【作者】 刘晓东；

【导师】 范益民；

【作者基本信息】 山西医科大学 ， 神经外科学， 2011， 博士

【摘要】 研究背景逆转胶质瘤耐药成为治疗胶质瘤的重要策略，但对胶质瘤耐药发生机制的认识仍存在争议。肿瘤耐药与其高表达耐药基因相关的看法达成了共识；组成肿瘤组织的细胞存在异质性，即由不同亚型细胞组成也是众所周知的事实，然而早先认为相关耐药基因表达是群体细胞的作用，而较少注意到因细胞异质性引发的差别。近年来肿瘤干细胞学说的提出，为解释肿瘤异质性导致肿瘤分子生物学行为差异提供了理论依据，为研究肿瘤细胞生物学特性、解释肿瘤耐药的原因等提供了新思路。凋亡是细胞在一定生理或病理条件下程序性死亡的过程，对生物维持组织稳态和个体生长发育有重要的调控作用，凋亡是涉及细胞层面的一个重要生物学行为，由于大多数化疗药物是通过诱导肿瘤细胞凋亡而发挥作用，所以细胞凋亡通路的异常改变是肿瘤耐药的又一重要机制。逆转肿瘤耐药的研究需要我们重视凋亡调控在肿瘤细胞产生耐药性中的地位及其作用机制。从胶质瘤干细胞层面寻找耐药根源，寻找关键基因、明确凋亡通路异常机制及其相互关系是增强肿瘤对化疗药物敏感性研究的有效途径。本项目在既往研究基础上，从胶质瘤干细胞异质性入手，以胶质瘤凋亡相关的p53上调凋亡调控因子（PUMA）为切入点，以凋亡通路为主线，应用基因转染、RT-PCR、Western-blot方法，从体内体外干预实验探讨PUMA在增强胶质瘤细胞对化疗药物替莫唑胺敏感性的作用机制，即PUMA高表达或激活PUMA促进胶质瘤细胞凋亡并逆转耐药；PUMA可能调控机制：①PUMA维持细胞抗凋亡与促凋亡系统的平衡，其功能缺失，导致凋亡系统失衡，从而发生耐药；②PUMA低表达影响细胞生长周期，使得细胞能够避开化疗药物作用的有效周期，导致细胞耐药；③PUMA调控耐药相关蛋白表达，PUMA对耐药分子的调控异常与耐药发生密切相关。④激活处于G0/G1期的胶质瘤干细胞，使其进入化疗药物的有效作用周期，导致细胞内凋亡触发。本研究从细胞异质性及凋亡调控两方面寻找胶质瘤耐药的根源，并提出PUMA逆转胶质瘤耐药的调控通路模式，为防治胶质瘤化疗耐药提供新思路。研究目的：1.探讨促凋亡基因PUMA在人类脑胶质瘤中的表达，及其与Bcl-2、Bax蛋白表达的相关性。2.探讨体外转染PUMA的人类脑胶质瘤细胞对替莫唑胺敏感性增强的作用机制。3.通过体内实验探讨外源性PUMA对人类脑胶质瘤细胞生长的影响及其增强胶质瘤细胞对替莫唑胺（TMZ）敏感性的机制。4.探讨基于CD133免疫原型的胶质瘤干细胞耐药性的差异以及PUMA改变胶质瘤细胞对TMZ敏感性的研究。研究方法：1.应用免疫印迹法(Western blotting.WB)，检测10例正常脑组织及52例脑胶质瘤瘤组织中PUMA、Bcl-2、Bax蛋白的表达，通过计算吸光度比值求得各蛋白相对表达量并统计分析结果。2.将人类脑胶质瘤细胞U87MG细胞分为正常对照组、空载体组和实验组进行细胞培养，分别将MOI=50的空载体腺病毒（Ad-△BH3）和携带PUMA的重组腺病毒（Ad-PUMA）转染U87MG细胞，转染后用MTT比色法测定各组细胞的存活率并计算IC50，流式细胞仪检测细胞周期的变化，应用TUNEL法检测细胞的凋亡情况。Western blot检测凋亡相关蛋白P53、Bax的表达。3.建立人类脑胶质瘤细胞株U87MG裸鼠皮下移植瘤模型，将胶质瘤裸鼠随机分为四组，于建模2周后，分别自腹腔注射生理盐水100μl (对照组，n=8)、2×108 pfu/100μlAd-PUMA ( PUMA组，n=8)、TMZ 10mg/kg (TMZ组，n= 8)和2×108 pfu/100μl Ad-PUMA+ TMZ 10mg/kg (联合组，n= 8)。治疗20天后处死裸鼠，剖腹测量原位肿瘤体积、抑瘤率。TUNEL检测肿瘤细胞凋亡情况。半定量RT-PCR和Western blot检测DNA损伤修复蛋白MGMTmRNA和MGMT蛋白表达情况。4.将分选出的CD133+和CD133- U87细胞分别进行传代培养，应用WB，RT-PCR，基因转染流式细胞分析等方法观察外源性PUMA、TMZ干预前后两群细胞的生长变化情况。研究结果：1. PUMA蛋白、Bax、Bcl-2蛋白表达在Ⅱ、Ⅲ、Ⅳ级胶质瘤中与正常脑组织有统计学意义(P<0.01)，Ⅰ级胶质瘤与对照组表达无统计学意义（P>0.05）。病理分级与PUMA、Bax蛋白和Bcl-2蛋白表达相关(rPUMA=-0.925,rBax =-0.931,rBcl-2=0.945,P均<0.001)。PUMA和Bax、Bcl-2蛋白的表达相关(r1=0.963,r2=-0.937 P＜0.001)。2.外源性PUMA基因在Ad-PUMA转染U87MG 48h后，随着时间延长，PUMA表达使U87MG细胞的增殖能力降低并诱导凋亡，转染24、48、72h的生长抑制率分别为17.3%、35.6%、43.3%，凋亡率分别为20.3%、31.4%、45.4%。正常对照组、Ad-PUMA、Ad-△BH3组细胞的TMZ IC50分别为(15±1.9)、(2.3±0.14)和(14.4±1.6)μmol/L。PUMA表达的U87MG细胞DNA合成受到抑制，周期阻滞在G2期；Western blot检测显示P53表达在各组中差别无统计学意义（P>0.05）、PUMA和Bax在实验组中表达较对照组强，差别有统计学意义（P<0.01）。3.治疗20天时对照组、PUMA组、TMZ组、联合组诱发肿瘤瘤体积分别为(3.68±0.16)、(2.63±0.22)、(2.13±0.16)、(0.97±0.18)cm3四组体积进行两两比较差别有统计学意义(P均<0.05)。治疗组抑瘤率分别为28.5%、42.1%、73.6%，两两比较差别有统计学意义(P均<0.05)。TUNEL染色并计算凋亡指数（AI）：对照组(2.0±1.2)%、Ad-PUMA组(11.4±2.6)%、TMZ组(7.6±3.2)%、联合组(20.6±8.6)%进行两两比较差别有统计学意义(P均<0.05)。