人参皂苷Rg3通过下调去乙酰化酶3的表达影响黑色素瘤细胞增殖及凋亡作用的初步研究
发布时间：2018-08-01 10:56
【摘要】：目的 人参是我国名贵的中药，其主要功效成分人参皂苷Rg3(以下简称Rg3)。Rg3是我国首先应用于临床抗肿瘤转移治疗的中药参一胶囊的主要活性成分。研究表明Rg3具有抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡、抑制肿瘤细胞粘附、侵润和转移,抑制肿瘤新生血管形成、抗淋巴道转移、逆转肿瘤细胞多药耐药性等等多种作用。 表观遗传学包括DNA的甲基化，组蛋白修饰，染色质重塑等。其中，组蛋白的共价修饰占有重要地位。组蛋白的共价修饰方式包括泛素化、磷酸化、乙酰化、甲基化等，它们可单独或协同调节基因转录，在肿瘤的发生发展中起到重要中所用。组蛋白的乙酰化修饰包括乙酰基化和去乙酰基化分别由乙酰基转移酶（histoneacetyltransferases，HATs）和组蛋白去乙酰化酶（histone deacetylases，HDACs）来调控。HDACs可乙酰化不同种类的细胞核转录因子和蛋白等，抑制多种抑癌蛋白的表达且与多种癌基因密切关联，进而导致细胞过度增殖进而导致肿瘤发生。 黑色素瘤一种恶性度较高的肿瘤，其发病机制的研究有很多，但乙酰化水平研究较少。本实验采用黑色素瘤细胞（mescl-28细胞系），观察人参皂苷Rg3对mescl-28的增殖及凋亡情况检测HDAC3的变化。 方法 体外培养人黑色素瘤mescl-28细胞。将实验分成4组（对照组，25μmol/l Rg3组,50μmol/l Rg3组,100μmol/l Rg3组)，CCK8法检测Rg3对细胞的生长抑制；倒置显微镜观察细胞在不同浓度的Rg3下的形态学变化；TRITC法检测不同浓度Rg3作用下HDAC3的变化；Western blot方法检测细胞增殖抗原（PCNA）、HDAC3、凋亡蛋白Bcl-2及Caspase-3的表达；RT-PCR检测PCNA及HDAC3的表达。 结果 1.CCK8检测50μmol/lRg3对细胞生长抑制作用，在24小时，48小时，72小时的抑制率分别为18.4%、23.1%、27.0%。 2.倒置显微镜观察不同浓度下细胞的生长情况。对照组细胞贴壁良好，，饱满，有棱角成不规则多边形。Rg3组随着药物浓度的增大细胞略为变圆，形态改变，数量逐渐变少。浓度大的可见细胞碎片。 3.不同浓度的药物作用下PCNA的变化，可见随着Rg3作用浓度的增大PCNA在基因和蛋白方面的表达均下降。 4．Western blot方法检测Bcl-2及Caspase-3的表达，可见随着Rg3浓度的增大，Bcl-2及Caspase-3的表达下降。5.hoechst32258染色检测不同药物浓度下细胞凋亡情况，可见随着药物浓度的增大，细胞核破碎较多。 5.TRITC发检测不同浓度下HDAC3的变化，可见随着Rg3作用浓度的增大，HDAC3表达逐渐变少。 6.Western blot及RT-PCR方法检测HDAC3，结果显示随着Rg3作用浓度的增大，HDAC3的表达逐渐减少。 结论 人参皂苷Rg3抑制黑色素瘤细胞mescl-28的生长，人参皂苷Rg3抑制HDAC3的表达。人参皂苷Rg3促进黑色素瘤细胞的凋亡。
[Abstract]:Objective ginsenoside Rg3 (Rg3) .Rg3 is one of the most valuable traditional Chinese medicines in China. The results show that Rg3 can inhibit tumor cell proliferation, induce apoptosis, inhibit tumor cell adhesion, invasion and metastasis, inhibit tumor angiogenesis, resist lymphatic metastasis, reverse multidrug resistance of tumor cells and so on. Epigenetics includes DNA methylation, histone modification, chromatin remodeling, etc. Among them, covalent modification of histone plays an important role. The covalent modification of histone includes ubiquitin phosphorylation acetylation methylation and so on. They can regulate gene transcription independently or cooperatively and play an important role in tumorigenesis and development. Acetylation modification of histone includes acetylation and deacetylation, which are regulated by histone acetyltransferase (HATs) and histone deacetyltransferase (HDACs) respectively. It inhibits the expression of many tumor suppressor proteins and is closely related to many oncogenes, which leads to excessive cell proliferation and tumorigenesis. Melanoma is a kind of tumor with high degree of malignancy. There are many studies on the pathogenesis of melanoma, but less on acetylation level. The proliferation and apoptosis of mescl-28 induced by ginsenoside Rg3 were observed by using melanoma cell line (mescl-28 cell line). Methods Human melanoma mescl-28 cells were cultured in vitro. The experiment was divided into 4 groups (control group, 25 渭 mol/l Rg3 group, 50 渭 mol/l Rg3 group, 100 渭 mol/l Rg3 group). CCK8 method was used to detect the growth inhibition of Rg3 on cells, and the morphological changes of cells under different concentrations of Rg3 were observed by inverted microscope. The expression of apoptotic protein Bcl-2 and Caspase-3 was detected by Western blot. The expression of PCNA and HDAC3 was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results the inhibitory effect of 50 渭 mol/lRg3 on cell growth was detected by 1.CCK8. The inhibitory rates of 50 渭 mol/lRg3 at 24 h and 48 h at 72 h were 18.4 / 23.1and 27.02. respectively. The growth of cells at different concentrations was observed by inverted microscope. In the control group, the cells adhered to the wall well and were plump. The cells in the control group became round with the increase of drug concentration, the number of cells changed and the number gradually decreased with the increase of drug concentration. High concentration of visible cell fragments. 3. The changes of PCNA under different concentrations of drugs showed that the expression of PCNA in gene and protein decreased with the increase of the concentration of Rg3. The expression of Bcl-2 and Caspase-3 was detected by 4.Western blot method. It can be seen that the expression of Bcl-2 and Caspase-3 decreased with the increase of Rg3 concentration. 5. The apoptosis of cells was detected under different drug concentration with the increase of Rg3 concentration, and the nuclear fragmentation was observed with the increase of drug concentration. The changes of HDAC3 in different concentrations were detected by 5.TRITC hair assay, and the expression of Bcl 2 and Caspase-3 decreased with the increase of Rg3 concentration. 6.Western blot and RT-PCR methods were used to detect HDAC3. The results showed that the expression of HDAC3 decreased with the increase of Rg3 concentration. Conclusion ginsenoside Rg3 inhibits the growth of mescl-28 and ginsenoside Rg3 inhibits the expression of HDAC3 in melanoma cells. Ginsenoside Rg3 promotes the apoptosis of melanoma cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.5
