【作者】 赵小美；

【导师】 仲平； 孙国涛；

【作者基本信息】 河南大学 ， 药物分析学， 2011， 硕士

【摘要】 背景目的:神经胶质瘤是神经系统最常见的恶性肿瘤,具有生长迅速、浸润性生长、恶性程度高、病程短、手术后易复发、预后很差等特点,严重威胁着人民群众的健康。神经胶质瘤发生的原因以及发生发展的机制还有很多待研究之处。肿瘤的发生发展与基因改变密切相关,而且存在一个基因改变和病变效应的不断积累、演化的过程。而基因改变中,癌基因和肿瘤抑制基因发挥的作用最为关键。肿瘤细胞中癌基因可以通过多种方式异常激活,同样,抑癌基因也可以通过多种方式异常失活。同时,不同个体肿瘤又在基因改变、药物敏感性上具有很大差异。抑癌基因TP53的表达产物p53蛋白在细胞周期的调控、细胞基因组完整性的维持、诱发细胞分化和凋亡中起着重要的作用。p53信号通路在肿瘤发生发展中发挥着相当重要的作用。P53信号通路在神经胶质瘤发生发展中也扮演着极其关键的作用。MDM2对p53蛋白水平具有重要的调控作用,而且这种调控作用与TP53基因状态密切相关,因此,本研究着重探讨MDM2特异性小分子抑制剂Nutlin-3对具有不同TP53基因状态的神经胶质瘤的作用和机制,为Nutlin-3的临床应用以及神经胶质瘤的个体化治疗提供实验依据。实验方法:采用RT-PCR方法克隆出神经胶质瘤细胞系U87、A172和U251中TP53基因转录出的cDNA,采用DNA测序方法确定其基因是否有改变。在此基础上,应用Nutlin-3处理三种细胞,使用MTT方法测定细胞活力,明确Nutlin-3对三种细胞作用的差异;采用MTT法检测Nutlin-3联合PI3K抑制剂LY294002应用对三种胶质瘤的作用。应用RT-PCR定量方法确定三种细胞用药前后p53以及p53靶基因MDM2、BAX和P21 mRNA表达量的差异,应用Western-blot方法检测p53蛋白、多聚腺苷二磷酸核糖聚合酶(PARP)和核增殖抗原(PCNA)用药前后量的变化,应用Oligonucleotide pull-down方法检测三种细胞中p53蛋白的DNA结合活性。实验结果:1、克隆到的TP53基因cDNA片段经测序后确定,神经胶质瘤细胞系U87中TP53基因没有突变,属于野生型;而A172细胞系发生N239S改变,U251细胞系发生R273H改变。2、MTT细胞活力测定实验显示:MDM2抑制剂Nutlin-3能够明显抑制神经胶质瘤细胞U87(25μM,抑制率为36.97%)、A172(25μM,61.41%)的生长,而对U251(25μM,﹣18.27%)细胞没有明显作用;PI3K抑制剂LY294002对三种胶质瘤细胞在较高浓度下均有作用;Nutlin-3联合LY294002处理,抑制神经胶质瘤细胞生长作用明显强于单独使用Nutlin-3或LY294002。3、RT-PCR半定量实验显示:使用Nutlin-3处理前后,神经胶质瘤U251细胞p53 mRNA表达水平没有明显变化,而处理后U87、A172细胞中p53 mRNA表达水平显著升高(p<0.01);用药处理前后,U251细胞中p53靶基因P21和BAX mRNA表达水平没有太大变化,MDM2基因mRNA表达水平略有升高,但没有统计学意义。而处理后U87细胞中p53靶基因MDM2,P21和BAX mRNA表达水平明显升高(p<0.05);A172细胞中p53靶基因MDM2,P21 mRNA表达水平显著升高(p<0.01),BAX mRNA表达水平明显升高(p<0.05)。4、Western-blot实验表明:无药物处理时,神经胶质瘤U251细胞中p53蛋白水平显著高于U87和A172细胞(p<0.01);Nutlin-3处理后,U251细胞中p53蛋白水平略有提高但没有统计学意义。而用药处理后U87、A172细胞中p53蛋白水平显著升高(p<0.01),A172细胞升高尤其显著;药物处理后,细胞凋亡标志蛋白PARP在U87和U251细胞中没有明显变化,而在A172细胞中检测到少量caspase切割后片段;细胞增殖标志蛋白PCNA在U251和A172细胞中的水平没有明显变化,而在U87细胞中显著降低(p<0.01)。5、Oligonucleotide pull-down实验显示:U87细胞中野生型p53能够结合特定DNA序列,而且A172细胞中N239S改变型p53蛋白亦具备结合活性,只有U251细胞中R273H突变型p53蛋白不具备DNA结合活性。结论:1、神经胶质瘤细胞TP53基因状态不同,MDM2特异性小分子抑制剂Nutlin-3的作用有明显差别,提示临床治疗依据TP53基因状态决定MDM2抑制剂使用的必要性。2、PI3K抑制剂在较大浓度下对三种神经胶质瘤均有一定作用,MDM2抑制剂联合PI3K抑制剂的作用明显增加。3、A172细胞中TP53基因的改变不影响MDM2抑制剂的作用,说明该改变并没有改变p53蛋白的功能,Oligonucleotide pull-down实验进一步支持该结论。4、MDM2抑制剂通过抑制神经胶质瘤细胞增殖(PCNA改变)、促进神经胶质瘤细胞凋亡(BAX改变及PARP变化)发挥作用。5、MDM2抑制剂处理减少p53降解,p53蛋白水平增加导致靶基因表达水平增加,增加的MDM2会消掉MDM2抑制剂的部分作用,而增加的P21会进一步抑制MDM2的作用,提示p53-MDM2反馈调控环在肿瘤治疗中的重要性。更多还原

