外周T细胞淋巴瘤中转录因子GATA3与JAK-STAT通路相关性的体外研究

发布时间：2018-01-26 11:55

本文关键词： 外周T细胞淋巴瘤 GATA3 JAK-STAT通路相关性　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：背景和目的外周T细胞淋巴瘤(PTCL)是一组少见、异质性很强的非霍奇金淋巴瘤,侵袭性强、预后较差,暂无规范有效的治疗方案。寻找新的分子标记物并探索其在成瘤过程中的具体机制,对于新型靶向药物的研发具有重要的意义。近年来国外基于高通量基因组层面的研究结果显示转录因子GATA3对于PTCL的发生发展可能起到促进作用,而广泛参与体内生理过程的JAK-STAT信号通路对于多种肿瘤的形成和侵袭密切相关。过敏性疾病的研究显示二者之间具有一定调控的作用。本研究拟通过在体外条件下探究GATA3和JAK-STAT通路之间相互的关系,为进一步对新型靶向药物的研究提供新的思路。方法在体外条件下通过慢病毒转染法建立GATA3过表达的Hut78 T细胞淋巴瘤细胞系,并结合之前已有的GATA3敲低细胞系,在RNA和蛋白层面检测GATA3水平变化条件下JAK-STAT通路的成员STAT1、STAT6的变化情况。再反过来通过使用JAK-STAT通路特异性抑制剂及siRNA干扰的方法分别降低细胞的STAT1、 STAT6水平,检测GATA3水平的变化。结果对T淋巴瘤细胞系Hut78应用慢病毒转染技术建立GATA3过表达的细胞系Hut78-pLV-GATA3,并结合已获得的GATA3敲低的细胞系Hut78-shGATA3进行STAT1和STAT6水平检测。与对照组相比,随着GATA3水平降低,在RNA和蛋白层面STAT1水平均升高,STAT6水平均降低；而随着GATA3水平升高,在RNA和蛋白层面STAT1水平均降低,RNA层面STAT6水平升高,但蛋白水平有所下降。使用JAK2抑制剂AG490处理Hut78细胞系,在RNA和蛋白层面均有GATA3和STAT6的升高和STAT1水平的降低；使用STAT1抑制剂Fludarabine处理使STAT1水平降低后,STAT6的RNA水平升高,蛋白水平变化轻度下降,GATA3的RNA水平也有下降,蛋白水平无明显变化。使用siRNA对Hut78细胞系的STAT6进行干扰后,随着STAT6水平的下降,STAT1的RNA水平明显升高,蛋白水平变化不明显；GATA3 RNA和蛋白水平均明显下降。结论在PTCL中,GATA3水平的变化不能完全解释STAT1和STAT6水平的变化,提示GATA3直接位于JAK-STAT通路上游的可能性不大。而STAT水平变化后,GATA3水平在RNA和蛋白水平均有所改变,提示GATA3位于STAT下游的可能性较大。
[Abstract]:Background and objective Peripheral T-cell lymphoma (PTCLs) is a rare heterogeneous non-Hodgkin 's lymphoma with strong invasion and poor prognosis. Find new molecular markers and explore their specific mechanisms in tumorigenesis. In recent years, foreign research based on high-throughput genome level results show that transcription factor GATA3 may play a role in the development of PTCL. The JAK-STAT signaling pathway, which is widely involved in physiological processes in vivo, is closely related to the formation and invasion of many kinds of tumors. To explore the relationship between GATA3 and JAK-STAT pathway in vitro. Methods GATA3 over-expressed Hut78 T cell lymphoma cell lines were established by lentivirus transfection in vitro. Combined with previous GATA3 knockout cell lines, the STAT1 of JAK-STAT pathway was detected at the RNA and protein levels. The changes of STAT6. In turn, the levels of STAT1 and STAT6 were decreased by using JAK-STAT pathway specific inhibitor and siRNA interference, respectively. Results Lentivirus transfection technique was used to establish GATA3 overexpression cell line Hut78-pLV-GATA3 in T lymphoma cell line Hut78. The levels of STAT1 and STAT6 were detected by combining the obtained GATA3 knockout cell line Hut78-shGATA3. Compared with the control group, the level of GATA3 decreased. The levels of STAT1 increased and STAT6 decreased in both RNA and protein levels. However, with the increase of GATA3 level, the level of STAT1 in both RNA and protein level decreased and STAT6 level increased. JAK2 inhibitor AG490 was used to treat Hut78 cell line. The levels of GATA3 and STAT6 increased and the level of STAT1 decreased at the level of RNA and protein. After treatment with STAT1 inhibitor Fludarabine, the RNA level of STAT6 increased and the protein level decreased slightly after STAT1 level decreased. The RNA level of GATA3 also decreased, but the protein level did not change significantly. After siRNA was used to interfere with the STAT6 of Hut78 cell line, the level of STAT6 decreased with the decrease of STAT6 level. The RNA level of STAT1 increased significantly, but the protein level did not change significantly. Conclusion the changes of GATA3 level in PTCL can not fully explain the changes of STAT1 and STAT6 levels. The results suggest that it is not possible for GATA3 to be located directly upstream of JAK-STAT pathway, but the level of GATA3 changes in RNA and protein after the change of STAT level. It suggests that GATA3 is more likely to be located downstream of STAT.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.1

【相似文献】