通补消积饮对人肺腺癌A549细胞增殖与凋亡影响的研究
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摘要
目的：研究通补消积饮含药血清对人肺腺癌A549细胞增殖与凋亡及其相关基因PCNA、Survivin表达的影响,探讨通补消积饮治疗肺癌的作用机制。
方法：(1)应用倒置显微镜观察不同含药血清作用于A549细胞后对其形态学的影响。(2)采用MTT法观察各组含药血清对A549细胞增殖的影响。(3)分别采用RT-PCR法检测A549细胞PCNA mRNA、Survivin mRNA的表达和Western-blot法检测A549细胞PCNA蛋白、Survivin蛋白的表达。(4)应用Annexin V/PI双染色流式法检测各组对A549细胞凋亡的影响。
结果：(1)通补消积饮含药血清作用于A549细胞24h和48h时,有些细胞体积变小,形体固缩,细胞变成不规则状,细胞膜完整,少许细胞脱落。也有少数细胞体积增大,细胞肿胀、细胞膜破裂,呈坏死状。72h时很多细胞形体固缩,体积变小,细胞变成不规则状。细胞肿胀、破裂坏死的细胞也有所增加。(2)分别观察通补消积饮低、中、高剂量含药血清作用于A549细胞24h、48h、72h抑制率,实验结果显示：与同时间点空白血清组比较,差异有显著的统计学意义(P＜0.01),说明通补消积饮对A549细胞具有明显的增殖抑制作用。其增殖抑制作用在一定时间内与剂量一时间呈正相关关系,即随着作用时间的延长和血清药物剂量的增加,其增殖抑制作用明显增强。通补消积饮高剂量组作用72h,达到其最大抑制率,为38.63%。(3)分别观察通补消积饮低、中、高剂量含药血清作用于A549细胞24h、48h、72h凋亡率,实验结果显示：通补消积饮低、中、高剂量含药血清组与同时间点空白血清组比较,差异有显著的统计学意义(P＜0.01),说明通补消积饮对A549细胞具有明显的诱导凋亡作用。其诱导凋亡作用在一定时间内与剂量一时间呈正相关关系,即随着作用时间的延长和血清药物剂量的增加,其诱导凋亡作用明显增强。通补消积饮高剂量组作用72h,达到其最大诱导凋亡率,为36.92%左右。(4)应用RT-PCR法检测结果表明,通补消积饮含药血清作用于A549细胞48h后,PCNA mRNA及Survivin mRNA表达显著下降,且随着通补消积饮剂量的增加,PCNA mRNA及Survivin mRNA表达逐渐下调,并表现出明显剂量依赖性,与空白血清组比较差异有统计学意义(P＜0.05)。经过通补消积饮含药血清作用于A549细胞48h后,Western-blot方法检测结果显示,随着通补消积饮剂量的增加,PCNA蛋白及Survivin蛋白表达逐渐下调。在蛋白水平上用Western-blot方法检测的结果与RT-PCR方法检测的结果是一致的。
结论：(1)通补消积饮对A549细胞有明显的增殖抑制作用,在一定的时间内表现出明显的量-效关系。(2)通补消积饮能有效下调PCNA基因在A549细胞中的表达,这可能是其抑制增殖作用的机制之一。(3)通补消积饮对A549细胞有明显的诱导凋亡作用,在一定的时间内表现出明显的量-效关系。(4)通补消积饮能有效下调Survivin基因在A549细胞中的表达,这可能是其诱导凋亡作用的机制之一。
Objectives:To investigate the influence of the Tongbu-Xiaoji decoction to the human lung adenocarcinoma cell line A549cells, and the expression of PCNA and Survivin which are correlated with proliferation and apoptosis. To discuss the mechanism of the Tongbu-Xiaoji decoction to lung carcinoma.
Methods:(1) The cell morphological effect of Tongbu-Xiaoji decoction to the human lung adenocarcinoma cell line A549cells was observed with inverted microscope.(2) The sensitivity and growth curve of the contained herb-serum to A549cells were observed with MTT.(3) The mRNA expression of PCNA and Survivin in cells were evaluated by RT-PCR, and the protein expression of PCNA and Survivin in cells were evaluated by Western blot.(4) The different categories of A549cell lines were stained with PI and Annexin V, and were evaluated by flowcytomety.
