【作者】 王跃红；

【导师】 赵文清； 李文玲；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 博士

【摘要】 恶性胶质瘤是一种最常见的中枢神经系统恶性肿瘤,常规治疗的效果均不理想,目前还没有非常有效的治疗方法。近年来,随着分子生物学、分子遗传学的发展,基因治疗的研究发展较快,为恶性胶质瘤的治疗开辟了新的道路。白细胞介素24（interleukin-24,IL-24）是白介素10细胞因子家族中的新成员,最初发现IL-24基因在终末分化的人类黑色素瘤中高度表达,因此也被称为黑色素瘤分化相关因子-7（melanoma differentiation-associated gene-7,mda-7）基因。研究发现IL-24对人多种肿瘤细胞（包括肺癌、前列腺癌、乳腺癌、卵巢癌、胰腺癌等）均有选择性抑制作用,因此成为基因治疗的候选基因。此外,IL-24与化疗、放疗相结合的研究亦有报道,比如Nishikawa于2001年首先报道将IL-24基因转染入人非小细胞肺癌的细胞后,可增强此肿瘤细胞对放射治疗的敏感性,而同时接受照射的人正常成纤维细胞则未受此放射线照射的影响。Chada也报道:对于人乳腺癌细胞,IL-24和放疗、化疗相结合后有协同和增效作用。本研究应用载有IL-24基因的新型重组腺病毒（Ad5F35-IL24）感染人胶质瘤细胞系U251和胶质瘤组织,并与化疗药替尼泊甙进行联合干预研究,观察IL-24基因对胶质瘤细胞在体外和体内的影响以及IL-24与化疗药卫盟联合作用是否相互协同增效,并探讨其可能的作用机制。第一部分Ad5F35-IL24重组腺病毒的构建目的:构建重组腺病毒Ad5F35-IL24。方法:先用HindIII和SalI双酶切ATCC质粒,得到目的条带,然后再用HindIII和SalI双酶切pDC316质粒,得到线性化的载体条带,接着进行片断连接,转化,筛选得到重组的pDC316-IL24质粒。随后用PCR和酶切的方法对重组pDC316-IL24质粒进行鉴定,经过此两者鉴定可能正确的IL-24重组质粒在全自动核酸分析仪上进行序列测定。接着制备感受态细胞,进行转化,随后大量制备质粒DNA;之后用双质粒共转染293细胞,同源重组产生重组腺病毒Ad5F35-IL24,再用PCR方法对其鉴定。然后进行病毒扩增、纯化,再用PCR方法对其鉴定后,按照公式:VP/ml=OD₂₆₀×1.1×10¹²进行滴度测定。结果:成功构建成载有IL-24基因的重组pDC316-IL24质粒,并用PCR、酶切、序列测定和BLAST的方法进行了鉴定;获得了携带IL-24基因的大肠杆菌菌株;成功构建成载有IL-24基因的增殖能力缺陷的新型重组腺病毒Ad5F35-IL24,并进行了扩增和纯化,且用PCR的方法进行了鉴定;并按照公式:VP/ml=OD₂₆₀×1.1×10¹²测定了病毒的滴度,其滴度为2.8×10¹¹vp/ml。结论:构建成载有IL-24基因的增殖能力缺陷的新型重组腺病毒Ad5F35-IL24,其滴度为2.8×1011vp/ml。第二部分白细胞介素24抑制胶质瘤细胞生长的体内外实验研究目的:观察IL-24基因对在体外和体内胶质瘤细胞的影响,并探讨IL-24基因对胶质瘤的治疗作用及其可能的作用机制。方法:先原代培养人胎脑星形胶质细胞,然后用载有人白细胞介素24cDNA的新型重组腺病毒（Ad5F35-IL24）感染人胎脑星形胶质细胞和人胶质瘤细胞系U251,体外观察Ad5F35-IL24对它们生长的影响;建立人胶质瘤动物模型,瘤内注射Ad5F35-IL24,观察肿瘤生长情况;用流式细胞仪检测细胞和组织的凋亡及其Bax/Bcl-2的表达情况。结果:Ad5F35-IL24感染人胎脑星形胶质细胞、U251细胞和肿瘤组织后IL-24蛋白高表达,人胎脑星形胶质细胞的生长未受到明显抑制（P>0.05）,U251细胞和组织的生长受到明显抑制（P<0.05）,其凋亡率明显升高（P<0.05）,Bax/Bcl-2也明显升高（P<0.05）。结论:白细胞介素24具有选择性抑制人胶质瘤细胞系U251生长和诱导凋亡作用。Ad5F35-IL24转导白细胞介素24基因可能是一种有效的胶质瘤基因治疗途径。第三部分白细胞介素24在体内外抑制胶质瘤细胞生长的机制实验研究目的:探讨IL-24基因抑制胶质瘤细胞生长的可能的作用机制。方法:先用MTT法检测加入2-氨基嘌呤后U251细胞的抑制率,再用流式细胞仪检测加入2-氨基嘌呤后U251细胞的凋亡率和Bax/Bcl-2蛋白的表达情况,接着再用western blot检测加或未加2-氨基嘌呤的U251细胞和各组肿瘤组织中IL-24蛋白、一些信号蛋白如PKR、p-PKR、p38-MAPK、p-p38-MAPK、促凋亡蛋白BAX和抗凋亡蛋白Bcl-2的表达情况。随后用免疫组织化学染色检测各组肿瘤组织的血管密度和bFGF、VEGF的表达情况。结果:通过Western Blot发现:IL-24对U251的选择性诱导凋亡作用可能是通过上调并激活其PKR蛋白（P<0.01）,随后又激活p38MAPK途径而实现的。接着又通过应用2-AP阻断了PKR蛋白的激活后,其下游p38MAPK的激活和凋亡也被阻断,这进一步证实了其作用机制。通过免疫组化技术,发现IL-24治疗组的微血管生成明显减少（P<0.05）,且其VEGF和bFGF表达也明显减少（P<0.05）。结论:IL-24选择性的抑制U251细胞和胶质瘤生长的机制是通过上调并激活PKR蛋白,随后又激活p38MAPK途径而实现的。IL-24还可抑制胶质瘤微血管生成,进而抑制肿瘤增殖。其机制可能与IL-24诱导胶质瘤组织中VEGF和bFGF表达的减少有关。第四部分白细胞介素24对胶质瘤细胞化疗敏感性影响的体内外实验研究目的:观察IL-24与化疗药卫盟联合作用是否相互协同增效,并探讨其可能的作用机制。方法:先用MTT法检测IL-24和替尼泊甙联合干预后的U251细胞抑制率,再用Hochest33258法检测其凋亡率,接着用流式细胞仪检测其Bcl-2和Bax蛋白的表达情况。随后用Western blot分析IL-24和替尼泊甙联合干预后的U251细胞的IL-24、PKR、p-PKR、p38-MAPK、p-p38-MAPK、Bcl-2、BAX蛋白表达水平。还采用TUNEL法测定各组肿瘤组织的凋亡情况,再用流式细胞仪检测其Bcl-2和Bax蛋白的表达情况。接着用免疫组织化学染色检测各组肿瘤组织的血管密度和bFGF、VEGF的表达情况。