重组腺病毒三突变型低氧诱导因子-1α治疗兔后肢缺血的安全性初步研究
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摘要
背景
缺血性疾病通常由动脉狭窄或闭塞引起,包括外周梗死性血管病、冠状动脉梗死等。临床上主要采用内科药物、血管成形术或外科搭桥术治疗,但仍然存在诸多问题,例如介入后的再狭窄、旁路移植术后的血管再闭塞,以及严重广泛的弥漫性病变难于用上述传统的三种方法治疗等。因此必须寻求其它更有效的治疗方法。“治疗性血管新生(therapeutic angiogenesis)"治疗缺血性疾病是近年来国内外研究的一个热点。治疗性血管新生,就是通过应用血管生长因子或其基因,促进缺血组织的血管新生和侧支血管形成,使缺血得到改善的新的治疗方法。近十余年来,研究者们一直在寻找一种有效的刺激新生血管形成的基因。研究最多的是血管内皮生长因子(vascular endothelial growth factor, VEGF)和成纤维细胞生长因子(fibroblast growth factor,FGF)。但研究资料却显示VEGF可导致新生血管不成熟、组织水肿、血管渗漏、炎症反应重,甚至可能促进动脉硬化进展及血管瘤形成等；FGF治疗则与蛋白尿等有关。因此,人们期望寻找一种能诱导生理功能完整的血管新生且安全的基因。
人低氧诱导因子-1α(Hypoxia inducible factor 1α, HIF-1α)作为低氧或缺氧应答反应中“总开关”基因而日益受到重视。它可调控下游60多种与血管生长、血管张力、糖代谢、红细胞生成及干细胞诱导分化等相关的基因,包括血管内皮生长因子(VEGF)及其受体(Flt-1),血管生成素(Ang)1,2,4,血小板衍生生长因子(PDGF)等。HIF-1α通过促进VEGF、Ang-2、Ang-4、PDGF等基因转录促进血管新生,这些因子相互协同、相互影响,有望生成生理功能完整的血管。但是,在常氧状态下,HIF-1α容易降解,主要与常氧状态下HIF-1α氧依赖降解结构区域(oxygen-dependent degradation domain,ODDD) Pro564和Pro402发生羟基化导致其水解有关,经实验研究,突变其中任一位点的单突变型HIF-1α在常氧下都可以得到表达,不过双突变型HIF-1α则是近乎一种组成型表达；而Asn804突变则可以促进靶基因的转录。
目前,国内外实验证实将野生型或突变型HIF-1α基因通过腺病毒、质粒或者疱疹病毒等载体转染可以促进鼠、兔等动物缺血肢体或心脏血管新生,但涉及HIF-1α在活体表达的安全性评价则鲜见报道,而重组腺病毒三突变型低氧诱导因子-1α在体实验更是未见相关报道。
因此,本课题组前期已将HIF-1α的564、402、803三个位点联合定点突变,成功构建了腺病毒介导的三突变型HIF-1α-Trip (Ad-HIF-1α-Ala402-Ala564-Ala803),使其在常氧下能够稳定高效表达；体外研究显示Ad-HIF-1α-Trip对hMVECs增殖效应较Ad-HIF-1α-nature强,且HIF-1α蛋白表达水平高。本课题组在上述实验的基础上,为进一步明确Ad-HIF-1α-Trip应用于动物体内表达是否安全,将Ad-HIF-1α-Trip转染于兔急性后肢缺血模型中,观察其在动物体内表达可能出现的毒副作用,对其安全性进行初步评价,为进一步进行与人类相似的灵长目动物实验及临床试验提供依据。
目的
基因药物治疗时安全性和有效性同等重要,为了深入研究Ad-HIF-1α-Trip基因在动物体内表达是否安全,本课题将Ad-HIF-1α-Trip转染于兔急性后肢缺血模型中,观察其在动物体内表达时可能出现的毒副作用,对其安全性进行初步评价。
方法
第一部分：将本课题组先前构建的重组腺病毒载体Ad-HIF-1α-Trip、空腺病毒载体Ad-Blank,在HEK293A细胞内大量扩增,经氯化铯密度梯度离心法纯化,终点稀释法测定病毒滴度；提取纯化后病毒DNA,采用PCR法及基因测序对三突变型HIF-1α基因信息进行鉴定。
第二部分：1、构建急性兔后肢缺血模型,造模成功后即将模型兔随机分3组,每组6只,分别为Ad-HIF-1α-Trip组、Ad-Blank组和生理盐水(NS)组。分别以Ad-HIF-1α-Trip(2.0×1010 pfu)、Ad-Blank(2.0×1010pfu)、和生理盐水(NS0.5ml)肌注术侧后肢骨骼肌。
2、本实验周期为28天。每天观测各组实验兔一般药物反应；术前及术后7d、28d行血常规和肝肾功能检测及心电图检查；动物观察28d后全部处死,均做大体解剖,检查肌肉、心、肝、肺、肾、睾丸等组织脏器是否有大体病变,上述所有脏器均取材制作常规组织病理切片,镜检。
结果
第一部分：在HEK293A细胞内扩增后获得重组腺病毒Ad-HIF-1α-Trip、空腺病毒载体Ad-Blank,纯化后终点稀释法测得的滴度分别为2.5×1012 pfu/ml, 4.0×1012 pfu/ml。重组腺病毒转染HEK293A细胞后24h即可出现病变效应(CPE)。提取Ad-HIF-1α-Trip DNA, PCR检测HIF-1α基因得到预期的380bp、460bp、214bp的目的片段,且DNA测序结果正确,符合动物实验要求。
