【作者】 郑克彬；

【导师】 焦保华；

【作者基本信息】 河北医科大学 ， 外科学， 2011， 博士

【摘要】 脑胶质瘤作为人类最常见的原发性颅内肿瘤,有很高的发病率和死亡率。约占成人颅内原发肿瘤的30%-50%,近年来发病率还有增加的趋势。恶性肿瘤的常规治疗手段,如手术、放疗、化疗、生物治疗等对胶质瘤的治疗,效果甚微,胶质瘤患者的平均生存期仍较短。迄今的治疗方法都不能从根本上治疗这一致命的肿瘤。因而新疗法的探索成为热点课题。基因治疗作为治疗疾病的一种新手段,正愈来愈受到人们的重视和关注。常用的基因治疗的方法包括基因置换和增补缺陷基因。将抑癌基因转移肿瘤细胞是直接杀伤或抑制肿瘤细胞的最有效的基因治疗的措施之一。PTEN(phosphatase and tensin homologue deleted on chromosome 10)抑癌基因,即第10号染色体缺失的与张力蛋白同源的基因,是新近发现的一个具有双特异性的抑癌基因,在胶质瘤干细胞的表达谱中缺失。PTEN基因位于10号染色体q23.3,有9个外显子和8个内含子。第5个外显子编码第122和123位氨基酸,该编码序列与蛋白质丝氨酸/苏氨酸磷酸酶和蛋白质酪氨酸磷酸酶催化中心具有同源序列，表明该区域具有双特异性磷酸酶功能。PTEN蛋白所具有的双特异性磷酸酯酶的活性在抑制肿瘤中具有极其重要的作用,除了能使磷酸丝氨酸/苏氨酸和酪氨酸去磷酸化,发挥蛋白磷酸酯酶的作用外,还能通过抑制1-磷酸酰肌醇-3-激酶(P13K)的产物磷酸酰肌醇3,4,5-三磷酸(PIP3)的磷酸化,发挥脂性磷酸脂酶的功能,在P13K信号传导通路中起负性调节作用。PTEN直接使PIP3的D3位去磷酸化,这是PTEN抑制细胞生长和促进凋亡的重要机制。PTEN蛋白的表达能抑制P13K激酶信号通路介导的下游事件,如：PKB/Akt细胞存活增殖信号的激活,促进G1细胞周期转换P70S6-激酶的激活。PTEN基因已经成为有市场前景的抑癌基因。为进一步探讨PTEN基因及蛋白在脑胶质瘤中的作用,我们通过临床研究和基础研究两方面深入分析了PTEN在脑胶质瘤中的作用机制。通过研究我们检测了PTEN基因水平以及蛋白水平在脑胶质瘤中的表达以及与细胞凋亡的关系。通过基础研究我们将外源性野生型PTEN转染胶质瘤细胞系,观察野生型PTEN在体外及体内对胶质瘤细胞的治疗作用,并检测其可能的作用机制,为脑胶质瘤的基因治疗提供理论依据。系统的研究PTEN-PI3K-Akt-NF-KB以及下游靶基因的信号传导通路,在国内外尚无报道。第一部分PTEN在正常脑组织及脑胶质瘤中的表达与细胞凋亡关系的研究目的：探讨PTEN (phosphatase and tensin homology deleted on chromosome ten)在人脑胶质瘤及正常脑组织中的表达以及与细胞凋亡的关系。方法：应用免疫组织化学SP及Western blot检测人正常脑组织及人脑胶质瘤组织中PTEN蛋白水平的表达,半定量RT-PCR法检测人正常脑组织和人脑胶质瘤中PTEN mRNA水平的表达,以及TUNEL法检测人正常脑组织和人脑胶质瘤中细胞凋亡的情况。结果：1 PTEN在正常人脑组织标本中过度表达,在胶质瘤组织标本之中有表达,且随着级别的增加表达的阳性率降低(Figl),它们从正常到高级别的阳性率分别为100%、85.7%、63.6%、50%(table.1),低级别胶质瘤(I-II)和高级别胶质瘤(Ⅲ、Ⅳ)之间具有统计学意义(P<0.05)。2 PTEN在蛋白水平均为正常脑组织表达高于人脑胶质瘤,随着胶质瘤级别的升高,PTEN蛋白表达水平明显降低(Fig2),其相对表达量分别为0.667±0.203、0.475±0.169、0.237±0.087、0.153±0.046(table2),胶质瘤各级别之间表达有显著差异(P<0.05)。3 PTEN在mRNA水平均为正常脑组织表达高于人脑胶质瘤,随着胶质瘤级别的升高,PTENmRNA表达水平明显降低(Fig2),其相对表达量分别为3.767±0.865、2.530±0.619、1.853±0.436、0.688±0.140(table2),胶质瘤各级别之间表达有显著差异(P<0.05)。4人正常脑组织中细胞凋亡率明显低于人脑胶质瘤,两组之间表达有显著差异(P<0.05)(Fig4),且随着级别的增加表达的细胞凋亡率降低,它们从正常到高级别的凋亡率分别为3.2±0.5%、46.4±6.7%、37.2±5.2%、21.2±5.1%(table.4),细胞凋亡率与胶质瘤级别的升高以及PTEN mRNA及蛋白表达水平密切相关(P<0.05),随着PTEN mRNA及蛋白表达水平表达升高,细胞凋亡率随之下降。结论：PTEN mRNA及蛋白表达水平在人脑胶质瘤中随级别的升高表达下降,PTEN与细胞凋亡密切相关,可能成为其诊疗的一个新的指示靶点。第二部分PTEN基因重组腺病毒对胶质瘤细胞株增殖、凋亡以及细胞周期的影响目的：探讨抑癌基因PTEN对人胶质瘤U251、U87、A172细胞系增殖、凋亡及细胞周期的影响,并对其可能的作用机制进行探讨。方法：将携带PTEN及绿色荧光蛋白的重组腺病毒(Ad-PTEN-GFP)或空载体腺病毒(Ad- GFP)转染人胶质瘤U251、U87、A172细胞系。MTT检测细胞生长曲线；TUNEL法检测细胞凋亡；流式细胞仪检测Ad-PTEN-GFP转染后细胞周期分布以及细胞凋亡率；半定量PCR(RT- PCR)检测PTEN、CIAP1、CIAP2、XIAP、Survivin mRNA水平变化；Western Blot检测PTEN、AKT、p-AKT、IκB、P65、CIAP1、CIAP2、XIAP、Survivin、CyclinD1、P27、Bcl-2、Bax蛋白水平变化。结果：1携带PTEN腺病毒转染的检测结果Ad-PTEN感染胶质瘤细胞后,细胞在第三天达到最高转染率,当感染复数(M.O.I)为100时,荧光显微镜镜下显示细胞的荧光表达率均为80%以上(Figl、2、3),符合基因治疗对载体的要求。2 MTT检测细胞生长曲线在MOI=100时,Ad-PTEN-GFP转染组与Ad-GFP转染组各时间段细胞吸光度相比,增殖活性程度均降低(P<0.01 Fig 7),差异具有统计学意义。PTEN增高后明显降低胶质瘤细胞株的增殖活性。3 TUNEL法检测细胞凋亡在MOI=100时,腺病毒转染U251、U87、A172细胞系后,分别取1d、3d、5d后转染Ad-PTEN-GFP组细胞凋亡比例均明显高于未转染组,且于第5天,细胞凋亡比率最高(Fig 6、9 P<0.01)。4流式细胞仪检测细胞凋亡以及细胞周期变化Ad-PTEN-GFP转染U251、U87、A172细胞后与Ad-GFP组相比细胞凋亡率逐渐增加,空白对照和无义序列组相比凋亡率明显增加,提示PTEN基因表达上调明显诱导胶质瘤细胞株的凋亡(Fig 10)。