本文選題：LV3-si-hOTUD7B + pEGFP-hOTUD7B　； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：目的:乳腺癌對(duì)世界女性造成的身心傷害早已不容樂(lè)觀。乳腺癌的發(fā)生、發(fā)展是由多基因、多通路、多反應(yīng)相互作用的結(jié)果,其發(fā)病率和病死率呈現(xiàn)逐年上升的趨勢(shì)。腫瘤的增殖速率、發(fā)生轉(zhuǎn)移及轉(zhuǎn)移部位不同直接影響著乳腺癌患者的生存期。近年來(lái)乳腺癌患者的發(fā)病年齡趨向于年輕化,發(fā)生侵襲、轉(zhuǎn)移的機(jī)率大大增加,導(dǎo)致許多患者就診時(shí)已經(jīng)失去了手術(shù)時(shí)機(jī),直接影響著患者總生存期。所以抑制腫瘤的生長(zhǎng)、阻斷腫瘤的轉(zhuǎn)移,對(duì)提高患者的生存率、改善生活質(zhì)量發(fā)揮著至關(guān)重要的作用。然而腫瘤血管生成是引發(fā)惡性腫瘤發(fā)生、發(fā)展、轉(zhuǎn)移的根本源頭,所以抑制乳腺癌細(xì)胞的血管生成對(duì)治療乳腺癌具有極其重大的意義。其中核因子-Kappa B(NF-κB)可調(diào)節(jié)血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)的表達(dá),而去泛素化酶對(duì)于NF-κB的調(diào)節(jié)至關(guān)重要。有研究發(fā)現(xiàn)NF-κB不僅能夠在胃癌、甲狀腺癌、乳腺癌中促進(jìn)VEGF的表達(dá),還能夠促進(jìn)淋巴結(jié)的轉(zhuǎn)移。去泛素化酶OTUD7B能夠阻止NF-κB激活,而NF-κB能夠調(diào)節(jié)癌細(xì)胞遷移、侵襲、血管內(nèi)皮生長(zhǎng)因子的表達(dá)等。所以明確去泛素化酶OTUD7B與乳腺癌細(xì)胞增殖、遷移、凋亡及VEGF表達(dá)之間的關(guān)系,可能為治療乳腺癌提供新的方法。方法:構(gòu)建慢病毒小干擾RNA沉默載體(LV3-si-h OTUD7B)及對(duì)照載體(LV3-NC),構(gòu)建帶有綠色熒光蛋白標(biāo)簽的人OTUD7B表達(dá)質(zhì)粒(pEGFP-h OTUD7B)及對(duì)照質(zhì)粒(pEGFP-CI)。用包含LV3-si-h OTUD7B、LV3-NC、pEGFP-h OTUD7B、pEGFP-CI慢病毒液分別感染MCF-7細(xì)胞。MTS法檢測(cè)OTUD7B對(duì)MCF-7細(xì)胞增殖的影響;細(xì)胞劃痕實(shí)驗(yàn)評(píng)價(jià)OTUD7B對(duì)MCF-7細(xì)胞遷移能力的影響;流式細(xì)胞術(shù)PI染色法檢測(cè)OTUD7B對(duì)MCF-7細(xì)胞周期的影響;蛋白質(zhì)印跡法(Western blot)檢測(cè)OTUD7B對(duì)MCF-7細(xì)胞細(xì)胞質(zhì)NF-κBP65和細(xì)胞核NF-κBP65的表達(dá)水平的調(diào)節(jié);RT-PCR檢測(cè)OTUD7B對(duì)VEGF m RNA的相對(duì)表達(dá)水平的影響;ELISA檢測(cè)OTUD7B對(duì)MCF-7乳腺癌細(xì)胞分泌VEGF的調(diào)節(jié)。應(yīng)用SPSS19.0統(tǒng)計(jì)軟件進(jìn)行處理,所有數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(?)表示,均數(shù)比較采用t檢驗(yàn)或單因素方差分析(One-way ANOVA),以P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:通過(guò)MTS法檢測(cè)細(xì)胞增殖顯示,轉(zhuǎn)染pEGFP-h OTUD7B后,MCF-7乳腺癌細(xì)胞增殖受到抑制,轉(zhuǎn)染LV3-si-h OTUD7B后,MCF-7乳腺癌細(xì)胞增殖得到促進(jìn)。細(xì)胞劃痕實(shí)驗(yàn)表明,轉(zhuǎn)染pEGFP-h OTUD7B后,MCF-7乳腺癌細(xì)胞遷移受到抑制,轉(zhuǎn)染LV3-si-h OTUD7B后,MCF-7乳腺癌細(xì)胞遷移能力升高。流式細(xì)胞術(shù)PI染色檢測(cè)細(xì)胞周期結(jié)果提示pEGFP-h OTUD7B可調(diào)節(jié)MCF-7細(xì)胞周期阻滯在G1期。Western blot結(jié)果分析顯示,與陰性對(duì)照組及正常對(duì)照組相比,pEGFP-h OTUD7B能夠下調(diào)MCF-7乳腺癌細(xì)胞核蛋白NF-κBP65的表達(dá)水平,而細(xì)胞質(zhì)內(nèi)NF-κBP65卻無(wú)明顯差異。RT-PCR結(jié)果提示,在m RNA水平,pEGFP-h OTUD7B抑制了VEGF的表達(dá)。ELISA法檢測(cè)MCF-7細(xì)胞條件培養(yǎng)基中的VEGF含量結(jié)果顯示,與陰性對(duì)照組及正常對(duì)照組相比,pEGFP-h OTUD7B組條件培養(yǎng)基中VEGF含量低。結(jié)論:OTUD7B能夠抑制MCF-7乳腺癌細(xì)胞增殖、遷移能力,可調(diào)節(jié)MCF-7乳腺癌細(xì)胞阻滯于G1期,同時(shí)還可抑制MCF-7乳腺癌細(xì)胞對(duì)VEGF的表達(dá)和分泌。
[Abstract]:Objective: breast cancer in the world women's physical and psychological harm caused already is not optimistic. Breast cancer development by multiple genes, multiple channels, multiple reaction interaction, the incidence rate and mortality rate is rising year after year. The proliferation rate of tumor, metastasis and metastasis of breast cancer directly affects different parts the survival of the patients. In recent years, the age of onset of breast cancer patients tend to be younger, invasion, metastasis rate increased significantly, resulting in many patients have lost the opportunity of operation, directly affect the overall survival of patients. So the inhibition of tumor growth, tumor metastasis, to improve the survival rate of the patients. Play a crucial role in improving the quality of life. However, tumor angiogenesis is caused by malignant tumor occurrence, development and metastasis of the fundamental source, so the inhibition of breast