【作者】 彭雍；

【导师】 蒋宇钢；

【作者基本信息】 中南大学 ， 神经外科， 2011， 博士

【摘要】 目的胶质母细胞瘤或多形性胶质母细胞瘤(glioblastoma multiforme, GBM)是最为常见,恶性程度最高的胶质瘤类型。同其他肿瘤一样,GBM的发生、发展以及对各种辅助治疗方式的抵抗与肿瘤细胞内多基因的调节及多种因素相互作用有关。基因治疗被认为是各种恶性肿瘤非常有前景的治疗方式,慢病毒载体介导的基因治疗因其较高的转导效率,较好的操控性,既可感染分裂期也可感染非分裂期细胞,并可将外源基因整合入目的细胞基因组中,长期稳定地表达外源基因,是基因治疗中非常有力的工具；慢病毒载体还可介导RNA干扰,从而持续稳定地抑制靶基因的表达。凋亡是一种程序性细胞死亡,并且已得到了非常广泛而深入地研究。一般认为坏死是一个不可调控的、非程序性的细胞死亡方式,然而,研究证实,坏死可能至少部分是通过程序调控而形成的,这种细胞死亡被命名为程序性坏死。RIP3,属于RIP家族成员,是Ser/Thr蛋白激酶,因其结构中含有RIP家族同源激酶结构域而得名。RIP3在人体多个正常组织中有表达,而在多种恶性肿瘤细胞系中却表达下降或缺失,研究发现,RIP3是程序性细胞坏死过程中的关键蛋白。GBM的发生、发展以及对放化疗的抵抗与肿瘤细胞凋亡功能失调有重要的关系。虽然凋亡过程有缺陷,几乎所有的GBM均表现出不同程度的瘤内坏死。另外,某些抗肿瘤的辅助治疗方式通过诱导肿瘤坏死对多种恶性肿瘤产生良好的治疗效应。诱导肿瘤细胞坏死从而清除肿瘤细胞在近年来逐渐得到重视。然而,通过诱导肿瘤细胞坏死来治疗恶性肿瘤的研究鲜有报道,且尚未有坏死关键因子RIP3对恶性胶质瘤作用的研究。我们通过构建重组人RIP3慢病毒过表达载体和慢病毒shRNA于二扰载体,转导GBM细胞株U251,调控RIP3基因在GBM细胞中的表达,研究RIP3对恶性胶质瘤细胞增殖、死亡的影响及对临床标准的恶性胶质瘤化疗药替莫唑胺敏感性的影响,并探讨其作用机制,为RIP3在胶质母细胞瘤治疗中的应用提供依据。方法1.使用Gateway技术构建人RIP3慢病毒过表达载体。使用酶切连接技术构建RIP3慢病毒十扰载体。2.携带人RIP3基因的重组HIV-1慢病毒与携带RIP3shRNA序列的慢病毒分别转导野生型U251(U251-WT)细胞株,通过G418筛选转导后的U251,Western blot鉴定RIP3的表达情况。3.使用Annexin V/PI双染法检测U251-WT细胞、U251-RIP3细胞,U251-shRIP3细胞的死亡情况,进一步使用广谱Caspase抑制剂zVAD验证死亡方式。4.使用CellROXTM Deep Red试剂检测U251-WT细胞、U251-RIP3细胞,U251-shRIP3细胞中ROS的表达水平,研究ROS在RIP3诱导的细胞死亡中的作用。5.ELISA法检测上述U251-WT细胞、U251-RIP3细胞,U251-shRIP3细胞中的TNF-a的分泌情况,研究其在RIP3诱导的细胞死亡中的作用。6.MTT检测上述U251-WT细胞、U251-RIP3细胞,U251-shRIP3细胞的生长情况。7. Western blot法检测上述三株细胞中PI3K/Akt信号通路中关键因子Akt的磷酸化情况。8.MTT法检测上述三株细胞对TMZ的敏感性差异。使用Annexin V/PI双染法检测U251-WT细胞、U251-RIP3细胞在temozolomide作用下的死亡情况。9. Western blot法检测U251-WT, U251-RIP3, U251-shRIP3中PARP-1的表达情况。结果1.成功构建RIP3慢病毒过表达载体和慢病毒shRNA干扰载体,并与第四代包装质粒共转导包装细胞,获得高达3x108 TU/ml的病毒颗粒。2.携带人RIP3基因的慢病毒与携带RIP3shRNA序列的慢病毒分别转导U251-WT细胞株,通过G418筛选,分别获得稳定过表达人RIP3基因和RIP3被干扰的细胞株(U251：RIP3及U251-shRIP3)。3.过表达RIP3的U251细胞出现肿胀样死亡,且这种死亡不被广谱的caspase抑制剂阻断, Annexin V/PI检测进一步证实为死亡细胞PI强阳性。过表达RIP3的U251细胞死亡率明显高于U251-WT及U251-shRIP3,且RIP3过表达诱导U251细胞死亡需要依赖ROS的产生,使用ROS清除剂BHA作用后,其死亡明显减少。4.与野生型相比,过表达RIP3的U251细胞TNF-α分泌明显增加。而U251-WT与U251-shRIP3的TNF-α分泌无明显差别。5.在替莫唑胺(TMZ)作用下,过表达RIP3的U251细胞增殖能力明显低于U251-shRIP3和U251-WT细胞。且U251-RIP3的细胞死亡率明显高于U251-WT细胞。6.相比U251-WT和U251-shRIP3细胞,过表达RIP3的U251细胞株对TMZ作用的敏感性增加,这种作用与RIP3影响PARP-1的表达有关。结论1.RIP3可诱导U251出现坏死。RIP3诱导U251细胞TNF-α分泌增加,进一步增加ROS产生从而导致细胞坏死。RIP3通过抑制PI3K/Akt信号通路而影响U251的增殖、生长。RIP3是GBM基因治疗的一个新的的靶分子。2.RIP3通过影响PARP-1的表达调控U251对TMZ的敏感性,RIP3是一种新的调控胶质母细胞瘤药物敏感性的靶分子。更多还原

