抑癌基因PDCD5在脑神经胶质瘤中的表达及其对化疗药物敏感性影响研究

发布时间：2018-06-05 01:02

本文选题：PDCD5 + 脑神经胶质瘤　；参考：《吉林大学》2015年博士论文

【摘要】：目的脑神经胶质瘤是一种常见的中枢神经系统的恶性肿瘤，发病率居所有神经系统肿瘤首位，约占35%~60%。随着医学技术的不断发展，以手术治疗为主，结合术后放疗、化疗的个体化综合治疗已成为胶质瘤治疗的主要手段，但临床上相当一部分患者对常规化疗的敏感性较差，以往认为是因血脑屏障影响化疗药物到达肿瘤细胞。近年来研究证实，脑神经胶质瘤化疗效果受诸多方面原因的影响，其中化疗药物的耐药性是导致化疗失败的重要原因之一。因此，如何提高胶质瘤患者对化疗药物的敏感性，寻找更有效的治疗靶点逐渐成为胶质瘤术后辅助治疗的重点课题。胶质瘤分子遗传学研究发现，几乎所有类型的胶质瘤均伴有不同程度的癌基因的激活以及抑癌基因的下调甚至缺失，而学者研究认为上述基因的突变不仅参与肿瘤发生发展，还与肿瘤耐药性密切相关。近年来，越来越多的基因治疗进入临床试验阶段，而脑神经胶质瘤基因靶向治疗为脑胶质瘤的治疗提供了新的思路。程序性细胞死亡-5（PDCD5）是一种新发现的促凋亡因子，与基因缺失突变体的构建及其相关功能密切相关，且可能参与促凋亡活性的功能结构域的编码过程。同时学者发现PDCD5在影响肿瘤组织的表达中也具有重要作用。现已有试验证实，PDCD5在胃癌、卵巢癌、肾透明癌等多种肿瘤疾病中的表达水平明显下降，且与肿瘤预后有密切联系，但其在脑神经胶质瘤中的表达水平，对神经胶质瘤发生、发展的影响，能否影响胶质瘤的化疗敏感性并作为胶质瘤靶基因治疗的可行性，目前尚缺乏有力证据。为了明确抑癌基因PDCD5在脑神经胶质瘤中的表达及其对化疗药物的敏感性，将PDCD5发展成为相对安全、有效的基因治疗靶点提供实验依据，本文主要进行了以下两方面的研究。 1.抑癌基因PDCD5在脑神经胶质瘤中的表达及其作用。 2.抑癌基因PDCD5表达对胶质瘤细胞生长及化疗敏感性的影响。方法一、抑癌基因PDCD5在脑神经胶质瘤中的表达及其作用 1.收集吉林大学第一临床医院医院神经外科2009年1月-2012年12月收治的神经胶质瘤患者石蜡包埋组织共86例，同时收集2013年1月-2014年12月在吉林大学第一临床医院神经外科接受手术治疗的脑神经胶质瘤患者的新鲜标本30例，癌旁组织标本1例。 2.分别利用RT-PCR、Western Blot及免疫组化方法检测神经胶质瘤细胞系U87、U251及原发性神经胶质瘤组织中PDCD5mRNA及蛋白质的表达情况。 3.比较分析脑神经胶质瘤患者PDCD5的表达与胶质瘤临床病理生理学特点的相关性，利用Kaplan-Meier生存曲线分析PDCD5的低表达或缺失与预后之间的关系。二、抑癌基因PDCD5表达对胶质瘤细胞生长及化疗敏感性的影响 1.采用不同浓度的化疗药物替莫唑胺（TMZ）处理胶质瘤细胞系U87及U251，应用MTT法绘制细胞生长曲线，分析PDCD5的表达水平对胶质瘤细胞系生长率的影响，及与不同浓度TMZ化疗敏感性的关系。 2.利用基因转染技术将PDCD5重组表达载体转染胶质瘤细胞系U87，应用RT-PCR及Western Blot及免疫组化的方法检测转染后PDCD5的表达，筛选稳定克隆的U87-PDCD5细胞，并利用MTT法比较U87-PDCD5和未转染的U87的细胞生存率，分析外源性PDCD5过表达对脑神经胶质瘤化疗敏感性的影响。 3.根据目的基因PDCD5设计合成PDCD5特异性siRNA靶点，并将siRNA转染高表达PDCD5的胶质瘤细胞系U251及稳定转染的U87-PDCD5，采用RT-PCR及WesternBlot的方法评价干扰效果，分析siRNA沉默后PDCD5的表达对胶质瘤细胞系化疗敏感性的影响。 4.建立脑胶质瘤细胞系U87裸鼠皮下荷瘤模型，将模型随机分为4组，分别注射生理盐水（对照组，n=5），TMZ（TMZ组，n=5），外源性PDCD5重组表达载体（PDCD5组，n=5），TMZ+外源性PDCD5重组表达载体（联合组，n=5）。治疗20d后断颈处死所有裸鼠，切取肿瘤组织并测量组织体积、称重。采用RT-PCR、Western Blot方法检测体内PDCD5的表达，分析PDCD5联合TMZ对脑神经胶质瘤生长的影响。结果一、抑癌基因PDCD5在脑神经胶质瘤中的表达及其作用 1. PDCD5mRNA在脑神经胶质瘤细胞系、原发性胶质瘤中的表达 RT-PCR结果显示，PDCD5mRNA在U87、U251均有不同程度的表达，且PDCD5mRNA在U87中的相对表达量明显低于在U251中的相对表达量，差异有统计学意义（P0.05）。与癌旁组织比较，PDCD5mRNA在原发性脑神经胶质瘤中的表达出现不同程度的下调，甚至缺失，差异有统计学意义（P0.05）。 2. PDCD5蛋白在脑神经胶质瘤细胞系、原发性胶质瘤中的表达 Western Blot结果显示，PDCD5蛋白在U87中的表达较弱，明显低于在U251中的表达。癌旁组织PDCD5蛋白的表达水平较高，而原发性脑神经胶质瘤PDCD5蛋白的表达水平则不同程度的下调，甚至缺失。免疫组化结果显示，在癌旁组织中，PDCD5阳性表达主要位于胶质细胞核及细胞浆内，呈棕黄色颗粒，其分布表现为明显的异质性，而在脑神经胶质瘤组织中，PDCD5染色较弱，甚至呈阴性表达。 