本文選題：Syk + siRNA��； 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：研究背景外周T細胞淋巴瘤是一組異質(zhì)性強,臨床表現(xiàn)和生物學行為都極具侵襲性的惡性腫瘤,約占非霍奇金氏淋巴瘤的15%,且亞洲人群發(fā)病率高于西方國家人群。該組疾病對傳統(tǒng)CHOP化學療法反應不佳,療效差,長期生存率低。外周T細胞淋巴瘤的新型療法亟待被提出。目前,國內(nèi)外研究結(jié)果顯示Syk基因在B細胞淋巴瘤中過表達,且Syk口服抑制劑已被研發(fā),目前已投入臨床應用。故而Syk的過表達是否與外周T細胞淋巴瘤的發(fā)生發(fā)展有關,且能否成為其治療的一個新靶點成為了研究熱門。本課題組前期研究結(jié)果顯示,Syk基因在正常外周T淋巴細胞中表達缺失,而在外周T細胞淋巴瘤中的表達增加,且收集的臨床標本中,其免疫組織化學結(jié)果顯示Syk蛋白的表達在大部分組織中呈陽性,與國內(nèi)外的研究結(jié)果一致。本實驗繼續(xù)深入研究外周T細胞淋巴瘤中Syk基因的作用,從細胞生物學行為揭示Syk基因?qū)ν庵躎細胞淋巴瘤細胞的影響。研究目的探討siRNA抑制脾酪氨酸激酶(Syk)基因后對外周T細胞淋巴瘤細胞株HUT-78細胞增殖與凋亡的影響。研究方法1、針對Syk基因特異靶點設計3條siRNA(siRNA-1、siRNA-2和siRNA-3),采用電穿孔法轉(zhuǎn)染外周T細胞淋巴瘤細胞株HUT-78,同時設Mock組(即空白對照組)和siRNA-NC組(即陰性對照組);2、轉(zhuǎn)染48 h后,分別用RT-PCR和Western blot技術檢測Syk m RNA和蛋白的表達水平,篩選出最有效的Syk siRNA;3、Syk siRNA轉(zhuǎn)染HUT-78后分別用軟瓊脂克隆形成實驗檢測Syk下調(diào)后HUT-78細胞的克隆形成能力,MTT法檢測干擾24,48,72 h后細胞增殖情況,流式細胞術檢測Syk下調(diào)后HUT-78細胞的凋亡情況。研究結(jié)果1、RT-PCR和Western blot的結(jié)果顯示,與Mock組和siRNA-NC組相比,3條siRNA均能有效降低HUT-78細胞中Syk m RNA和蛋白的表達,其中Syk siRNA-1抑制效果最明顯。2、克隆形成實驗顯示,下調(diào)Syk基因表達后HUT-78細胞的克隆形成能力與Mock組和siRNA-NC組相比明顯下降(P0.05);3、MTT實驗結(jié)果顯示,下調(diào)Syk基因表達后HUT-78細胞的增殖能力與Mock組和siRNA-NC組相比顯著下降(P0.05);4、流式細胞術結(jié)果顯示,下調(diào)Syk基因表達后HUT-78細胞的凋亡比例約37%,明顯高于Mock組和siRNA-NC組(P0.05)。結(jié)論siRNA下調(diào)Syk基因表達后可抑制外周T細胞淋巴瘤細胞的增殖、克隆形成,促進凋亡,推測Syk基因在外周T細胞淋巴瘤的發(fā)生發(fā)展中發(fā)揮重要作用,有可能成為外周T細胞淋巴瘤基因治療的新靶點。
[Abstract]:Background Peripheral T-cell lymphoma is a group of highly heterogeneous malignant tumors with very aggressive clinical manifestations and biological behaviors, accounting for about 15% of non-Hodgkin 's lymphoma, and the incidence of T cell lymphoma in Asia is higher than that in western countries. The disease had poor response to conventional chop chemotherapy, poor efficacy and low long-term survival rate. A new therapy for peripheral T cell lymphoma needs to be proposed. At present, the results of domestic and foreign studies show that Syk gene is overexpressed in B-cell lymphoma, and Syk oral inhibitor has been developed, and has been applied in clinical practice. Therefore, whether the overexpression of Syk is related to the occurrence and development of peripheral T cell lymphoma and whether it can become a new target for its treatment has become a hot topic. Our previous study results showed that Syk gene expression was absent in normal peripheral T lymphocytes, but increased in peripheral T cell lymphoma. The results of immunohistochemistry showed that Syk protein was positive in most tissues, which was consistent with the domestic and foreign research results. In this study, the role of Syk gene in peripheral T cell lymphoma was further studied, and the effect of Syk gene on peripheral T cell lymphoma cells was revealed from the cell biological behavior. Objective to investigate the effects of siRNA on proliferation and apoptosis of peripheral T cell lymphoma cell line HUT-78 after inhibition of spleen tyrosine kinase (Syk) gene. Methods 1. Three siRNA-1siRNA-2 and siRNA-3s were designed for Syk gene specific targets. The peripheral T-cell lymphoma cell lines HUT-78were transfected by electroporation. The cells were divided into Mock group (blank control group) and siRNA-NC group (negative control group, 48 h after transfection). The expression of Syk mRNA and protein was detected by RT-PCR and Western blot, respectively. After transfection of Syk siRNA3 and Syk siRNA into HUT-78, the colony forming ability of HUT-78 cells after Syk down-regulation was detected by soft Agar clone formation assay. MTT assay was used to detect the proliferation of HUT-78 cells after interfering with 244872 h. Apoptosis of HUT-78 cells after Syk downregulation was detected by flow cytometry. The results of RT-PCR and Western blot showed that compared with Mock group and siRNA-NC group, three siRNAs could effectively reduce the expression of Syk mRNA and protein in HUT-78 cells. Compared with Mock group and siRNA-NC group, the colony forming ability of HUT-78 cells decreased significantly after down-regulation of Syk gene expression. The results of MTT assay showed that the proliferation ability of HUT-78 cells after down-regulation of Syk gene expression was significantly lower than that of Mock group and siRNA-NC group. The percentage of apoptosis in HUT-78 cells after down-regulation of Syk gene expression was 37%, which was significantly higher than that in Mock group and siRNA-NC group. Conclusion down-regulation of Syk gene expression by siRNA can inhibit proliferation, clone formation and promote apoptosis of peripheral T cell lymphoma cells. It is suggested that Syk gene plays an important role in the occurrence and development of peripheral T cell lymphoma. It may be a new target for gene therapy of peripheral T cell lymphoma.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.1

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相關期刊論文 前2條

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本文編號：2020426