【作者】 何文凯；

【导师】 吴平生；

【作者基本信息】 南方医科大学 ， 心血管内科（专业学位）， 2014， 博士

【摘要】 研究背景随着人口老龄化社会的到来及体力劳动普遍减少的生活方式改变,近年肢体慢性缺血性疾病的发病率有增高趋势并表现出严重后果,如患侧肢体疼痛、溃疡、坏死,最终需要截肢。抗凝、抗血小板聚集、降脂、稳定斑块是近年来针对缺血性外周动脉疾病的常规药物治疗措施,但对于重症患者效果欠佳。血管腔内介入治疗虽然发展迅速,但存在术后再狭窄等问题,尤其对于远端血管狭窄闭塞、狭窄病变弥漫的患者效果不佳,影响临床进一步推广应用,故而有必要寻找新的治疗途径。治疗性血管生成(therapeutic angiogenesis)有望为慢性缺血性外周动脉疾病的治疗提供新的途径。当机体组织器官在缺血发生时,体内促血管新生的相关因子及其受体的表达自动上调,从而促进缺血局部侧支循环的形成以进行代偿,但该代偿作用不足以为缺血组织器官提供有效的血液供应,因此会引起一系列与缺血相关的不适症状、体征、生化及病理学改变,如外周动脉狭窄、闭塞引起的间歇性跛行,患侧肢体皮温下降,感觉障碍等等。为改变这种自然代偿的不足,治疗性血管生成通过外源性补充促血管生成因子促进缺血局部组织的血管新生、加强血流灌注,以期达到减轻缺血症状、改善预后的作用。最早应用于缺血性疾病治疗性血管新生领域的血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)、成纤维细胞生长因子(fibroblast growth factor, FGF)在动物实验及Ⅰ期临床试验阶段均取得了较为满意的结果,但在Ⅱ期临床研究中分别因组织水肿和蛋白尿的发生而影响了其进一步在临床应用。故而有必要寻找更为合适的促血管生成因子。低氧诱导因子一1(hypoxia inducible factor1, HIF-1)作为血管生成的总开关基因(master "switch")引起了研究者的重视。HIF-1是由a和β两个亚基组成,以异源性二聚体的形式存在。HIF-1α是专一受氧浓度调节的HIF-1亚单位,决定HIF-1的活性。HIF-1β (已知的芳香烃受体核转移蛋白,ARNT)为HIF-1的组成性表达,在常氧下持续表达且不降解,可与多种bHLH-PAS蛋白形成二聚体,结合DNA以启动转录。在血管生成基因治疗领域,研究的重点是HIF-1α。HIF-1α与p结合后,从胞浆转位到核内并激活下游靶基因。经HIF-1α直接调控参与血管新生的基因有30多种：如：VEGF、诱导型NO合酶(inducible Nitric Oxide Synthase, iNOS)、VEGF受体1(VEGF receptor1, Flt-1)、血管生成素-1(Angiopoietin-1,Ang-1)、Ang-2Ang-4、血小板衍生生长因子(Platelet-derived growth factor、以及血小板生长因子(placenta growth factor, PIGF)中的其他成员。在HIF-1α的诱导下,有望使下游诸多靶基因协同作用,在功能上彼此互补,从而达到使用单基因治疗诱导满意促血管生成作用的效果。HIF-1α在常氧下的半衰期很短,而且转录活性明显降低。该现象的发生在于：HIF-1α结构域中含有两个重要区域,分别为氧依赖降解区(Oxygen Dependent Degradation Domain, ODDD)和C端反式活化结构域(C terminal trans-activation domain, C-TAD)。常氧下,脯氨酰羟化酶作用于ODDD区的402位和564位两个脯氨酸(Pro)残基使其发生羟基化作用(这两个残基是否羟基化决定其蛋白稳定性),导致HIF-1α在数分钟内迅速发生降解。常氧下,C-TAD结构域内803位点上的天门冬酰胺(Asn803)被羟化,会造成激活下游靶基因的能力下降。为了使HIF-1α在常氧条件下不发生降解,高效稳定表达,发挥临床所需的促血管生成作用,本课题组在前期工作中对上述三个位点进行了点突变,先后成功构建了腺病毒介导的单突变体(Ad-HIF-1α-402、Ad-HIF-1α-564、 Ad-HIF-1α-803)、双突变体(Ad-HIF-1α-564/803)及三突变体(Ad-HIF-1α-Trip),从而使其在常氧环境下能高效、稳定表达。本课题组的前期研究表明,Ad-HIF-1α-Trip在离体实验中与其它突变体对比稳定性及转录活性最好。动物实验在体研究表明,Ad-HIF-1α-Trip能促进兔急性缺血肢体血流灌注、血管新生,明显改善预后。目的本研究通过建立兔慢性后肢缺血模型,采用局部肌肉注射的方式,将Ad-HIF-1α突变体转染于兔慢性缺血后肢局部,研究Ad-HIF-1α突变体对下游促血管生成基因表达的影响及对慢性缺血后肢的促血管生成作用；比较不同突变体促血管生成的作用强弱；对Ad-HIF-1α突变体转染兔慢性缺血后肢的部分安全性进行观察。方法1.将本课题组前期构建的重组腺病毒HIF-1α野生型(Ad-HIF-1α-Nature)、重组腺病毒HIF-1α双突变体(Ad-HIF-1α-564/803)、重组腺病毒HIF-1α三突变体(Ad-HIF-1α-Trip)在HEK293A细胞中进行病毒扩增；氯化铯浓度梯度离心透析纯化；终点稀释法测定病毒滴度；对重组腺病毒HIF-1α突变体进行基因测序鉴定。2.建立兔慢性后肢模型,随机分成4组：生理盐水组(Saline组)、Ad-HIF-1α-Nature组、Ad-HIF-1α-564/803组、Ad-HIF-1α-Trip组。兔缺血后肢局部通过肌肉注射方式进行病毒转染或给予生理盐水。基因转染后7天行Real-time PCR检测HIF-1α及其下游基因VEGF、bFGF的表达；基因转染后0天、7天,14天,28天,对兔后肢进行对比超声(CEU)、血管超声、小腿血压多普勒检查,了解缺血后肢血流灌注情况；基因转染后第28天,切取兔缺血后肢肌肉进行CD31免疫组化检查,计数微血管密度(MVD)。3.建立兔慢性后肢缺血模型,随机分成4组：生理盐水组、Ad-HIF-1α-Nature组、Ad-HIF-1α-564/803组、Ad-HIF-1α-Trip组。兔缺血后肢局部通过肌肉注射方式进行病毒转染或给予生理盐水。基因转染前当天(0天)及转染后48小时、72小时、7天、14天、28天抽取静脉血测定血常规、脑钠肽前体(NT-proBNP)、肝功能(谷草转氨酶、谷丙转氨酶)、肾功能(血清肌酐)。4.应用SPSS13.0统计软件进行数据分析,计量数据以x±s表示。