3. PDCD5在脑神经胶质瘤的表达状态和脑神经胶质瘤的病理分期相关性 RT-PCR结果显示，PDCD5mRNA相对表达量在高级别脑神经胶质瘤中的表达明显低于低级别，差异有统计学意义（P0.05）。免疫组化结果显示，脑神经胶质瘤中组织中PDCD5蛋白的表达随病理分级增高而减低，差异均有统计学意义（P0.05），而与年龄、性别、组织分型无关（P0.05）。 4. PDCD5在脑神经胶质瘤中的低表达或缺失与预后的相关性 Kaplan-Meier生存分析结果显示，所有患者平均生存时间19.6个月（95%CI:17.3~21.8），其中低表达组平均生存时间17.5个月（95%CI:15.1~19.9），高表达组平均生存时间23.7个月（95%CI:19.5~27.8），经log-rank检验，低表达组的生存时间显著短于高表达组，差异有统计学意义（χ2=4.94，P0.05）。二、抑癌基因PDCD5表达对胶质瘤细胞生长及化疗敏感性的影响 1. PDCD5表达与胶质瘤细胞生长及化疗敏感性的关系 MTT法结果显示，当TMZ浓度在0~110μmol/L范围时，细胞生存率与TMZ浓度呈剂量依赖性，随着TMZ浓度的升高，U87和U251细胞的相对生存率均随之下降，，且U251细胞生存率下降的更为明显，说明U251对TMZ的敏感性高于U87。当TMZ浓度大于110μmol/L时，生存率变化不大。 2.外源性PDCD5过表达对神经胶质瘤细胞化疗敏感性的影响 RT-PCR结果显示，U87-PDCD5细胞中PDCD5mRNA的表达明显高于未转染U87，且PDCD5mRNA在U87-PDCD5中的相对表达量明显高于在未转染U87中的相对表达量，差异有统计学意义（P0.05）。Western Blot结果显示，U87-PDCD5的蛋白质水平明显高于未转染U87中PDCD5。MTT法结果显示，当TMZ浓度在0~110μmol/L范围时，细胞生存率与TMZ浓度呈剂量依赖性，随着TMZ浓度的升高，U87和U87-PDCD5细胞的相对生存率均随之下降，且U87-PDCD5细胞生存率下降的更为明显，说明其对TMZ的敏感性高于U87。当TMZ浓度大于110μmol/L时，生存率变化不大。 3. siRNA沉默PDCD5表达对神经胶质瘤细胞生长及化疗敏感性的影响 RT-PCR结果显示，转染PDCD5siRNA后，与对照组比较，U251、U87-PDCD5胶质瘤细胞内PDCD5mRNA的表达均明显下降，差异有统计学意义（P0.05）。Western Blot结果显示，转染PDCD5siRNA后，U251、U87-PDCD5胶质瘤细胞内PDCD5蛋白的表达明显下降，与对照组比较，差异有统计学意义（P0.05）。转染后采用MTT法检测细胞的生长率，结果显示，随着TMZ浓度的升高，U87-PDCD5-siRNA和U251-siRNA细胞的相对生存率均随之下降，而U87-PDCD5-siRNA、U251-siRNA均明显高于对照组，即对TMZ的化疗敏感性降低。 4. PDCD5联合TMZ对脑神经胶质瘤生长的影响联合组肿瘤体积、体重分别为（1.78±0.07mm3、0.21±0.03g）均明显低于对照组（3.46±0.06mm3、0.58±0.04g）、TMZ组（2.73±0.13mm3、0.37±0.06g）、PDCD5组（2.34±0.11mm3、0.34±0.05g），差异均有统计学意义（P0.05）。经两两比较，各组肿瘤体积、体重依次为对照组 TMZ组、PDCD5组联合组，其中TMZ组、PDCD5组间比较，差异无统计学意义（P0.05）。RT-PCR、Western Blot方法检测结果显示，含PDCD5载体的联合组和PDCD5组PDCD5mRNA及蛋白的表达高于对照组和TMZ组，表明PDCD5在荷瘤模型体内成功表达。结论 1. PDCD5mRNA和蛋白在脑神经胶质瘤患者中表达明显下调甚至缺失，且PDCD5的表达随病理分级增高而减低，而与年龄、性别、组织分型无关，PDCD5低表达或缺失的脑神经胶质瘤患者的生存时间显著短于高表达者。 2. PDCD5过表达可明显抑制胶质瘤细胞的生长，增强脑神经胶质瘤对化疗药物TMZ的化疗敏感性，沉默表达PDCD5后脑神经胶质瘤对TMZ的敏感性明显降低；PDCD5与TMZ联合应用可更好地抑制脑神经胶质瘤的生长，有望成为一种新的临床治疗靶点。
[Abstract]:objective
Brain glioma is a common malignant tumor of the central nervous system. The incidence of neuroglioma is the first one in all nervous system tumors. It accounts for about 35%~60%. with the continuous development of medical technology. Surgery is the main treatment, combined with postoperative radiotherapy, individualized chemotherapy has become the main method for the treatment of glioma, but it is quite a part of the