半定量RT-PCR和Western blot检测显示MGMT mRNA和MGMT蛋白在TMZ组中表达最高、与其他三组比较差异有统计学意义(P均< 0.05)。4. CD133阳性免疫磁珠分选出胶质瘤干细胞，CD133+胶质瘤干细胞IC50较CD133-胶质瘤干细胞高，CD133+胶质瘤干细胞对替莫唑胺不敏感。CD133+胶质瘤干细胞IC50较CD133-胶质瘤干细胞低(P < 0.05)。将分选出的CD133+胶质瘤干细胞进行分组干预，检测细胞凋亡率，联合组细胞凋亡率与其他组比较差别有显著统计学意义(P < 0.05)结论：1. PUMA可能是胶质瘤的一个重要的预后标记物。PUMA可通过促进肿瘤细胞凋亡，对脑胶质瘤的发生、发展起作用；PUMA可能与凋亡相关基因Bax、Bcl-2在脑胶质瘤的凋亡中分别起协同和拮抗作用。2. Ad-PUMA转染可有效增强人类脑胶质瘤细胞U87MG细胞对替莫唑胺的敏感性，抑制人类脑胶质瘤细胞U87MG细胞的增殖，通过诱导细胞G2期阻滞促进其凋亡，促凋亡机制不依赖于P53。3. Ad-PUMA联合TMZ具有协同抑制胶质瘤生长作用，其机制可能与Ad-PUMA促进凋亡和抑制MGMT表达有关。4. Ad-PUMA联合TMZ可以促进耐药的责任细胞胶质瘤干细胞凋亡，从而增强胶质瘤对化疗敏感性。更多还原

【Abstract】 BACKGROUNDThe reversion of glioma drug-resistance become the important strategy for treatment glioma.But the understanding of mechanisms of glioma drug-resistance occurs remains a matter ofdebate. There is a consensus between glioma drug resistance and high expression drug-resistancegenes. In certain physiological or pathological conditions, apoptosis is programmed cell deathprocess. Apoptosis regulating biological tissue in maintaining steady and individual growth playsan important role. Since most chemotherapy drugs by inducing tumor cell apoptosis is ratherplay a role, so the cell apoptosis pathways of reversion of tumor drug-resistance is anotherimportant mechanism. The tumor drug-resistance research requires that we attach importance tothe apoptosis control mechanism of tumor cell. Looking for key genes is to enhance research inan effective way between apoptosis pathways and tumor drug-resistance. The project is to raisethe p53 up-regulated modulator of apoptosis (PUMA) as the breakthrough point, use of genetransfection, RT - PCR, the Western blot methods, from in vivo and in vitro studies, to explorethe mechanism of PUMA enhance glioma cell chemotherapy drug sensitivity. PUMA highexpression or the PUMA activated promote cell apoptosis and reverse gliomas drug-resistance.PUMA probable control mechanisms: (1) The PUMA lower expression avoid chemotherapydrugs through affectting cell growth cycle; (2) PUMA maintaining cell antiapoptotic andpromote apoptosis system balance, its function lack leads to apoptosis system imbalance to causedrug-resistant; (3) Drug-resistant molecular regulation abnormalities would be closely relatedwith drug resistance. This study try to look for the root of glioma drug resistance from cellapoptosis level, prove the regulation pathways mode of PUMA enhancing glioma drugsensitivity and provide a new approach to prevent and control glioma chemotherapy drugresistance, (4) Activation in G0 / G1 phase of gliomas stem cells, make its into