第二部分：在观察期间各组动物精神、食欲良好,大便成形,尿液无浑浊,体温约在38℃。术后28d三组兔体重均有所增加,无一动物死亡；心电图检查显示三组兔均无心律失常发生,P波、QRS波、T波、ST段无异常；血常规、肝肾功能检测,重复测量数据方差分析结果显示,血常规WBC计数在day0、day7和day28之间有显著差异(F=11.257,P=0.000),三个处理组间无显著性差异(F=0.500,P=0.617),且两者不存在交互作用(F=0.333,P=0.969), WBC计数在day7最高,但在正常值范围内,day28则下降至接近day0水平；One-Way ANOVA结果显示,day0、day7、day28各组间WBC计数无显著差异(F=0.138,P=0.872；F=0.273,P=0.765；F=0.648,P=0.537)。肝功能AST、ALT值在day0、day7和day28之间有显著差异(F=8.525,P=0.001；F=25.547,P=0.000),三个处理组间无显著性差异(F=2.752,P=0.096;F=0.056,P=0.946),且ALT、AST值均不存在组别与时间点的交互作用(F=1.238,P=0.316;F=2.311,P=0.081),AST、ALT值均在day7最高,但在正常值范围内,day28则下降至接近day0水平；One-Way ANOVA结果显示,day0、day7.day28各组间AST值均无显著差异(F=1.484,P=0.258;F=1.733,P=0.210;F=1.898,P=0.184)；day0、day7、day28各组间ALT值亦均无显著差异(F=0.612,P=0.555；F=2.620,P=0.127；F=2.297,P=0.135)。其余指标未见明显异常,各时间点间对比、各组间对比均无显著差异(P>0.05),同一时间点各组间对比亦无显著差异(P>0.05)。病理组织学检查,三组兔心、肝、肺、肾、睾丸、Ad-HIF-1α转染部位肌肉色泽、体积正常；组织切片光镜下观察,结构正常,无明显水肿,无明显炎性细胞侵润,未见癌细胞。
结论
第一部分：本课题组先前构建的重组腺病毒载体Ad-HIF-1α-Trip.空腺病毒载体Ad-Blank扩增后获得较高的滴度,PCR法及基因测序鉴定符合预期目标,为下一步进行动物实验提供了保证。
第二部分：兔急性缺血模型局部一次性肌肉注射剂量为2.0×1010 pfu的Ad-HIF-1α-Trip,无明显毒副作用；Ad-HIF-1α-Trip(2.0×1010pfu)应用于兔活体是相对安全的。
Background
Vascular ischemic disease is usually caused by arterial stenosis or occlusion, including lower limb ischemia, coronary infarction, and etc. Now, the traditional therapy of vascular ischemic disease includes medication, operation and interventional therapy, but the therapeutic effect is inadequate. A significant proportion of patients having symptoms such as complete vascular occlusion, obstruction of the arteriole after operation and interventional therapy, are refractory to medical treatment. It is necessary to find an alternative strategy for the treatment of ischemic disease. Such patients are potential candidates for alternative forms of revascularization, like therapeutic angiogenesis. The recent research has found that angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and FGF, which play an critical role in physiologic and pathologic angiogenesis, could lead to neovascularization and promote collateral artery development in animal models of hindlimb ischemia. VEGF and FGF have been studied deeply, and have shown serious side effects such as edema, inflammation, leakage of vascular, vascular tumor and etc. So people try to explore a growth factor which induces mature and safe angiogenesis.