clinic. The sensitivity of the patients to conventional chemotherapy is poor, which was previously thought to be due to the influence of the blood brain barrier to the tumor cells. In recent years, the study has confirmed that the effect of chemotherapy on brain glioma is affected by many factors, and the drug resistance of chemotherapy is one of the major causes of chemotherapy failure. Therefore, how to improve the glioma patients The sensitivity of chemotherapeutic drugs and the search for more effective therapeutic targets have gradually become a key topic for postoperative adjuvant therapy for glioma. The molecular genetic study of glioma has found that almost all types of gliomas are associated with the activation of different degrees of oncogenes and the downregulation or even deletion of the tumor suppressor genes. The mutation is not only involved in the development of tumor, but also closely related to the drug resistance. In recent years, more and more gene therapy has entered the clinical trial stage, and the gene targeting therapy of brain glioma provides a new way of thinking for the treatment of glioma.
Programmed cell death -5 (PDCD5) is a newly discovered proapoptotic factor, which is closely related to the construction of gene deletion mutants and their related functions, and may be involved in the coding process of the functional domain of the apoptotic activity. At the same time, scholars have found that PDCD5 also plays an important role in the expression of tumor tissue. Now, experiments have been proved that PDCD 5 the level of expression in cancer, ovarian cancer and renal clear cancer is obviously decreased, and it has a close relationship with the prognosis of tumor. However, the expression level in brain glioma, the effect on the occurrence and development of glioma, whether it affects the chemensitivity of glioma and the feasibility of treating glioma as the target gene of glioma, There is still a lack of strong evidence.
In order to clarify the expression of tumor suppressor gene PDCD5 in brain glioma and its sensitivity to chemotherapeutic drugs, the development of PDCD5 as a relatively safe and effective gene therapy target provides experimental basis. The following two aspects are mainly carried out in this paper.
