研究背景与目的： 神经胶质瘤(glioma)具有发病率高、病情进展快、难治愈和病死率高的特征，是最常见的颅内肿瘤，也是最难攻克的神经系统恶性肿瘤。神经胶质瘤呈浸润性生长，肿瘤组织与正常脑组织边界模糊、不易辨别，病灶难以彻底切除导致手术治疗效果不理想，因此目前神经胶质瘤的治疗方式是以手术切除为基础，结合放射治疗和化学治疗。然而在治疗过程中，肿瘤细胞对放射治疗、化学治疗出现不同程度的抵抗或耐受，使放射治疗、化学治疗效果不佳。面对重重障碍，基因治疗有望成为一种更有效的治疗神经胶质瘤的方法。 肿瘤基因治疗的策略之一就是通过载体介导转入外源性目的基因，利用其表达产物激活肿瘤细胞内促凋亡途径，诱导肿瘤细胞凋亡而发挥抗肿瘤作用。基因治疗的载体可分为病毒载体和非病毒载体，病毒载体是基因治疗的常用载体，目的基因随病毒载体在靶细胞中稳定、持续的表达。与化学药物或蛋白制剂相比，基因治疗纯度高、副作用少、不易发生耐受，且载体表达具有特异性、持续性等，从而解决了临床上长期用药带来的问题。 已有的研究发现某些细菌毒素可通过调控肿瘤细胞的增殖、分化和凋亡，杀死肿瘤细胞，在肿瘤基因治疗方面显示出良好的应用潜力。白喉毒素(diphtheria toxin，DT)是由β-棒状杆菌噬菌体所编码的外毒素，由A亚单位和B亚单位组成。A亚单位负责杀伤细胞，B亚单位负责识别靶细胞。DT的细胞毒性强，一个毒素分子就能杀死一个细胞，故常作为基因治疗中的毒性分子。在对病毒载体进行包装的过程中，DT的强毒性会杀死包装细胞(如AD293、HEK293和293A细胞)，因而难以获得携带DT基因的腺病毒。 CRM197 (cross-reacting material 197， CRM197)是由白喉毒素第52位的甘氨酸突变为谷氨酸而形成，细胞毒性明显降低，但仍能抑制细胞蛋白质的合成，导致靶细胞死亡。已有学者将CRM197蛋白用于治疗卵巢癌、口腔癌、急性T淋巴细胞性白血病、结肠癌和乳腺癌等。目前CRM197蛋白治疗卵巢癌的研究已进入I期临床实验阶段。虽然CRM197被成功用于多种肿瘤的治疗，但之前的研究都只针对CRM197蛋白，目前还未查见腺病毒介导CRM197基因治疗肿瘤的研究报道。本研究尝试用腺病毒介导CRM197基因治疗人类神经胶质瘤，期望找到治疗神经胶质瘤的新方法。本研究通过构建pAd-CRM197腺病毒重组质粒，获得CRM197重组腺病毒(AdCRM197)，观察其对U87、U251、H4胶质瘤细胞的抑制作用和裸鼠移植瘤(U87、U251)动物模型的治疗作用，分析其与HB-EGF (heparin-binding epidermal growth factor-like growth factor， HB-EGF)和EF2-ADP-核糖基化的关系，深入探索其抗神经胶质瘤的作用机制。 研究方法： 1.构建CRM197原核表达质粒，转化大肠杆菌BL21(DE3)，IPTG诱导CRM197蛋白表达，并通过SDS-PAGE及Western blot对蛋白进行鉴定。利用金属螯合亲和层析柱纯化CRM197蛋白，纯化的蛋白免疫BALB/C小鼠后，获得抗CRM197多克隆抗体。 2.将CRM197基因插入pShuttle-CMV穿梭载体，构建pShuttle-CMVCRM197重组穿梭载体。pShuttle-CMV-CRM197重组穿梭载体经Pwel线性化，转入含有骨架质粒AdEasy-1的大肠杆菌BJ5183中，利用同源重组技术构建pAd-CRM197腺病毒重组质粒。pAd-CRM197经PacI线性化，转染包装细胞AD293以得到AdCRM197。扩增、收集、纯化AdCRM197重组腺病毒并检测病毒滴度。 3.PCR鉴定AdCRM197的稳定性。用Western blot方法和抗CRM197多克隆抗体鉴定靶细胞中CRM197蛋白的表达。 4.将AdCRM197作用U87、U25K H4细胞，MTT检测AdCRM197对肿瘤细胞生长的影响；Hoechst染色、流式细胞技术检测AdCRM197诱导肿瘤细胞凋亡的情况；Western blot方法检测AdCRM197对凋亡相关蛋白表达的影响。 5.构建pGreen-p53-RNAi慢病毒干扰质粒，经三质粒系统共转染293T细胞以获得p53-RNAi重组慢病毒。将p53-RNAi重组慢病毒感染U87细胞，通过puromycin筛选获得p53-RNAi-U87 稳定细胞株。将AdCRM197作用 p53-RNAi-U87细胞和U87细胞，MTT检测AdCRM197对两种细胞生长的影响；Western blot方法检测细胞中p53的表达。 6.构建pCDH-CMV-mEF2-GFP+Puro慢病毒质粒，经三质粒系统共转染293T细胞以获得mEF2重组慢病毒。将mEF2重组慢病毒感染U87细胞，再通过puromycin筛选获得mEF2-U87稳定细胞株。将AdCRM 197作用mEF2-U87细胞和U87细胞，MTT检测AdCRM197对细胞生长的影响；Western blot方法检测细胞中p53、HB-EGF的表达。 7.将AdCRM197应用于裸鼠移植瘤动物模型的治疗性实验，TUNEL荧光显色检测肿瘤组织的凋亡情况；Ki67免疫组化染色检测AdCRM197对肿瘤组织增殖的影响；观察裸鼠的生存率和生存时间、胶质瘤瘤体的生长速度以及AdCRM197治疗的毒副作用。 研究结果： 1.双酶切鉴定及DNA序列测定表明pET-32a(+)-CRM197原核表达质粒己成功构建。pET-32a(+)-CRM197 转化大肠杆菌 BL21(DE3)后，经 16 ℃，0.1 mmol/L IPTG诱导6 h， SDS-PAGE及Western blot结果证实宿主菌中有CRM197蛋白的表达。通过金属螯合亲和层析柱获得纯化的CRM197蛋白。纯化蛋白免疫BALB/C小鼠后，获得效价为1：16，000的抗CRM197多克隆抗体。 2.PCR鉴定及DNA序列测定表明CRM197基因成功插入pShuttle-CMV穿梭载体，PacI酶切鉴定证实pAd-CRM197腺病毒重组质粒构建成功。在病毒包装和扩增过程中，AD293细胞病变明显，表明AdCRM197包装成功，其滴度为7.5×10¹⁰PFU/mL。 3.对第2代和第6代AdCRM197进行PCR鉴定，结果显示AdCRM197稳定性良好，CRM197基因持续、稳定存在。Western blot结果证实AdCRM 197在AD293细胞中稳定表达CRM197蛋白。 4.体外实验：AdCRM197作用U87、U251和H4细胞后，MTT结果显示U87细胞增殖的抑制率为30%， U251和H4细胞增殖的抑制率分别为28%、33.4%。与空白对照组(0.1%)、空病毒组(0.3%)相比，AdCRM197明显抑制胶质瘤细胞的生长。AdCRM197作用后，U87、U251、H4细胞生长缓慢，细胞变圆、皱缩、脱落、空泡；Hoechst 33258染色显示凋亡细胞增多且细胞核出现核固缩、碎块状致密浓染及凋亡小体；流式细胞术提示U87细胞的凋亡率为15.5%， U251和H4细胞的凋亡率分别为14.5%、20.1%。与空白对照组和空病毒组相比， AdCRM197明显促进胶质瘤细胞凋亡。 5.Western blot检测发现，AdCRM197作用后U87细胞中Cytc、Smac、Bax、 Cleaved Caspase-9、Cleaved Caspase-3、p53、HB-EGF表达上调。 6.成功构建pGreen-p53-RNAi慢病毒干扰质粒，转染293T细胞后获得p53-RNAi重组慢病毒。慢病毒感染U87细胞后，Western blot显示p53表达被干扰，证实p53-RNAi-U87稳定细胞株成功构建。MTT结果显示p53-RNAi-U87细胞对AdCRM197具有明显抗性。