【作者】 叶红星；

【导师】 袁贤瑞；

【作者基本信息】 中南大学 ， 神经外科， 2009， 博士

【摘要】 【研究背景】目前恶性胶质瘤的治疗仍然面临巨大挑战,寻找比传统治疗更有效的治疗手段一直备受各国神经科学工作者的关注。血管生成不仅在个体发育、损伤修复、组织再生等生理过程中扮演重要角色,同时它也是肿瘤生长和转移过程中的一个重要步骤。由于肿瘤内皮细胞遗传上稳定,不易产生耐药,抗肿瘤血管生成治疗已成为肿瘤治疗领域很有前途的治疗方法之一。目前已有多种抑制新生血管生成的药物用于抗肿瘤研究。肿瘤抑素（Tumstatin）是继血管生成抑素（Angiostatin）和内皮抑素（Endostatin）之后,新发现的源于血管基膜Ⅳ型胶原α3链的内源性血管生成抑制因子,具有明显的抑制肿瘤血管生成和诱导其内皮细胞调亡的活性。由于采用血管生成抑制因子直接投药的方式治疗肿瘤存在所需剂量过大,同时生产有完整活性的血管生成抑制因子存在技术困难,因此通过基因转染的方法提供有完整生物活性的血管生成抑制因子在体内持续、较高浓度的表达,是肿瘤抗血管生成治疗的理想策略。利用全长Tumstatin片段抑制胶质瘤血管生成的基因治疗实验目前国内外尚未见报导;故本课题试图克隆人Tumstatin基因,构建其真核细胞表达载体（pcDNA3.1-Tumstatin）;观察Tumstatin体外抗血管生成的生物活性及了解此基因转染对U251胶质瘤细胞株的自身增殖有无影响,并观察Tumstatin在肿瘤局部持续、较高浓度表达对U251裸鼠移植瘤生长有无抑制作用,探讨Tumstatin基因治疗胶质瘤的可行性。目的构建抗血管生成的人肿瘤抑素（Tumstatin）基因真核表达载体,为后续体外Tumstatin转染U251细胞及其在荷瘤裸鼠（BALB/N）的体内表达创造条件。方法1、从HEK293细胞提取总RNA,通过RT-PCR扩增出Tumstatin基因。2、将该基因导入克隆载体pUC57-T中,转化大肠杆菌DH5α进行扩增,并提取纯化,对所获重组质粒进行酶切鉴定和DNA序列分析鉴定。3、用内切酶NheI和NotI对重组质粒pUC57-Tumstatin双酶切,并纯化目的基因片段,同时用内切酶NheI和NotI对质粒pcDNA3.1（-）进行双酶切。取纯化的目的基因片段与表达载体pcDNA3.1（-）在T₄连接酶的作用下进行连接反应。经菌落PCR、酶切鉴定和DNA测序证实后,制备和纯化重组质粒。结果1、提取的RNA经电泳有明显的28S、18S、5S三条带,分光光度法测得A₂60/A₂80=1.8933,提示RNA无明显降解;RT-PCR产物电泳显示在750bp处有一特异性条带。2、重组质粒pUC57-TumstatinDNA测序结果显示插入的目的基因与GenBank收录的人Tumstatin基因cDNA序列完全一致,无任何突变。3、重组质粒pcDNA3.1-Tumstatin经酶切鉴定后,DNA序列分析结果与GenBank收录的人Tumstatin基因cDNA序列亦完全一致,无任何突变。分光光度法测得此重组质粒溶液A₂60/A₂80=1.823。结论成功克隆了人Tumstatin基因并构建了Tumstatin基因的真核表达载体pcDNA3.1-Tumstatin。目的观察血管生成抑制因子Tumstatin体外抗血管生成的生物功能及了解此基因转染对于U251胶质瘤细胞株的自身增殖有无影响。方法以脂质体法将分泌型真核表达载体pcDNA3.1-Tumstatin转染至人胶质瘤细胞株U251,并用G418筛选出稳定转染的细胞株,观察转染后的U251细胞的生物学特性（MTT法观察U251细胞株的增殖活性、流式细胞仪检测其细胞周期）,并以Western blot检测培养上清中Tumstatin的分泌表达,进一步提取U251细胞的培养上清液,加至脐静脉内皮细胞（HUVECs）的培养液中,采用MTT法观察HUVECs的增殖活性、细胞划痕法检测其迁移能力、体外管腔形成抑制实验检测其成管能力及流式早期细胞凋亡检测法检测HUVECs的早期细胞凋亡率。结果1、成功获得可表达Tumstatin的U251细胞株,转染组U251细胞的增殖活性及细胞周期与未转染组的无显著差别（P＞0.05）;2、Tumstatin在体外不能抑制HUVECs的迁移,但它能显著抑制HUVECs的增殖,诱导HUVECs细胞凋亡及抑制其形成毛细血管的能力。结论Tumstatin基因转染对U251细胞自身增殖无显著影响,Tumstatin在体外有较强的抗血管生成的活性。目的探讨Tumstatin抑制U251裸鼠移植瘤生长的作用。方法BALB/N裸鼠随机分成三组,每组10只,然后将未转染的U251细胞株及pcDNA3.1-Tumstatin稳定转染和pcDNA3.1稳定转染的U251细胞株各分别以1×10⁷/只接种裸鼠。成瘤后每隔一周测量移植瘤体积一次,于接种后第28天处死裸鼠,将获取的移植瘤标本称重后,做肿瘤切片HE染色、TUNEL法检测肿瘤细胞凋亡、CD31免疫组化检测MVD。结果1、pcDNA3.1-Tumstatin组移植瘤的平均体积及瘤重都比pcDNA3.1组及未转染组低（p＜0.05）。2、肿瘤切片HE染色三组无明显区别,但pcDNA3.1-Tumstatin组肿瘤细胞平均凋亡率比其它两组高（26.60±2.02 vs.11.21±1.57,9.69±0.86,P＜0.05）,同时微血管密度（MVD）比其它两组低（5.62±1.32 vs.23.84±1.71,29.33±4.45,P＜0.05）。结论人Tumstatin有抑制U251移植瘤生长的作用,通过抑制肿瘤血管生成而间接诱发肿瘤细胞凋亡可能是其主要机制。更多还原

【Abstract】 [Background]Treatment of malignant gliomas remains a formidable challenge and it deserves special attention to search for more effective therapeutic methods.Angiogenesis is required for a variety of physiological processes such as development,wound repair,and tissue regeneration.Moreover,it is also one major pathogenic step in the process of tumor growth and metastasis.Because nonmalignant endothelial cells are genetically stable and unlikely to mutate into drug-resistant variants,anti-tumor angiogenesis therapy has become a promising method in cancer treatment. Several anti-angiogenic