化痰散结中药甲肿消对甲状腺细胞凋亡影响的实验研究
摘要
目的
甲状腺肿大广泛存在于各种类型的甲状腺疾病中,主要是由于甲状腺细胞增殖＞凋亡而引起的。中医治疗甲状腺肿大具有几千年的历史,而化痰散结法为基本治法,在临床上有较好的疗效。化痰散结中药分为富碘中药和非富碘两类,目前对非富碘中药作用机制研究尚少,有待进一步的阐明。通过研究化痰散结组方中药甲肿消(组方:夏枯草等)对甲状腺细胞凋亡的影响,探讨其治疗甲状腺肿大的作用机理,以期为中医临床应用化痰散结法治疗甲状腺肿大提供基础研究支撑。
方法
制备甲肿消含药动物血清,干预甲状腺FRTL细胞株,研究含药血清对FRTL细胞株凋亡的影响。实验共分六组:A:正常组、B:空白血清对照组(1%空白血清)、C:小剂量组(1%含药血清)、D:中剂量组(5%含药血清)、E:大剂量组(10%含药血清)、F:谷光甘肽(GSHNO)诱导凋亡对照组(2%的GSHNO)。收集各组细胞,分别提取RNA和蛋白。采用实时荧光定量PCR法检测Bax、Bcl-2的mRNA表达,Western-Blot免疫印迹法检测其蛋白表达情况。采用SPSS11.5软件分析各组细胞Bax、Bcl-2 mRNA和蛋白的表达数据,统计方法主要包括单因素方差分析、LSD-t检验、Dunnett-t检验、Tamhane检验。
结果
甲肿消大剂量和中剂量组能使甲状腺细胞促凋亡基因Bax的mRNA和蛋白的表达水平明显升高;各中药组Bcl-2的mRNA和蛋白表达未见统计学差异性;各中药组Bax/Bcl-2比值均有升高趋势,中剂量组差异显著(p＜0.05);GSHNO诱导凋亡对照组的Bax mRNA和蛋白表达以及Bax/Bcl-2比值均明显升高。
结论
化痰散结中药甲肿消有促进甲状腺细胞凋亡的作用,可以上调促凋亡基因Bax的表达,提示甲肿消治疗甲状腺肿大可能是通过促进甲状腺细胞凋亡而实现,而Bax可能是其主要作用位点。
Objective
Thyromegaly exists abroadly in the thyroid diseases,it is the reason of thyroid cell proliferation＞cell apoptosis.The treatment of thyromegaly in TCM had a history of several thousand years,the principle of removing phlegm and resovling mass is the basic treatment and have an effective cure for thyromegaly in clinic.It contains iodine-rich herbs and None rich iodine herbs.Now we know little about the mechanism of None rich iodine herbs and it needs further research.Through investigating the effect of removing phlegm and resovling mass Herbs Jiazhongxiao (including Spica prunellae and so on)on the apoptosis and expressions of bax、bcl-2 in FRTL cells,to explore the therapeutic mechanism of thyromegaly of the removing phlegm and resovling mass Herbs.And it is supposed to provide some basic research for the therapy of thyromegaly in Traditional Chinese Medicine.
Methods
Rats'Thyroid FRTL cells was interfered by Serum of Chinese Herbs of Removing phlegm and Resovling mass in vitro.Determined the effect of its herbs-containing serum on the apoptosis of FRTL cells.The cells were divided into six groups:A: normal group;B:blank group(containing 1%non-herbs serum);Tested groups are C、D、E:low、middling and high dose groups(containing1%、5%、10%herbs serum respectively);F:postived control group(GSHNO induce apoptosis group containing 2%GSHNO).The protein and mRNA were extracted from each group's cells separately.mRNA expressions of Bax and Bcl-2 were detected with real-time quantitative RT-PCR.The protein expressions were tested by Western Blot.We used SPSS11.5 to analyze the data.The main statistic methods contained ANOVA、LSD-t、Dunnett-t and Tamhane Tests.
Results
The mRNA and protein expressions of Bax increased distinctly in the high dose group and middling group.The ratio of Bax/Bcl-2 all increased in the three Chinese herbs groups,but remarkably only in the ZJ group(P＜0.05).Bax mRNA and protein expressions was obvious in the GSHNO group.
Conclusion
This study showed that Chinese Herbs of removing phlegm and resovling mass can promote the apoptosis of thyroid cells and increase the pro-apoptosis gene Bax, which may be related to the mechanism of Jiazhongxiao's therapeutical effect on thyromegaly,and gene Bax may be the key point.
