本文關(guān)鍵詞： 超保守RNA uc.133 淋巴瘤 RSRC1　出處：《揚州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]淋巴瘤在我國是比較常見的惡性腫瘤之一,近幾年來它的發(fā)病率呈上升趨勢,盡管臨床經(jīng)過一系列的治療可以獲得一定的成效,但依然存在難治和復(fù)發(fā)的病例。淋巴瘤的預(yù)后不良,所以分子靶向治療的研究極其重要。超保守RNA是一種新的長鏈非編碼RNA,近幾年來許多人研究證明超保守RNA在多種腫瘤的發(fā)生、轉(zhuǎn)移、侵襲以及預(yù)后有著相當(dāng)密切的關(guān)系。本課題主要研究uc.133在小鼠和人的B細(xì)胞淋巴瘤與其正常相關(guān)組織中的表達(dá)情況,并研究uc.133在B細(xì)胞淋巴瘤發(fā)生發(fā)展中的作用及機(jī)制。[方法]1.采取 4-羥基他莫昔芬(4-hydroxytamoxifen,4-OHT)分別激活 p53 0min、1Omin、15min、30min的小鼠的腫瘤組織,分別用qRT-PCR驗證前期基因芯片篩選出的4個UCRs的表達(dá)水平。并對其在小鼠B細(xì)胞淋巴瘤與小鼠正常骨髓B細(xì)胞中的表達(dá)情況進(jìn)行比較,篩選差異性表達(dá)比較明顯的UCR。2.構(gòu)建差異性表達(dá)比較明顯的uc.133的逆轉(zhuǎn)錄病毒表達(dá)質(zhì)粒及MSCV空載體表達(dá)質(zhì)粒,然后分別感染小鼠B細(xì)胞淋巴瘤38B9細(xì)胞株,人B細(xì)胞淋巴瘤Romas細(xì)胞株,嘌呤霉素篩選后使細(xì)胞感染率達(dá)90%以上,使uc.133在小鼠和人的B細(xì)胞淋巴瘤細(xì)胞株中過表達(dá)。3.體外檢測38B9細(xì)胞與Romas細(xì)胞過表達(dá)uc.133后的增殖及凋亡情況;體內(nèi)將過表達(dá)uc.133的細(xì)胞注射至BALB/c小鼠和裸鼠皮下,觀察腫瘤的生長情況,一般兩周左右取出小鼠腫瘤組織做Western Blot檢測uc.133基因序列所在的宿主基因RSRC1蛋白的表達(dá)及病理實驗。[結(jié)果]1.qRT-PCR 檢測顯示 uc.133、uc.268、uc.316A、uc.95 在 p53 激活 Omin、1Omin、15min、30min后的表達(dá)水平上調(diào),而其在小鼠B細(xì)胞淋巴瘤與小鼠正常骨髓B細(xì)胞中的比較,表達(dá)具有差異性(p0.05),其中uc.133下調(diào)最明顯;2.將差異性最明顯的uc.133質(zhì)粒及MSCV空載體轉(zhuǎn)染293T細(xì)胞包裝得到病毒,然后病毒分別感染38B9細(xì)胞和Romas細(xì)胞,嘌呤霉素篩選后使細(xì)胞感染率達(dá)96%。3.體外檢測38B9細(xì)胞和Romas細(xì)胞過表達(dá)uc.133后與對照組比較,細(xì)胞增殖緩慢,凋亡增多。體內(nèi)檢測到38B9細(xì)胞過表達(dá)uc.133后腫瘤組織與對照組比較明顯變小,而Romas細(xì)胞過表達(dá)uc.133后未形成腫瘤。Western Blot檢測到38B9細(xì)胞與Romas細(xì)胞過表達(dá)uc.133后RSRC1的表達(dá)減少。[結(jié)論]uc.133在小鼠B細(xì)胞淋巴瘤發(fā)生發(fā)展中起著抑癌作用。
[Abstract]:[objective] Lymphoma is one of the most common malignant tumors in China. In recent years, the incidence of lymphoma is on the rise, although a series of clinical treatment can achieve some results. But there are still refractory and relapsing cases. The prognosis of lymphoma is poor. Therefore, the study of molecular targeting therapy is extremely important. Ultra-conservative RNA is a new type of long-chain non-coding RNAs. In recent years, many studies have proved that ultra-conservative RNA is involved in the occurrence and metastasis of many kinds of tumors. Invasion and prognosis are closely related. In this study, we studied the expression of uc.133 in mouse and human B-cell lymphoma and its normal tissues. To study the role and mechanism of uc.133 in the development of B-cell lymphoma. [methods] 1.The tumor tissues of mice were activated by 4-hydroxytamoxifenol 4-OHT-4-hydroxytamoxifende (4-hydroxytamoxifenn 4-OHT4) for 30 min, respectively. QRT-PCR was used to verify the expression level of 4 UCRs in mouse B-cell lymphoma and normal bone marrow B cells. To construct the retroviral expression plasmid and MSCV empty vector expression plasmid of uc.133, and then infect the murine B cell lymphoma 38B9 cell line and human B cell lymphoma Romas cell line, respectively. The infection rate of purine mycin reached more than 90%, and uc.133 was overexpressed in murine and human B-cell lymphoma cell lines. The proliferation and apoptosis of 38B9 and Romas cells were detected in vitro. The cells expressing uc.133 were injected subcutaneously into BALB/c mice and nude mice to observe the growth of tumor. The expression of RSRC1 protein, the host gene of uc.133 gene sequence, was detected by Western Blot at about two weeks. [results] 1. QRT-PCR showed that the expression level of uc.133nuc.268Auc.316Auc.95 was up-regulated 30 minutes after the activation of p53. Compared with normal bone marrow B cells of mice, the expression of B cell lymphoma is different from that of normal bone marrow B cells, in which uc.133 down-regulation is the most obvious. The most distinct uc.133 plasmid and MSCV empty vector are transfected into 293T cells to obtain the virus. Then the virus was infected with 38B9 cells and Romas cells respectively, and the infection rate reached 96. 3.The proliferation of 38B9 cells and Romas cells in vitro was slower than that of the control group. Apoptosis increased. After 38 B9 cells were detected to overexpression uc.133 in vivo, the tumor tissues were significantly smaller than those in the control group. However, after overexpression of uc.133 in Romas cells, no tumor was formed. Western Blot showed that the expression of RSRC1 in 38B9 cells and Romas cells was decreased after overexpression of uc.133. [conclusion] uc.133 plays an inhibitory role in the carcinogenesis and development of B-cell lymphoma in mice.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.1
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本文編號：1497573