脂联素对人乳腺癌MCF-7细胞生物学行为的影响及其所诱导的凋亡与自噬相关性的研究

发布时间：2018-01-15 05:21

  本文关键词：脂联素对人乳腺癌MCF-7细胞生物学行为的影响及其所诱导的凋亡与自噬相关性的研究　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： Acrp30 MCF-7细胞 增殖 自噬 凋亡

【摘要】：目的研究全长脂联素(Acrp30)在人乳腺癌MCF-7细胞增殖、迁移、自噬、凋亡中的作用,并探讨自噬与脂联素诱导的凋亡之间的关系。方法1.体外培养MCF-7细胞,分为对照组(未加Acrp30)细胞,加入Acrp30组(Acrp30浓度分别为25、50、100、200 ng/ml),各组细胞进行常规培养。四亚基偶氮唑盐(MTT)比色法检测各组细胞的OD490nm值及生长抑制率(%)=生长抑制率(%)=(OD对照组-ODAcrp30)/OD对照组×100%,了解细胞增殖情况;根据MTT结果,选取最适全长Acrp30干预浓度为100ng/ml。2.将乳腺癌细胞分为对照组,Acrp30组(加100 ng/ml Acrp30),分别常规培养0,24,48,72h,倒置显微镜观察两组细胞生长情况,包括细胞形态、贴壁、汇合率等;划痕试验了解细胞迁移能力;Western blot法检测LC3-II、LC3-I蛋白的表达情况评价细胞自噬水平;3.常规培养MCF-7细胞,分为对照组,3-MA组(加入3-MA即3-甲基腺嘌呤浓度为2mmol/l),Acrp30组(加100 ng/ml Acrp30),3-MA+Acrp 30组(2mmol/l 3-MA预先处理细胞1h后再加入100ng/ml Acrp30),培养0、24、48、72h,Annexin V-FITC染色结合细胞,流式细胞仪检测不同时间点各组细胞的总凋亡率情况,培养24小时行Western blot法检测LC3II与LC3I蛋白的表达情况。结果1.1 MTT结果显示:OD490nm值方面:培养24h时各组细胞OD490nm值无明显差异(P0.05);48h后,各组细胞OD490nm值随着Acrp30浓度的增加,逐渐下降,但差异无统计学意义(P0.05);72h后,与对照组相比,50、100、200 ng/ml Acrp30组OD490nm值显著降低(P0.05),而100ng/ml Acrp30与加200ng/ml Acrp30组OD490nm值无明显统计学意义(P0.05)。1.2各组细胞的生长抑制率:各组细胞24h生长抑制率无明显差异(P0.05);培养48h后,与对照组和25ng/ml组相比,200ng/ml组生长抑制率明显增高(P0.05),但是200ng/ml Acrp30组与100ng/ml Acrp30组无明显差异(P0.05);培养72h后,与对照组相比,100、200ng/ml Acrp30组生长抑制率明显高于对照组(P0.05),而100ng/ml Acrp30与200ng/ml Acrp30组细胞的生长抑制率无明显差异(P0.05)。(2)倒置显微镜观察细胞生长情况:随着培养时间的延长,100ng/ml Acrp30组培养72h时,细胞对照组汇合率降低,细胞贴壁欠好,细胞形态成不规则形,培养液中可见大量漂浮的细胞。(3)划痕抑制实验结果显示:与对照组相比,24 h Acrp30组细胞爬行距离差异无显著性(P0.05),48 h、72 h Acrp30组爬行距离明显减少(P0.01);(4)Western blot结果:与对照组相比,Acrp30组LC3-II/LC3-I比值24、48 h显著提高(P0.05),72 h有所升高但无统计学意义(P0.05);(5)流式细胞仪检测凋亡率及western blot结果显示:培养24h,各组凋亡率无明显差异(P0.05);培养48h时,与对照组及3-MA组相比,Acrp30组和3-MA+Acrp30组总凋亡率明显增加(P0.01),而Acrp30组与3-MA+Acrp 30组总凋亡率无明显差异;培养72h时,与对照组及3-MA组相比,Acrp30组和3-MA+Acrp 30组总凋亡率明显增加(P0.05),与Acrp30组相比,3-MA+Acrp 30组总凋亡率显著升高(P0.05),3-MA组总凋亡率略高于对照组,但差异无明显统计学意义(P0.05);24h western blot结果提示,与对照组相比,3-MA组LC3II/LC3I有所下降,但无统计学意义(P0.05),Acrp30组和3-MA+Acrp 30组LC3II/LC3I明显升高(P0.05);与Acrp 30组相比3-MA+Acrp 30组LC3II/LC3I明显升高(P0.01);结论(1)Acrp30可抑制MCF-7细胞的增殖和迁移,诱导细胞的自噬和凋亡;(2)抑制细胞自噬可促进Acrp30对MCF-7细胞凋亡的诱导。
[Abstract]:Objective to study the full-length adiponectin (Acrp30) on the proliferation of human breast cancer MCF-7 cell migration, autophagy, apoptosis, and to explore the relationship between autophagy and apoptosis induced by adiponectin. Methods MCF-7 cells were cultured in vitro for 1., divided into control group (without Acrp30) cells into Acrp30 group (Acrp30 concentration was 25,50100200 ng/ml), cells of each group were cultured. The four subunit thiazolyl tetrazolium (MTT) assay to detect the cells than the OD490nm value and growth inhibition rate (%) = growth inhibition rate (%) = (OD group -ODAcrp30) /OD control group * 100%, understand the cell proliferation; according to the results of MTT, select the suitable concentration of 100ng/ml.2. intervention full-length Acrp30 breast cancer cells were divided into control group, Acrp30 group (ng/ml Acrp30 100), respectively, cultured 0,24,48,72h, inverted microscope observation of the two groups including cell growth, cell morphology, adherence, convergence rate; scratch test Understand the cell migration ability; detection of LC3-II Western by blot, the expression level of autophagy evaluation LC3-I protein; 3. of cultured MCF-7 cells were divided into control group, 3-MA group (adding 3-MA 3- methyladenine concentration was 2mmol/l), group Acrp30 (with 100 ng/ml Acrp30 3-MA+Acrp (2mmol/l), 30 groups of 3-MA pretreated cells 1H after adding 100ng/ml Acrp30, Annexin) 0,24,48,72h culture, V-FITC staining combined with detection of total cells, apoptosis cells at different time points was the rate of flow