發(fā)布時間：2018-06-05 01:02

  本文選題：PDCD5 + 腦神經(jīng)膠質瘤�。� 參考：《吉林大學》2015年博士論文

【摘要】：目的 腦神經(jīng)膠質瘤是一種常見的中樞神經(jīng)系統(tǒng)的惡性腫瘤，發(fā)病率居所有神經(jīng)系統(tǒng)腫瘤首位，約占35%~60%。隨著醫(yī)學技術的不斷發(fā)展，以手術治療為主，結合術后放療、化療的個體化綜合治療已成為膠質瘤治療的主要手段，但臨床上相當一部分患者對常規(guī)化療的敏感性較差，以往認為是因血腦屏障影響化療藥物到達腫瘤細胞。近年來研究證實，腦神經(jīng)膠質瘤化療效果受諸多方面原因的影響，其中化療藥物的耐藥性是導致化療失敗的重要原因之一。因此，如何提高膠質瘤患者對化療藥物的敏感性，尋找更有效的治療靶點逐漸成為膠質瘤術后輔助治療的重點課題。膠質瘤分子遺傳學研究發(fā)現(xiàn)，幾乎所有類型的膠質瘤均伴有不同程度的癌基因的激活以及抑癌基因的下調甚至缺失，而學者研究認為上述基因的突變不僅參與腫瘤發(fā)生發(fā)展，還與腫瘤耐藥性密切相關。近年來，越來越多的基因治療進入臨床試驗階段，而腦神經(jīng)膠質瘤基因靶向治療為腦膠質瘤的治療提供了新的思路。 程序性細胞死亡-5（PDCD5）是一種新發(fā)現(xiàn)的促凋亡因子，與基因缺失突變體的構建及其相關功能密切相關，且可能參與促凋亡活性的功能結構域的編碼過程。同時學者發(fā)現(xiàn)PDCD5在影響腫瘤組織的表達中也具有重要作用。現(xiàn)已有試驗證實，PDCD5在胃癌、卵巢癌、腎透明癌等多種腫瘤疾病中的表達水平明顯下降，且與腫瘤預后有密切聯(lián)系，但其在腦神經(jīng)膠質瘤中的表達水平，對神經(jīng)膠質瘤發(fā)生、發(fā)展的影響，能否影響膠質瘤的化療敏感性并作為膠質瘤靶基因治療的可行性，目前尚缺乏有力證據(jù)。 為了明確抑癌基因PDCD5在腦神經(jīng)膠質瘤中的表達及其對化療藥物的敏感性，將PDCD5發(fā)展成為相對安全、有效的基因治療靶點提供實驗依據(jù)，本文主要進行了以下兩方面的研究。 1.抑癌基因PDCD5在腦神經(jīng)膠質瘤中的表達及其作用。 2.抑癌基因PDCD5表達對膠質瘤細胞生長及化療敏感性的影響。 方法 一、抑癌基因PDCD5在腦神經(jīng)膠質瘤中的表達及其作用 1.收集吉林大學第一臨床醫(yī)院醫(yī)院神經(jīng)外科2009年1月-2012年12月收治的神經(jīng)膠質瘤患者石蠟包埋組織共86例，同時收集2013年1月-2014年12月在吉林大學第一臨床醫(yī)院神經(jīng)外科接受手術治療的腦神經(jīng)膠質瘤患者的新鮮標本30例，癌旁組織標本1例。 2.分別利用RT-PCR、Western Blot及免疫組化方法檢測神經(jīng)膠質瘤細胞系U87、U251及原發(fā)性神經(jīng)膠質瘤組織中PDCD5mRNA及蛋白質的表達情況。 3.比較分析腦神經(jīng)膠質瘤患者PDCD5的表達與膠質瘤臨床病理生理學特點的相關性，利用Kaplan-Meier生存曲線分析PDCD5的低表達或缺失與預后之間的關系。 二、抑癌基因PDCD5表達對膠質瘤細胞生長及化療敏感性的影響 1.采用不同濃度的化療藥物替莫唑胺（TMZ）處理膠質瘤細胞系U87及U251，應用MTT法繪制細胞生長曲線，分析PDCD5的表達水平對膠質瘤細胞系生長率的影響，及與不同濃度TMZ化療敏感性的關系。 2.