Result:(1) There were some cell morphological change of herb serum containing Tongbu-Xiaoji decoction treating the A549cell line in24-48hours, cell volume became small, cell became irregular and apart of cells fall off. The cell numbers of A549reduced materially in72hours, and some cell began to confluence, the cell straining became deep, cell body began smaller.(2) The herb serum containing Tongbu-Xiaoji decoction showed growth inhibiting visibly to cell line A549. And there were dose-dependent and time-dependent manner in some time point,that is the proliferation was significantly enhanced with increasing reaction time and serum drug dose.The optimal affection time of herb serum containing Tongbu-Xiaoji decoction on cell was in72hours. The inhibition rate was38.63%.(3)The herb serum containing Tongbu-Xiaoji decoction showed apoptosis inducing ability to human lung adenocarcinoma in cell line A549. A549cells were treated by Tongbu-Xiaoji decoction with low、median and high dose medicated serum, Compared with that of blank group, there was a significant dlfference(P<0.01).Tongbu-Xiaoji decoction shows a significant apoptosis inducing effect to A549,and the affection shows a dose-dependent and time-dependent manner,that is the apoptosis inducing effect was significantly enhanced with increasing reaction time and serum drug dose.The optimal affection time of herb serum containing Tongbu-Xiaoji decoction on cell was in72hours. The apoptosis inducing rate was36.92%.(4) From the results of RT-PCR,A549cells were treated by Tongbu-Xiaoji decoction with48h, the relative values of PCNA mRNA expression and Survivin mRNA expression were reduced significantly(P<0.05),and the affection shows a dose-dependent manner. The protein expression of PCNA and Survivin are as the mRNA expression, Tongbu-Xiaoji decoction restrains the protein expression of PCNA and Survivin of A549cells significantly(P<0.05),and the affection shows a dose-dependent manner.
Conclusions:(1) Tongbu-Xiaoji decoction significantly inhibited the proliferation of to A549cells, and the effect had an obvious dose-effect relationship in a certain period of time.(2) Tongbu-Xiaoji decoction restrains the mRNA and protein expression of PCNA to A549cells significantly.(3) Tongbu-Xiaoji decoction has the apoptosis-inducing effect to A549cells.(4) Tongbu-Xiaoji decoction restrains the mRNA and protein expression of Survivin to A549cells significantly.
方法：(1)应用倒置显微镜观察不同含药血清作用于A549细胞后对其形态学的影响。(2)采用MTT法观察各组含药血清对A549细胞增殖的影响。(3)分别采用RT-PCR法检测A549细胞PCNA mRNA、Survivin mRNA的表达和Western-blot法检测A549细胞PCNA蛋白、Survivin蛋白的表达。(4)应用Annexin V/PI双染色流式法检测各组对A549细胞凋亡的影响。
结果：(1)通补消积饮含药血清作用于A549细胞24h和48h时,有些细胞体积变小,形体固缩,细胞变成不规则状,细胞膜完整,少许细胞脱落。也有少数细胞体积增大,细胞肿胀、细胞膜破裂,呈坏死状。72h时很多细胞形体固缩,体积变小,细胞变成不规则状。细胞肿胀、破裂坏死的细胞也有所增加。(2)分别观察通补消积饮低、中、高剂量含药血清作用于A549细胞24h、48h、72h抑制率,实验结果显示：与同时间点空白血清组比较,差异有显著的统计学意义(P＜0.01),说明通补消积饮对A549细胞具有明显的增殖抑制作用。其增殖抑制作用在一定时间内与剂量一时间呈正相关关系,即随着作用时间的延长和血清药物剂量的增加,其增殖抑制作用明显增强。通补消积饮高剂量组作用72h,达到其最大抑制率,为38.63%。(3)分别观察通补消积饮低、中、高剂量含药血清作用于A549细胞24h、48h、72h凋亡率,实验结果显示：通补消积饮低、中、高剂量含药血清组与同时间点空白血清组比较,差异有显著的统计学意义(P＜0.01),说明通补消积饮对A549细胞具有明显的诱导凋亡作用。其诱导凋亡作用在一定时间内与剂量一时间呈正相关关系,即随着作用时间的延长和血清药物剂量的增加,其诱导凋亡作用明显增强。通补消积饮高剂量组作用72h,达到其最大诱导凋亡率,为36.92%左右。(4)应用RT-PCR法检测结果表明,通补消积饮含药血清作用于A549细胞48h后,PCNA mRNA及Survivin mRNA表达显著下降,且随着通补消积饮剂量的增加,PCNA mRNA及Survivin mRNA表达逐渐下调,并表现出明显剂量依赖性,与空白血清组比较差异有统计学意义(P＜0.05)。经过通补消积饮含药血清作用于A549细胞48h后,Western-blot方法检测结果显示,随着通补消积饮剂量的增加,PCNA蛋白及Survivin蛋白表达逐渐下调。在蛋白水平上用Western-blot方法检测的结果与RT-PCR方法检测的结果是一致的。