多个时间点血常规、血生化检查、多普勒小腿血压标化值、CEU所测得(A×p)标化值与超声心动图所测得髂内动脉血流量标化值(Q1/2)采用重复测量分析,组间同一时间点的对比及组内不同时间点的对比采用单向方差分析,方差齐时组间多重比较采用LSD法,方差不齐时采用Games-Howell检验；免疫组化MVD计数、Real-time PCR所得HIF-1α mRNA、VEGF、bFGF在mRNA水平相对表达量进行单向方差分析。P<0.05差异有统计学意义。结果1.在HEK293A细胞内反复扩增后获得重组腺病毒Ad-HIF-1α-Nature、 Ad-HIF-1α-564/803、Ad-HIF-1α-Trip。终点稀释法测得的滴度分别为3.1×102PFU/ml、1.9×1012PFU/ml、2.5×1012PFU/m。提取所扩增病毒DNA后进行基因测序,目的信息完整,无变异或丢失。所扩增病毒能满足动物实验要求。2.实验兔慢性后肢缺血模型基因转染后7天,Real-time PCR结果显示：四组HIF-1α、VEGF、bFGF的mRNA水平表达由高至低依次为Ad-HIF-1α-Trip、 Ad-HIF-1α-564/803Ad-HIF-1α-Nature、Saline组(P<0.01)。但Ad-HIF-1α-Nature组与Saline组无显著差异。3.实验兔血常规及血生化结果显示：四组实验兔基因转染从初始到转染后28天均未出现血常规及心功能、肝功能、肾功能的明显异常变化。4.多普勒小腿血压测量、CEU、血管超声结果显示：各组缺血患肢血流灌注均呈现随时间而增长的趋势,HIF-1α突变体血流灌注恢复程度明显快于其它各组,尤以Ad-HIF-1α-Trip组增长最为迅速,在基因转染后7天、14天、28天,四组间均存在显著差异(均为P<0.01),但Ad-HIF-1α-Nature组与Saline组无显著差异。5.实验兔慢性后肢缺血模型基因转染后28天病理切片免疫组化CD31染色结果显示：四组微血管密度(MVD)计数由多到少依次为Ad-HIF-1α-Trip、 Ad-HIF-1α-564/803、Ad-HIF-1α-Nature、Saline组(P<0.01)。但Ad-HIF-1α-Nature组与Saline组无显著差异。6.至基因转染后28天,血常规、脑钠肽前体(NT-proBNP)、肝功能(谷草转氨酶、谷丙转氨酶)、肾功能(血清肌酐)均未出现明显异常。结论1.重组腺病毒HIF-1α突变体(Ad-HIF-1α-Trip、Ad-HIF-1α-564/803)经肌肉注射局部转染后在兔慢性缺血后肢能良好表达,并可促进下游VEGF、bFGF的表达；从而促进兔慢性缺血后肢血管生成,增强缺血组织血流灌注,尤其以Ad-HIF-1α-Trip的作用最为显著。2.通过肌肉注射的方式进行转染,重组腺病毒HIF-1α突变体(Ad-HIF-1α-Trip、Ad-HIF-1α-564/803)未引起靶外重要脏器：心脏、肝脏、肾脏的功能改变。3.局部肌肉注射转染Ad-HIF-1α-Trip有望为治疗外周慢性缺血性疾病提供新的途径。更多还原

【Abstract】 IntroductionBecause the changes of diet and the arrival of the aging society, the incidence of chronic peripheral ischemic diseases is increasing these years. Severe disabling symptoms of ischemic limbs related to ischemia are paid more attention, such as claudication, resting pain, and loss of tissue integrity in the distal limbs. Despite great advances in classic drugs, surgical and percutaneous revascularization techniques, treatment of patients with serious peripheral ischemic disease remains a significant clinical challenge. Currently available approaches for treating critical ischemic peripheral vascular disease are not satisfied. The occurrence of restenosis after revascularization discourages the application of this treatment. Furthermore, there is not an effective treatment for the distal and diffuse vascular stenosis. Other effective treatment is needed.Therapeutic angiogenesis maybe bring brightness for the patients. When the ischemia is induced by the stenosis or occlusion of blood vascular, the expressions of angiogenesis-related cytokines and the related acceptors are increased to induce the revascularization of blood perfusion, which are often less sufficient to improve the angiogenesis, blood perfusion and ischemic symptoms. Therapeutic angiogenesis is a procedure that prompts the formation of blood vessels, improve blood perfusion and alleviate the ischemic symptoms by giving exogenous angiogenic