【Abstract】 Background and objectiveGlioma is one of the most common malignant tμMors in central nervous system. Malignant gliomas are characterized by its invasiveness and dissemination, resulting in frequent tμMor recurrence after surgical resection and/or conventional chemotherapy and radiation therapy. Current combinations of surgical therapy, radiotherapy and chemotherapy regimens do not significantly improve long-term survival of the patients with malignant glioma. The abnormal behaviors demonstrated by cancer cells are the result of a series of mutations in key regulatory genes. The cells become progressively more abnormal as more genes become damaged. The transition from a normal, healthy cell to a cancer cell is step-wise progression that requires genetic changes in several different oncogenes and tμMor suppressors. The growing awareness of differences in inter-individual response to radiochemical exposure calls for a new approach in the therapy and follow-up of cancer patients. P53 acts as a tμMor suppressor in many tμMor types, induces growth arrest or apoptosis depending on the physiological circμMstances and cell type. TP53 is frequently mutated or inactivated in about 60% of cancers. P53 is ubiquitinated by MDM2, which leads to proteasomal degradation. The goal is to improve the therapy results of Nutlin-3 by evaluating inter-individual differences in TP53 status.MethodsGlioma cell lines U87, A172 and U251 were used to evaluate the effects of MDM2 inhibitor Nutlin-3. First, the whole coding sequence of the p53 gene was cloned from cDNA by RT-PCR and sequenced. Second, the MTT assay was used to measure the activity of cell proliferation. Third, the effects of combination of Nutlin-3 and PI3k inhibitor, LY294002, were detected. Fourth, after the drug treatment, the mRNA levels of p53 and the target genes of p53, MDM2, P21 and BAX were compared with the control by semi-quantitative RT-PCR. Fifth, Western-blots were used to detect the changes of p53, PARP and PCNA protein after the treatment. Sixth, the specific DNA binding capacity of p53 protein in three cell lines was evaluated by oligonucleotide pull-down.Results1. The cloned coding sequence of p53 was sequenced. The results indicated that TP53 gene in U87 is wild-type, but N239S in A172, R273H in U251.2. MTT assays showed that Nutlin-3 can significantly inhibit the growth of U87 (25μM, inhibition rate 36.97%) and A172 (61.41%), but no effects on U251. The PI3K inhibitor, LY294002, can inhibit all three cells under high concentration. The effects of combination of Nutlin-3 and LY294002 are stronger than alone.3. Semi-quantitative RT-PCR analysis indicated that the p53 mRNA level didn’t change with Nutlin-3 treatment in U251 cells, and increased remarkably in U87 and A172 (p<0.01). After the treatment, the expression levels of p53 target genes, P21 and BAX, maintained, but that of MDM2 increased in U251. The Nutlin-3 treatment improved the expression of all the genes in U87 and A172.4. Without drug treatment, the protein level of p53 is higher in U251 than in U87 and A172. After the treatment, the p53 protein didn’t change in U251 and increased significantly in U87 and A172. The cleavage of poly (ADP-ribose) polymerase (PARP) - a biochemical marker for apoptosis - was induced by Nutlin-3 in A172 .The PCNA (Proliferating Cell Nuclear Antigen) decreased in U87, but not changed in U251 and A172.5. Oligonucleotide pull-down analysis showed that wild-type p53 in U87 and N239S p53 in A172 can effectively binding DNA, but R273H p53 in U251.Conclusions1. The effects of MDM2 inhibitor Nutlin-3 are different on gliomas with different TP53 status, which indicated the importance for the use of Nutlin-3 in clinic.2. The PI3K inhibitor has effects on gliomas under high concentration. And combination of MDM2 inhibitor and PI3K inhibitor give more strong effects.3. The genetic change in A172 doesn’t influence the effects of Nutlin-3, which indicates that N239S p53 have normal function. And oligonucleatide pull-down analysid supported this conclusion.4. The mechanisms of MDM2 inhibitor are inhibition of proliferation (PCNA change) and promotion of apoptosis (BAX change and PARP change).5. MDM2 inhibitor inhibits the degradation of p53, which promotes the expression of MDM2, and increased MDM2 will improve the degradation of p53, increased P21 will inhibit the effects of MDM2. Collectively, our results indicate that the p53-MDM2 feedback cycle play important role in glioma therapy.更多还原