Western blot结果显示，AdCRM197不影响p53RNAi-U87细胞中p53的表达，但上调U87细胞p53的表达。 7.成功构建pCDH-CMV-mEF2-GFP+Puro慢病毒质粒，获得mEF2重组慢病毒。慢病毒感染U87细胞后获得mEF2-U87稳定细胞株。MTT显示mEF2-U87细胞对AdCRM197具有明显的抗性。Western blot显示AdCRM197不影响mEF2-U87细胞p53、HB-EGF的表达，但上调U87细胞p53和HB-EGF的表达。 8.与对照组裸鼠相比，经AdCRM197治疗的裸鼠移植瘤体积缩小，生存时间延长，肿瘤组织细胞凋亡率增加，肿瘤组织细胞Ki67表达量下调以及未观察到明显的副作用。 研究结论： 1.本研究成功构建AdCRM197重组腺病毒，并利用腺病毒载体介导CRM197基因对神经胶质瘤进行实验研究。 2.体内、体外实验结果都表明AdCRM197能明显抑制神经胶质瘤细胞生长，促进肿瘤细胞凋亡。 3.研究结果揭示p53在AdCRM197诱导胶质瘤细胞凋亡中起关键作用，还证实p53的上调是因为EF2-ADP-核糖基化，而不是因为AdCRM197抑制了HB-EGF。 AdCRM197通过EF2的ADP-核糖基化，激活p53进而激活线粒体凋亡途径，诱导肿瘤细胞凋亡。 本研究结果为神经胶质瘤的基因治疗提供新的思路，为腺病毒载体介导CRM197基因的进一步应用奠定了实验基础，对探索腺病毒载体介导的基因治疗具有重要意义，并具有良好的临床应用价值。 关键词：CRM197；神经胶质瘤；腺病毒；细胞凋亡；p53

 Background and objective Glioma is the most common and malignant brain tumor with characteristics of poor prognosis and high rates of morbidity and mortality. In addition, it is the most major challenge in central nervous system (CNS) cancer therapy. Glioma tends to infiltrate and grow into normal brain tissue, which can result in incomplete tumor removal through surgery. Therefore, the treatment of glioma is a combination of multifaceted therapies including surgery resection, radiotherapy and chemotherapy. However, the treatment resistance and tolerance in cancer therapy drastically reduce the efficacy of radiotherapy and chemotherapy. With endeavor of its development for complementing other strategies, gene therapy is gradually considered as an attractive treatment for gliomas. The vector-associated exogenous target gene transduction is one of the major strategies because of its activation of apoptosis pathway to trigger tumor cell apoptosis. Viral vector and non-viral vector are two main carriers applied in gene therapy. Specially, viral vector is more common in gene therapy due to its potency of continuous target gene express in transduced cells .Gene therapy has higher purity, less side effects and low tolerance incidence than chemicals or protein preparation, so gene therapy can solve clinical problems posed by long-term medication. The finding of bacterial toxin on tumor cell killing, regulation of tumor cell proliferation, apoptosis and differentiation discloses its great potential on tumor gene therapy. Diphtheria toxin (DT), encoded by a particular bacteriophage of Corynebacterium diphtheriae, is composed of A and B fragments. The A fragment plays a role on tumor cell killing and the B fragment functions as target cell recognition. One molecule of DT could kill a cell, and thus the strong toxicity of DT can be used as a toxic molecule in gene therapy. However, the strong toxicity can cause the death of the packaging cells (such as AD293, HEK293 and 293A cells) in the process of virus package, which leads to insufficient harvest of adenovirus carrying DT gene. CRM197 (the cross-reacting material 197) is a type of DT mutant. The substitution of its glutamic acid at position 52 for glycine significantly reduces its toxicity and meanwhile retains its inhibition to cellular protein synthesis. CRM197 has been used in the treatment of ovarian cancer, oral cancer, T-Cell acute lymphoblastic leukemia, colon cancer, breast cancer and so on. Currently, CRM197 associated ovarian cancer treatment has been in human phase-I trial. Till now most of CRM19 contribution