嵌合型重组腺病毒Ad5F35介导的碱性成纤维细胞生长因子基因治疗兔耳缺血性慢性创面的实验研究

发布时间：2018-07-15 11:47

【摘要】： 研究背景：慢性创面是外科常见疾病,病因繁多、病理复杂,组织再生能力差,愈合困难。在改善全身状态,控制原发病的基础上,目前慢性创面的治疗方式除了手术清除坏死组织外,主要是依靠外用药物或敷料进行的。缺血是慢性创面发生形成的一个重要原因,因此刺激血管新生,改善血运,增强组织再生能力,促进伤口愈合,成为慢性创面修复的一个研究重点。最近引人注目的是生物活性生长因子如TGFbeta、bFGF以及神经传递因子CGRP(calcintonin-gene related peptide)和asoactive intestinal polypeptide的应用。bFGF作为重要的血管生成刺激因子和目前已知最强的促有丝分裂素,不仅可以促使多种细胞分裂增生、趋化,促进血管新生,而且在组织修复、创面愈合过程中发挥了重要作用。将bFGF蛋白因子直接作用于创面,其治疗作用有限,且易被创面内各种酶降解；持续性的直接给药,成本太高。而通过载体介导的基因转然方法将bFGF蛋白因子应用于创面发挥作用,能持续改善血运、增强组织再生能力,促进伤口愈合,现已成为国内外研究的热点。但目前生长因子的基因治疗策略,都存在转然效率低下、载体靶向特异性差,基因表达时间短等缺点,影响了其疗效。因此,本研究旨在,设计构建新型嵌合型重组腺病毒,提高其转染效率、改善靶向性,增强bFGF的基因表达,并将嵌合型重组腺病毒用以介导bFGF作用于兔耳慢性缺血性创面。研究目的：应用兔耳缺血慢性创面模型,用经改造的具有高效感染能力和靶向表达能力的腺病毒载体,介导人碱性成纤维细胞生长因子(bFGF)的表达,研究其是否可以促进血管新生,增强组织细胞再生,促进慢性创面愈合,对慢性创面是否具有保护作用。并分别比较评价其和传统腺病毒介导的bFGF、外源性bFGF对创面愈合的效果差异及影响。研究内容和方法： 1.构建、鉴定和扩增重组嵌合型腺病毒Ad5F35-ET-bFGF。通过PCR的方法扩增EDN1启动子将其插入前期构建完成的pDC316-bFGF腺病毒穿梭载体得到pDC316ET-bFGF,将穿梭质粒pDC316ET-bFGF与Ad5/F35嵌合型腺病毒骨架质粒pPE3-F35共转染至包装细胞得到病毒空斑,经PCR方法和基因测序鉴定得到正确的重组克隆即Ad5F35-ET-bFGF,然后经过反复感染293细胞扩增得到一定滴度的腺病毒,经氯化铯密度梯度离心法纯化病毒,最后通过计算TCID50换算出最终病毒滴度。 2.兔耳缺血慢性创面模型的建立和重组腺病毒在创面的表达。在新西兰白兔兔耳背侧制成直径为1cm的一个圆形皮肤缺损区,直至暴露软骨。以肉眼大体观察、HE染色、创面愈合率计算、兔耳皮温测定、静脉血气分析及荧光免疫检测等评价该模型的可靠性和可行性及鉴定重组腺病毒在创面内的表达情况。 3.嵌合型重组腺病毒介导的bFGF对兔耳慢性创面的基因治疗。实验分为5组：Ad5F35-ET-bFGF组、Ad5bFGF组、Ad5F35-ET-EGFP组、外源性bFGF组和空白对照组。术后第1、3、5、7、9、14d,使用微量注射器多点(分10点)于每只兔耳创面周缘皮下分别注射实验试剂：Ad5F35-ET-bFGF处理组每个创面注入108pfu/ml的重组嵌合型腺病毒Ad5F35-ET-bFGF,病毒用磷酸盐缓冲液(PBS)稀释至总量lml,每点0.1ml；Ad5-bFGF处理组使用同样方法注入10pfu/ml的Ad5-bFGF lml; Ad5F35-ET-EGFP组使用同样方法注入108pfu/ml的Ad5-bFGF 1ml;外源性bFGF处理组使用同样方法注入108pfu/ml的重组牛bFGF lml;空白对照组不予干预。通过大体观察、HE染色和免疫组化、PCR、Western等方法检测bFGF在创面中的表达,评价构建的重组嵌合型腺病毒介导的bFGF和传统腺病毒介导的bFGF、外源性bFGF对创面愈合的不同效果及影响。研究结果： 1.通过同源重组技术,构建制备了END1启动子引导的携带bFGF基因,具有高度感染和特异靶向表达能力的嵌合型增殖腺病毒Ad5F35-ET-bFGF,测定病毒滴度为5.56×1010pfu/ml。 2.制备了兔耳缺血慢性创面模型,通过大体观察、HE染色、创面愈合率计算证实兔耳缺血创面模型可靠、稳定、可行性高。荧光免疫方法说明重组腺病毒成功转染于创面组织,在细胞内呈阳性表达,且表达时间至少持续2周。 3.HE染色、免疫组织化学说明Ad5F35-ET-bFGF在慢性创面组织中持续性高表达；Wsetern Blot半定量检测证明bFGF蛋白在创面组织内过量表达至少持续2周。荧光定量PCR检测结果证明转染后创面内bFGFmRNA水平明显增加。所有结果均显示Ad5F35-ET-bFGF对慢性创面的治疗效果要优于其它实验组和对照组。结论：本课题的研究,建立了兔耳缺血慢性创面模型,在构建具有高感染效率和特异靶向表达能力的嵌合型重组腺病毒Ad5F35-ET基础上,介导bFGF基因治疗缺血慢性创面。证实其对慢性创面具有良好的治疗效果,优于传统腺病毒和外源性生长因子。实验中改进的一些研究方法,产生的特色和创新技术,也代表了未来bFGF基因治疗的发展方向。
[Abstract]:Research background:
Chronic wound is a common surgical disease. There are many causes, complicated pathology, poor tissue regeneration and difficult healing. On the basis of improving the state of the whole body and controlling the original disease, the treatment of chronic wounds is mainly based on the external use of drugs or dressings except the operation to remove the necrotic tissue. Ischemia is a chronic wound. It is important to stimulate angiogenesis, improve blood transport, enhance tissue regeneration, and promote wound healing. It has become a focus of research on chronic wound repair. Recently, it is noticeable that bioactive growth factors such as TGFbeta, bFGF and neurotransmitter CGRP (calcintonin-gene related peptide) and asoactive intestinal Polype The application of ptide, as an important angiogenic stimulator and the strongest known mitogen, can not only promote the proliferation, chemotaxis and angiogenesis of many cells, but also play an important role in tissue repair and wound healing. The effect of bFGF egg white factor on the wound is limited. It is easy to be degraded by various enzymes in the wound; the cost of continuous direct administration is too high. And the bFGF protein factor is applied to the wound by the vector mediated gene transfer method, which can improve the blood transport, enhance the ability of tissue regeneration and promote the healing of the wound. The therapeutic strategies have the disadvantages of low efficiency, poor target specificity and short gene expression time. Therefore, this study aims to design and construct a new chimeric recombinant adenovirus to improve the transfection efficiency, improve the targeting and enhance the gene expression of bFGF, and use the chimeric recombinant adenovirus to mediate bFGF Chronic ischemic wound in rabbit ears.
The purpose of the study is:
The expression of human basic fibroblast growth factor (bFGF) was mediated by the modified model of rabbit ear ischemia and chronic wound surface, and the expression of human basic fibroblast growth factor (bFGF) was mediated, and whether it could promote angiogenesis, enhance the regeneration of tissue cells, promote the healing of chronic wounds, and protect the chronic wounds. The effects of bFGF and traditional bFGF on wound healing were compared and compared.