利用基因轉染技術將PDCD5重組表達載體轉染膠質瘤細胞系U87，應用RT-PCR及Western Blot及免疫組化的方法檢測轉染后PDCD5的表達，篩選穩(wěn)定克隆的U87-PDCD5細胞，并利用MTT法比較U87-PDCD5和未轉染的U87的細胞生存率，分析外源性PDCD5過表達對腦神經(jīng)膠質瘤化療敏感性的影響。 3.根據(jù)目的基因PDCD5設計合成PDCD5特異性siRNA靶點，并將siRNA轉染高表達PDCD5的膠質瘤細胞系U251及穩(wěn)定轉染的U87-PDCD5，采用RT-PCR及WesternBlot的方法評價干擾效果，分析siRNA沉默后PDCD5的表達對膠質瘤細胞系化療敏感性的影響。 4.建立腦膠質瘤細胞系U87裸鼠皮下荷瘤模型，將模型隨機分為4組，分別注射生理鹽水（對照組，n=5），TMZ（TMZ組，n=5），外源性PDCD5重組表達載體（PDCD5組，n=5），TMZ+外源性PDCD5重組表達載體（聯(lián)合組，n=5）。治療20d后斷頸處死所有裸鼠，切取腫瘤組織并測量組織體積、稱重。采用RT-PCR、Western Blot方法檢測體內PDCD5的表達，分析PDCD5聯(lián)合TMZ對腦神經(jīng)膠質瘤生長的影響。 結果 一、抑癌基因PDCD5在腦神經(jīng)膠質瘤中的表達及其作用 1. PDCD5mRNA在腦神經(jīng)膠質瘤細胞系、原發(fā)性膠質瘤中的表達 RT-PCR結果顯示，PDCD5mRNA在U87、U251均有不同程度的表達，且PDCD5mRNA在U87中的相對表達量明顯低于在U251中的相對表達量，差異有統(tǒng)計學意義（P0.05）。與癌旁組織比較，PDCD5mRNA在原發(fā)性腦神經(jīng)膠質瘤中的表達出現(xiàn)不同程度的下調，甚至缺失，差異有統(tǒng)計學意義（P0.05）。 2. PDCD5蛋白在腦神經(jīng)膠質瘤細胞系、原發(fā)性膠質瘤中的表達 Western Blot結果顯示，PDCD5蛋白在U87中的表達較弱，明顯低于在U251中的表達。癌旁組織PDCD5蛋白的表達水平較高，而原發(fā)性腦神經(jīng)膠質瘤PDCD5蛋白的表達水平則不同程度的下調，甚至缺失。免疫組化結果顯示，在癌旁組織中，PDCD5陽性表達主要位于膠質細胞核及細胞漿內，呈棕黃色顆粒，其分布表現(xiàn)為明顯的異質性，而在腦神經(jīng)膠質瘤組織中，PDCD5染色較弱，甚至呈陰性表達。 3. PDCD5在腦神經(jīng)膠質瘤的表達狀態(tài)和腦神經(jīng)膠質瘤的病理分期相關性 RT-PCR結果顯示，PDCD5mRNA相對表達量在高級別腦神經(jīng)膠質瘤中的表達明顯低于低級別，差異有統(tǒng)計學意義（P0.05）。免疫組化結果顯示，腦神經(jīng)膠質瘤中組織中PDCD5蛋白的表達隨病理分級增高而減低，差異均有統(tǒng)計學意義（P0.05），而與年齡、性別、組織分型無關（P0.05）。 4. PDCD5在腦神經(jīng)膠質瘤中的低表達或缺失與預后的相關性 Kaplan-Meier生存分析結果顯示，所有患者平均生存時間19.6個月（95%CI:17.3~21.8），其中低表達組平均生存時間17.5個月（95%CI:15.1~19.9），高表達組平均生存時間23.7個月（95%CI:19.5~27.8），經(jīng)log-rank檢驗，低表達組的生存時間顯著短于高表達組，差異有統(tǒng)計學意義（χ2=4.94，P0.05）。 二、抑癌基因PDCD5表達對膠質瘤細胞生長及化療敏感性的影響 1. PDCD5表達與膠質瘤細胞生長及化療敏感性的關系 MTT法結果顯示，當TMZ濃度在0~110μmol/L范圍時，細胞生存率與TMZ濃度呈劑量依賴性，隨著TMZ濃度的升高，U87和U251細胞的相對生存率均隨之下降，，且U251細胞生存率下降的更為明顯，說明U251對TMZ的敏感性高于U87。