结论：(1)通补消积饮对A549细胞有明显的增殖抑制作用,在一定的时间内表现出明显的量-效关系。(2)通补消积饮能有效下调PCNA基因在A549细胞中的表达,这可能是其抑制增殖作用的机制之一。(3)通补消积饮对A549细胞有明显的诱导凋亡作用,在一定的时间内表现出明显的量-效关系。(4)通补消积饮能有效下调Survivin基因在A549细胞中的表达,这可能是其诱导凋亡作用的机制之一。
Objectives:To investigate the influence of the Tongbu-Xiaoji decoction to the human lung adenocarcinoma cell line A549cells, and the expression of PCNA and Survivin which are correlated with proliferation and apoptosis. To discuss the mechanism of the Tongbu-Xiaoji decoction to lung carcinoma.
Methods:(1) The cell morphological effect of Tongbu-Xiaoji decoction to the human lung adenocarcinoma cell line A549cells was observed with inverted microscope.(2) The sensitivity and growth curve of the contained herb-serum to A549cells were observed with MTT.(3) The mRNA expression of PCNA and Survivin in cells were evaluated by RT-PCR, and the protein expression of PCNA and Survivin in cells were evaluated by Western blot.(4) The different categories of A549cell lines were stained with PI and Annexin V, and were evaluated by flowcytomety.
Result:(1) There were some cell morphological change of herb serum containing Tongbu-Xiaoji decoction treating the A549cell line in24-48hours, cell volume became small, cell became irregular and apart of cells fall off. The cell numbers of A549reduced materially in72hours, and some cell began to confluence, the cell straining became deep, cell body began smaller.(2) The herb serum containing Tongbu-Xiaoji decoction showed growth inhibiting visibly to cell line A549. And there were dose-dependent and time-dependent manner in some time point,that is the proliferation was significantly enhanced with increasing reaction time and serum drug dose.The optimal affection time of herb serum containing Tongbu-Xiaoji decoction on cell was in72hours. The inhibition rate was38.63%.(3)The herb serum containing Tongbu-Xiaoji decoction showed apoptosis inducing ability to human lung adenocarcinoma in cell line A549. A549cells were treated by Tongbu-Xiaoji decoction with low、median and high dose medicated serum, Compared with that of blank group, there was a significant dlfference(P<0.01).Tongbu-Xiaoji decoction shows a significant apoptosis inducing effect to A549,and the affection shows a dose-dependent and time-dependent manner,that is the apoptosis inducing effect was significantly enhanced with increasing reaction time and serum drug dose.The optimal affection time of herb serum containing Tongbu-Xiaoji decoction on cell was in72hours. The apoptosis inducing rate was36.92%.(4) From the results of RT-PCR,A549cells were treated by Tongbu-Xiaoji decoction with48h, the relative values of PCNA mRNA expression and Survivin mRNA expression were reduced significantly(P<0.05),and the affection shows a dose-dependent manner. The protein expression of PCNA and Survivin are as the mRNA expression, Tongbu-Xiaoji decoction restrains the protein expression of PCNA and Survivin of A549cells significantly(P<0.05),and the affection shows a dose-dependent manner.
Conclusions:(1) Tongbu-Xiaoji decoction significantly inhibited the proliferation of to A549cells, and the effect had an obvious dose-effect relationship in a certain period of time.(2) Tongbu-Xiaoji decoction restrains the mRNA and protein expression of PCNA to A549cells significantly.(3) Tongbu-Xiaoji decoction has the apoptosis-inducing effect to A549cells.(4) Tongbu-Xiaoji decoction restrains the mRNA and protein expression of Survivin to A549cells significantly.
引文
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