發(fā)布時(shí)間：2018-08-01 10:56

【摘要】：目的 人參是我國(guó)名貴的中藥，其主要功效成分人參皂苷Rg3(以下簡(jiǎn)稱Rg3)。Rg3是我國(guó)首先應(yīng)用于臨床抗腫瘤轉(zhuǎn)移治療的中藥參一膠囊的主要活性成分。研究表明Rg3具有抑制腫瘤細(xì)胞增殖、誘導(dǎo)腫瘤細(xì)胞凋亡、抑制腫瘤細(xì)胞粘附、侵潤(rùn)和轉(zhuǎn)移,抑制腫瘤新生血管形成、抗淋巴道轉(zhuǎn)移、逆轉(zhuǎn)腫瘤細(xì)胞多藥耐藥性等等多種作用。 表觀遺傳學(xué)包括DNA的甲基化，組蛋白修飾，染色質(zhì)重塑等。其中，組蛋白的共價(jià)修飾占有重要地位。組蛋白的共價(jià)修飾方式包括泛素化、磷酸化、乙酰化、甲基化等，它們可單獨(dú)或協(xié)同調(diào)節(jié)基因轉(zhuǎn)錄，在腫瘤的發(fā)生發(fā)展中起到重要中所用。組蛋白的乙�；揎棸ㄒ阴；腿ヒ阴；謩e由乙酰基轉(zhuǎn)移酶（histoneacetyltransferases，HATs）和組蛋白去乙酰化酶（histone deacetylases，HDACs）來(lái)調(diào)控。HDACs可乙酰化不同種類的細(xì)胞核轉(zhuǎn)錄因子和蛋白等，抑制多種抑癌蛋白的表達(dá)且與多種癌基因密切關(guān)聯(lián)，進(jìn)而導(dǎo)致細(xì)胞過(guò)度增殖進(jìn)而導(dǎo)致腫瘤發(fā)生。 黑色素瘤一種惡性度較高的腫瘤，其發(fā)病機(jī)制的研究有很多，但乙酰化水平研究較少。本實(shí)驗(yàn)采用黑色素瘤細(xì)胞（mescl-28細(xì)胞系），觀察人參皂苷Rg3對(duì)mescl-28的增殖及凋亡情況檢測(cè)HDAC3的變化。 方法 體外培養(yǎng)人黑色素瘤mescl-28細(xì)胞。將實(shí)驗(yàn)分成4組（對(duì)照組，25μmol/l Rg3組,50μmol/l Rg3組,100μmol/l Rg3組)，CCK8法檢測(cè)Rg3對(duì)細(xì)胞的生長(zhǎng)抑制；倒置顯微鏡觀察細(xì)胞在不同濃度的Rg3下的形態(tài)學(xué)變化；TRITC法檢測(cè)不同濃度Rg3作用下HDAC3的變化；Western blot方法檢測(cè)細(xì)胞增殖抗原（PCNA）、HDAC3、凋亡蛋白Bcl-2及Caspase-3的表達(dá)；RT-PCR檢測(cè)PCNA及HDAC3的表達(dá)。 結(jié)果 1.CCK8檢測(cè)50μmol/lRg3對(duì)細(xì)胞生長(zhǎng)抑制作用，在24小時(shí)，48小時(shí)，72小時(shí)的抑制率分別為18.4%、23.1%、27.0%。 2.倒置顯微鏡觀察不同濃度下細(xì)胞的生長(zhǎng)情況。對(duì)照組細(xì)胞貼壁良好，，飽滿，有棱角成不規(guī)則多邊形。Rg3組隨著藥物濃度的增大細(xì)胞略為變圓，形態(tài)改變，數(shù)量逐漸變少。濃度大的可見(jiàn)細(xì)胞碎片。 3.不同濃度的藥物作用下PCNA的變化，可見(jiàn)隨著Rg3作用濃度的增大PCNA在基因和蛋白方面的表達(dá)均下降。 4．Western blot方法檢測(cè)Bcl-2及Caspase-3的表達(dá)，可見(jiàn)隨著Rg3濃度的增大，Bcl-2及Caspase-3的表達(dá)下降。5.hoechst32258染色檢測(cè)不同藥物濃度下細(xì)胞凋亡情況，可見(jiàn)隨著藥物濃度的增大，細(xì)胞核破碎較多。 5.TRITC發(fā)檢測(cè)不同濃度下HDAC3的變化，可見(jiàn)隨著Rg3作用濃度的增大，HDAC3表達(dá)逐漸變少。 6.Western blot及RT-PCR方法檢測(cè)HDAC3，結(jié)果顯示隨著Rg3作用濃度的增大，HDAC3的表達(dá)逐漸減少。 結(jié)論 人參皂苷Rg3抑制黑色素瘤細(xì)胞mescl-28的生長(zhǎng)，人參皂苷Rg3抑制HDAC3的表達(dá)。人參皂苷Rg3促進(jìn)黑色素瘤細(xì)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.5

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