本文關(guān)鍵詞：脂聯(lián)素對人乳腺癌MCF-7細胞生物學行為的影響及其所誘導(dǎo)的凋亡與自噬相關(guān)性的研究　出處：《安徽醫(yī)科大學》2015年碩士論文　論文類型：學位論文

  更多相關(guān)文章： Acrp30 MCF-7細胞 增殖 自噬 凋亡

【摘要】：目的研究全長脂聯(lián)素(Acrp30)在人乳腺癌MCF-7細胞增殖、遷移、自噬、凋亡中的作用,并探討自噬與脂聯(lián)素誘導(dǎo)的凋亡之間的關(guān)系。方法1.體外培養(yǎng)MCF-7細胞,分為對照組(未加Acrp30)細胞,加入Acrp30組(Acrp30濃度分別為25、50、100、200 ng/ml),各組細胞進行常規(guī)培養(yǎng)。四亞基偶氮唑鹽(MTT)比色法檢測各組細胞的OD490nm值及生長抑制率(%)=生長抑制率(%)=(OD對照組-ODAcrp30)/OD對照組×100%,了解細胞增殖情況;根據(jù)MTT結(jié)果,選取最適全長Acrp30干預(yù)濃度為100ng/ml。2.將乳腺癌細胞分為對照組,Acrp30組(加100 ng/ml Acrp30),分別常規(guī)培養(yǎng)0,24,48,72h,倒置顯微鏡觀察兩組細胞生長情況,包括細胞形態(tài)、貼壁、匯合率等;劃痕試驗了解細胞遷移能力;Western blot法檢測LC3-II、LC3-I蛋白的表達情況評價細胞自噬水平;3.常規(guī)培養(yǎng)MCF-7細胞,分為對照組,3-MA組(加入3-MA即3-甲基腺嘌呤濃度為2mmol/l),Acrp30組(加100 ng/ml Acrp30),3-MA+Acrp 30組(2mmol/l 3-MA預(yù)先處理細胞1h后再加入100ng/ml Acrp30),培養(yǎng)0、24、48、72h,Annexin V-FITC染色結(jié)合細胞,流式細胞儀檢測不同時間點各組細胞的總凋亡率情況,培養(yǎng)24小時行Western blot法檢測LC3II與LC3I蛋白的表達情況。結(jié)果1.1 MTT結(jié)果顯示:OD490nm值方面:培養(yǎng)24h時各組細胞OD490nm值無明顯差異(P0.05);48h后,各組細胞OD490nm值隨著Acrp30濃度的增加,逐漸下降,但差異無統(tǒng)計學意義(P0.05);72h后,與對照組相比,50、100、200 ng/ml Acrp30組OD490nm值顯著降低(P0.05),而100ng/ml Acrp30與加200ng/ml Acrp30組OD490nm值無明顯統(tǒng)計學意義(P0.05)。1.2各組細胞的生長抑制率:各組細胞24h生長抑制率無明顯差異(P0.05);培養(yǎng)48h后,與對照組和25ng/ml組相比,200ng/ml組生長抑制率明顯增高(P0.05),但是200ng/ml Acrp30組與100ng/ml Acrp30組無明顯差異(P0.05);培養(yǎng)72h后,與對照組相比,100、200ng/ml Acrp30組生長抑制率明顯高于對照組(P0.05),而100ng/ml Acrp30與200ng/ml Acrp30組細胞的生長抑制率無明顯差異(P0.05)。(2)倒置顯微鏡觀察細胞生長情況:隨著培養(yǎng)時間的延長,100ng/ml Acrp30組培養(yǎng)72h時,細胞對照組匯合率降低,細胞貼壁欠好,細胞形態(tài)成不規(guī)則形,培養(yǎng)液中可見大量漂浮的細胞。(3)劃痕抑制實驗結(jié)果顯示:與對照組相比,24 h Acrp30組細胞爬行距離差異無顯著性(P0.05),48 h、72 h Acrp30組爬行距離明顯減少(P0.01);(4)Western blot結(jié)果:與對照組相比,Acrp30組LC3-II/LC3-I比值24、48 h顯著提高(P0.05),72 h有所升高但無統(tǒng)計學意義(P0.05);(5)流式細胞儀檢測凋亡率及western blot結(jié)果顯示:培養(yǎng)24h,各組凋亡率無明顯差異(P0.05);培養(yǎng)48h時,與對照組及3-MA組相比,Acrp30組和3-MA+Acrp30組總凋亡率明顯增加(P0.01),而Acrp30組與3-MA+Acrp 30組總凋亡率無明顯差異;培養(yǎng)72h時,與對照組及3-MA組相比,Acrp30組和3-MA+Acrp 30組總凋亡率明顯增加(P0.05),與Acrp30組相比,3-MA+Acrp 30組總凋亡率顯著升高(P0.05),3-MA組總凋亡率略高于對照組,但差異無明顯統(tǒng)計學意義(P0.05);24h western blot結(jié)果提示,與對照組相比,3-MA組LC3II/LC3I有所下降,但無統(tǒng)計學意義(P0.05),Acrp30組和3-MA+Acrp 30組LC3II/LC3I明顯升高(P0.05);與Acrp 30組相比3-MA+Acrp 30組LC3II/LC3I明顯升高(P0.01);結(jié)論(1)Acrp30可抑制MCF-7細胞的增殖和遷移,誘導(dǎo)細胞的自噬和凋亡;(2)抑制細胞自噬可促進Acrp30對MCF-7細胞凋亡的誘導(dǎo)。
[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.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R737.9

【參考文獻】

相關(guān)期刊論文 前3條

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

2 毋飛飛;王佑民;王瓊;許敏;胡紅琳;;重組人球狀脂聯(lián)素抑制MCF-7細胞生長及NF-κB的相關(guān)性[J];安徽醫(yī)科大學學報;2014年02期

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

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本文編號：1426969