with above-mentioned.
The change of cell cycle phase at 24 hour after intervention is analyzed by flow cytometry.Final concentrations of TPs are 0,40,60,80ug/ml.
The changes of the apoptosis at 24 hour and 48 hour after intervention is inspected by acridine orange/ethidium bromide(AO/EB) fluorescent staining method.Final concentrations of TPs are also 0,40,60,80ug/ml.
By scratche assay,we observe the change of cell migration at 0h,4h,8h,12h and 24h after intervention with different concentrations of TPs.Final concentrations of TPs are also 0,40,60,80ug/ml.
We conduct the transwell assay.We know the change of cell invision force when PC-3M cell is intervented for 24h and 48h with different concentrations of TPs.Final concentrations of TPs are 0,40,60,80ug/ml.
Besides,the transcription and translation of the genes of cyclin D1,caspase-3, survivin,MMP-2,TIMP-2 in carcinoma cells are observed by RT-PCR and western blot.Final concentrations of TPs are 0,40,60,80ug/ml.
Data shown here are from representative experiments repeated at least three times with similar results.The data are shown asχ±s.With SPSS 17.0 statistical software,the data are analysised as t-test for independent samples and analysis of variance for the single factor and multiple sets(a=0.05).
Results:
After being treated with tea polyphenols solution,the number,morphology and state of human androgen-independent prostate cancer PC-3M cell are all changed appropriately.Compared with control group,dosing groups cell proliferation is slower,and cell is weak.Under the microscope,the cell number decreases and cell is slender and collapse.In some local visual fields,fine filamentous "pseudopodia" is visiable.Cell transmittance is weakened,and grey-black particles increase in cytoplasm.
The results of MTT assay about cell inhibition incidence are shown in Talbe 1.1,from which we can see that as TPs’s concentrations being elevated and time being extended,proliferation inhibitory effect of TPs on the PC-3M cell has gradually enhanced.
AO/EB staining reflects the apoptosis rate after PC-3M cell is treated with TPs at different concentrations as shown in Talbe 1.2,from which we can see that partial cells present the morphological change of apoptosis under the fluorescent microscope, and the apoptotic rates of the treatment groups increase respectively.
Flow cytometry reflects cell cycle changes after prostate cancer PC-3M cell is treated with TPs for 24h,as shown in Talbe 1.3,from which we can see that the cells in S and G2/M phase increase, the cells in G0/G1 phase decrease.It demonstrates that the cell cycle of experimental group is blocked in the G1 phase,and proliferation index decrease.
As shown in the scratch assay,as TPs concentration increasing, the lateral transfer distances of PC-3M cells decrease.And there is also change about cell morphology.Especially when the TPs concentrations is up to 80ug/ml,the cell on the edge of scratch creases and become round.It indicates that the TPs can attenuate the ablity of migration of prostate cancer PC-3M cell.
It is shown in the photo of transwell assay and Talbe 1.4 that PC-3M cells which penetrated through the matrix and migrated to under-surface decrease after treatment with TPs.It explains that PC-3M cell invasion forces weaken.
In part 1 of experiment,RT-PCR and Western blot result shows the transcription and translation of caspase-3 gene is up-rugulated,and the transcription and translation of cyclin D1 and survivin gene are down-rugulated.The above results in a certain range are time-dependent and dose-dependent,which to a certain extent explain and validate the phenomenons that TPs can cause prostate cancer PC-3M cell cycle arrest,proliferation depreciation and promotion of cell apoptosis.
In part 2 of experiment,RT-PCR and Western blot result shows the transcription and translation of MMP-2 genes is down-rugulated,and the transcription and translation of TIMP-2 gene is up-rugulated.The above results in a certain range is time-dependent and(or) dose-dependent,which to a certain extent explain and validate the phenomenons that TPs can weaken prostate cancer PC-3M invasion.
Conclusion:
TPs can inhibit the proliferation of human androgen-independent prostate cancer PC-3M cell,induce the apoptosis and weaken PC-3M cell migration and invasion force.The transcription and translation of up-regulation of caspase-3 and TIMP-2 gene,and of down-regulation of cyclin D1,survivin and MMP-2 gene may one of its molecular mechanisms.
