本文選題：結(jié)腸癌　切入點：黃芪注射液　出處：《甘肅中醫(yī)藥大學(xué)(原名：甘肅中醫(yī)學(xué)院)》2015年碩士論文

【摘要】：目的:研究不同濃度黃芪注射液(或)氟尿嘧啶(5-FU)單獨/聯(lián)合應(yīng)用對結(jié)腸癌Lo Vo細胞生長的影響及其機制。方法:(1)采用MTT法測定結(jié)腸癌Lo Vo細胞的生長曲線;(2)采用MTT法測定結(jié)腸癌Lo Vo細胞在不同濃度的黃芪注射液、5-FU、黃芪注射液聯(lián)合5-FU單獨或共同干預(yù)下的生長抑制率;(3)采用流式細胞術(shù)檢測黃芪注射液、5-FU、黃芪注射液聯(lián)合5-FU作用于Lo Vo細胞的細胞周期變化;(4)實時定量PCR檢測bcl-2、bax、P-gp(P-糖蛋白)的m RNA水平表達變化;(5)免疫細胞化學(xué)法檢測bcl-2、bax及P-gp蛋白水平表達情況;采用Ki-67免疫細胞化學(xué)及免疫熒光法檢測細胞的增殖活性。結(jié)果:(1)MTT細胞增殖活性檢測結(jié)果顯示黃芪注射液作用于結(jié)腸癌Lo Vo細胞,其濃度為0.25g/ml時對細胞有增殖作用,濃度為0.5-2g/ml時可明顯抑制結(jié)腸癌Lo Vo細胞的生長,并且隨著濃度的增大和作用時間的延長,對Lo Vo細胞生長抑制作用增強(P0.05);不同濃度的5-FU可明顯抑制結(jié)腸癌Lo Vo細胞的生長,并隨著濃度的增大和作用時間的增長,抑制腫瘤生長效果增強(P0.0);在用藥不同時間點,黃芪注射液1g/ml與5-FU 10mg/l聯(lián)合用藥比單用黃芪注射液或單用5-FU抑制結(jié)腸癌Lo Vo細胞生長的作用更強(P0.05)。(2)流式細胞術(shù)檢測細胞周期結(jié)果顯示黃芪注射液、5-FU作用于結(jié)腸癌Lo Vo細胞后,G0/G1期細胞比例上升,S期細胞比例下降,聯(lián)合用藥組這一變化趨勢更顯著(P0.05)。(3)實時定量PCR檢測bcl-2、bax、P-gp的m RNA水平表達,結(jié)果顯示:黃芪注射液使結(jié)腸癌Lo Vo細胞中bax基因表達下調(diào)差異無統(tǒng)計學(xué)意義(P0.05),bcl-2基因表達上調(diào),P-gp基因表達下調(diào),差異有統(tǒng)計學(xué)意義(P0.05);5-Fu使Lo Vo細胞中bcl-2、P-gp基因表達下調(diào),bax基因表達上調(diào),差異有統(tǒng)計學(xué)意義(P0.05);聯(lián)合用藥組較單用黃芪注射液或單用5-FU對bcl-2、P-gp基因表達下調(diào),bax基因表達上調(diào)作用明顯增強(P0.05)。(4)免疫細胞化學(xué)法檢測bcl-2、bax、P-gp、細胞增殖蛋白Ki-67的表達,結(jié)果顯示空白對照組與黃芪注射液組對比bax、bcl-2蛋白的表達水平差異無統(tǒng)計學(xué)意義(P0.05),P-gp、Ki-67蛋白的表達水平差異有統(tǒng)計學(xué)意義(P0.05);空白對照組與5-FU組對比bax、bcl-2、P-gp、Ki-67蛋白的表達水平差異有統(tǒng)計學(xué)意義(P0.05);5-FU組與聯(lián)合用藥組對比bcl-2蛋白表達水平差異無統(tǒng)計學(xué)意義(P0.05),bax、P-gp、Ki-67蛋白表達水平差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:(1)黃芪注射液濃度為1g/ml時,抑制結(jié)腸癌Lo Vo細胞增殖作用最強,濃度過高可能產(chǎn)生藥物毒性,而在低濃度0.25g/ml時可促進腫瘤細胞增殖。(2)黃芪注射液濃度為0.5-2g/ml或5-FU單用可抑制結(jié)腸癌Lo Vo細胞的生長,且與時間、劑量呈正相關(guān)性;二者聯(lián)合應(yīng)用對結(jié)腸癌Lo Vo細胞的生長抑制明顯優(yōu)于單用黃芪注射液或5-FU,且在共同用藥48小時后具有顯著協(xié)同作用。(3)黃芪注射液聯(lián)合5-FU抑制結(jié)腸癌生長的作用機制可能是:阻滯腫瘤細胞周期,抑制細胞增殖,下調(diào)bcl-2、P-gp,上調(diào)bax表達,誘導(dǎo)結(jié)腸癌細胞凋亡。
[Abstract]:Objective: to study the effect of different concentrations of astragalus injection (or fluorouracil 5-FU) on the growth of colon cancer Lo Vo cells and its mechanism. Methods: MTT assay was used to determine the growth curve of colon cancer Lo Vo cells. MTT assay was used to detect the growth inhibition rate of Lo Vo cells in different concentrations of Astragalus membranaceus injection (Astragalus membranaceus injection) and Astragalus injection (Astragalus injection combined with 5-FU). Flow cytometry was used to detect Astragalus injection 5-FU and Astragalus injection combined with 5-FU. Cell cycle changes of Lo Vo cells: (1) Real-time quantitative PCR was used to detect the expression of m RNA in Bcl-2B ~ (2 +) P-gpP- glycoprotein); (5) Immunocytochemistry was used to detect the expression of bcl-2mbax and P-gp protein. Ki-67 immunocytochemistry and immunofluorescence were used to detect the proliferative activity of the cells. Results the results showed that Astragalus membranaceus injection acted on Lo Vo cells of colon cancer, and the concentration of Astragalus membranaceus injection was 0.25g/ml. The growth of Lo Vo cells was inhibited by 0.5-2g/ml, and the growth inhibition of Lo Vo cells was enhanced with the increase of concentration and the time of treatment, and the growth of Lo Vo cells was inhibited by 5-FU at different concentrations. With the increase of the concentration and the increase of the time of action, the effect of inhibiting tumor growth was enhanced (P0.00. 