【摘要】：目的:觀察苦參堿對(duì)肝癌細(xì)胞的抑制效果及機(jī)制。方法:選取HepG2肝癌細(xì)胞系,MTT篩選苦參堿作用于HepG2肝癌細(xì)胞的濃度梯度,并觀察苦參堿對(duì)HepG2肝癌細(xì)胞形態(tài)學(xué)的影響。實(shí)驗(yàn)分為空白對(duì)照組、實(shí)驗(yàn)組、陽(yáng)性對(duì)照組3組,空白對(duì)照組加入不含藥的細(xì)胞培養(yǎng)基,實(shí)驗(yàn)組分別加入苦參堿,使其終濃度為1、2、4 mg/mL,陽(yáng)性對(duì)照組加用終濃度為20umol/L的ERK信號(hào)通路阻斷劑PD98059,培養(yǎng)24小時(shí)后,MTT檢測(cè)細(xì)胞增殖、Transwell小室檢測(cè)細(xì)胞遷移數(shù)目、逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)ERK信號(hào)通路上的關(guān)鍵蛋白ERK的核酸表達(dá)變化,蛋白質(zhì)免疫印跡法(Western-Blot)檢測(cè)ERK、p-ERK蛋白質(zhì)表達(dá)變化。結(jié)果:1.細(xì)胞形態(tài)學(xué)變化:空白對(duì)照組細(xì)胞排列緊密,呈梭形,形態(tài)飽滿,貼壁良好;實(shí)驗(yàn)組細(xì)胞形態(tài)隨著濃度升高細(xì)胞密度逐漸減低,貼壁不牢,并在高濃度時(shí)出現(xiàn)壞死的細(xì)胞碎片。2.MTT結(jié)果顯示,苦參堿對(duì)HepG2細(xì)胞增殖的抑制作用具有時(shí)間和濃度依賴性。并且在24h時(shí)間段內(nèi)1 mg/m L、2 mg/mL、4 mg/mL濃度時(shí)苦參堿對(duì)HepG2作用最敏感。3.細(xì)胞遷移能力變化:實(shí)驗(yàn)組與空白對(duì)照組相比,1 mg/m L、2 mg/m L、4mg/m L組細(xì)胞遷移能力顯著減少(P0.01),4 mg/m L與陽(yáng)性對(duì)照組比較無(wú)差異(P0.05)。4.ERK核酸表達(dá):經(jīng)苦參堿作用后ERK mRNA的表達(dá)量顯著下降,實(shí)驗(yàn)組中4 mg/m L、2 mg/m L與空白對(duì)照組相比有差異(P0.05);實(shí)驗(yàn)組與陽(yáng)性對(duì)照組比較核酸表達(dá)量未下降,4mg/m L、2mg/m L有差異(P0.05)。5.ERK和p-ERK蛋白的表達(dá):實(shí)驗(yàn)組與空白對(duì)照組相比,p-ERK蛋白表達(dá)量在2 mg/m L、4 mg/mL濃度時(shí)下調(diào)顯著,有統(tǒng)計(jì)學(xué)意義(P0.01),ERK蛋白表達(dá)量在2 mg/m L、4 mg/m L濃度時(shí)下調(diào)顯著,有統(tǒng)計(jì)學(xué)差異(P0.05);與陽(yáng)性對(duì)照組相比,1 mg/m L、2 mg/mL苦參堿作用后p-ERK蛋白下調(diào)量不及PD98059(P0.05),且1 mg/mL具有顯著性差異(P0.01)。結(jié)論:1.苦參堿可抑制HepG2肝癌細(xì)胞的增殖和遷移,其具體機(jī)制可能通過(guò)下調(diào)ERK信號(hào)轉(zhuǎn)導(dǎo)通路中的關(guān)鍵蛋白p-ERK和ERK蛋白的表達(dá)。2.苦參堿對(duì)HepG2肝癌細(xì)胞的抑制作用具有時(shí)間和濃度依賴性。
[Abstract]:Objective: to observe the inhibitory effect and mechanism of Matrine on HCC cells. Methods: HepG2 HCC cell line was selected and MTT was selected to screen the concentration gradient of Matrine on HepG2 HCC cells, and the effect of Matrine on the morphology of HepG2 HCC cells was observed. The experiment was divided into three groups: blank control group, experimental group and positive control group. The blank control group was supplemented with non-drug cell culture medium, and the experimental group was added with Matrine to make the final concentration of Matrine 1, 2, 4 mg/mL,. The positive control group was cultured with ERK signal pathway blocker PD98059, with final concentration of 20umol/L for 24 hours. The proliferation of cells was detected by MTT and the number of cell migration was detected by Transwell chamber. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the nucleic acid expression of the key protein ERK in ERK signaling pathway, and Western-Blot was used to detect the expression of ERK,p-ERK protein. Result: 1. Morphological changes of cells in the blank control group: the cells in the blank control group were closely arranged, fusiform, full in shape and good adherent to the wall. The cell morphology of the experimental group decreased gradually with the increase of concentration, the adherent cell density was not firm, and the cell fragments appeared at high concentration. 2. The results showed that the inhibitory effect of Matrine on the proliferation of HepG2 cells was time-and concentration-dependent. At the concentration of 1 mg/mL, 2 mg/mL,4 mg/mL in 24 h, Matrine was the most sensitive to HepG2. The changes of cell migration ability: compared with the blank control group, the cell migration ability of the experimental group was significantly lower than that of the blank control group (1 mg/m / L, 2 mg/m / L, 4 mg 路L) (P 0.01). There was no significant difference in the expression of ERK mRNA between the 4 mg/m / L group and the positive control group (P 0.05). 4. The expression of ERK nucleic acid: the expression of ERK decreased significantly after treatment with Matrine, and the expression of ERK in the experimental group was 4 mg/m / L. 2There was significant difference between mg/m / L and blank control group (P 0.05). Compared with the positive control group, the expression of nucleic acid in the experimental group was not decreased, but the expression of 4mg/m L and 2 mg / L was different (P 0.05). 5. The expression of ERK and p-ERK protein in the experimental group was higher than that in the blank control group. The expression of p-ERK protein decreased significantly at the concentration of 2 mg/mL and 4 mg/mL, which was statistically significant (P 0.01). The expression of), ERK protein decreased significantly at the concentration of 2 mg/mL and 4 mg/mL (P 0.05). Compared with the positive control group, the down regulation of p-ERK protein in 1 mg/mL and 2 mg/mL Matrine was lower than that in PD98059 (P 0.05), and there was significant difference in 1 mg/mL (P 0.01). Conclusion: 1. Matrine can inhibit the proliferation and migration of HepG2 hepatocellular carcinoma cells, and its mechanism may be by down-regulating the expression of p-ERK and ERK proteins in ERK signal transduction pathway. 2. The inhibitory effect of Matrine on HepG2 HCC cells was time-and concentration-dependent.
【學(xué)位授予單位】：甘肅中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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