本文编号：2157344
[Abstract]:Objective ginsenoside Rg3 (Rg3) .Rg3 is one of the most valuable traditional Chinese medicines in China. The results show that Rg3 can inhibit tumor cell proliferation, induce apoptosis, inhibit tumor cell adhesion, invasion and metastasis, inhibit tumor angiogenesis, resist lymphatic metastasis, reverse multidrug resistance of tumor cells and so on. Epigenetics includes DNA methylation, histone modification, chromatin remodeling, etc. Among them, covalent modification of histone plays an important role. The covalent modification of histone includes ubiquitin phosphorylation acetylation methylation and so on. They can regulate gene transcription independently or cooperatively and play an important role in tumorigenesis and development. Acetylation modification of histone includes acetylation and deacetylation, which are regulated by histone acetyltransferase (HATs) and histone deacetyltransferase (HDACs) respectively. It inhibits the expression of many tumor suppressor proteins and is closely related to many oncogenes, which leads to excessive cell proliferation and tumorigenesis. Melanoma is a kind of tumor with high degree of malignancy. There are many studies on the pathogenesis of melanoma, but less on acetylation level. The proliferation and apoptosis of mescl-28 induced by ginsenoside Rg3 were observed by using melanoma cell line (mescl-28 cell line). Methods Human melanoma mescl-28 cells were cultured in vitro. The experiment was divided into 4 groups (control group, 25 渭 mol/l Rg3 group, 50 渭 mol/l Rg3 group, 100 渭 mol/l Rg3 group). CCK8 method was used to detect the growth inhibition of Rg3 on cells, and the morphological changes of cells under different concentrations of Rg3 were observed by inverted microscope. The expression of apoptotic protein Bcl-2 and Caspase-3 was detected by Western blot. The expression of PCNA and HDAC3 was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results the inhibitory effect of 50 渭 mol/lRg3 on cell growth was detected by 1.CCK8. The inhibitory rates of 50 渭 mol/lRg3 at 24 h and 48 h at 72 h were 18.4 / 23.1and 27.02. respectively. The growth of cells at different concentrations was observed by inverted microscope. In the control group, the cells adhered to the wall well and were plump. The cells in the control group became round with the increase of drug concentration, the number of cells changed and the number gradually decreased with the increase of drug concentration. High concentration of visible cell fragments. 3. The changes of PCNA under different concentrations of drugs showed that the expression of PCNA in gene and protein decreased with the increase of the concentration of Rg3. The expression of Bcl-2 and Caspase-3 was detected by 4.Western blot method. It can be seen that the expression of Bcl-2 and Caspase-3 decreased with the increase of Rg3 concentration. 5. The apoptosis of cells was detected under different drug concentration with the increase of Rg3 concentration, and the nuclear fragmentation was observed with the increase of drug concentration. The changes of HDAC3 in different concentrations were detected by 5.TRITC hair assay, and the expression of Bcl 2 and Caspase-3 decreased with the increase of Rg3 concentration. 6.Western blot and RT-PCR methods were used to detect HDAC3. The results showed that the expression of HDAC3 decreased with the increase of Rg3 concentration. Conclusion ginsenoside Rg3 inhibits the growth of mescl-28 and ginsenoside Rg3 inhibits the expression of HDAC3 in melanoma cells. Ginsenoside Rg3 promotes the apoptosis of melanoma cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.5
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本文编号：2157344
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