in various tumor therapy are based on purified CRM197 protein, and adenovirus mediated CRM197 therapy is rarely reported. In this study, we attempted to apply adenovirus mediated CRM197 into human glioma therapy and aimed to develop a new method for glioma treatment. For this purpose, pAd-CRM197 plasmid was constructed to generate AdCRM197. To test anti-glioma effect of AdCRM197, U87, U251, H4 glioma cells were used to detect its inhibitory effect, the nude mouse xenograft animal models were established for observing its therapeutic efficacy, and the analysis of its correlation with HB-EGF and EF2-ADP-ribosylation was performed for further exploring its mechanism of anti-glioma. Methods: 1.After CRM197 prokaryotic expression plasmid was constructed and transformed into E.coli BL21(DE3), the recombinant CRM197 protein was expressed after IPTG induction. The expression of CRM197 protein was identified by SDS-PAGE and Western blot. To obtain anti-CRM 197 polyclonal antibodies, BALB/C mice were vaccinated with the CRM197 protein which was purified by metal chelating affinity chromatography column. 2.CRM197 gene was inserted into pShuttle-CMV vector for constructing pShuttle-CMV-CRM197. After linearized by PmeI, pShuttle-CMV-CRM197 was transformed into E.coli BJ5183 containing pAdEasy-1 backbone. In E.coli BJ5183, pAd-CRM197 was created through homologous recombination. In order to transfect to AD293 cells, pAd-CRM197 was linearized with PacI. The titer of AdCRM197 was measured after amplification and purification of AdCRM197. 3.The stability of AdCRM197 was identified by PCR. The expression of CRM197 protein in target cells was detected by Western blot. 4.After AdCRM197 was used to treat U87, U251 and H4 cells, the cell survival rate was valued via MTT assay and cell apoptosis was detected by Hoechst staining and flow cytometry. In the meantime, the protein expression was confirmed by Western blot. 5.After pGreen-p53-RNAi lentiviral interfering plasmid was constructed and transfected into 293T cells, p53-RNAi recombinant lentivirus was obtained. U87 cells were infected by p53-RNAi recombinant lentivirus in order to obtain P53-RNAi-U87 stable cell line. Afterward AdCRM197 was used to treat p53-RNAi-U87 cells and U87 cells, the cell growth was tested by MTT and the expression of p53 protein was detected by Western blot. 6.After pCDH-CMV-mEF2-GFP+Puro lentiviral plasmid was constructed and transfected into 293T cells, mEF2 recombinant lentivirus were obtained. U87 cells were infected by mEF2 recombinant lentivirus in order to obtain mEF2-U87 stable cell line. After that, AdCRM197 was used to treat mEF2-U87 and U87 cells, the cell growth was tested by MTT, the expression of p53 and HB-EGF protein was detected by Western blot. 7. Post vitro study, AdCRM197 was used to treat xenograft mouse model. Tumor apoptosis in tissue was examined by TUNEL, and cell proliferation, cell survival time, the growth rate of glioma and the side effects were observed by Ki67 immunohistochemical staining. Results: 1.The results of double enzyme digestion and DNA sequencing evidenced the success of pET-32a(+)-CRM197 construction. Post the transformation of pET-32a(+)-CRM197 into E.coli BL21(DE3), the expression of CRM197 protein was induced for 6 hours at 16℃ with 0.1 mmol/L IPTG, and confirmed by SDS-PAGE and Western blot. Followed purification via metal chelating affinity chromatography column, protein was used to immunize BALB/C mice for obtaining anti-CRM 197. The titer of antibody was 1:16,000. 