结果:通过MTT法发现IL-24和替尼泊甙联合干预组U251细胞的抑制率明显增高（P<0.001）,通过Hochest33258法和TUNEL法发现联合干预组U251细胞和肿瘤组织的凋亡率也明显增高（P<0.001）,通过流式细胞技术发现联合干预组U251细胞和肿瘤组织的Bax/Bcl-2升高（P<0.05）,通过Western Blot发现联合治疗组的PKR蛋白的表达和活化以及其下游的靶分子p38MAPK的表达和活化都明显增高（P<0.001）。通过免疫组化技术,发现联合治疗组的微血管生成明显减少（P<0.05）,且其VEGF和bFGF表达也明显减少（P<0.05）。结论:IL-24和替尼泊甙对U251细胞和胶质瘤具有协同杀伤和诱导凋亡的作用,其机制与IL-24诱导凋亡通路有关,还可能与促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的比例的升高有关。此外,联合治疗还可抑制胶质瘤微血管生成,进而抑制肿瘤增殖。其机制可能与IL-24诱导胶质瘤组织中VEGF和bFGF表达的减少有关。实验证明:IL-24和卫盟联合治疗胶质瘤是可取的治疗方案。更多还原

【Abstract】 Malignant gliomas are the most common brain tumors in humans, and current treatments fail to provide long-term management of these tumors. Gene therapy has developed swiftly along with the development of molecular biology and molecular genetics for the past few years. It established a new road for the therapy of malignant gliomas. Interleukin-24 is a new member of interleukin-10 family. Interleukin-24 is discovered overexpression in human melanoma during terminal differentiation. It is called melanoma differentiation-associated gene-7.Several groups have demonstrated that Ad-mda7 （using Ad-mda7） induces apoptosis in a wide range of cancer cells （lung, prostate, breast, ovary, pancreas）, so, it has become candidate gene of gene therapy. In addition, there are some researches about combination of interleukin-24 and radiotherapy or chemotherapy. For example, Nishikawa first discovered interleukin-24 can enhance the radiosensitizing effect of non-small cell lung cancer （NSCLC） cell lines. Chada discovered the combination of interleukin-24 and radiotherapy or chemotherapy has synergistic action.Human glioma cell line U251 and glioma tissue were infected with a new type recombine adenovirus included interleukin-24 in this study. We have to observe the influence of interleukin-24 to glioma cell in vitro and in vivo. it was studied that Combination of interleukin-24 and chemotherapy medicine VM-26 is synergy or no synergy. We approached its potential mechanism of the action.Part one Construction of recombinant adenovirus Ad5F35-IL24Objective: to construct recombinant adenovirus Ad5F35-IL24.Methods: When plasmids of ATCC were cut by both HindIII and SalI enzymes, the goal bands were obtained. Plasmids of pDC316 were cut by both HindIII and SalI enzymes. Subsequently, after some fragments were connected, transformed and filtered, reorganized plasmids of pDC316 were obtained. Thereafter it was identified with PCR and enzymes cut. Then the plasmids were carried out sequence determination in whole automatic nucleic acid analyzer. Subsequently competent cells were prepared and transformed. After large-scale preparation of plasmid DNA, Recombined adenoviruses Ad5F35-IL24 were generated with homologous recombination by cotransfection of 293 cells