0). The effect of Astragalus injection 1g/ml combined with 5-FU 10mg/l on the growth of colon cancer Lo Vo cells was stronger than that of Astragalus membranaceus injection alone or 5-FU alone. Flow cytometry analysis showed that Astragalus injection 5-FU acted on Lo Lo colon cancer cells. The percentage of cells in G _ 0 / G _ 1 phase after Vo cells increased, and the proportion of cells in S phase decreased. In combination group, the trend of this change was more significant (P0.05, P < 0.05). Real-time quantitative PCR was used to detect the expression of m RNA in bcl-2nb axon P-gp. The results showed that there was no significant difference in the down-regulation of bax gene expression in human colon cancer Lo Vo cells induced by Astragalus membranaceus injection (Astragalus membranaceus injection). The expression of bcl-2 gene was up-regulated and the expression of P-gp gene was down-regulated. The difference was statistically significant (P 0.05) and 5-Fu could down-regulate the expression of bcl-2n P-gp gene in Lo Vo cells, and up-regulate the expression of Bcl-2P -gp gene in Lo Vo cells. In combination group, the up-regulation of bcl-2nP-gp gene expression was significantly enhanced by P0.05P0.05, and the expression of bcl-2na-bax-P-gp, cell proliferation protein Ki-67 was detected by immunocytochemistry, and the expression of Bcl-2OP-gp and Bcl-2OP-gp protein were detected by immunocytochemistry, and the expression of Bcl-2OP-gp and Bcl-2OP-gp were detected by immunocytochemistry. The results showed that there was no significant difference in the expression of bcl-2 protein between the blank control group and the astragalus injection group. There was a significant difference in the expression level of the protein between the blank control group and the astragalus injection group. There was a significant difference in the expression level of the protein between the blank control group and the astragalus injection group, and the difference between the blank control group and the 5-FU group in the expression of baxbcl-2P-gppki-67 protein. There was no significant difference in the expression of bcl-2 protein between the two groups. There was no significant difference in the expression of bcl-2 protein between the P0.05-FU group and the combined treatment group. There was a significant difference in the expression of P-gpki-67 protein between the two groups. Conclusion the concentration of Astragalus membranaceus injection is 1g/ml. The inhibitory effect on the proliferation of colon cancer Lo Vo cells was strongest, and too high concentration might produce drug toxicity, while at low concentration of 0.25g/ml, the concentration of Astragalus membranaceus injection was 0.5-2g/ml or 5-FU alone to inhibit the growth of colon cancer Lo Vo cells, and the concentration of Astragalus membranaceus injection alone could inhibit the growth of colon cancer Lo Vo cells. There was a positive correlation with time and dose. The inhibitory effect of Astragalus membranaceus injection and 5-FU on the growth of colonic cancer Lo Vo cells was better than that of Astragalus membranaceus injection alone or 5-FU injection alone, and there was a significant synergistic effect of Astragalus injection combined with 5-FU on the inhibition of colon cancer growth. It could be that the tumor cell cycle is blocked, Inhibition of cell proliferation, down-regulation of bcl-2 P-gp, up-regulation of bax expression and induction of apoptosis in colon cancer cells.
【學(xué)位授予單位】：甘肅中醫(yī)藥大學(xué)(原名：甘肅中醫(yī)學(xué)院)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.35

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