细胞周期分析显示,当Ad-PTEN-GFP转染U251、U87、A172细胞后5天,G1期细胞比例增加,S期细胞比例降低,细胞周期阻滞于G1期(Fig 8)；5 RT-PCR检测PTEN以及IAP家族1mRNA水平以M.O.I=100转染U251、U87、A172细胞系1、3、5天,Ad-PTEN-GFP组与Ad-GFP组的CIAP1、CIAP2、XIAP、Survivin mRNA表达水平相比表达明显降低,且于第3天IAP家族mRNA表达水平降至最低水平。IAP家族mRNA表达水平与PTEN mRNA表达水平呈负相关改变(Fig 4、5；table 3、4 P<0.01)。6 Western blot检测PTEN及相关靶基因的蛋白水平以M.O.I=100转染U251、U87、A172细胞系1、3、5天,Ad-PTEN-GFP组与Ad-GFP组的AKT、p-Akt、IκB、P65、IAps、CyclinD1、P27、Bcl-2家族蛋白表达水平相比明显变化,且其蛋白表达水平与PTEN蛋白表达水平密切相关(Fig 5,table 4)。结论：高表达PTEN能够明显抑制人胶质瘤U251、U87、A172细胞系增殖并诱导凋亡,其可能与PTEN-PI3K/AKT-NF-κB信号通路被激活有关,并通过此通路调控多种凋亡相关因子家族,如IAPs及Bcl-2家族,并通过调控细胞周期相关因子导致细胞周期阻滞。第三部分PTEN基因重组腺病毒抑制裸鼠胶质瘤生长的实验研究目的：探讨Ad-PTEN对人胶质瘤荷瘤裸鼠的治疗作用,并对其可能的作用机制进行探讨。方法：建立裸鼠胶质瘤模型；将携带Ad-PTEN-GFP或空Ad-GFP注射治疗肿瘤,观察肿瘤生长情况及裸鼠生存期,TUNEL法检测细胞凋亡；免疫组化检查PTEN及P65蛋白水平变化。结果：1各组肿瘤生长情况及裸鼠存活时间U251细胞以7×105接种裸鼠后20天左右均可见肿瘤结节形成,瘤体内注射5次,Ad-PTEN组肿瘤体积生长明显变慢,较对照组体积小,治疗6周后肿瘤体积抑制率82.50%,与对照组相比有显著性差异(p<0.05 Fig5)。U251和Ad-GFP台疗组裸鼠肿瘤生长迅速,生存状态较差,均在移植后28-80天内相继死亡,两组裸鼠的平均生存时间分别54±7天和58±8天,Ad-PTEN治疗组裸鼠的生存状态明显好于另外两组,肿瘤生长缓慢直至移植后120天观察结束仍有2只裸鼠存活,平均生存时间103±10天,生成时间显著延长(Fig6)。2各组中目的蛋白及凋亡的表达免疫组化显示PTEN及P65蛋白主要表达于细胞核,偶有胞浆表达,着色后成棕黄色、棕褐色颗粒。其Ad-PTEN治疗组中PTEN表达明显增高,P65表达明显降低,细胞凋亡增加(Fig1-3),其表达差异有统计学意义。结论：各组细胞接种裸鼠后均可见移植瘤的生长,Ad-PTEN治疗后能明显抑制肿瘤在裸鼠内的生长,延长裸鼠的生存期,免疫组化结果提示PTEN可能通过PTEN-P65通路体内促瘤细胞凋亡。更多还原

【Abstract】 As the most common primary intracranial primary tumor, glioma has high morbidity and mortality. It accounts for 30%-50% of the adult intracranial, which is increasing in recent years. The conventional treatments of malignant tumor, such as surgery, radiation therapy, chemotherapy, biological therapy, all are not effective for gliomas. Therefore, the patients’average survival period is still short. So far this deadly cancer cannot be cured fundamentally. Thus the new therapy becomes a big task. As a fresh treatment for a disease, gene therapy is concerned by more and more people. Common gene therapy includes gene replacement and faulty gene supplement. Transfer tumor cells by tumor-suppressor gene are the most effective gene therapy to kill or inhibit tumor cells.PTEN (phosphates and tensing homologue deleted on chromosome 10) the tumor-suppressor genes, which are lack of chromosome 10 and homologous with tension protein genes, are one of the tumor suppressor genes with double specificity. It is discovered recently and lacked in the expression of gliomas stem cells. PTEN genes are located in chromosome 10 q23.3, including 9 explicit the son, and 8 introns. The 5th explicit son are coded the 122 and 123 amino acids. This coding sequence has the same sequence with protein serine/threonine phosphates and protein tyrosine phosphates catalytic center, so this region has double specificity phosphates’function. This function plays an important role in inhibiting tumor, which not only can dephosphorylated in phosphoric acid serine/threonine and tyrosine as protein phosphatase, but also can inhibit 1-3 instill acylating phosphate-the product of PI3K) kinase (3,4,5 inositol acylating phosphate 3 phosphoric acid (PIP3)-the phosphorylation of phosphate ester and plays a negative regulatory role in the function