本文關(guān)鍵詞：脂聯(lián)素對(duì)人乳腺癌MCF-7細(xì)胞生物學(xué)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(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Acrp30 MCF-7細(xì)胞 增殖 自噬 凋亡

【摘要】：目的研究全長(zhǎng)脂聯(lián)素(Acrp30)在人乳腺癌MCF-7細(xì)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(xì)胞,分為對(duì)照組(未加Acrp30)細(xì)胞,加入Acrp30組(Acrp30濃度分別為25、50、100、200 ng/ml),各組細(xì)胞進(jìn)行常規(guī)培養(yǎng)。四亞基偶氮唑鹽(MTT)比色法檢測(cè)各組細(xì)胞的OD490nm值及生長(zhǎng)抑制率(%)=生長(zhǎng)抑制率(%)=(OD對(duì)照組-ODAcrp30)/OD對(duì)照組×100%,了解細(xì)胞增殖情況;根據(jù)MTT結(jié)果,選取最適全長(zhǎng)Acrp30干預(yù)濃度為100ng/ml。2.將乳腺癌細(xì)胞分為對(duì)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(xì)胞生長(zhǎng)情況,包括細(xì)胞形態(tài)、貼壁、匯合率等;劃痕試驗(yàn)了解細(xì)胞遷移能力;Western blot法檢測(cè)LC3-II、LC3-I蛋白的表達(dá)情況評(píng)價(jià)細(xì)胞自噬水平;3.常規(guī)培養(yǎng)MCF-7細(xì)胞,分為對(duì)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(xì)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(xì)胞,流式細(xì)胞儀檢測(cè)不同時(shí)間點(diǎn)各組細(xì)胞的總凋亡率情況,培養(yǎng)24小時(shí)行Western blot法檢測(cè)LC3II與LC3I蛋白的表達(dá)情況。結(jié)果1.1 MTT結(jié)果顯示:OD490nm值方面:培養(yǎng)24h時(shí)各組細(xì)胞OD490nm值無(wú)明顯差異(P0.05);48h后,各組細(xì)胞OD490nm值隨著Acrp30濃度的增加,逐漸下降,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);72h后,與對(duì)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(wú)明顯統(tǒng)計(jì)學(xué)意義(P0.05)。1.2各組細(xì)胞的生長(zhǎng)抑制率:各組細(xì)胞24h生長(zhǎng)抑制率無(wú)明顯差異(P0.05);培養(yǎng)48h后,與對(duì)照組和25ng/ml組相比,200ng/ml組生長(zhǎng)抑制率明顯增高(P0.05),但是200ng/ml Acrp30組與100ng/ml Acrp30組無(wú)明顯差異(P0.05);培養(yǎng)72h后,與對(duì)照組相比,100、200ng/ml Acrp30組生長(zhǎng)抑制率明顯高于對(duì)照組(P0.05),而100ng/ml Acrp30與200ng/ml Acrp30組細(xì)胞的生長(zhǎng)抑制率無(wú)明顯差異(P0.05)。(2)倒置顯微鏡觀察細(xì)胞生長(zhǎng)情況:隨著培養(yǎng)時(shí)間的延長(zhǎng),100ng/ml Acrp30組培養(yǎng)72h時(shí),細(xì)胞對(duì)照組匯合率降低,細(xì)胞貼壁欠好,細(xì)胞形態(tài)成不規(guī)則形,培養(yǎng)液中可見(jiàn)大量漂浮的細(xì)胞。(3)劃痕抑制實(shí)驗(yàn)結(jié)果顯示:與對(duì)照組相比,24 h Acrp30組細(xì)胞爬行距離差異無(wú)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(duì)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(wú)統(tǒng)計(jì)學(xué)意義(P0.05);(5)流式細(xì)胞儀檢測(cè)凋亡率及western blot結(jié)果顯示:培養(yǎng)24h,各組凋亡率無(wú)明顯差異(P0.05);培養(yǎng)48h時(shí),與對(duì)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(wú)明顯差異;培養(yǎng)72h時(shí),與對(duì)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(duì)照組,但差異無(wú)明顯統(tǒng)計(jì)學(xué)意義(P0.05);24h western blot結(jié)果提示,與對(duì)照組相比,3-MA組LC3II/LC3I有所下降,但無(wú)統(tǒng)計(jì)學(xué)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(xì)胞的增殖和遷移,誘導(dǎo)細(xì)胞的自噬和凋亡;(2)抑制細(xì)胞自噬可促進(jìn)Acrp30對(duì)MCF-7細(xì)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(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

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(xì)胞生長(zhǎng)及NF-κB的相關(guān)性[J];安徽醫(yī)科大學(xué)學(xué)報(bào);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(hào)：1426969