【摘要】：目的:DLC(Deleted in liver cancer)家族基因是新近發(fā)現(xiàn)抑癌基因家族,研究證實(shí)該家族基因在多種腫瘤組織的低表達(dá)或表達(dá)缺失。DLCs蛋白在多種腫瘤的發(fā)生發(fā)展中可能發(fā)揮重要作用。本研究旨在探討DLC-3對(duì)乳腺癌MCF-7細(xì)胞的增殖、遷移及凋亡生物學(xué)行為的影響,觀察DLC-3在腫瘤發(fā)生和發(fā)展的過(guò)程中可能起到的作用。方法:(1)通過(guò)脂質(zhì)體搭載目的基因DLC-3,進(jìn)行瞬時(shí)轉(zhuǎn)染使得MCF-7細(xì)胞中的DLC-3表達(dá)水平顯著提高,得到過(guò)表達(dá)組細(xì)胞樣本;以相同條件對(duì)MCF-7細(xì)胞進(jìn)行空白質(zhì)粒的轉(zhuǎn)染,得到陰性對(duì)照組細(xì)胞樣本;以常規(guī)培養(yǎng)的MCF-7細(xì)胞作為實(shí)驗(yàn)的空白對(duì)照組。轉(zhuǎn)染操作后8h進(jìn)行換液,常規(guī)培養(yǎng)48h后,于熒光顯微鏡下觀察熒光蛋白表達(dá)情況,收集各樣本細(xì)胞用于進(jìn)一步實(shí)驗(yàn)(2)利用Real-time PCR技術(shù)對(duì)收集的各組樣本細(xì)胞進(jìn)行DLC-3的表達(dá)水平檢測(cè),以陰性對(duì)照組ΔCt值為矯正,2-ΔΔCt法進(jìn)行相對(duì)定量分析。(3)CCK-8法檢測(cè)各樣本MCF-7細(xì)胞的增殖水平;進(jìn)行細(xì)胞劃痕實(shí)驗(yàn)觀察各樣本MCF-7細(xì)胞遷移能力的差異;利用流式細(xì)胞術(shù)進(jìn)行各樣本MCF-7細(xì)胞中凋亡細(xì)胞比例的比較。結(jié)果:(1)過(guò)表達(dá)組1、2中的DLC-3相對(duì)表達(dá)量2-ΔΔCt值與空白組比較均存在顯著差異(p0.01),瞬時(shí)轉(zhuǎn)染后過(guò)表達(dá)組樣本細(xì)胞中的DLC-3表達(dá)水平顯著升高;(2)CCK-8法檢測(cè)得到:過(guò)表達(dá)組MCF-7細(xì)胞的增殖水平在培養(yǎng)12h、24h、48h后均低于陰性對(duì)照組和空白組樣本水平,比較顯示有顯著差異(p0.01),陰性對(duì)照組和空白組樣本的增殖水平不存在有統(tǒng)計(jì)學(xué)意義差異(3)細(xì)胞劃痕實(shí)驗(yàn)顯示:劃痕處理后12h,過(guò)表達(dá)組樣本的愈合率與空白組比較存在顯著差異(p0.01),過(guò)表達(dá)組與陰性對(duì)照組,陰性對(duì)照與空白組之間愈合率比較差異無(wú)統(tǒng)計(jì)學(xué)意義;劃痕處理48h后,過(guò)表達(dá)組樣本的愈合率與陰性對(duì)照組以及空白組樣本比較均存在顯著差異(p0.01),陰性對(duì)照組與空白組樣本的愈合率比較不存在有統(tǒng)計(jì)學(xué)意義的差異(4)流式細(xì)胞術(shù)檢測(cè)得到:過(guò)表達(dá)組樣本中凋亡細(xì)胞所占比率均高于陰性對(duì)照組和空白組樣本(p0.05),陰性對(duì)照組和空白組樣本凋亡細(xì)胞比率之間不存在差異(p0.05)。結(jié)論:通過(guò)現(xiàn)象學(xué)實(shí)驗(yàn)發(fā)現(xiàn)乳腺癌MCF-7細(xì)胞中DLC-3表達(dá)水平提高可以降低細(xì)胞增殖水平,抑制其細(xì)胞的遷移能力,可以促進(jìn)其細(xì)胞發(fā)生凋亡。
[Abstract]:Objective\ *\ The purpose of this study was to investigate the effects of DLC-3 on the biological behavior of proliferation, migration and apoptosis of breast cancer MCF-7 cells, and to observe the possible role of DLC-3 in the development of breast cancer. Methods: (1) transient transfection of the target gene DLC-3 by liposome increased the expression level of DLC-3 in MCF-7 cells, obtained samples of over-expressed cells, and transfected MCF-7 cells with blank plasmids under the same conditions. Cell samples of negative control group were obtained, and MCF-7 cells were cultured as blank control group. After 8 hours of transfection and 48 hours of conventional culture, the expression of fluorescent protein was observed under fluorescence microscope. Each sample cell was collected for further experiment (2) Real-time PCR technique was used to detect the expression of DLC-3 in each group of cells. The relative quantitative analysis was performed with 螖 Ct value of negative control group as correction 2- 螖 Ct method. (3) the proliferation of MCF-7 cells in each sample was detected by CCK-8 method, and the migration ability of MCF-7 cells in each sample was observed by cell scratch test. Flow cytometry was used to compare the percentage of apoptotic cells in MCF-7 cells. Results: (1) there was significant difference in the relative expression of DLC-3 between the overexpression group and the blank group (p0.01), and the expression of DLC-3 in the overexpression group was significantly higher than that in the control group (p0.01). (2) the results of CCK-8 method showed that: MCF-7 cells in the over-expressed group were detected by CCK-8 method, and the expression level of DLC-3 in the overexpression group was significantly higher than that in the control group (p0.01). The level of proliferation in the control group and the blank group was lower than that in the negative control group and the blank group after 12 h culture for 24 h or 48 h. Comparison showed significant difference (p0.01). There was no significant difference in proliferative level between negative control group and blank group. (3) Cell scratch test showed that the healing rate of overexpression group was higher than that of blank group 12 hours after scratch treatment. In the significant difference (p0.01), the overexpression group and the negative control group, There was no significant difference in the healing rate between the negative control group and the blank group. There was significant difference in the healing rate between the overexpression group and the negative control group and the blank group (p0.01). There was no significant difference in the healing rate between the negative control group and the blank group (4) flow cytometry was used to detect the healing rate. The results showed that the percentage of apoptotic cells in overexpression group was higher than that in negative control group and blank group (p0.05), but there was no difference between negative control group and blank group (p0.05). Conclusion: the increase of DLC-3 expression in breast cancer MCF-7 cells can reduce the proliferation of MCF-7 cells, inhibit the migration ability of MCF-7 cells and promote the apoptosis of MCF-7 cells.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.2

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