另外,前列腺癌组织包含两种癌细胞类型:激素依赖性和激素非依赖性,这导致中晚期前列腺癌治疗的短期效果与中长期效果差距甚大。
茶叶中的主要成分:茶多酚(TPs)具有多种生物活性,因此尝试用TPs来抗治前列腺癌便在情理之中。
本实验选用人雄激素非依赖性前列腺癌PC-3M细胞,设立对照组与实验组,倒置显微镜观察TPs处理后细胞数量与形态变化;采用MTT比色法检测TPs对雄激素非依赖性前列腺肿瘤PC-3M细胞增殖的影响;吖啶橙/溴化乙锭细胞染色法观察诱导细胞凋亡情况;流式细胞术分析细胞周期;划痕实验观察细胞迁移力;细胞侵袭实验观察细胞侵袭能力;RT-PCR和Western blot检测cyclin D1、Caspase-3、survivin、MMP-2、TIMP-2基因的转录与表达。
结果表明,与对照组比较,加药组细胞增殖速度减慢,细胞状态变差。在镜下表现为数量减少,且细胞细瘦、皱缩,局部可见细丝状“伪足”形成,细胞透光性减弱,细胞质内灰黑色颗粒状物增多。MTT法示实验组对PC-3M细胞的增殖抑制作用增强;细胞染色法示实验组凋亡率增高;流式细胞术显示细胞阻滞于G1期,增殖指数降低;划痕实验示茶多酚使PC-3M细胞迁移力下降;细胞侵袭实验示PC-3M细胞侵袭力减弱;RT-PCR与Western blot示Caspase-3、TIMP-2基因转录与翻译上调,而cyclin D1、survivin、MMP-2基因转录与翻译下调,且上述结果在一定范围内呈时间、剂量依赖性。
由此我们得出结论:茶多酚对人雄激素非依赖性前列腺癌PC-3M细胞具有阻滞细胞周期、抑制增殖、促进凋亡,减弱PC-3M细胞侵袭力的作用,上述作用与上调Caspase-3、TIMP-2基因,下调cyclin D1、survivin、MMP-2基因的转录与翻译有关。
Objective:
Tea,as a traditional health drink in China, has lasted for over three thousand years,and it is one of the three world's favourest beverages for it's speciality on health care.Today it's still popular.It has became more and more scientists' concerns that the habit of drinking tea regularly has advantages of keeping human health.Prostate cancer is the second largest cancer that result of the death,only second to lung cancer in men in western countries.The prostate tumor incidence in China,Japan and other asiatic countries in which peoples have the long-term habits of drinking tea is far lower than the west.Whether this difference is relative with the habits of drinking tea in above-mentioned zones?Many findings of experiments in vivo and in vitro have indicated that the main ingredient of tea:tea polyphenols(TPs) is anticarcinogenic,so it is reasonable that more and more investigators try to cure the tumor with the TPs.Our experiment also try to investigate the regulatory effect of tea polyphenol on proliferation inhibition apoptosis and invasion force of human androgen-independent prostate cancer PC-3M cells.
Methods:
The androgen-independent prostate cancer cells PC-3M is cultured in Iscove's Modified Dulbecco's Medium(IMDM) containing 10% of fetal bovine serum,and is incubatored in incubation at 37℃, 5% CO2 and constant humidity.
When cell fusion reachs about 80%,intervention treatment is conducted.The PC-3M cells are divided into treatment group and control group.
At 24 hour and 48 hour after intervention,we observe the change about cell number,morphology and state by phase contrast microscope.Final concentrations of TPs are 0,20,40,60,80,100ug/ml.
The inhibitions of cell proliferation at 24 hour,48 hour and 72 hour after intervention are detected by MTT assay.Final concentrations of TPs are same with above-mentioned.
The change of cell cycle phase at 24 hour after intervention is analyzed by flow cytometry.Final concentrations of TPs are 0,40,60,80ug/ml.