Hypoxia inducible factor 1(HIF-1), the "master switch" gene, is a heterodimeric transcription factor that plays a key role in the cellular adaptive response to hypoxia. HIF-1 participates a series of physiological and pathological processes such as erythropoiesis and angiogenesis by modulating the transcription of more than 60 target genes including VEGF and its receptors (Flt-1), angiopoietin (Ang) 1,2,4, platelet-derived growth factor (PDGF) and etc. It has been demonstrated in the preclinical studies that gene therapy with HIF-la may result in physiologically functional neovascularization. So HIF-1αis considered to be one of the most prospective genes of angiogenesis. HIF-1 is consisted of a constitutively HIF-1βsubunit and a HIF-la subunit. However, HIF-1αproteins decay rapidly under normoxic conditions. It was related to the prolyl hydroxylation of the two proline residus Pro564 and Pro402 in the oxygen dependent degradation domain(ODDD) of HIF-1α. Researches have shown that the mutation in either single Pro402 or Pro564 of HIF-1αcan be expressed under normoxic conditions. The activity of transcription of HIF-1αwhich is related to the hydroxylation of Asn803 and mutation of Asn804 can promote the transcription of target genes.
Experiments at home and abroad have confirmed that wild-type HIF-la gene can promote angiogenesis of ischemic limb of the rats, rabbits and other animals by adenovirus, or herpes virus vector plasmid. However, there are rare reports about the safety evaluation of HIF-1αexpression in vivo and safety evaluation of expression of recombinant adenovirus mediated HIF-la of triple mutant in vivo is not seen in any reports now.
Therefore, we have finished the mutation of Pro402,Pro564 and Asn803 in HIF-1αgene and successfully constructed the adenovirus vector of HIF-1αof triple mutant (Ad-HIF-1α-Trip) in order to get high levels of gene expression under normoxic condition. Experiments identify that multiplier effect mediated by Ad-HIF-1α-Trip is stronger than that mediated by Ad-HIF-1αin hMVECs. To clarify the safety of expression of Ad-HIF-1α-Trip in animals, we will transfect Ad-HIF-1α-Trip in rabbit models with acute hindlimb ischemia and observe the potential side effects in vivo.
Objective
To further study the safety of expression of Ad-HIF-la-Trip in animals, we transfect Ad-HIF-la-Trip in rabbit model with acute hindlimb ischemia and observe the potential side effects in vivo.
Method
(1) The first part of this study:Recombinant adenovirus Ad-HIF-la-Trip and Ad-Blank were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions, then the adenoviral titer was determined by End-Point Dilution Assay. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and DNA sequence analysis.
(2)The second part of this study:①Rabbit models of acute ischemic hindlimb were made, which were divided into 3 groups (6 each) randomly and the Ad-HIF-1α-Trip (2.0×1010 pfu), Ad-Blank (2.0×1010 pfu) and normal saline(NS 0.5ml) were administered respectively intramuscularly into the ischemic limb.②Toxicity was assessed by observing the general drug reaction and testing blood count, liver and kidney function and examining ECG on the day just before (day 0) and on the day7,28 after vector delivery. All animals were sacrificed 28d after injection and the histopathological changes of the muscles, heart, liver, lung, kidney and testicle were observed.
Result
(1) The first part of this study:The last titer of Ad-HIF-la-Trip, Ad-Blank was 2.5×1012pfu/ml,4.0×1012 pfu/ml respectively.HEK293A cells could be infected by recombinant adenovirus, and cytopathic effect appeared just after 24 hours. The DNA of HIF-la-Trip gene was extracted to confirm the presence of three mutant points by PCR, and the sizes of PCR products were 380bp,460bp and 214bp respectively, the result of DNA sequence analysis was correct.