cancer cell to treat angiogenesis Has the extremely significant significance of treatment of breast cancer. The nuclear factor -Kappa B (NF- K B) can regulate vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) and the expression of deubiquitinase for regulating vital NF- kappa B. Studies have found that NF- K B can not only in gastric cancer, thyroid cancer, expression the promotion of VEGF in breast cancer, can also promote lymph node metastasis. Deubiquitinase OTUD7B prevented NF- kappa B activation, and NF- K B can regulate cancer cell migration, invasion, vascular endothelial growth factor expression. So clearly deubiquitinase OTUD7B and breast cancer cell proliferation, migration, the relationship between the expression of VEGF and apoptosis, may provide a new method for the treatment of breast cancer. Methods: to construct lentiviral vector silencing by small interfering RNA (LV3-si-h OTUD7B) and control vector (LV3-NC), constructed a green fluorescent protein tag OTUD7B expression plasmid (pE GFP-h OTUD7B) and the control plasmid (pEGFP-CI) containing LV3-si-h OTUD7B. LV3-NC, pEGFP-h, OTUD7B, pEGFP-CI lentivirus infected MCF-7 cells was detected by.MTS OTUD7B on the proliferation of MCF-7 cells; evaluation of cell scratch test OTUD7B on MCF-7 cells migration effects; flow cytometry with PI staining was detected on OTUD7B MCF-7 cell cycle; Western blotting (Western blot) regulating the level of OTUD7B expression was detected in MCF-7 cell cytoplasm and nucleus of NF- NF- kappa BP65 kappa BP65; the relative expression level of RT-PCR effects on VEGF M detection of OTUD7B RNA; ELISA OTUD7B VEGF on the secretion regulation of detection of human breast cancer cell line MCF-7 by statistical software SPSS19.0. For processing, all data to mean + standard deviation (?) said, were compared by t test or ANOVA (One-way ANOVA), with P0.05 as the difference was statistically significant. Results: Cell proliferation was detected by MTS method showed that the transfection of pEGFP-h OTUD7B, the proliferation of MCF-7 breast cancer cells was inhibited by transfection of LV3-si-h, OTUD7B, MCF-7 breast cancer cell proliferation was promoted. The scratch test indicates that the cells transfected with pEGFP-h, OTUD7B, MCF-7 in breast cancer cell migration was inhibited after transfection of LV3-si-h, OTUD7B, MCF-7 in breast cancer cell migration.. PI staining flow cytometry to detect the cell cycle results suggest that pEGFP-h OTUD7B may regulate MCF-7 cell cycle arrest in G1 phase,.Western blot results showed that compared with the negative control group and normal control group, the expression level of pEGFP-h OTUD7B could downregulate MCF-7 breast cancer cell nuclear protein NF- kappa BP65, and cytosolic NF- kappa BP65 but no significant difference of.RT-PCR results suggest that m in RNA level, pEGFP-h OTUD7B inhibited the expression of VEGF.ELISA detected by MCF-7 cell conditioned culture medium VEGF content. The results show that, with the negative control group and normal control group compared to pEGFP-h group OTUD7B conditioned medium with low content of VEGF. Conclusion: OTUD7B can inhibit MCF-7 proliferation, migration of breast cancer cells, can regulate breast cancer MCF-7 cell arrest in G1 phase, and can also inhibit the secretion of MCF-7 breast cancer cells on the expression of VEGF.

【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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