uc.133抑制B细胞淋巴瘤形成的机制研究

发布时间：2018-02-09 09:06

  本文关键词： 超保守RNA uc.133 淋巴瘤 RSRC1　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]淋巴瘤在我国是比较常见的恶性肿瘤之一,近几年来它的发病率呈上升趋势,尽管临床经过一系列的治疗可以获得一定的成效,但依然存在难治和复发的病例。淋巴瘤的预后不良,所以分子靶向治疗的研究极其重要。超保守RNA是一种新的长链非编码RNA,近几年来许多人研究证明超保守RNA在多种肿瘤的发生、转移、侵袭以及预后有着相当密切的关系。本课题主要研究uc.133在小鼠和人的B细胞淋巴瘤与其正常相关组织中的表达情况,并研究uc.133在B细胞淋巴瘤发生发展中的作用及机制。[方法]1.采取 4-羟基他莫昔芬(4-hydroxytamoxifen,4-OHT)分别激活 p53 0min、1Omin、15min、30min的小鼠的肿瘤组织,分别用qRT-PCR验证前期基因芯片筛选出的4个UCRs的表达水平。并对其在小鼠B细胞淋巴瘤与小鼠正常骨髓B细胞中的表达情况进行比较,筛选差异性表达比较明显的UCR。2.构建差异性表达比较明显的uc.133的逆转录病毒表达质粒及MSCV空载体表达质粒,然后分别感染小鼠B细胞淋巴瘤38B9细胞株,人B细胞淋巴瘤Romas细胞株,嘌呤霉素筛选后使细胞感染率达90%以上,使uc.133在小鼠和人的B细胞淋巴瘤细胞株中过表达。3.体外检测38B9细胞与Romas细胞过表达uc.133后的增殖及凋亡情况;体内将过表达uc.133的细胞注射至BALB/c小鼠和裸鼠皮下,观察肿瘤的生长情况,一般两周左右取出小鼠肿瘤组织做Western Blot检测uc.133基因序列所在的宿主基因RSRC1蛋白的表达及病理实验。[结果]1.qRT-PCR 检测显示 uc.133、uc.268、uc.316A、uc.95 在 p53 激活 Omin、1Omin、15min、30min后的表达水平上调,而其在小鼠B细胞淋巴瘤与小鼠正常骨髓B细胞中的比较,表达具有差异性(p0.05),其中uc.133下调最明显;2.将差异性最明显的uc.133质粒及MSCV空载体转染293T细胞包装得到病毒,然后病毒分别感染38B9细胞和Romas细胞,嘌呤霉素筛选后使细胞感染率达96%。3.体外检测38B9细胞和Romas细胞过表达uc.133后与对照组比较,细胞增殖缓慢,凋亡增多。体内检测到38B9细胞过表达uc.133后肿瘤组织与对照组比较明显变小,而Romas细胞过表达uc.133后未形成肿瘤。Western Blot检测到38B9细胞与Romas细胞过表达uc.133后RSRC1的表达减少。[结论]uc.133在小鼠B细胞淋巴瘤发生发展中起着抑癌作用。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
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本文编号：1497573