(2) The second part of this study:Animals have generally a good appetite, normal stool and urine, and no fever during the observation period. No animl died. Electrocardiogram examination showed that arrhythmia, P wave, QRSwave, T wave and ST segment were normal. There was statistical significance in terms of WBC counts at different time points (F=11.257, P=0.000) and there was no significance in factor of groups (F=0.500,P=0.617), and there was no intercept effect between factors of group and time (F=0.333,P=0.969). WBC counts are the highest at the day7 but values are the normal range. WBC counts of day28 decreased to the levels which are similar to those of day0; One-Way ANOVA results showed that the WBC count was no significant difference in factor of groups at the same time (F=0.138,P=0.872; F=0.273,P=0.765; F=0.648,P=0.537). There was statistical significance in factor of time of values of AST, ALT (F=8.525, P=0.001; F=25.547, P=0.000 respectively) and there was no significance in factor of groups (F=2.752, P=0.096; F=0.056, P=0.946 respectively), and there was no intercept effect between factors of group and time (F=1.238, P=0.316; F=2.311, P=0.081 respectively). Values of AST, ALT are the highest at the day7, but in the normal range. The values of AST and ALT of day28 decreased the levels similar to those of day0. One-Way ANOVA results showed that the values of AST was no significant difference in factor of groups at the same time (F=1.484,P=0.258; F=1.733,P=0.210;F=1.898,P=0.184 respectively) and the values of ALT was no significant difference in factor of groups at the same time (F=0.612,P=0.555;F=2.620, P=0.127;F=2.297, P=0.135).There was no significance in factors of group and time in other indicators (P>0.05). The color, size of the muscles, heart, liver, lung, kidney and testicle were normal, and no inflammatory cells, tumor or malignant cells were seen in the rabbits of Ad-HIF-la-Trip group.
Conclusion
(1) Recombinant adenovirus Ad-HIF-1α-Trip were amplified and purified with high titer.
(2) Intramuscular injection of Ad-HIF-1α-Trip at single dose of 2.0×1010 pfu is safe and Ad-HIF-1α-Trip can be safely used to rabbits.
缺血性疾病通常由动脉狭窄或闭塞引起,包括外周梗死性血管病、冠状动脉梗死等。临床上主要采用内科药物、血管成形术或外科搭桥术治疗,但仍然存在诸多问题,例如介入后的再狭窄、旁路移植术后的血管再闭塞,以及严重广泛的弥漫性病变难于用上述传统的三种方法治疗等。因此必须寻求其它更有效的治疗方法。“治疗性血管新生(therapeutic angiogenesis)"治疗缺血性疾病是近年来国内外研究的一个热点。治疗性血管新生,就是通过应用血管生长因子或其基因,促进缺血组织的血管新生和侧支血管形成,使缺血得到改善的新的治疗方法。近十余年来,研究者们一直在寻找一种有效的刺激新生血管形成的基因。研究最多的是血管内皮生长因子(vascular endothelial growth factor, VEGF)和成纤维细胞生长因子(fibroblast growth factor,FGF)。但研究资料却显示VEGF可导致新生血管不成熟、组织水肿、血管渗漏、炎症反应重,甚至可能促进动脉硬化进展及血管瘤形成等；FGF治疗则与蛋白尿等有关。因此,人们期望寻找一种能诱导生理功能完整的血管新生且安全的基因。