2.The results of PCR and DNA sequencing indicated the effective insertion of CRM197 gene into pShuttle-CMV. The pAd-CRM197 plasmid was confirmed by the enzyme assay. During virus were packaged and amplificated, CPE was obvious in AD293 cells, indicating that AdCRM197 packaging was successful. The titer of recombinant adenovirus was 7.5×10¹⁰PFU/mL. 3.The results of PCR displayed that AdCRM197 and CRM197 gene were stable. As well, Western blot confirmed that CRM197 protein was expressed in AD293 cells after AdCRM197 treatment. 4.Following AdCRM197 treatment, MTT presented 30% inhibition rate in U87 cells and 28% and 33.4% inhibition rate in U251 and H4 cells respectively in comparison with control group (0.1%) and empty virus group (0.3%). AdCRM197 significantly inhibited the growth of glioma cells. U87, U251 and H4 cells grew slowly, and morphological shape was round, shriveled, peeled and vacuolated. Hoechst 33258 staining showed the increase of apoptotic cells, and the appearance of karyopyknosis and apoptotic body in the nucleus. Flow cytometry suggested that the apoptosis rate of U87, U251 and H4 cells was 15.5%, 14.5% and 20.1%, respectively. Compared with the control group and empty virus group, AdCRM197 significantly promoted apoptosis. 5.The expression of Cytc, Smac, Bax, Cleaved Caspase-9, Cleaved Caspase-3, p53 and HB-EGF were up-regulated in U87 cells treated by AdCRM197. 6.p53-RNAi lentivirus was harvested. p53-RNAi-U87 stable cell line was obtained successfully. After p53-RNAi-U87 and U87 cells were treated by AdCRM197, p53-RNAi-U87 cells were significantly resistant to AdCRM197, and p53 expression was not up-regulated, but p53 expression of U87 cells was up-regulated. 7.Post mEF2 recombinant lentivirus synthesis, mEF2-U87 stable cell line was obtained successfully. After mEF2-U87 and U87 cells were treated by AdCRM197, mEF2-U87 cells were significantly resistant to AdCRM197, p53 was not upregulated in mEF2-U87 cells, but p53 and HB-EGF was up-regulated in U87 cells. 8.Compared with control group, AdCRM197 inhibited tumor growth and cell proliferation, raised survival rate, and promoted apoptosis. Conclusion: 1.AdCRM197 recombinant adenovirus was successfully constructed and used to treat glioma. 2.AdCRM197 significantly inhibited the growth of glioma cells and promoted apoptosis in vivo and in vitro. 3.The key function of p53 was revealed in the apoptosis induced by AdCRM197. p53 up-regulation was brought by EF2-ADP-ribosylation and was not caused by AdCRM197 induced HB-EGF inhibition. AdCRM197 activates p53 pathway through EF2-ADP-ribosylation and leads to cell death by mean of activation of mitochondrial apoptosis pathway. These experimental results may provide new insights of CRM197 gene therapy, establish a foundation for application of adenovirus vector mediated gene CRM197. It shows great significance in the exploration of adenovirus vectors mediated gene therapy and has a good application potential in clinic development. Keyword: CRM197; glioma; adenovirus; apoptosis; p53