本文選題：乳腺癌 + p53基因　； 參考：《安徽醫(yī)科大學(xué)》2015年碩士論文

【摘要】：研究目的自1979年p53基因被發(fā)現(xiàn)以來,其結(jié)構(gòu)與功能已被廣泛認(rèn)知,其轉(zhuǎn)錄后編碼的p53蛋白已成為腫瘤細(xì)胞發(fā)生發(fā)展過程中重要的參與者。在100多種人類腫瘤中,有60%以上的腫瘤發(fā)生與p53基因突變有關(guān),目前已知人乳腺癌p53基因突變率為20%,而其余80%的乳腺癌均含有野生型的p53,與胃癌、大腸癌、肺癌等腫瘤類型不同,乳腺癌發(fā)生和發(fā)展的原因除了抑癌基因p53的基因突變外,大部分是由于基因序列正常的p53(野生型p53)沒有發(fā)揮應(yīng)有的作用所致。通過腺病毒載體將外源性腫瘤抑制基因(p53基因)導(dǎo)入腫瘤內(nèi),通過細(xì)胞凋亡、旁觀者效應(yīng)、抑制多藥耐藥基因表達(dá)等多種抗腫瘤機(jī)制,抑制腫瘤組織的生長,促進(jìn)腫瘤細(xì)胞的凋亡,已成為一種新的腫瘤治療方向。p53和雌激素受體(ER)都是乳腺癌發(fā)生發(fā)展重要的因子,研究發(fā)現(xiàn),p53能促進(jìn)ER表達(dá),反過來,ER可拮抗p53的促凋亡作用,抑制p53介導(dǎo)的細(xì)胞凋亡反應(yīng)。氟維司群(fulvestrant)是全新的乳腺癌內(nèi)分泌治療藥物,稱為選擇性雌激素受體下調(diào)劑(Selective Estrogen Receptor Downregulators, SERDs),能夠高親和力地結(jié)合阻斷并下調(diào)雌激素受體(ER)的數(shù)量,使激素受體上的轉(zhuǎn)錄活性區(qū)域AF1和AF2均失活,并加速了雌激素受體功能的喪失。我們通過聯(lián)合應(yīng)用重組人p53腺病毒和氟維司群,觀察乳腺癌細(xì)胞系MCF-7的形態(tài)學(xué)變化、p53蛋白和ER蛋白的表達(dá)水平、MCF-7細(xì)胞的凋亡情況以及增殖抑制率等,探討氟維司群是否能阻斷并下調(diào)ER,解除其對p53的抑制作用,觀察兩者是否具有協(xié)同作用,為基礎(chǔ)研究與臨床乳腺癌治療的新方法開拓新的方向。研究方法1. 比較各組細(xì)胞的生長及形態(tài)學(xué)變化：設(shè)對照組(空載病毒組)、氟維司群(1μmol/1)組、Ad-p53 (50MOI)組、Ad-p53 (50MOI)+氟維司群(1μmol/l)組作用于MCF-7細(xì)胞72h后,倒置顯微鏡觀察不同實(shí)驗(yàn)組細(xì)胞的生長情況以及形態(tài)學(xué)變化。2. 比較各組細(xì)胞的增殖抑制率：設(shè)對照組(空載病毒組)、氟維司群(1μmol/l)組、Ad-p53 (50MOI)組、Ad-p53 (50MOI)+氟維司群(1μmol/l)組作用于MCF-7細(xì)胞72h后,比較各實(shí)驗(yàn)組細(xì)胞的增殖抑制率。3. 比較各組細(xì)胞的凋亡率：設(shè)對照組(空載病毒組)、氟維司群(1μmol/l)組、Ad-p53 (50MOI)組、Ad-p53 (50MOI)+氟維司群(1μmol/l)組作用于MCF-7細(xì)胞72h后,用流式細(xì)胞儀AnnexinV-FITC/PI雙標(biāo)法檢測分析不同處理組MCF-7細(xì)胞的凋亡情況。4. 比較各組細(xì)胞p53和ER蛋白的表達(dá)水平：設(shè)對照組(空載病毒組)、氟維司群(1μmol/l)組、Ad-p53 (50MOI)組、Ad-p53 (50MOI)+氟維司群(1μmol/l)組作用于MCF-7細(xì)胞72h后,檢測各實(shí)驗(yàn)組細(xì)胞p53和ER蛋白的表達(dá)情況。研究結(jié)果1.倒置顯微鏡觀察細(xì)胞生長狀態(tài)：對照組MCF-7細(xì)胞正常貼壁生長,培養(yǎng)基清澈無渾濁,細(xì)胞生長狀態(tài)佳,氟維司群組細(xì)胞貼壁生長可,培養(yǎng)基稍渾濁,出現(xiàn)一定的懸浮細(xì)胞；Ad-p53組可見貼壁細(xì)胞生長狀態(tài)不佳,細(xì)胞密度較低,細(xì)胞形態(tài)不規(guī)則,出現(xiàn)細(xì)胞碎片、細(xì)胞脫落等細(xì)胞凋亡的改變；Ad-p53與氟維司群聯(lián)合作用于MCF-7細(xì)胞后,高倍鏡下可見散在生長細(xì)胞,密度較低,貼壁細(xì)胞數(shù)量大幅減少,細(xì)胞出現(xiàn)皺縮,懸浮細(xì)胞數(shù)目更多,細(xì)胞培養(yǎng)液出現(xiàn)渾濁。2.流式細(xì)胞術(shù)檢測細(xì)胞凋亡結(jié)果：對照組、氟維司群組、Ad-p53組、 Ad-p53+氟維司群組中細(xì)胞凋亡率分別是(6.3±1.5)%、(13.3±1.2)%、(14.5±2.9)%和(38.1±5.9)%。3.MTS法檢測細(xì)胞增殖抑制率結(jié)果：50 MOI Ad-p53和氟維司群分別處理MCF-7細(xì)胞24"96 h后,增殖抑制率分別為(8.21±0.54)%、(28.5±1.42)%、(50.14±0.78)%、(58.25±2.92)%和(9.73±1.68)%、(25.26±0.82)%、(35.25±3.94)%、(46.37±2.56)%；氟維司群與Ad-p53聯(lián)合作用24～96h后,細(xì)胞的增殖抑制率為(12.42±1.76)%、(35.20±0.58)%、(62.08±2.56)%及(75.43%±3.56)%。4.蛋白印跡法(Western blot)檢測結(jié)果：對照組、氟維司群組中均有p53蛋白的低表達(dá)；Ad-p53組、Ad-p53+氟維司群組中p53蛋白高表達(dá)；與對照組相比,Ad-p53組細(xì)胞的ER蛋白表達(dá)增加,但是聯(lián)合用藥組ER蛋白表達(dá)降低。研究結(jié)論1.Ad-p53感染乳腺癌MCF-7后,細(xì)胞生長緩慢且形態(tài)學(xué)發(fā)生明顯改變,且當(dāng)與氟維司群聯(lián)合使用時(shí)細(xì)胞生長幾乎停止,細(xì)胞密度極低,調(diào)亡細(xì)胞更多,細(xì)胞碎片更多,細(xì)胞凋亡現(xiàn)象更明顯。2.Ad-p53或氟維司群單獨(dú)作用乳腺癌MCF-7細(xì)胞后,MTS法檢測細(xì)胞的各時(shí)期增殖抑制率均增加；當(dāng)Ad-p53與氟維司群聯(lián)合作用于MCF-7細(xì)胞,24～96 h的各時(shí)期MCF-7細(xì)胞的增殖抑制率均高于各單藥組。3.Ad-p53或氟維司群單藥均可明顯誘導(dǎo)乳腺癌MCF-7細(xì)胞發(fā)生凋亡增加,聯(lián)合用藥組誘導(dǎo)細(xì)胞凋亡作用更加明顯。4.對照組、氟維司群組中均有p53蛋白的低表達(dá)；Ad-p53組、Ad-p53+氟維司群組中p53蛋白高表達(dá),說明腺病毒介導(dǎo)的p53基因成功轉(zhuǎn)入MCF-7細(xì)胞中并有效表達(dá),但是Ad-p53組和聯(lián)合組蛋白表達(dá)沒有明顯差異；與對照組相比,Ad-p53組細(xì)胞的ER蛋白表達(dá)增加,但是聯(lián)合用藥組ER蛋白表達(dá)降低。