人低氧诱导因子-1α(Hypoxia inducible factor 1α, HIF-1α)作为低氧或缺氧应答反应中“总开关”基因而日益受到重视。它可调控下游60多种与血管生长、血管张力、糖代谢、红细胞生成及干细胞诱导分化等相关的基因,包括血管内皮生长因子(VEGF)及其受体(Flt-1),血管生成素(Ang)1,2,4,血小板衍生生长因子(PDGF)等。HIF-1α通过促进VEGF、Ang-2、Ang-4、PDGF等基因转录促进血管新生,这些因子相互协同、相互影响,有望生成生理功能完整的血管。但是,在常氧状态下,HIF-1α容易降解,主要与常氧状态下HIF-1α氧依赖降解结构区域(oxygen-dependent degradation domain,ODDD) Pro564和Pro402发生羟基化导致其水解有关,经实验研究,突变其中任一位点的单突变型HIF-1α在常氧下都可以得到表达,不过双突变型HIF-1α则是近乎一种组成型表达；而Asn804突变则可以促进靶基因的转录。
目前,国内外实验证实将野生型或突变型HIF-1α基因通过腺病毒、质粒或者疱疹病毒等载体转染可以促进鼠、兔等动物缺血肢体或心脏血管新生,但涉及HIF-1α在活体表达的安全性评价则鲜见报道,而重组腺病毒三突变型低氧诱导因子-1α在体实验更是未见相关报道。
因此,本课题组前期已将HIF-1α的564、402、803三个位点联合定点突变,成功构建了腺病毒介导的三突变型HIF-1α-Trip (Ad-HIF-1α-Ala402-Ala564-Ala803),使其在常氧下能够稳定高效表达；体外研究显示Ad-HIF-1α-Trip对hMVECs增殖效应较Ad-HIF-1α-nature强,且HIF-1α蛋白表达水平高。本课题组在上述实验的基础上,为进一步明确Ad-HIF-1α-Trip应用于动物体内表达是否安全,将Ad-HIF-1α-Trip转染于兔急性后肢缺血模型中,观察其在动物体内表达可能出现的毒副作用,对其安全性进行初步评价,为进一步进行与人类相似的灵长目动物实验及临床试验提供依据。
目的
基因药物治疗时安全性和有效性同等重要,为了深入研究Ad-HIF-1α-Trip基因在动物体内表达是否安全,本课题将Ad-HIF-1α-Trip转染于兔急性后肢缺血模型中,观察其在动物体内表达时可能出现的毒副作用,对其安全性进行初步评价。
方法
第一部分：将本课题组先前构建的重组腺病毒载体Ad-HIF-1α-Trip、空腺病毒载体Ad-Blank,在HEK293A细胞内大量扩增,经氯化铯密度梯度离心法纯化,终点稀释法测定病毒滴度；提取纯化后病毒DNA,采用PCR法及基因测序对三突变型HIF-1α基因信息进行鉴定。
第二部分：1、构建急性兔后肢缺血模型,造模成功后即将模型兔随机分3组,每组6只,分别为Ad-HIF-1α-Trip组、Ad-Blank组和生理盐水(NS)组。分别以Ad-HIF-1α-Trip(2.0×1010 pfu)、Ad-Blank(2.0×1010pfu)、和生理盐水(NS0.5ml)肌注术侧后肢骨骼肌。
2、本实验周期为28天。每天观测各组实验兔一般药物反应；术前及术后7d、28d行血常规和肝肾功能检测及心电图检查；动物观察28d后全部处死,均做大体解剖,检查肌肉、心、肝、肺、肾、睾丸等组织脏器是否有大体病变,上述所有脏器均取材制作常规组织病理切片,镜检。
结果
第一部分：在HEK293A细胞内扩增后获得重组腺病毒Ad-HIF-1α-Trip、空腺病毒载体Ad-Blank,纯化后终点稀释法测得的滴度分别为2.5×1012 pfu/ml, 4.0×1012 pfu/ml。重组腺病毒转染HEK293A细胞后24h即可出现病变效应(CPE)。提取Ad-HIF-1α-Trip DNA, PCR检测HIF-1α基因得到预期的380bp、460bp、214bp的目的片段,且DNA测序结果正确,符合动物实验要求。
第二部分：在观察期间各组动物精神、食欲良好,大便成形,尿液无浑浊,体温约在38℃。术后28d三组兔体重均有所增加,无一动物死亡；心电图检查显示三组兔均无心律失常发生,P波、QRS波、T波、ST段无异常；血常规、肝肾功能检测,重复测量数据方差分析结果显示,血常规WBC计数在day0、day7和day28之间有显著差异(F=11.257,P=0.000),三个处理组间无显著性差异(F=0.500,P=0.617),且两者不存在交互作用(F=0.333,P=0.969), WBC计数在day7最高,但在正常值范围内,day28则下降至接近day0水平；One-Way ANOVA结果显示,day0、day7、day28各组间WBC计数无显著差异(F=0.138,P=0.872；F=0.273,P=0.765；F=0.648,P=0.537)。肝功能AST、ALT值在day0、day7和day28之间有显著差异(F=8.525,P=0.001；F=25.547,P=0.000),三个处理组间无显著性差异(F=2.752,P=0.096;F=0.056,P=0.946),且ALT、AST值均不存在组别与时间点的交互作用(F=1.238,P=0.316;F=2.311,P=0.081),AST、ALT值均在day7最高,但在正常值范围内,day28则下降至接近day0水平；One-Way ANOVA结果显示,day0、day7.day28各组间AST值均无显著差异(F=1.484,P=0.258;F=1.733,P=0.210;F=1.898,P=0.184)；day0、day7、day28各组间ALT值亦均无显著差异(F=0.612,P=0.555；F=2.620,P=0.127；F=2.297,P=0.135)。其余指标未见明显异常,各时间点间对比、各组间对比均无显著差异(P>0.05),同一时间点各组间对比亦无显著差异(P>0.05)。病理组织学检查,三组兔心、肝、肺、肾、睾丸、Ad-HIF-1α转染部位肌肉色泽、体积正常；组织切片光镜下观察,结构正常,无明显水肿,无明显炎性细胞侵润,未见癌细胞。
结论
第一部分：本课题组先前构建的重组腺病毒载体Ad-HIF-1α-Trip.空腺病毒载体Ad-Blank扩增后获得较高的滴度,PCR法及基因测序鉴定符合预期目标,为下一步进行动物实验提供了保证。
第二部分：兔急性缺血模型局部一次性肌肉注射剂量为2.0×1010 pfu的Ad-HIF-1α-Trip,无明显毒副作用；Ad-HIF-1α-Trip(2.0×1010pfu)应用于兔活体是相对安全的。
Background