factors.Vascular endothelial growth factor(VEGF) and fibroblast growth factor (FGF) were the genes first be used for ischemic disease in preclinical and Phase Ⅰ clinical studies and achieved some satisfactory results. However, the results of Phase Ⅱ clinical trials were somewhat disappointing and hampered the further study. VEGF induced the formation of immature blood vessels, but leading to tissue edema and other adverse reactions. Proteinuria occurred in some patients with ischemic disease treated with FGF. It is necessary to find other genes more suitable for clinical application to promote angiogenesis closing to the physiological processes. Hypoxia inducible factor-1(hypoxia inducible factor-1, HIF-1) attracted the researchers’ attention as a’master switch’gene.HIF-1is a heterodimeric transcription factor composed of a and β subunits. The a subunit is the focal point of the research. The a subunit is unique to the hypoxic response and determines the activity of HIF-1. The β subunit is the constitutive composition of HIF-1and not subject to oxygen dependent regulation. Its main function is to form two mer with a variety of b HLH-PAS proteins, which binding DNA to initiate transcription. HIF-1α binding with HIF-1β and coactivators shifting from the cytoplasm to the nucleus, lead to the enhanced expression of angiogenic genes and initiate transcription. HIF-1α can directly control a variety of downstream target genes, which related to blood vessel growth, such as VEGF, inducible Nitric Oxide Synthase (iNOS), VEGF receptor1(Flt-1), Angiopoietin-1(Ang-1), Ang-2, Ang-4, Platelet-derived growth factor (PDGF) and placenta growth factor (PIGF). In the induction of HIF-1α, it is hopeful to achieve a more satisfactory angiogenesis result because of the complement of the domnstream target genes. In normoxia, the half life of HIF-1α is very short with reduced transcription activity. HIF-1α contains one oxygen-dependent degradation domain (ODDD) and two transactivation activation domains (TAD), naming N-TAD and C-TAD. ODDD mediates the oxygen-regulated stability, and two transactivation activation domains (TAD), N-TAD and C-TAD. The rapidly degradation of HIF-1α is achieved by the prolyl hydroxylases at residues P402and P564within ODDD in several minutes. The inactivation of transcription happens by an asparaginyl hydroxylase at residue N803in the C-TAD.Theoretically, mutation of Pro402, Pro564and Asn803in HIF-la will enhance its stability and transcriptional activity, furthermore, lead to improved angiogenic effects. Our study team had successfully constructed the recombinants of adenovirus-mediated hypoxia inducible factor-la by one, two or three point-mutations of Pro402, Pro564and Asn803. Our previous studies showed that,Our previous studies