proteins have been identified that supress the growth of cancer.Tumstatin,the NC1 domain ofα3 chain of typeⅣcollagen,was identified as prossessing anti-angiogenic activity.Therapy with endogenous angiogenesis inhibitor is not clinically practical because of the high doses required and the difficulty in producing them in a fully functional state.Alternatively,It is an ideal strategy that if endogenous angiogenesis inhibitors（such as angiostatin or endostatin） can be expressed in vivo by gene transfection method and provided therapeutic levels of the functionally actively proteins.We cloned the cDNA of human tumstatin gene and constructed a recombinant plasmid which expressed tumstatin in eukaryotic cells.U251 glioma cells were transfected with this recombinant plasmid and transplanted into the nude mice’s armpits.Then the inhibitory effect of Tumstatin on the growth of U251 glioma xenograft nude mice model was investigated. PartⅠConstruction of eukaryotic expression vector of human tumstatin geneObjective To construct the eukaryotic expression vector of human antiangiogenic tumstatin gene,which will be used in the next section experiments,including transfection U251 cell line with tumstatin in vitro and its expression in BALB/N mice.Methods 1.Total RNA was extracted from HEK293 cell lines, then the target gene were amplified by RT-PCR technique.2.The target gene was linked to pUC57-T vector,and transformed into E.coli DH5α.The purified plasmid,named as pUC57-Tumstatin,was identified by restriction endonuclease and the sequence of the target gene was identified by dideoxy chain termination method.3.pUC57-Tumstatin and pcDNA3.1（-） were digested by enzyme NheI and NotI respectively,then coupled reaction between the target gene and pcDNA3.1（-） segment was carried out by T4 DNA joinase.The recombinant plasmid,named as pcDNA3.1-Tumstatin,was identified by restriction endonuclease and the sequence of the target gene was identified by dideoxy chain termination method,then the recombinant plasmid was amplified and purified.Results 1.Total RNA of HEK293 cells was extracted successfully. Electrophoresis and OD value ratio(A₂₆₀/A₂₈₀=l.8933) demonstrated the integrity of RNA.A fragment of DNA about 750bp was expanded in reverse-transcript polymerase chain reaction.2.The target gene in the recombinant plasmid pUC57-Tumstatin was comfirmed to be the cDNA of Tumstatin by sequencing.plasmid pUC57-Tumstatin was expanded and purified.3.The target gene in the recombinant plasmid pcDNA3.1-Tumstatin was also comfirmed to be the cDNA of Tumstatin by sequencing.This plasmid was expanded and purified.Conclusion The target gene was cloned successfully.Its sequence was completely consistent with that Human tumstatin gene listed in GenBank database.pcDNA3.1-Tumstatin,a recombinant eukaryonic expression plasmid,was constructed with the corrected sequence.Objective To study the antiangiogenic functions of Tumstatin and to explore the biological influence of the genetic-modification with Tumstatin