Vascular ischemic disease is usually caused by arterial stenosis or occlusion, including lower limb ischemia, coronary infarction, and etc. Now, the traditional therapy of vascular ischemic disease includes medication, operation and interventional therapy, but the therapeutic effect is inadequate. A significant proportion of patients having symptoms such as complete vascular occlusion, obstruction of the arteriole after operation and interventional therapy, are refractory to medical treatment. It is necessary to find an alternative strategy for the treatment of ischemic disease. Such patients are potential candidates for alternative forms of revascularization, like therapeutic angiogenesis. The recent research has found that angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and FGF, which play an critical role in physiologic and pathologic angiogenesis, could lead to neovascularization and promote collateral artery development in animal models of hindlimb ischemia. VEGF and FGF have been studied deeply, and have shown serious side effects such as edema, inflammation, leakage of vascular, vascular tumor and etc. So people try to explore a growth factor which induces mature and safe angiogenesis.
Hypoxia inducible factor 1(HIF-1), the "master switch" gene, is a heterodimeric transcription factor that plays a key role in the cellular adaptive response to hypoxia. HIF-1 participates a series of physiological and pathological processes such as erythropoiesis and angiogenesis by modulating the transcription of more than 60 target genes including VEGF and its receptors (Flt-1), angiopoietin (Ang) 1,2,4, platelet-derived growth factor (PDGF) and etc. It has been demonstrated in the preclinical studies that gene therapy with HIF-la may result in physiologically functional neovascularization. So HIF-1αis considered to be one of the most prospective genes of angiogenesis. HIF-1 is consisted of a constitutively HIF-1βsubunit and a HIF-la subunit. However, HIF-1αproteins decay rapidly under normoxic conditions. It was related to the prolyl hydroxylation of the two proline residus Pro564 and Pro402 in the oxygen dependent degradation domain(ODDD) of HIF-1α. Researches have shown that the mutation in either single Pro402 or Pro564 of HIF-1αcan be expressed under normoxic conditions. The activity of transcription of HIF-1αwhich is related to the hydroxylation of Asn803 and mutation of Asn804 can promote the transcription of target genes.
Experiments at home and abroad have confirmed that wild-type HIF-la gene can promote angiogenesis of ischemic limb of the rats, rabbits and other animals by adenovirus, or herpes virus vector plasmid. However, there are rare reports about the safety evaluation of HIF-1αexpression in vivo and safety evaluation of expression of recombinant adenovirus mediated HIF-la of triple mutant in vivo is not seen in any reports now.
