本文選題：宮頸癌 + LRAT基因　； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：背景與目的宮頸癌是婦科最常見惡性腫瘤之一,除HPV感染以外,被感染細(xì)胞的基因出現(xiàn)異常表達(dá)才能最終導(dǎo)致宮頸癌的發(fā)生發(fā)展。前期研究首次通過RNA-Seq技術(shù),檢測宮頸癌與癌旁組織的差異基因,發(fā)現(xiàn)視黃醇代謝相關(guān)基因LRAT和RDH12在宮頸癌組織和細(xì)胞中的表達(dá)明顯低于癌旁組織。已有的體外實(shí)驗(yàn)證實(shí):構(gòu)建LRAT及RDH12過表達(dá)載體并轉(zhuǎn)染宮頸癌細(xì)胞系,瞬時轉(zhuǎn)染LRAT和RDH12后,能夠抑制宮頸癌細(xì)胞的增殖、侵襲和遷移,阻滯宮頸癌細(xì)胞周期,增加凋亡率。這一現(xiàn)象與抑制MAPK及NF-kB信號通路有關(guān)。當(dāng)LRAT及RDH12基因的表達(dá)升高時,宮頸癌細(xì)胞中MAPK及NF-kB通路中相關(guān)蛋白的表達(dá)水平有所下降。本課題擬在組織層面上,通過動物體內(nèi)實(shí)驗(yàn),進(jìn)一步驗(yàn)證LRAT、RDH12基因與MAPK及NF-kB信號通路的關(guān)系。通過建立宮頸癌細(xì)胞的裸鼠移植瘤模型,分析穩(wěn)定轉(zhuǎn)染LRAT和RDH12基因的兩種Hela細(xì)胞,在裸鼠中的成瘤情況。并對移植瘤組織的病理進(jìn)行分析,探討LRAT/RDH12與宮頸癌的臨床病理因素的關(guān)系,旨在闡明LRAT/RDH12基因在宮頸癌進(jìn)展中的作用及機(jī)制,為宮頸癌的防治提供新的理論支持和分子指標(biāo)。方法一、培養(yǎng)穩(wěn)定轉(zhuǎn)染的細(xì)胞株:利用重組質(zhì)粒,培養(yǎng)并獲得穩(wěn)定轉(zhuǎn)染LRAT及RDH12基因的兩種Hela細(xì)胞株。二、構(gòu)建Hela細(xì)胞的裸鼠移植瘤模型:利用未做處理的對照組Hela細(xì)胞,探索裸鼠中最佳的Hela細(xì)胞成瘤濃度。將Hela細(xì)胞配置成5×106/mL、1×107/mL、5×107/mL和1×108/mL的單細(xì)胞懸液,4個濃度梯度下進(jìn)行成瘤濃度探索。三、觀察并記錄裸鼠中Hela細(xì)胞的成瘤情況,記錄腫瘤體積大小,繪制腫瘤生長曲線。四、對裸鼠皮下移植瘤進(jìn)行取材,進(jìn)行免疫組化切片。分析病理組織中,MAPK通路中p38及NF-kB通路中p50、p65的表達(dá)情況。結(jié)果培養(yǎng)并獲得穩(wěn)定轉(zhuǎn)染LRAT、RDH12基因的兩種Hela細(xì)胞系。經(jīng)過qPCR驗(yàn)證,穩(wěn)轉(zhuǎn)的LRAT基因過表達(dá)倍數(shù)是空白對照的29360倍,穩(wěn)轉(zhuǎn)的RDH12基因過表達(dá)倍數(shù)是空白對照的39631倍。對培養(yǎng)獲得的穩(wěn)轉(zhuǎn)細(xì)胞進(jìn)行實(shí)驗(yàn),發(fā)現(xiàn)穩(wěn)轉(zhuǎn)LRAT、RDH12基因的兩種Hela細(xì)胞中,P38、P50、P65蛋白的表達(dá)水平均下調(diào)。裸鼠成瘤模型建立的實(shí)驗(yàn)中,成功找出Hela細(xì)胞在裸鼠中的最佳成瘤濃度為5×107/mL,并順利構(gòu)建Hela細(xì)胞的裸鼠移植瘤模型。觀察腫瘤生長變化情況,對腫瘤大小進(jìn)行比對分析,穩(wěn)轉(zhuǎn)LRAT、RDH12基因的兩組Hela細(xì)胞,在裸鼠模型中的成瘤速度及腫瘤生長速度均慢于對照組,且LRAT組受抑制程度較RDH12組明顯。對3組同周數(shù)的瘤體組織分別進(jìn)行免疫組化染色實(shí)驗(yàn)及Western實(shí)驗(yàn),表明在LRAT、RDH12兩組實(shí)驗(yàn)組中,P38、P50、P65蛋白的表達(dá)水平均下降。LRAT、RDH12的過表達(dá),會抑制MAPK、NF-kB通路的活性。結(jié)論LRAT與RDH12基因的過表達(dá),能夠有效抑制宮頸癌組織的生長,這一現(xiàn)象與抑制MAPK、NF-kB信號上的相關(guān)蛋白表達(dá)水平有關(guān),過表達(dá)LRAT、RDH12基因可以抑制宮頸癌的發(fā)展,這兩個基因有望成為宮頸癌治療的新靶點(diǎn)。
[Abstract]:Background & objective Cervical cancer is one of the most common malignant tumors in gynecology. In addition to HPV infection, abnormal gene expression in infected cells can eventually lead to the occurrence and development of cervical cancer. For the first time, RNA-Seq technique was used to detect the differentially expressed genes of retinol metabolism related genes LRAT and RDH12 in cervical cancer tissues and adjacent tissues, and the expression of LRAT and RDH12 genes in cervical cancer tissues and cells was significantly lower than that in adjacent tissues. It has been confirmed in vitro that LRAT and RDH12 overexpression vectors were constructed and transfected into cervical cancer cell lines. After transient transfection of LRAT and RDH12, LRAT and RDH12 could inhibit the proliferation, invasion and migration of cervical cancer cells, block the cell cycle and increase the apoptosis rate. This phenomenon is related to the inhibition of MAPK and NF-kB signaling pathways. When the expression of LRAT and RDH12 gene increased, the expression of MAPK