[Abstract]:Its structure and function have been widely recognized since the discovery of the p53 gene in 1979. The post transcriptional encoded p53 protein has become an important participant in the development of tumor cells. In more than 100 human tumors, more than 60% of the tumors are associated with the mutation of the p53 gene. At present, the mutation rate of p53 gene in human breast cancer is known. For 20%, the other 80% breast cancers all contain wild type p53, which are different from cancer types such as gastric cancer, large intestine cancer and lung cancer. The cause of breast cancer is due to the gene mutation of tumor suppressor gene p53, most of which is caused by the normal p53 (wild type p53) in the gene sequence. The tumor suppressor gene (p53 gene) is introduced into the tumor, and through the apoptosis, bystander effect, inhibition of multidrug resistance gene expression and other anti-tumor mechanisms, inhibiting the growth of tumor tissue and promoting the apoptosis of tumor cells, it has become a new tumor treatment direction.P53 and estrogen receptor (ER) are the important causes of the development of breast cancer. The study found that p53 can promote the expression of ER, in turn, ER can antagonize the apoptotic effect of p53 and inhibit the apoptosis response mediated by p53. The fluavinic group (fulvestrant) is a new endocrine therapy drug for breast cancer, known as the selective estrogen receptor (Selective Estrogen Receptor Downregulators, SERDs), and is capable of high affinity. Combined with the number of blocking and down-regulation of estrogen receptor (ER), both AF1 and AF2 were inactivated in the transcriptional active region of the hormone receptor and the loss of the function of estrogen receptor was accelerated. We observed the morphological changes of MCF-7 in the breast cancer cell line, the expression of p53 protein and ER protein by combining the recombinant human p53 adenovirus and the fluinosus group, MCF, MCF. -7 cell apoptosis and proliferation inhibition rate and so on, to explore whether the fluvia group can block and downregulate ER, relieve its inhibitory effect on p53, observe the synergistic effect between the two, and explore new directions for the new methods of basic research and clinical breast cancer treatment. Method 1. compare the growth and morphological changes of cells in each group: set up Control group (unloaded virus group), fluinus group (1 mol/1) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI) + fluidus group (1 mol/l) group after MCF-7 cell 72h, inverted microscope observation of the growth of cells in different experimental groups and morphological changes.2. compared each group of cell proliferation inhibition rate: set control group (empty virus group), fluinus Group (1 mol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI) + fluflund group (1 mu) group acted on MCF-7 cell 72h, compared the proliferation inhibition rate of each experimental group, compared the apoptosis rate of each group: the control group (empty virus group), the fluflex group (1 mu mol/l) group, Ad-p53 (50MOI) group, 1 micron group (1 mu) group. After MCF-7 cell 72h, the apoptosis of MCF-7 cells in different treatment groups was detected and analyzed by flow cytometry AnnexinV-FITC/PI double standard.4. comparison of the expression level of p53 and ER protein in each group: the control group (empty virus group), the fluflex group (1 mu mol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI) + fluflex group (1 mu) group acted on the After F-7 cell 72h, the expression of p53 and ER protein in all the experimental