The changes of the apoptosis at 24 hour and 48 hour after intervention is inspected by acridine orange/ethidium bromide(AO/EB) fluorescent staining method.Final concentrations of TPs are also 0,40,60,80ug/ml.
By scratche assay,we observe the change of cell migration at 0h,4h,8h,12h and 24h after intervention with different concentrations of TPs.Final concentrations of TPs are also 0,40,60,80ug/ml.
We conduct the transwell assay.We know the change of cell invision force when PC-3M cell is intervented for 24h and 48h with different concentrations of TPs.Final concentrations of TPs are 0,40,60,80ug/ml.
Besides,the transcription and translation of the genes of cyclin D1,caspase-3, survivin,MMP-2,TIMP-2 in carcinoma cells are observed by RT-PCR and western blot.Final concentrations of TPs are 0,40,60,80ug/ml.
Data shown here are from representative experiments repeated at least three times with similar results.The data are shown asχ±s.With SPSS 17.0 statistical software,the data are analysised as t-test for independent samples and analysis of variance for the single factor and multiple sets(a=0.05).
Results:
After being treated with tea polyphenols solution,the number,morphology and state of human androgen-independent prostate cancer PC-3M cell are all changed appropriately.Compared with control group,dosing groups cell proliferation is slower,and cell is weak.Under the microscope,the cell number decreases and cell is slender and collapse.In some local visual fields,fine filamentous "pseudopodia" is visiable.Cell transmittance is weakened,and grey-black particles increase in cytoplasm.
The results of MTT assay about cell inhibition incidence are shown in Talbe 1.1,from which we can see that as TPs’s concentrations being elevated and time being extended,proliferation inhibitory effect of TPs on the PC-3M cell has gradually enhanced.
AO/EB staining reflects the apoptosis rate after PC-3M cell is treated with TPs at different concentrations as shown in Talbe 1.2,from which we can see that partial cells present the morphological change of apoptosis under the fluorescent microscope, and the apoptotic rates of the treatment groups increase respectively.
Flow cytometry reflects cell cycle changes after prostate cancer PC-3M cell is treated with TPs for 24h,as shown in Talbe 1.3,from which we can see that the cells in S and G2/M phase increase, the cells in G0/G1 phase decrease.It demonstrates that the cell cycle of experimental group is blocked in the G1 phase,and proliferation index decrease.
As shown in the scratch assay,as TPs concentration increasing, the lateral transfer distances of PC-3M cells decrease.And there is also change about cell morphology.Especially when the TPs concentrations is up to 80ug/ml,the cell on the edge of scratch creases and become round.It indicates that the TPs can attenuate the ablity of migration of prostate cancer PC-3M cell.
It is shown in the photo of transwell assay and Talbe 1.4 that PC-3M cells which penetrated through the matrix and migrated to under-surface decrease after treatment with TPs.It explains that PC-3M cell invasion forces weaken.
In part 1 of experiment,RT-PCR and Western blot result shows the transcription and translation of caspase-3 gene is up-rugulated,and the transcription and translation of cyclin D1 and survivin gene are down-rugulated.The above results in a certain range are time-dependent and dose-dependent,which to a certain extent explain and validate the phenomenons that TPs can cause prostate cancer PC-3M cell cycle arrest,proliferation depreciation and promotion of cell apoptosis.
In part 2 of experiment,RT-PCR and Western blot result shows the transcription and translation of MMP-2 genes is down-rugulated,and the transcription and translation of TIMP-2 gene is up-rugulated.The above results in a certain range is time-dependent and(or) dose-dependent,which to a certain extent explain and validate the phenomenons that TPs can weaken prostate cancer PC-3M invasion.
Conclusion:
TPs can inhibit the proliferation of human androgen-independent prostate cancer PC-3M cell,induce the apoptosis and weaken PC-3M cell migration and invasion force.The transcription and translation of up-regulation of caspase-3 and TIMP-2 gene,and of down-regulation of cyclin D1,survivin and MMP-2 gene may one of its molecular mechanisms.
引文
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