showed that, the recombinant adenovirus containing the three-point(402/564/803) mutation of HIF-la gene(HTF-1α-trip) expressed better stability and transcriptional activity than single and two points mutations in vitro. In acute ischemic peripheral vascular study in rabbit, Ad-HIF-1α-trip showed significant effects to improve blood perfusion, angiogenesis and ameliorate the prognosis.PurposeThe present study was undertaken to test the hypothesis whether the recombinant adenovirus-mediated HIF-1α mutants could effect the expression of the downstream angiogenic genes in the chronic ischemic limb of the rabbit by intramuscular injection; whether Ad-HIF-1α-Trip is more effective than Ad-HIF-1α-564/803to promote angiogenesis. We also investigated the partial safety of the adenovirus-mediated HIF-la mutants transfection in the rabbit model of hind limb ischemiaMethods1. In the first stage of our study, Ad-HIF-1α-Nature and the mutants of Ad-HIF-1α (Ad-HIF-1α-564/803and Ad-HIF-1α-Trip) were amplified in HEK293A cells and purified by ultracentrifugation in CsCl concentration gradient solutions. The viral titers were evaluated by end-point dilution method. Furthermore, the adenovirus was determined by gene sequencing2.7days after ligation of the left femoral artery,40Zealand Whites Rabbits were randomly divided into4groups immediately:Saline group, Ad-HIF-1α-Nature group, Ad-HIF-1α-564/803group and Ad-HIF-1α-Trip group. Saline or recombinant adenovirus-mediated HIF-1α was given by intramuscular injection.7days after treated with corresponding intramuscular injection, Real-time PCR was chosen to evaluate the expression of HIF-1α and its downstream genes (VEGF and bFGF); On the day of gene transfection and7,14as well as28days after gene transfection, contrast enhanced ultrasound (CEU), vascular ultrasound and Doppler calf blood pressure measurement were used to estimate the blood perfusion of hindlimb and the blood flow of internal iliac artery. On the28th day, immunohistochemistry of CD31was used to evaluate the degree of micro vascular density (MVD).3. The chronic peripheral ischemic models of rabbits were divided into four groups. The day before gene transfection (Oday) and48hours,72hours,7days,14days,28days after gene transfection, venous blood was drew off to evaluate the blood routine count, the NT-proBNP,the liver function and renal function.4. SPSS13.0statistical software was used for data analysis. Measurement data were showed by x±s. Measurement data, such as blood routine count, the NT-proBNP, the liver function, and renal function, the normalized value of A×β, the normalized blood flow of internal iliac artery and the normalized calf blood pressure were analysised by reapeated measurement or one-way ANOVA. Multiple comparisons between