groups was detected. Results 1. inverted microscope was used to observe the cell growth state: the MCF-7 cells in the control group were normally adhered to the wall, the culture medium was clear and no turbidity, the cell growth state was good, the cell wall growth of the fluorine group group, the medium turbidity in the culture medium, and the certain suspended cells; A In group d-p53, the growth of adherent cells was poor, cell density was low, cell morphology was irregular, cell debris, cell fall and other cell apoptosis changes. After the combination of Ad-p53 and fluinisus group, the growth cells were scattered under the high magnification microscope, the density was low, the number of adherent cells decreased significantly, and the cell crinkled, and the cells crinkled and suspended. The number of floating cells was more, and the apoptotic results were detected by turbid.2. flow cytometry in the cell culture solution: the apoptosis rates in the control group, the flufir group, the Ad-p53 group and the Ad-p53+ flock group were (6.3 + 1.5)%, (13.3 + 1.2)%, (14.5 + 2.9)% and (38.1 + 5.9)%.3.MTS method to detect the cell proliferation inhibition rate: 50 MOI Ad-p53 and fluorine. The proliferation inhibition rate of MCF-7 cells was (8.21 + 0.54)%, (28.5 + 1.42)%, (50.14 + 0.78)%, (58.25 + 2.92)%, (58.25 + 2.92)% and (9.73 + 1.68)%, (25.26 + 1.68)%, (58.25 + 0.78)%, (58.25 + 2.92)%, (50.14 +%)%, and (28.5 + 1.42)%. % and (75.43% + 3.56)%.4. blot method (Western blot) test results: the control group, the low expression of p53 protein in the control group, and the high expression of p53 protein in the group Ad-p53 and the Ad-p53+ fluovers group; and the ER protein expression in the Ad-p53 group was increased compared with the control group, but the expression of ER protein in the combined group was reduced. The conclusion 1.Ad-p of the study was 1.Ad-p. 53 after MCF-7 infection of breast cancer, the cell growth is slow and the morphology changes obviously. And when combined with the fluinfill group, the cell growth almost stops, the cell density is very low, the cell number is more, the cell debris is more, the apoptosis phenomenon is more obvious that.2.Ad-p53 or the fluinfill group acts on the MCF-7 cells of breast cancer alone, and the MTS method is used to detect the cells. The proliferation inhibition rate increased at all times. When Ad-p53 and fluvia were combined with MCF-7 cells, the proliferation inhibition rate of MCF-7 cells in each period of 24~96 h was higher than that of the single drug group.3.Ad-p53 or the single drug of fluodex group, which could induce the increase of apoptosis of MCF-7 cells in breast cancer, and the combined drug group could induce apoptosis more obviously. In the.4. control group, the low expression of p53 protein in the fluovers group and the high expression of p53 protein in the group Ad-p53 and the Ad-p53+ fluovers group showed that the adenovirus mediated p53 gene was successfully transferred into MCF-7 cells and expressed effectively, but there was no significant difference in the expression of the protein in the Ad-p53 group and the combined histone. Compared with the control group, the ER protein table of the Ad-p53 group cells was compared with the control group. The expression of ER decreased in combination group.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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