當TMZ濃度大于110μmol/L時，生存率變化不大。 2.外源性PDCD5過表達對神經(jīng)膠質瘤細胞化療敏感性的影響 RT-PCR結果顯示，U87-PDCD5細胞中PDCD5mRNA的表達明顯高于未轉染U87，且PDCD5mRNA在U87-PDCD5中的相對表達量明顯高于在未轉染U87中的相對表達量，差異有統(tǒng)計學意義（P0.05）。Western Blot結果顯示，U87-PDCD5的蛋白質水平明顯高于未轉染U87中PDCD5。MTT法結果顯示，當TMZ濃度在0~110μmol/L范圍時，細胞生存率與TMZ濃度呈劑量依賴性，隨著TMZ濃度的升高，U87和U87-PDCD5細胞的相對生存率均隨之下降，且U87-PDCD5細胞生存率下降的更為明顯，說明其對TMZ的敏感性高于U87。當TMZ濃度大于110μmol/L時，生存率變化不大。 3. siRNA沉默PDCD5表達對神經(jīng)膠質瘤細胞生長及化療敏感性的影響 RT-PCR結果顯示，轉染PDCD5siRNA后，與對照組比較，U251、U87-PDCD5膠質瘤細胞內PDCD5mRNA的表達均明顯下降，差異有統(tǒng)計學意義（P0.05）。Western Blot結果顯示，轉染PDCD5siRNA后，U251、U87-PDCD5膠質瘤細胞內PDCD5蛋白的表達明顯下降，與對照組比較，差異有統(tǒng)計學意義（P0.05）。轉染后采用MTT法檢測細胞的生長率，結果顯示，隨著TMZ濃度的升高，U87-PDCD5-siRNA和U251-siRNA細胞的相對生存率均隨之下降，而U87-PDCD5-siRNA、U251-siRNA均明顯高于對照組，即對TMZ的化療敏感性降低。 4. PDCD5聯(lián)合TMZ對腦神經(jīng)膠質瘤生長的影響 聯(lián)合組腫瘤體積、體重分別為（1.78±0.07mm3、0.21±0.03g）均明顯低于對照組（3.46±0.06mm3、0.58±0.04g）、TMZ組（2.73±0.13mm3、0.37±0.06g）、PDCD5組（2.34±0.11mm3、0.34±0.05g），差異均有統(tǒng)計學意義（P0.05）。經(jīng)兩兩比較，各組腫瘤體積、體重依次為對照組 TMZ組、PDCD5組聯(lián)合組，其中TMZ組、PDCD5組間比較，差異無統(tǒng)計學意義（P0.05）。RT-PCR、Western Blot方法檢測結果顯示，含PDCD5載體的聯(lián)合組和PDCD5組PDCD5mRNA及蛋白的表達高于對照組和TMZ組，表明PDCD5在荷瘤模型體內成功表達。 結論 1. PDCD5mRNA和蛋白在腦神經(jīng)膠質瘤患者中表達明顯下調甚至缺失，且PDCD5的表達隨病理分級增高而減低，而與年齡、性別、組織分型無關，PDCD5低表達或缺失的腦神經(jīng)膠質瘤患者的生存時間顯著短于高表達者。 2. PDCD5過表達可明顯抑制膠質瘤細胞的生長，增強腦神經(jīng)膠質瘤對化療藥物TMZ的化療敏感性，沉默表達PDCD5后腦神經(jīng)膠質瘤對TMZ的敏感性明顯降低；PDCD5與TMZ聯(lián)合應用可更好地抑制腦神經(jīng)膠質瘤的生長，有望成為一種新的臨床治療靶點。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R739.41

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