【Abstract】 BackgroundGlioblastoma or glioblastoma multiforme (glioblastoma multiforme, GBM) is the most encountered and malignant variants of gliomas in clinical practice. As with other tumors, the pathogenesis, development and its resistance to adjuvant treatments of GBM are associated with regulation of multiple genes and interaction of various factors within the tumor cells. Gene therapy is considered as a promising antitumor treatment to various malignancies. Because it has high efficiency of gene transfer, better controllability and it can integrate desired genes into non-dividing cells and dividing cell with stable expression of transgene, lentiviral vector-mediated gene transfer is a very useful way for gene therapy. Lentiviral vector-mediated RNA interference also is widely used to achieve a stable gene silencing in various cell lines. Apoptosis is programmed cell death, and there has been a major emphasis in the literature on the apoptotic form of cell death. Just recently, necrotic cell death is considered as a regulated and controlled process, like apoptosis, termed necroptosis or programmed necrosis. RIP3, a RIP family member, is a Ser/Thr protein kinase and shares homology with the kinase domain within RIP family. It is found that RIP3 is a key protein during the process of programmed cell necrosis. Dysfunction of apoptosis is believed to underlie GBM tumorigenesis and resistance to chemotherapy and radiotherapy. Although there are defects in apoptotic process, nearly all GBMs have different degree of intratumoral necrosis, which does not seem to be associated with the tumor size. In addition, some antitumor therapies have therapeutic effects on a variety of malignant tumors through the induction of tumor cells necrosis. It is a novel way to achieve a better therapeutic effect through the induction of tumor cells necrosis and this idea has gain increasing attention in recent years. Yet, there are limited literature shed light on inducing tumor cell necrosis to combat malignant tumors and no research has been reported to study the RIP3function on GBM. We construct recombinant lentiviral overexpression of human RIP3 vector and shRIP3 vector to modulate the expression of RIP3 in GBM cell lines to study the function and mechanism of RIP3 on proliferation, death and chemosensitivity of GBM cells. This would further reveal the function of RIP3 on GBM and provide basis for applications of RIP3 in GBM treatment.Methods and ResultsConstructing a lentiviral expression vector containing EF1 A-driving human RIP3 gene using Gateway technology and a lentiviral shRNA vector targeting human RIP3 gene using ligation and packaging into viral particles with high titers 1x108 TU/ml. Transducing human RIP3 gene and shRNA targeting human RIP3 into U251 respectively and establishing stable cell lines. U251-RIP3 and U251-shRIP3 by selection with antibiotic. Western blotting showed that the expression lever of RIP3 in U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. Some U251-RIP3 cells exhibited swelling-like death during culturing. This phenomenon was not inhibited by pan-caspase inhibitor zVAD. Annexin V/PI assay confirmed the death of U251-RIP3 cells was PI positive. Annexin V/PI assay was conducted to study the death rate. Death rate of U251-shRJP3 and U251-WT cells has significant differences with U251-RIP3 cell by Annexin V/PI assay. But there is no significant difference between U251-shRIP3 cells and U251-WT cells. ROS lever was measured by CellROXTM Deep Red Reagent to study the role of ROS in RIP3 induced cell death. ROS level of U251-RIP3 cells is significantly higher than that of U251-shRIP3 cells and U251-WT cells. And U251-shRIP3 did not show a significant difference with U251-WT cells. Death of U251-RIP3 cells is ROS dependent and BHA can reduce the death rate. Recombinant TNF-a do not enhance the death rate of U251-RIP3 cells. ELISA measured the secretion of TNF-a among U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. TNF-a level of U251-RIP3 cells is significantly higher than that of U251-shRIP3 cells and U251-WT cells. And U251-shRIP3 cells did not show any difference with U251-WT cells. MTT colorimetric assay showed the proliferation of U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. The proliferation of U251-RIP3 was lower than that of U251-shRIP3 cells and U251-WT cells. Western blotting analyzed the average activation of Akt in U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. Our result showed that the Akt activation was suppressed in U251-RIP3 cells, whereas, both U251-shRIP3 cells and U251-WT cells had higher level of Akt activation and latter tow group has no significant differences. We analyzed the cell death in U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells in the treatment of temozolomide. The average of cell death rate in the treatment of temozolomide measured by flow cytometry. Death rate of U251-RIP3 cells is much higher than that of U251-WT cells in treatment of temozolomide. MTT colorimetric assay showed that ectopic overexpression of RIP3 in GBM cell line U251 increase its chemosensitivity to temozolomide. U251-shRIP3 cells and U251-WT cells had the same low sensitivity to temozolomide. Western blotting analysis showed that the PARP-1 expression lever in U251-RIP3 cells was lower than that of U251-shRIP3 cells and U251-WT cells. This suggests RIP3 enhance the chemosensitivity of U251 to temozolomide at least partially via its impact on PARP-1.ConclusionRIP3 induces necrosis in GBM cell line U251. RIP3 activates the secretion of TNF-a, and then increases ROS production to cause necrosis. RIP3 inhibits proliferation of U251 at least partially by suppressing the Akt pathway. We propose RIP3 is a novel target to GBM gene therapy. RIP3 increases U251 cells sensitivity to temozolomide via the impact on PARP-1, RIP3 may be a targeted molecular to modulate the chemosensitivity of GBM.更多还原