1. expression and role of tumor suppressor gene PDCD5 in brain glioma.
2. the effect of PDCD5 expression on growth and chemosensitivity of glioma cells.
Method
Expression and role of tumor suppressor gene PDCD5 in glioma
1. a total of 86 cases of paraffin embedded tissues from patients with glioma treated in the Department of Neurosurgery, the first clinical hospital of Jilin University, January 2009 -2012 December, were collected and 30 fresh specimens of brain neuroglioma patients were collected in December January 2013 -2014, the first clinical hospital of Jilin University. This is the 1 case.
2. the expression of PDCD5mRNA and protein in glioma cell lines U87, U251 and primary glioma tissues were detected by RT-PCR, Western Blot and immunohistochemistry respectively.
3. the correlation between the expression of PDCD5 and the clinicopathological characteristics of glioma was compared. The relationship between the low expression or deletion of PDCD5 and the prognosis was analyzed by the Kaplan-Meier survival curve.
Two, the effect of tumor suppressor gene PDCD5 expression on the growth and chemosensitivity of glioma cells
1. the glioma cell lines U87 and U251 were treated with different concentrations of timozolamine (TMZ). The cell growth curve was plotted by MTT method. The effect of PDCD5 expression level on the growth rate of glioma cell line and the relationship with the sensitivity of different concentrations of TMZ chemotherapy were analyzed.
2. the recombinant expression vector of PDCD5 was transfected into glioma cell line U87 by gene transfection technology. The expression of PDCD5 after transfection was detected by RT-PCR and Western Blot and immunohistochemical method, and the stable cloned U87-PDCD5 cells were screened. The cell survival rate of U87-PDCD5 and untransfected U87 was compared by MTT method, and the exogenous PDCD5 overexpression was analyzed. The effect of chemosensitivity of brain glioma.
3. the target gene of PDCD5 specific siRNA was designed according to the target gene PDCD5, and siRNA was transfected to the glioma cell line of high expression PDCD5 and the stable transfected U87-PDCD5. The interference effect was evaluated by RT-PCR and WesternBlot, and the effect of PDCD5 expression on the chemosensitivity of glioma cell lines after siRNA was analyzed.
4. subcutaneous tumor bearing tumor model of glioma cell line U87 was established. The models were randomly divided into 4 groups, including saline (control group, n=5), TMZ (TMZ group, n=5), exogenous PDCD5 recombinant expression vector (PDCD5 group, n=5), TMZ+ exogenous PDCD5 recombinant expression vector (joint group, n=5). The expression of PDCD5 in vivo was detected by RT-PCR and Western Blot, and the effect of PDCD5 combined with TMZ on the growth of glioma was analyzed.
Result
Expression and role of tumor suppressor gene PDCD5 in glioma
Expression of 1. PDCD5mRNA in glioma cell line and primary glioma
RT-PCR results showed that PDCD5mRNA was expressed in different degrees in U87 and U251, and the relative expression of PDCD5mRNA in U87 was significantly lower than that in U251, and the difference was statistically significant (P0.05). The expression of PDCD5mRNA in primary brain glioma was down to varying degrees, even missing, and the difference was found. There was a statistical significance (P0.05).
Expression of 2. PDCD5 protein in glioma cell line and primary glioma
The results of Western Blot showed that the expression of PDCD5 protein in U87 was weaker, obviously lower than that in U251. The expression level of PDCD5 protein in the paracancerous tissues was higher, while the expression level of PDCD5 protein in primary brain glioma was downregulated and even missing. The immunohistochemical staining showed that the positive expression of PDCD5 in the para cancerous tissues was the main expression. In the nucleus and cytoplasm of glial nuclei and cytoplasm, brown yellow granules are found, and their distribution is obviously heterogeneous, but in brain glioma tissue, PDCD5 staining is weak and even negative.
The correlation between the expression of 3. PDCD5 in glioma and pathological stage of glioma
RT-PCR results showed that the expression of relative expression of PDCD5mRNA in advanced brain glioma was significantly lower than that of low level, and the difference was statistically significant (P0.05). The immunohistochemical results showed that the expression of PDCD5 protein in the tissues of brain glioma decreased with the histopathological grade, and the difference was statistically significant (P0.05), but with age and sex. The tissue classification is independent (P0.05).