groups were analysied by LSD method or Games-Howell test. The count of MVD, the angiography score and the relative expression of HIF-1α mRNA were analysised by one-way ANOVA. The difference was statistically significant when P<0.05.Results1. The titers of Ad-HIF-1α-Nature, Ad-HIF-la-564/803and Ad-HIF-1α-Trip were3.1×1012PFU/ml,1.9×1012PFU/ml and2.5×1012PFU/ml respectively by End-point dilution method. The results of gene sequencing showed that the virus carried the integrity of mutated genetic information The virus amplified reached the requirement of our study.2. On the7th day after gene transfection in the chronic ischemic limb, the results of Real-time PCR showed that the expression of HIF-1α, VEGF and bFGF at mRNA level from higher to lower was Ad-HIF-1α-Trip group、Ad-HIF-1α-564/803group、 Ad-HIF-1α-Nature group and Saline group (P<0.01), but there was no statistical difference between Ad-HIF-1α-Nature group and Saline group.3. The blood routine test and serum biochemical tests showed that:the gene transfection did not cause serious impact on the blood cell count, the heart function, the liver function and renal function from the beginning to28days after gene transfection.4. The results of Doppler calf blood pressure measurement, the contrast enhanced ultrasound (CEU) and the vascular ultrasound showed that the blood perfusion and angiogenesis were improved in each group with the time prolonged. The mutants of Ad-HIF-1α (Ad-HIF-la-564/803, Ad-HIF-1α-Trip), especially Ad-HIF-1α-Trip showed outstanding effects of angiogenesis and improving blood perfusion in chronic peripheral ischemic limb at every time point (P<0.01), but there was no statistical difference between Ad-HIF-1α-Nature group and saline group.5. On the28th day after gene transfection, in the rabbit model of chronic ischemic hind limb, the angiography score of the mutants of Ad-HIF-1α groups (Ad-HIF-1α-564/803, Ad-HIF-1α-Trip), especially Ad-HIF-1α-Trip group, were higher than other groups, but there was no statistical difference between Ad-HIF-la-Nature group and Saline group. The result of MVD evaluated by CD31showed the same trend.Conclusion1. The mutants of Ad-HIF-1α (Ad-HIF-1α-564/803, Ad-HIF-1α-Trip) expressed well in the ischemic limb of rabbit by local intramuscular injection. They promoted revascularization and blood perfusion in the ischemic hindlimb of rabbit. Furthermore, the results of Ad-HIF-1α-Trip was more outstanding than Ad-HIF-1α-564/803.2. The mutants of Ad-HIF-1α (Ad-HIF-1α-564/803, Ad-HIF-1α-Trip) administrated by intramuscular injection didn’t damage the important organs.3. Intramuscular injection of Ad-HIF-1α-Trip is a feasible and effective treatment of chronic peripheral ischemic disease, which maybe be used clinically in the future hopefully.更多还原