chemotherapydrugs, leading to the effective period within cells apoptosis triggeredOBJECTIVE1. To study the expression of PUMA, Bcl-2 and Bax in human glioma cell and the relation among PUMA, Bcl-2 and Bax.2. To investigate the effect of PUMA on the sensitivity of human glioma U87MG cells totemozolomide and its related mechanisms3. To explore the inhibitive effects of Ad-PUMA combined with temozolomide on humanglioma cells growth in vivo experiments4. To investigate the differences of stem cells based on the glioma CD133 immune prototyperesistant to the effects of drug resistance PUMA..METHODS1. The expression of PUMA, Bcl-2 and Bax protein were examined by Western-blot analysis in52 cases of human glioma cell and 10 cases of normal brain tissues.2. U87MG cells under culture were divided into the normal control group, mock group andexperiment group. After 24 hours 0f culture, the mock and experiment group weretransfected with mock vector (Ad-△BH3) and the recombinant ademnrus carrying PUMA（Ad-PUMA）respectively at a multiplication of infection (MOI) of 50. The proliferationactivity of cells and IC50 were detected by MTT assay, the apoptosis effect and the changeof cell cycle determined by Hoechst stain and flow cytometry (FCM) technologyrespectively．The expression of related proteins was revealed by the method of Western blot.3. The nude mouse model with human glioma cells subcutaneous transplantation wasestablished. The mice were randomly divided into 4 groups to receive subcutaneousinjection at the 14th day separately with: Normal saline (control, n=8), Ad-PUMA2×108pfu/100μl (PUMA group, n=8), 10mg/kg TMZ (TMZ group, n=8) and 2×108pfu/100μlAd-PUMA + 10mg/kg TMZ (combined group, n=8). Mice were killed after 20daystreatment. Tumor volume, inhibition rates and apoptotic index (AI) were measured,meanwhile, apoptotic tumor cells were detected by TUNEL technology respectively．Theexpression of MGMT mRNA and MGMT protein were revealed by the methods of RT-PCRand Western blot.4. Sorting out CD133 + U87 cells, application CD133-WB, RT - PCR, gene carrying flowcytometric analysis method observation exogenous PUMA, TMZ before and after theintervention of cells to grow two changesRESULTS1. There were significant differences in the expression of PUMA, Bcl-2 and Bax protein between the control and gradeⅡ, gradeⅢor gradeⅣ(all P<0.01). There was no significantdifference in the expression of those protein between the control and gradeⅠ(P>0.05).There were correlations between the expression of PUMA, Bax or Bcl-2 and tumordifferentiation grade in human glioma (rPUMA=-0.925, rBax =-0.931, rBcl-2=0.945, allP<0.001). There were correlations between the expression of PUMA and Bax, Bcl-2 inhuman glioma (r1=0.963,r2=-0.937,P＜0.001).2. Exotic PUMA gene was expressed in U87MG cells transfected with Ad-PUMA, whichcould inhibit the proliferation of U87MG cells．