黄芪注射液联合5-FU对结肠癌LoVo细胞生长的影响

发布时间：2018-03-30 08:11

本文选题：结肠癌　切入点：黄芪注射液　出处：《甘肃中医药大学(原名：甘肃中医学院)》2015年硕士论文

【摘要】：目的:研究不同浓度黄芪注射液(或)氟尿嘧啶(5-FU)单独/联合应用对结肠癌Lo Vo细胞生长的影响及其机制。方法:(1)采用MTT法测定结肠癌Lo Vo细胞的生长曲线;(2)采用MTT法测定结肠癌Lo Vo细胞在不同浓度的黄芪注射液、5-FU、黄芪注射液联合5-FU单独或共同干预下的生长抑制率;(3)采用流式细胞术检测黄芪注射液、5-FU、黄芪注射液联合5-FU作用于Lo Vo细胞的细胞周期变化;(4)实时定量PCR检测bcl-2、bax、P-gp(P-糖蛋白)的m RNA水平表达变化;(5)免疫细胞化学法检测bcl-2、bax及P-gp蛋白水平表达情况;采用Ki-67免疫细胞化学及免疫荧光法检测细胞的增殖活性。结果:(1)MTT细胞增殖活性检测结果显示黄芪注射液作用于结肠癌Lo Vo细胞,其浓度为0.25g/ml时对细胞有增殖作用,浓度为0.5-2g/ml时可明显抑制结肠癌Lo Vo细胞的生长,并且随着浓度的增大和作用时间的延长,对Lo Vo细胞生长抑制作用增强(P0.05);不同浓度的5-FU可明显抑制结肠癌Lo Vo细胞的生长,并随着浓度的增大和作用时间的增长,抑制肿瘤生长效果增强(P0.0);在用药不同时间点,黄芪注射液1g/ml与5-FU 10mg/l联合用药比单用黄芪注射液或单用5-FU抑制结肠癌Lo Vo细胞生长的作用更强(P0.05)。(2)流式细胞术检测细胞周期结果显示黄芪注射液、5-FU作用于结肠癌Lo Vo细胞后,G0/G1期细胞比例上升,S期细胞比例下降,联合用药组这一变化趋势更显著(P0.05)。(3)实时定量PCR检测bcl-2、bax、P-gp的m RNA水平表达,结果显示:黄芪注射液使结肠癌Lo Vo细胞中bax基因表达下调差异无统计学意义(P0.05),bcl-2基因表达上调,P-gp基因表达下调,差异有统计学意义(P0.05);5-Fu使Lo Vo细胞中bcl-2、P-gp基因表达下调,bax基因表达上调,差异有统计学意义(P0.05);联合用药组较单用黄芪注射液或单用5-FU对bcl-2、P-gp基因表达下调,bax基因表达上调作用明显增强(P0.05)。(4)免疫细胞化学法检测bcl-2、bax、P-gp、细胞增殖蛋白Ki-67的表达,结果显示空白对照组与黄芪注射液组对比bax、bcl-2蛋白的表达水平差异无统计学意义(P0.05),P-gp、Ki-67蛋白的表达水平差异有统计学意义(P0.05);空白对照组与5-FU组对比bax、bcl-2、P-gp、Ki-67蛋白的表达水平差异有统计学意义(P0.05);5-FU组与联合用药组对比bcl-2蛋白表达水平差异无统计学意义(P0.05),bax、P-gp、Ki-67蛋白表达水平差异有统计学意义(P0.05)。结论:(1)黄芪注射液浓度为1g/ml时,抑制结肠癌Lo Vo细胞增殖作用最强,浓度过高可能产生药物毒性,而在低浓度0.25g/ml时可促进肿瘤细胞增殖。(2)黄芪注射液浓度为0.5-2g/ml或5-FU单用可抑制结肠癌Lo Vo细胞的生长,且与时间、剂量呈正相关性;二者联合应用对结肠癌Lo Vo细胞的生长抑制明显优于单用黄芪注射液或5-FU,且在共同用药48小时后具有显著协同作用。(3)黄芪注射液联合5-FU抑制结肠癌生长的作用机制可能是:阻滞肿瘤细胞周期,抑制细胞增殖,下调bcl-2、P-gp,上调bax表达,诱导结肠癌细胞凋亡。
[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.
【学位授予单位】：甘肃中医药大学(原名：甘肃中医学院)
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.35

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