of PI3K enzyme signaling pathways, as fat phosphatase. The dephosphorylation of D3 PIP3 is an important mechanism of inhibiting cell growth and promoting apoptosis. PTEN protein can restrain the activities of PI3K kinase signaling pathways, such as PKB/Akt cell survival proliferation signal activation which can promote the G1 cell cycle P70S6-kinase activation. PTEN gene has become a market prospect as one kind of tumor suppressor genes.To further explore the effect of PTEN genes and proteins in gliomas, we deeply analyzed the PTEN functions in gliomas through clinical research and basic research. Through the research we examined PTEN gene level and protein levels in gliomas and the relationship with cell apoptosis. Through the basic research we transferred the exogenous wild-type PTEN into glioma cells transfected, observed the therapeutic effect of wild-type PTEN to gliomas cells in vitro and in vivo, and detected the possibility for glioma, for providing the theory basis for gene therapy. The research of PTEN-PI3K-Akt-NF-κB and the downstream target genes signaling pathways have not been reported at home and abroad.1:The research of relationship between PTEN expression in the normal brain tissue and gliomas and cell apoptosisObjective:Explore the relationship between PTEN (phosphatase and tensin homology deleted on chromosome ten) expression in the normal brain tissue and gliomas and cell apoptosis.Methods:Test people normal brain tissue and the human.brain PTEN protein levels in gliomas organization expression using immunohistochemistry SP and Western blot; detect normal brain tissue and human brain mRNA level in gliomas PTEN expression using semi-quantitative RT-PCR; and detect normal brain tissue and cell apoptosis in human glioma using TUNEL.Result:1. PTEN is over-expressed in normal brain tissue samples, exists in gliomas tissue samples. The positive expression increased following the levels (Fig1),100%,85.7%,63.6%,50% for normal to higher-level (table 1). There is statistically significant between low-level gliomas (Ⅰ-Ⅱ), and high-level gliomas (Ⅲ,Ⅳ) (P< 0.05).2. PTEN protein expression level in normal brain is higher than in the human brain with gliomas. The higher level of gliomas, the lower PTEN protein expression obviously (Fig2), which showed 0.667±0.203, 0.475±0.169,0.237±0.087,0.153±0.046 relatively (table2). Furthermore glioma express has significant differences (P< 0.05).3. PTEN mRNA level in normal brain is higher than in the human brain with gliomas. The higher level of gliomas, the lower PTENmRNA expression obviously (Fig2), which showed 3.767±0.865.2.530±0.619、1.853±0.436、0.688±0.140 relatively (table2). Furthermore glioma express has significant differences (P< 0.05).4. Cell apoptosis rate in normal brain obviously lower than in human gliomas, the two groups express significant difference (P< 0.05) (Fig4). Moreover apoptosis rate decreased with the increase of the expression level, 3.2±0.5%、46.4±6.7%、37.2±5.2%、21.2±5.1% from normal