甲状腺肿大广泛存在于各种类型的甲状腺疾病中,主要是由于甲状腺细胞增殖＞凋亡而引起的。中医治疗甲状腺肿大具有几千年的历史,而化痰散结法为基本治法,在临床上有较好的疗效。化痰散结中药分为富碘中药和非富碘两类,目前对非富碘中药作用机制研究尚少,有待进一步的阐明。通过研究化痰散结组方中药甲肿消(组方:夏枯草等)对甲状腺细胞凋亡的影响,探讨其治疗甲状腺肿大的作用机理,以期为中医临床应用化痰散结法治疗甲状腺肿大提供基础研究支撑。
方法
制备甲肿消含药动物血清,干预甲状腺FRTL细胞株,研究含药血清对FRTL细胞株凋亡的影响。实验共分六组:A:正常组、B:空白血清对照组(1%空白血清)、C:小剂量组(1%含药血清)、D:中剂量组(5%含药血清)、E:大剂量组(10%含药血清)、F:谷光甘肽(GSHNO)诱导凋亡对照组(2%的GSHNO)。收集各组细胞,分别提取RNA和蛋白。采用实时荧光定量PCR法检测Bax、Bcl-2的mRNA表达,Western-Blot免疫印迹法检测其蛋白表达情况。采用SPSS11.5软件分析各组细胞Bax、Bcl-2 mRNA和蛋白的表达数据,统计方法主要包括单因素方差分析、LSD-t检验、Dunnett-t检验、Tamhane检验。
结果
甲肿消大剂量和中剂量组能使甲状腺细胞促凋亡基因Bax的mRNA和蛋白的表达水平明显升高;各中药组Bcl-2的mRNA和蛋白表达未见统计学差异性;各中药组Bax/Bcl-2比值均有升高趋势,中剂量组差异显著(p＜0.05);GSHNO诱导凋亡对照组的Bax mRNA和蛋白表达以及Bax/Bcl-2比值均明显升高。
结论
化痰散结中药甲肿消有促进甲状腺细胞凋亡的作用,可以上调促凋亡基因Bax的表达,提示甲肿消治疗甲状腺肿大可能是通过促进甲状腺细胞凋亡而实现,而Bax可能是其主要作用位点。
Objective
Thyromegaly exists abroadly in the thyroid diseases,it is the reason of thyroid cell proliferation＞cell apoptosis.The treatment of thyromegaly in TCM had a history of several thousand years,the principle of removing phlegm and resovling mass is the basic treatment and have an effective cure for thyromegaly in clinic.It contains iodine-rich herbs and None rich iodine herbs.Now we know little about the mechanism of None rich iodine herbs and it needs further research.Through investigating the effect of removing phlegm and resovling mass Herbs Jiazhongxiao (including Spica prunellae and so on)on the apoptosis and expressions of bax、bcl-2 in FRTL cells,to explore the therapeutic mechanism of thyromegaly of the removing phlegm and resovling mass Herbs.And it is supposed to provide some basic research for the therapy of thyromegaly in Traditional Chinese Medicine.
Methods
Rats'Thyroid FRTL cells was interfered by Serum of Chinese Herbs of Removing phlegm and Resovling mass in vitro.Determined the effect of its herbs-containing serum on the apoptosis of FRTL cells.The cells were divided into six groups:A: normal group;B:blank group(containing 1%non-herbs serum);Tested groups are C、D、E:low、middling and high dose groups(containing1%、5%、10%herbs serum respectively);F:postived control group(GSHNO induce apoptosis group containing 2%GSHNO).The protein and mRNA were extracted from each group's cells separately.mRNA expressions of Bax and Bcl-2 were detected with real-time quantitative RT-PCR.The protein expressions were tested by Western Blot.We used SPSS11.5 to analyze the data.The main statistic methods contained ANOVA、LSD-t、Dunnett-t and Tamhane Tests.
Results
The mRNA and protein expressions of Bax increased distinctly in the high dose group and middling group.The ratio of Bax/Bcl-2 all increased in the three Chinese herbs groups,but remarkably only in the ZJ group(P＜0.05).Bax mRNA and protein expressions was obvious in the GSHNO group.
Conclusion
This study showed that Chinese Herbs of removing phlegm and resovling mass can promote the apoptosis of thyroid cells and increase the pro-apoptosis gene Bax, which may be related to the mechanism of Jiazhongxiao's therapeutical effect on thyromegaly,and gene Bax may be the key point.
引文
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