Low expression or deletion of 4. PDCD5 in glioma and its correlation with prognosis
The Kaplan-Meier survival analysis showed that the average survival time of all the patients was 19.6 months (95%CI:17.3~21.8), of which the average survival time of the low expression group was 17.5 months (95%CI:15.1~19.9), the average survival time of the high expression group was 23.7 months (95%CI:19.5~27.8). The survival time of the low expression group was significantly shorter than that of the high expression group by log-rank test. Study significance (x 2=4.94, P0.05).
Two, the effect of tumor suppressor gene PDCD5 expression on the growth and chemosensitivity of glioma cells
1. the relationship between PDCD5 expression and growth and chemosensitivity of glioma cells
The results of MTT showed that when the concentration of TMZ was in the range of 0~110 mol/L, the cell survival rate and TMZ concentration were dose-dependent. With the increase of TMZ concentration, the relative survival rate of U87 and U251 cells decreased, and the survival rate of U251 cells decreased more obviously, indicating that the sensitivity of U251 to TMZ was higher than that of U87. when the concentration was greater than 110 mu. There is little change in the rate.
2. the effect of exogenous PDCD5 over expression on chemosensitivity of glioma cells
The results of RT-PCR showed that the expression of PDCD5mRNA in U87-PDCD5 cells was significantly higher than that of untransfected U87, and the relative expression of PDCD5mRNA in U87-PDCD5 was significantly higher than that in the untransfected U87. The difference was statistically significant (P0.05).Western Blot results showed that U87-PDCD5 protein level was significantly higher than that in the non transfected U87. The results showed that when the concentration of TMZ was in the range of 0~110 mol/L, the cell survival rate and the concentration of TMZ were dose-dependent. With the increase of TMZ concentration, the relative survival rate of U87 and U87-PDCD5 cells decreased, and the survival rate of U87-PDCD5 cells decreased more obviously, indicating that the sensitivity of the U87-PDCD5 cells to TMZ was higher than U87. when TMZ concentration was greater than 110 mu mol/L. There is little change in the survival rate.
Effect of 3. siRNA silencing PDCD5 expression on growth and chemosensitivity of glioma cells
RT-PCR results showed that after transfection of PDCD5siRNA, the expression of PDCD5mRNA in U251 and U87-PDCD5 glioma cells decreased significantly compared with the control group, and the difference was statistically significant (P0.05).Western Blot results showed that the expression of PDCD5 protein in U251 and U87-PDCD5 glioma cells decreased significantly after the transfection of PDCD5siRNA, and compared with the control group, there was a difference between the cells and the control group. The study significance (P0.05). After transfection, the cell growth rate was detected by MTT. The results showed that the relative survival rate of U87-PDCD5-siRNA and U251-siRNA cells decreased with the increase of TMZ concentration, and U87-PDCD5-siRNA, U251-siRNA were significantly higher than those of the control group, that is, the sensitivity of TMZ to chemotherapy was reduced.
Effect of 4. PDCD5 combined with TMZ on the growth of glioma
The volume of tumor and body weight (1.78 + 0.07mm3,0.21 + 0.03g) were significantly lower in the combined group than in the control group (3.46 + 0.06mm3,0.58 + 0.04g), TMZ group (2.73 + 0.13mm3,0.37 + 0.06g), PDCD5 group (2.34 + 0.11mm3,0.34 + 0.05g), and the difference was statistically significant (P0.05). After 22 comparison, the volume and body weight of each group were in the same group as the control group. In group TMZ and group PDCD5, the difference was not statistically significant (P0.05).RT-PCR, and the results of Western Blot assay showed that the expression of PDCD5mRNA and protein in the combination group containing PDCD5 carrier and PDCD5 group was higher than that of the control group and the TMZ group, indicating that PDCD5 was expressed in the tumor bearing model.
conclusion
The expression of 1. PDCD5mRNA and protein in the patients with brain glioma was obviously down and even missing, and the expression of PDCD5 decreased with the histopathological grade, but it was not related to age, sex and histological type. The survival time of the patients with brain glioma with low PDCD5 expression or deletion was significantly shorter than that of the high expression.
2. PDCD5 overexpression can obviously inhibit the growth of glioma cells, enhance the chemosensitivity of brain glioma to chemotherapeutic drug TMZ, and reduce the sensitivity of brain glioma to TMZ after silent expression of PDCD5, and the combination of PDCD5 and TMZ can better inhibit the growth of brain glioma, and it is expected to be a new therapeutic target. Point.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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