Therefore, we have finished the mutation of Pro402,Pro564 and Asn803 in HIF-1αgene and successfully constructed the adenovirus vector of HIF-1αof triple mutant (Ad-HIF-1α-Trip) in order to get high levels of gene expression under normoxic condition. Experiments identify that multiplier effect mediated by Ad-HIF-1α-Trip is stronger than that mediated by Ad-HIF-1αin hMVECs. To clarify the safety of expression of Ad-HIF-1α-Trip in animals, we will transfect Ad-HIF-1α-Trip in rabbit models with acute hindlimb ischemia and observe the potential side effects in vivo.
Objective
To further study the safety of expression of Ad-HIF-la-Trip in animals, we transfect Ad-HIF-la-Trip in rabbit model with acute hindlimb ischemia and observe the potential side effects in vivo.
Method
(1) The first part of this study:Recombinant adenovirus Ad-HIF-la-Trip and Ad-Blank were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions, then the adenoviral titer was determined by End-Point Dilution Assay. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and DNA sequence analysis.
(2)The second part of this study:①Rabbit models of acute ischemic hindlimb were made, which were divided into 3 groups (6 each) randomly and the Ad-HIF-1α-Trip (2.0×1010 pfu), Ad-Blank (2.0×1010 pfu) and normal saline(NS 0.5ml) were administered respectively intramuscularly into the ischemic limb.②Toxicity was assessed by observing the general drug reaction and testing blood count, liver and kidney function and examining ECG on the day just before (day 0) and on the day7,28 after vector delivery. All animals were sacrificed 28d after injection and the histopathological changes of the muscles, heart, liver, lung, kidney and testicle were observed.
Result
(1) The first part of this study:The last titer of Ad-HIF-la-Trip, Ad-Blank was 2.5×1012pfu/ml,4.0×1012 pfu/ml respectively.HEK293A cells could be infected by recombinant adenovirus, and cytopathic effect appeared just after 24 hours. The DNA of HIF-la-Trip gene was extracted to confirm the presence of three mutant points by PCR, and the sizes of PCR products were 380bp,460bp and 214bp respectively, the result of DNA sequence analysis was correct.
(2) The second part of this study:Animals have generally a good appetite, normal stool and urine, and no fever during the observation period. No animl died. Electrocardiogram examination showed that arrhythmia, P wave, QRSwave, T wave and ST segment were normal. There was statistical significance in terms of WBC counts at different time points (F=11.257, P=0.000) and there was no significance in factor of groups (F=0.500,P=0.617), and there was no intercept effect between factors of group and time (F=0.333,P=0.969). WBC counts are the highest at the day7 but values are the normal range. WBC counts of day28 decreased to the levels which are similar to those of day0; One-Way ANOVA results showed that the WBC count was no significant difference in factor of groups at the same time (F=0.138,P=0.872; F=0.273,P=0.765; F=0.648,P=0.537). There was statistical significance in factor of time of values of AST, ALT (F=8.525, P=0.001; F=25.547, P=0.000 respectively) and there was no significance in factor of groups (F=2.752, P=0.096; F=0.056, P=0.946 respectively), and there was no intercept effect between factors of group and time (F=1.238, P=0.316; F=2.311, P=0.081 respectively). Values of AST, ALT are the highest at the day7, but in the normal range. The values of AST and ALT of day28 decreased the levels similar to those of day0. One-Way ANOVA results showed that the values of AST was no significant difference in factor of groups at the same time (F=1.484,P=0.258; F=1.733,P=0.210;F=1.898,P=0.184 respectively) and the values of ALT was no significant difference in factor of groups at the same time (F=0.612,P=0.555;F=2.620, P=0.127;F=2.297, P=0.135).There was no significance in factors of group and time in other indicators (P>0.05). The color, size of the muscles, heart, liver, lung, kidney and testicle were normal, and no inflammatory cells, tumor or malignant cells were seen in the rabbits of Ad-HIF-la-Trip group.
Conclusion
(1) Recombinant adenovirus Ad-HIF-1α-Trip were amplified and purified with high titer.
(2) Intramuscular injection of Ad-HIF-1α-Trip at single dose of 2.0×1010 pfu is safe and Ad-HIF-1α-Trip can be safely used to rabbits.