cytometry, cultured for 24 hours with Western blot method to detect LC3II and LC3I protein expression. The results showed that OD490nm 1.1 MTT value: 24h cells were cultured the OD490nm value had no significant difference (P0.05); 48h, OD490nm cell value increased with the increase of the concentration of Acrp30 decreased gradually, but the difference was not statistically significant (P0.05); after 72h, compared with the control group, 50100200 ng/m L Acrp30 group OD490nm was significantly lower (P0.05), 100ng/ml Acrp30 and Acrp30 OD490nm plus 200ng/ml group had no obvious statistical significance (P0.05) in.1.2 cell growth inhibition rate of cells in each group: 24h growth inhibition rate had no significant difference (P0.05); after 48h, compared with control group and 25ng/ml group, 200ng/ml group growth the inhibition rate was significantly higher (P0.05), but the 200ng/ml Acrp30 100ng/ml group and Acrp30 group had no significant difference (P0.05); after 72h, compared with the control group, 100200ng/ml group Acrp30 growth inhibition rate was significantly higher than the control group (P0.05), 100ng/ml Acrp30 and 200ng/ml Acrp30 cell growth inhibition rate was no significant difference (P0.05). (2) the cell growth was observed by inverted microscope: with the prolongation of the culture time, the 100ng/ml Acrp30 group of cultured 72h cells in the control group, reduce the rate of convergence, adherent cells owe good cell morphology into irregular shape, medium Shows a large number of floating cells. (3) the scratch inhibition experiment results show that compared with the control group, 24 h group Acrp30 cells crawling distance had no significant difference (P0.05), 48 h, 72 h Acrp30 group significantly reduced the creeping distance (P0.01); (4) Western blot results: compared with the control group, Acrp30 the ratio of LC3-II/LC3-I 24,48 group H significantly increased (P0.05), 72 h increased but without statistical significance (P0.05); (5) flow cytometry was used to detect the apoptosis rate and Western blot results showed that the cultured 24h, the apoptotic rate was no significant difference (P0.05); 48h culture, compared with the control group and 3-MA group. Acrp30 group and 3-MA+Acrp30 group significantly increased the total apoptosis rate (P0.01), Acrp30 group and 3-MA+Acrp 30 group total apoptosis rate had no significant difference; culture of 72h, compared with the control group and 3-MA group, Acrp30 group and 3-MA+Acrp 30 group total apoptosis rate (P0.05) increased significantly, compared with the Acrp30 group, 3-MA+Acrp 30 group the apoptosis rate significantly With the increase of (P0.05) 3-MA group, the apoptosis rate was slightly higher than the control group, but the difference was not statistically significant (P0.05); 24h Western blot showed that, compared with the control group, 3-MA group, LC3II/LC3I decreased, but there was no statistical significance (P0.05), Acrp30 group and 3-MA+Acrp 30 group LC3II/LC3I increased significantly (P0.05); compared with the Acrp 30 group 3-MA+Acrp 30 group LC3II/LC3I increased significantly (P0.01); (1) conclusion Acrp30 can inhibit the proliferation and migration of MCF-7 cells, induce cell autophagy and apoptosis; (2) the inhibition of autophagy could promote cell apoptosis induced by Acrp30 on MCF-7.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

【参考文献】

相关期刊论文 前3条

1 ;Adipocytokines and breast cancer risk[J];Chinese Medical Journal;2007年18期

2 毋飞飞;王佑民;王琼;许敏;胡红琳;;重组人球状脂联素抑制MCF-7细胞生长及NF-κB的相关性[J];安徽医科大学学报;2014年02期

3 ;Adiponectin as a negative regulator in obesity-related mammary carcinogenesis[J];Cell Research;2007年04期

，

本文编号：1426969