to higher-level (table 4). There is close relationship between apoptosis rate, the gliomas level and mRNA and protein expression level PTEN (P< 0.05), because when PTEN mRNA and protein expression level express rise, cell apoptosis rate decrease.Conclusion:PTEN mRNA and protein expression level of in the brain with elevated levels of gliomas, express drop, closely related with PTEN cell apoptosis, may become a new instructions targets diagnosis..2:The influence of PTEN gene recombinant adenovirus for cell proliferation of glioma, apoptosis and the cell cycleObjective:Analyze the PTEN tumor-suppressor genes’influence to U251, U87, A172 gliomas, apoptosis and cell proliferation, anddiscuss the cycle of its possible mechanism.Methods:Transfer restructuring adenovirus (Ad-PTEN-GFP) carrying PTEN and green fluorescent protein or empty carrier adenovirus (Ad-GFP) to U251, U87, A172 glioma cell lines. Detect cell growth curve by MTT; detect apoptosis by TUNEL; detect cell cycle distribution and apoptosis rate after carrying Ad-PTEN-GFP by flow cytometric analysis ion; test PTEN,CIAP1, CIAP2 XIAP, Survivin mRNA level by half quantitative polymerase chain reaction (PCR) (RT-PCR); test PTEN、AKT、p-AKT、IκB、P65、CIAP1、CIAP2、XIAP、Survivin、CyclinD1、P27、Bcl-2、Bax protein levels by Western Blot.Result:1. Test results of Carrying PTEN adenovirusThe highest rate reached in the third day after Ad-PTEN infection.when infections (M.O.I) was 100 plural, the fluorescent expression rate were all more than 80%(Fig1,2,3), which accorded with the requirement of the carrier of gene therapy.2. The MTT detection for cell growth curveWhen the MOI=100, the proliferation activity degree reduced compared to spectrophotometry Ad-PTEN-GFP carrying group and Ad-GFP carrying group each time (P< 0.01 Fig 7), with a statistically significant difference. Increased PTEN significantly reduced glioma cell proliferation activity.3. TUNEL method to detect apoptosisWhen MOI=100, adenovirus carrying U251, U87, A172 cell lines, cell apoptosis proportion in which respectively taking 1d,3d,5d carrying Ad-PTEN-GFP group were significantly higher than not carrying group. In the fifth day, apoptosis rate was highest (Fig 6,9 P< 0.01).4. Detect cell apoptosis and cell cycle changes by streaming instrument Compared with Ad-GFP cell, Ad PTEN-GFP-carrying U251, U87, A172 cells, apoptosis rate increase gradually; compared with no righteous sequence, blank control group apoptosis rate significantly increased. It noticed that PTEN genes over-active obvious lead to glioma cell apoptosis (Fig 1). The cell cycle analysis show that 5 days after the Ad-PTEN-GFP carrying U251, U87, A172 cell, G1 phase cells increased, S period cells decreased, the cell cycle block proportion in G1 phase (Fig 8).5. mRNA level of PTEN and IAP family by RT-PCR detection when M.O.I= 100 in carrying U251, U87, A172 cell line 1,3,5 days, compared with Ad-GFP group, Ad-PTEN-GFP group’s CIAP2, CIAP1, XIAP, Survivin mRNA expression level was significantly