and NF-kB related proteins in cervical cancer cells decreased. At the tissue level, the relationship between LRATA RDH12 gene and MAPK and NF-kB signaling pathway was further verified by animal experiments in vivo. The tumor formation of two Hela cells stably transfected with LRAT and RDH12 gene in nude mice was analyzed by establishing the tumor model of nude mice. The relationship between LRATR / RDH12 and the clinicopathological factors of cervical cancer was discussed in order to elucidate the role and mechanism of LRATR RDH12 gene in the progression of cervical cancer and to provide new theoretical support and molecular index for the prevention and treatment of cervical cancer. Methods 1. Stable transfected cell lines: two Hela cell lines transfected stably with LRAT and RDH12 gene were obtained by using recombinant plasmid. Secondly, the model of Hela cell transplantation in nude mice was established. The optimal concentration of Hela cells in nude mice was explored by using untreated Hela cells. Hela cells were configured into 5 脳 10 6 / mL mLN 1 脳 10 7 / mL and 1 脳 10 8 / mL single cell suspensions. The tumor concentration was investigated under four concentration gradients. Third, the tumorigenesis of Hela cells in nude mice was observed and recorded, the tumor volume was recorded and the tumor growth curve was drawn. Fourthly, the subcutaneous transplanted tumor of nude mice was selected and immunohistochemical sections were made. The expression of p38 and NF-kB p50 p65 in MAPK pathway was analyzed. Results two Hela cell lines stably transfected with LRAT-RDH12 gene were obtained. The results of qPCR showed that the overexpression of LRAT gene was 29360 times higher than that of the control, and the overexpression of RDH12 gene was 39631 times higher than that of the control. The results showed that the expression level of P38-P50 / P65 protein was down-regulated in the two Hela cells transfected with LRAT-RDH12 gene. The optimal concentration of Hela cells in nude mice was found to be 5 脳 10 7 / mL, and the model of Hela cells was successfully constructed in nude mice. The tumor growth rate and tumor growth rate of two groups of Hela cells with stable transformation of LRATA RDH12 gene in nude mice were slower than those in control group, and the inhibition degree of LRAT group was significantly higher than that of RDH12 group. Immunohistochemical staining and Western blot analysis showed that the expression level of P38-P50 p65 protein decreased and the overexpression of RDH12 in LRAT-RDH12 group inhibited the activity of MAPK-NF-kB pathway. Conclusion the overexpression of LRAT and RDH12 gene can effectively inhibit the growth of cervical cancer. This phenomenon is related to the inhibition of the expression of related proteins in MAPK- NF-kB signal. Overexpression of LRAT-RDH12 gene can inhibit the development of cervical cancer. These two genes are expected to become a new target for cervical cancer treatment.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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