siRNA抑制Syk基因对外周T细胞淋巴瘤细胞增殖与凋亡的影响

发布时间：2018-06-15 03:33

本文选题：Syk + siRNA　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究背景外周T细胞淋巴瘤是一组异质性强,临床表现和生物学行为都极具侵袭性的恶性肿瘤,约占非霍奇金氏淋巴瘤的15%,且亚洲人群发病率高于西方国家人群。该组疾病对传统CHOP化学疗法反应不佳,疗效差,长期生存率低。外周T细胞淋巴瘤的新型疗法亟待被提出。目前,国内外研究结果显示Syk基因在B细胞淋巴瘤中过表达,且Syk口服抑制剂已被研发,目前已投入临床应用。故而Syk的过表达是否与外周T细胞淋巴瘤的发生发展有关,且能否成为其治疗的一个新靶点成为了研究热门。本课题组前期研究结果显示,Syk基因在正常外周T淋巴细胞中表达缺失,而在外周T细胞淋巴瘤中的表达增加,且收集的临床标本中,其免疫组织化学结果显示Syk蛋白的表达在大部分组织中呈阳性,与国内外的研究结果一致。本实验继续深入研究外周T细胞淋巴瘤中Syk基因的作用,从细胞生物学行为揭示Syk基因对外周T细胞淋巴瘤细胞的影响。研究目的探讨siRNA抑制脾酪氨酸激酶(Syk)基因后对外周T细胞淋巴瘤细胞株HUT-78细胞增殖与凋亡的影响。研究方法1、针对Syk基因特异靶点设计3条siRNA(siRNA-1、siRNA-2和siRNA-3),采用电穿孔法转染外周T细胞淋巴瘤细胞株HUT-78,同时设Mock组(即空白对照组)和siRNA-NC组(即阴性对照组);2、转染48 h后,分别用RT-PCR和Western blot技术检测Syk m RNA和蛋白的表达水平,筛选出最有效的Syk siRNA;3、Syk siRNA转染HUT-78后分别用软琼脂克隆形成实验检测Syk下调后HUT-78细胞的克隆形成能力,MTT法检测干扰24,48,72 h后细胞增殖情况,流式细胞术检测Syk下调后HUT-78细胞的凋亡情况。研究结果1、RT-PCR和Western blot的结果显示,与Mock组和siRNA-NC组相比,3条siRNA均能有效降低HUT-78细胞中Syk m RNA和蛋白的表达,其中Syk siRNA-1抑制效果最明显。2、克隆形成实验显示,下调Syk基因表达后HUT-78细胞的克隆形成能力与Mock组和siRNA-NC组相比明显下降(P0.05);3、MTT实验结果显示,下调Syk基因表达后HUT-78细胞的增殖能力与Mock组和siRNA-NC组相比显著下降(P0.05);4、流式细胞术结果显示,下调Syk基因表达后HUT-78细胞的凋亡比例约37%,明显高于Mock组和siRNA-NC组(P0.05)。结论siRNA下调Syk基因表达后可抑制外周T细胞淋巴瘤细胞的增殖、克隆形成,促进凋亡,推测Syk基因在外周T细胞淋巴瘤的发生发展中发挥重要作用,有可能成为外周T细胞淋巴瘤基因治疗的新靶点。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1

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