lower, and IAP family mRNA expression level is reduced to the minimum level on the third day. MRNA of IAP Expression level family and mRNA of PTEN Expression level were negatively correlated (Fig 4,5; table 3,4 P< 0.01).6. Western blot test PTEN and related target genes protein level When M.O.I= 100 in carrying U251, U87, A172 cell line 1,3,5 days, Ad-PTEN-GFP group protein expression level changed obviously compared to Ad-GFP group AKT、p-Akt、IκB、P65、IAps、CyclinD1、P27、Bcl-2 family, and the protein expression level was closely related to PTEN protein expression level (Fig 5, table 4).Conclusion:High expression PTEN can significantly inhibite gliomas U251, U87, A172 proliferation and apoptosis-oriented lines, which may be relevant to PTEN-PI3K/AKT-NF-κB signaling pathways activated. Through this channel regulation various factors related to family, such as apoptosis IAPs and Bcl-2 family, and through regulating the cell cycle related factors cause the cell cycle block.3:Experimental study of restructuring nude glioma growth by PTEN genesObjective:Study the therapeutic effect of nude tumor-burdened using the Ad-PTEN gliomas, and discuss its possible mechanism.Methods:Build nude glioma model, inject tumor carrying Ad-PTEN-GFP or empty Ad-GFP, observe tumor growth and nude survival, detect apoptosis using TUNEL method; Immunohistochemical check PTEN protein levels and P65 change.Results:1. Situation of tumor growth and survival time of nude U251 cells to 7×105 nude about 20 days after inoculation tumor nodules are visible form, inject inside 5 times, tumors Ad-PTEN group of tumor size obviously, a slow growth control volume small, the treatment for 6 weeks after tumor size inhibition rate, as compared with control 82.50% was significant difference (p <0.05 Fig5). U251 and Ad-GFP treatment group nude tumor growth and survival condition worse quickly after transplantation, two groups died off the average survival time in 28～80 days. Nude respectively to 54±7 days and 58±8days, Ad-PTEN nude treatment group was significantly better than living condition of the other two groups, tumor growth slow until 120 days after transplantation end still has 2 only observe nude alive, the mean survival time plus or minus 10 days,103 generate time significantly extend (Fig6).2. Each target protein and apoptosis in the expression Immunohistochemical display P65 protein mainly express PTEN and in the nucleus, with occasional cytoplasm after expression, shaded into tan, brown particles. Ad-PTEN treatment group significantly higher in P65 PTEN expression expression, reduced significantly, and apoptosis increase (Fig 1-3). Its expression differences with a statistical significance.Conclusion:Each cell nude are visible transplantation after inoculated the growth of tumors, Ad-PTEN after treatment can significantly inhibit the growth of tumors in nude, extend survival time. Immunohistochemical results suggest that PTEN possibly by PTEN-P65 pathways promote tumor cell apoptosis body.更多还原