Research contents and methods:
1. construction, identification and amplification of recombinant chimeric adenovirus Ad5F35-ET-bFGF., the EDN1 promoter was amplified by PCR to insert pDC316ET-bFGF into the early constructed pDC316-bFGF adenovirus shuttle vector, and the shuttle plasmid pDC316ET-bFGF and Ad5/F35 chimeric adenovirus matrix pPE3-F35 were co transfected to the packaged cell to obtain the virus. The correct recombinant clone, Ad5F35-ET-bFGF, was obtained by PCR method and gene sequencing. Then the adenovirus was amplified by repeated infection of 293 cells. The virus was purified by cesium density gradient centrifugation. Finally, the virus titer was converted to the final virus by calculating TCID50.
2. the model of rabbit ear ischemic chronic wound and the expression of recombinant adenovirus in the wound. A circular skin defect area with a diameter of 1cm was made in the ear dorsal side of New Zealand white rabbit. The gross observation, HE staining, wound healing rate, rabbit ear skin temperature measurement, venous blood gas analysis and fluorescence immunoassay were evaluated. The reliability and feasibility of the model and the expression of recombinant adenovirus in the wound were identified.
The gene therapy of 3. chimeric recombinant adenovirus mediated bFGF for rabbit ear chronic wound. The experiment was divided into 5 groups: group Ad5F35-ET-bFGF, group Ad5bFGF, group Ad5F35-ET-EGFP, exogenous bFGF group and blank control group. After operation, 1,3,5,7,9,14d, multipoint injector was used (10 points) to be injected subcutaneously on the periphery of each rabbit ear: Ad The recombinant chimeric adenovirus Ad5F35-ET-bFGF was injected into each wound of the 5F35-ET-bFGF treatment group, and the virus was diluted to the total amount of LML and 0.1ml with the phosphate buffer solution (PBS). The Ad5-bFGF treatment group used the same method to infuse 10pfu/ml Ad5-bFGF LML; Ad5F35-ET-EGFP group used the same method to inject 108pfu/ml. The F treatment group used the same method to infuse the recombinant bovine b FGF LML with the same method, and the blank control group did not intervene. Through gross observation, HE staining and immunohistochemistry, PCR, Western and other methods, the expression of bFGF in the wound was detected. The recombinant chimeric adenovirus mediated bFGF and common adenovirus mediated bFGF, and exogenous bFGF on the wound healing were evaluated. Different effects and effects.
The results of the study:
1. through the homologous recombination technology, the END1 promoter guided bFGF gene was constructed and the chimeric proliferating adenovirus Ad5F35-ET-bFGF with high infection and specific target expression was constructed, and the virus titer was measured to be 5.56 x 1010pfu/ml.
2. the model of rabbit ear ischemia and chronic wound was prepared. Through gross observation, HE staining, the calculation of wound healing rate confirmed that the rabbit ear ischemic wound model was reliable, stable and feasible. The fluorescent immunoassay showed that the recombinant adenovirus was transfected to the wound tissue successfully, and the expression was positive in the cell, and the expression time lasted for at least 2 weeks.
3.HE staining, immunohistochemistry showed that Ad5F35-ET-bFGF was highly expressed in the chronic wound tissue, and the Wsetern Blot semi quantitative detection showed that the excessive expression of bFGF protein in the wound tissue lasted for at least 2 weeks. The fluorescence quantitative PCR detection results showed that the bFGFmRNA level in the wound was obviously increased after the transfection. All the results showed Ad5F35-ET-bFGF. The therapeutic effect of chronic wounds is better than that of other experimental groups and control groups.
Conclusion:
On the basis of building a chimeric recombinant adenovirus Ad5F35-ET with high infection efficiency and specific target expression, bFGF gene is used to treat chronic ischemic wounds. It is proved that it has good therapeutic effect on chronic wound, which is better than traditional adenovirus and exogenous growth cause. Some of the improved research methods, characteristics and innovative technologies in the experiment also represent the future direction of bFGF gene therapy.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764

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