with a couple plasmids. After it be identified with PCR, Ad5F35-IL24 recombinant adenovirus species was amplified and purified. Then we assayed its titre according to formula: VP/ml=OD₂₆₀×1.1×10¹² after it be identified with PCR.Results: Reconstructed Plasmids of pDC316-IL24 containing IL-24 gene was successfully constructed and identified by PCR, enzymes cut and sequence determination. Escherichia coli strains containing IL-24 gene was obtained. Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, amplified, purified and identified with PCR. Its titre was assayed according to formula: VP/ml=OD₂₆₀×1.1×10¹², and the titer was 2.8×10¹¹vp/ml.Conclusions: Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, and the titer was 2.8×10¹¹vp/ml.Part two Interleukin 24 induces growth suppression of malignant glioma cells in vitro and in vivoObjective: We have to observe the influence of interleukin-24 to glioma cell in vitro and in vivo. We approached its therapeutic effect to malignant glioma and potential mechanism of action.Methods: when human fetal astrocytes were primary cultured sucessfully, They and U251 cells were transfected with Ad5F35-IL24. influence of human fetal astrocytes and U251 cells was observed in vitro by Ad5F35-IL24.While animal models of human glioma were established and Ad5F35-IL24 was injected into tumor nodules in vivo, the tumor growth was observed. Their apoptosis and Bax/Bcl-2 expression were detected with flow cytometer and TUNEL. Results: IL-24 proteins were expressed highly after inflection of human fetal astrocytes, U251 cells and tumor tissues with Ad5F35-IL24. growth of human fetal astrocytes wasn’t suppressed obviously（P>0.05）. While growth of U251 cells and xenograft tumors was suppressed heavily, their apoptosis and expression of Bax/Bcl-2 were elevated significantly（P<0.05）.Conclusion: Interleukin 24 selectively induces growth suppression and apoptosis in human glioma cells U251. Adenovirus-mediated IL24 gene therapy is a potential effective approach of malignant glioma.Part three Empirical study of mechanism about growth suppression of malignant glioma cells in vitro and in vivo by Interleukin 24Objective: to approach its potential mechanism of growth suppression of malignant glioma cells by Interleukin 24.Methods: After administration of 2-aminopurine to U251 cell, we detected inhibition rate with MTT, Apoptosis and expression of Bax/Bcl-2 protein with flow cytometer. We detected expression of IL-24, PKR, p-PKR, p38-MAPK, p-p38-MAPK, BAX, Bcl-2 protein of tumor tissues and U251 cell administrated and unadministrated by 2-aminopurine with western blot. Blood vessel density and expression of bFGF、VEGF would be assayed with immunohistochemistry,.Results: Western Blot analyses found that, IL-24 