The inhibition rate of proliferation 24, 48,72h after transfection was 17.3%, 35.6%, 43.3% and apoptosis rate was 20.3%, 31.4%,45.4% respectively. Results: The TMZ IC50 values of PBS group, Ad-PUMA and Ad-△BH3group cells were (15±1.9), (2.3±0.14) and (13.4±1.6)μmol/L respectively, with thesensitivity to the TMZ of Ad-PUMA group cells increased by 7.0 folds. PUMAoverexpressing U87MG cells showed the DNA synthesis was inhibited and arrested in G2phrase. The results of Western blot showed that after 72 hotms of transfection the PUMAand Bax protein showed increased expression (P<0.01). There was no significant differencein the expression of P53 among these groups (P>0.05).3. According to the order: control group, Ad-PUMA group, TMZ group, combined group,tumor volumes were (3.68±0.09)、(2.63±0.13)、(2.13±0.07)、(0.97±0.02) cm3 in 4 groupsrespectively; the inhibitive rates were 0, 28.5%, 42.1%, 73.6% respectively and the apoptoticindexes (AI) were (2.0±1.2) %, (11.4±2.6) %, (7.6±3.2) %, (20.6±8.6) %. The results ofWestern blot and RT-PCR showed that MGMT mRNA and MGMT protein levels in TMZgroup were higher than other groups (all P<0.01).4. The TMZ IC50 values of CD133 +glioma stem cell are higher than that of CD133- gliomastem cells. There was significant difference in apoptosis rate between CD133 +glioma stemcell and CD133- glioma stem cell. (all P < 0.05)CONCLUSION1. The expression of PUMA may be an important prognostic marker for human glioma. Theexpression of PUMA protein was correlated with tumor differentiation grade, could be usedas a valuable marker for the prognosis of human glioma.2. PUMA gene transfection greatly enhances the sensitivity of U87MG cells to TMZ-inducedapoptosis and can effectively inhibit the proliferation and promote the apoptosis of U87MG cells. The mechanism of apoptosis mainly relates to induce cell cycle G2 arrest and apoptosisin vitro.3. Ad-PUMA combined with TMZ greatly enhances the sensitivity of human glioma cells toTMZ and could effectively inhibit the proliferation and promote the apoptosis of gliomacells, its mechanism is probably related Ad-PUMA promote apoptosis and inhibit MGMTexpression.4. Ad - PUMA joint TMZ can promote glioma stem cell apoptosis, thus improving thesensitivity to chemotherapy gliomas.更多还原