cDNA on U251 cell line in vitro.Methods The pcDNA3.1-Tumstatin was transfected into the U251 cell with liposome,followed by G418 method to select definitely the positive-transformed clones.The proliferation ability of transfected cell line was measured by MTT assay and its cell cycle was analyzed by flow cytometry（FCM）.Then Western blot method was adopted to detect the protein expressed in cell culture medium.Finally,HUVECs was exposed to culture medium which was extracted from culture supernatant of positive-transformed U251 cells and contains tumstatin protein.The proliferation ability of HUVECs was measured by MTT assay,the migration ability of HUVECs was observed by the scratch assay,the endothelial tube formation ability of HUVECs was observed,and early cell apoptosis ratio of HUVECs was observed by FCM.Results 1.The U251 cell line with the character of tumstatin expression was obtained,meanwhile,the proliferation ability and cell cycle of the transfected U251 cell line was not significant different from the untransfected cell line（P>0.05）.2.Although tumstatin was found having no effects on migration of HUVECs,it was found having inhibitory effects on the proliferation of HUVECs,and it could inhibit endothelial tube formation and introduce endothelial cell apoptosis.Conclusion The transfection of Tumstatin into U251 cell line has no obvious influence on the proliferation of the transfected cells,and Tumstatin has antiangiogenic activity in vitro.Objective To investigate the inhibitory effect of Tumstatin on the growth of U251 glioma xenograft nude mice model.Methods BALB/N nude mice were divided into three groups at random,with 10 animals in each group.Then untransfected U251 cells and cells transfected with pcDNA3.1-Tumstatin/pcDNA3.1 were inoculated at the armpit of nude mices,the growth of the subcutaneously transplanted tumor was observed on 7th,14th,21st and 28th day after inoculation and the volumes were calculated.HE staining of xenografts of nude mice was conducted.Apoptosis of tumor cells was detected by terminal dUTP nick end labeling（TUNEL） technique and expression of CD31 was detected by immunohistochemical staining,then apoptosis index and microvessel density（MVD） was calculated among three groups respectively. Results 1.Compared to pcDNA3.1 group and untransfected group, the average volume and average weight of xenograft in pcDNA3.1-Tumstatin group was significant smaller（p<0.05）.2、There are no significant difference in HE staining of xenograft among three groups,but the tumor cell apoptotic index was significantly higher in pcDNA3.1-Tumstatin group than in the rest 2 groups（26.60±2.02 vs. 11.21±1.57 and 9.69±0.86,P<0.05）;meanwhile the MVD was significantly less in pcDNA3.1-Tumstatin group than in the rest 2 groups （5.62±1.32 vs.23.84±1.71 and 29.33±4.45,P<0.05）.Conclusion Human Tumstatin has the anti-tumor effect in the U251 xenograft.To inhibit tumor angiogenesis and indirectly induce tumor cell apoptosis may be the major mechanism.更多还原