selectively induced U251 cell apoptosis by up-regulating and activating PKR protein（P<0.01）, which would further activate p38MAPK pathway. After administration of 2-aminopurine blocked action of PKR protein, apoptosis and action of p38-MAPK were blocked. These further proved mechanism of action. We found that vascularization and expression of VEGF, bFGF is reduced obviously in tumor administrated IL-24 with Immunohistochemistry（P<0.05）.Conclusions: IL-24 selectively induced U251 cell and tumor apoptosis by up-regulating and activating PKR protein, which would further activate p38MAPK pathway. IL-24 inhibits glioma angiogenesis, thus inhibits tumor proliferation. The mechanism may involve the reduction of expression of VEGF and bFGF by IL-24 in gliomas. Part four Empirical study on effect of chemotherapy sensibility in malignant glioma cells in vitro and in vivo by Interleukin 24Objective: To observe that Combination of interleukin-24 and chemotherapy medicine VM-26 is synergy or no synergy and approach its potential mechanism of the action.Methods: After administration of IL-24 and VM-26 to U251 cell, we detected inhibition rate with MTT、Apoptosis with Hochest33258 and expression of Bax/Bcl-2 protein with flow cytometer. We detected expression of IL-24, PKR, p-PKR, p38-MAPK, p-p38-MAPK, BAX, Bcl-2 protein of U251 cell administrated by IL-24 and VM-26 with western blot. Apoptosis was assayed in all groups of tumors with TUNEL. expression of BAX, Bcl-2 protein was detected in all groups of tumors with flow cytometer. Blood vessel density and expression of bFGF、VEGF would be assayed with immunohistochemistry.Results: After administration of IL-24 and VM-26 to U251 cells and tumor tissues, inhibition rate of U251 cells was significantly raised up with MTT（P<0.001）, Apoptosis of U251cells and tumor tissues was significantly raised up with Hochest33258 and TUNEL（P<0.001）, expression of Bax/Bcl-2 protein was significantly raised up with flow cytometer（P<0.05）, expression and action of PKR and p38MAPK in U251 cells were significantly raised up with western blot（P<0.001）. vascularization and expression of VEGF, bFGFo is reduced obviously in tumor administrated by IL-24 and VM-26 with Immunohistochemistry（P<0.05）.Conclusions: L-24 and VM-26 synergisticly inhibit the proliferation and induce the apoptosis of U251 cells and glioma, which may be related to the apoptosis pathway and the increased ratio of the pro-apoptotic protein Bax and Anti-apoptosis protein Bcl-2. IL-24 and VM-26 inhibit glioma angiogenesis, thus inhibit tumor proliferation. The mechanism may involve the reduction of expression of VEGF and bFGF in gliomas. The experiment showed that combination administration of IL-24 and VM-26 are all desirable treatments for gliomas.更多还原