引文
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[2]金惠铭.治疗性血管新生研究的若干进展[J].国外医学.生理.病理科学与临床分册,2003(04)：331-333.
[3]Grines C L, Watkins M W, Helmer G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris.[J]. Circulation,2002,105(11):1291-1297.
[4]Makinen K, Manninen H, Hedman M, et al. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase Ⅱ study.[J]. Mol Ther,2002,6(1):127-133.
[5]Visconti R P, Richardson C D, Sato T N. Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF).[J]. Proc Natl Acad Sci U S A,2002,99(12):8219-8224.
[6]Fam N P, Verma S, Kutryk M, et al. Clinician guide to angiogenesis.[J]. Circulation,2003,108(21):2613-2618.
[7]Holash J, Maisonpierre P C, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.[J]. Science,1999,284(5422):1994-1998.
[8]Semenza G L, Roth P H, Fang H M, et al. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1.[J]. J Biol Chem,1994,269(38):23757-23763.
[9]Dery M A, Michaud M D, Richard D E. Hypoxia-inducible factor 1:regulation by hypoxic and non-hypoxic activators.[J]. Int J Biochem Cell Biol,2005,37(3):535-540.
[10]Fels D R, Koumenis C. HIF-1 alpha and p53:the ODD couple?[J]. Trends Biochem Sci,2005,30(8):426-429.
[11]Harris A L. Hypoxia--a key regulatory factor in tumour growth. [J]. Nat Rev Cancer,2002,2(1):38-47.
[12]Bracken C P, Whitelaw M L, Peet D J. The hypoxia-inducible factors:key transcriptional regulators of hypoxic responses. [J]. Cell Mol Life Sci,2003,60(7):1376-1393.
[13]Masson N, Willam C, Maxwell P H, et al. Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.[J]. EMBO J,2001,20(18):5197-5206.
[14]Jaakkola P, Mole D R, Tian Y M, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by 02-regulated prolyl hydroxylation.[J]. Science,2001,292(5516):468-472.
[15]Bruick R K, Mcknight S L. A conserved family of prolyl-4-hydroxylases that modify HIF.[J]. Science,2001,294(5545):1337-1340.
[16]Maxwell P H. Hypoxia-inducible factor as a physiological regulator.[J]. Exp Physiol,2005,90(6):791-797.
[17]Lando D, Peet D J, Whelan D A, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch.[J]. Science,2002,295(5556):858-861.
[18]Sang N, Fang J, Srinivas V, et al. Carboxyl-terminal transactivation activity of hypoxia-inducible factor 1 alpha is governed by a von Hippel-Lindau protein-independent, hydroxylation-regulated association with p300/CBP.[J]. Mol Cell Biol,2002,22(9):2984-2992.
[19]郭寿贵,吴平生,王月刚,等.人突变型低氧诱导因子1α腺病毒载体的构建及鉴定[J].第四军医大学学报,2005(17)：1614-1617.
[20]童锴,谢宜军,王月刚,等.人双突变型低氧诱导因子1α腺病毒载体的构建 及鉴定[J].南方医科大学学报,2007(04)：445-447.
[21]赖艳娴,刘城,王月刚,等.人3突变型低氧诱导因子1α真核表达载体和腺病毒表达载体的构建及鉴定[J].中国医科大学学报,2008(01)：86-89..
[22]刘城,吴平生,郭寿贵,等.腺病毒介导突变体HIF-1α基因对大鼠血管平滑肌细胞增殖的影响[J].广东医学,2007(06)：871-873.
[23]陈娓,张红亚,郭寿贵,等.腺病毒介导的人诱变型低氧诱导因子1α对大鼠后肢缺血模型血管新生的影响[J].解放军医学杂志,2008(05)：576-578.
[24]陈娓,童锴,张红亚,等.人突变型低氧诱导因子1α对大鼠后肢缺血模型血管新生的影响[J].第四军医大学学报,2008(15)：1360-1363.
[25]Anderson W F. Human gene therapy.[J]. Science,1992,256(5058):808-813.
[26]Marshall E. Gene therapy death prompts review of adenovirus vector. [J]. Science,1999,286(5448):2244-2245.
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