提高腺病毒对猪皮肤组织转染效率的研究

发布时间：2018-04-30 04:11

本文选题：CTLA4Ig + 重组腺病毒载体　；参考：《第三军医大学》2008年博士论文

【摘要】： 基因转染猪皮是由腺病毒载体介导CTLA4Ig基因转染活性猪皮肤组织而成,其移植于受体创面后,可在移植局部分泌CTLA4Ig蛋白,有效阻断T细胞活化所需的B7-CD28共刺激信号通道,从而抑制T细胞活化,形成移植局部免疫耐受,延长移植物存活时间,有效延缓了异种皮肤移植免疫排斥发生,同时避免了使用常规免疫抑制剂对免疫功能脆弱的大面积烧伤病人造成的副作用,是临床理想的活性生物敷料。但由于常用的5型腺病毒载体(Ad5)对猪皮肤组织的转染效率相对较低,需要进一步提高转染效率以提高治疗效果。基因转染效率是基因治疗的核心和关键,是当前基因治疗研究的热点。由于猪的皮肤组织结构十分致密,因此病毒载体的转染效率较低。为了达到足够的转染效果,需要使用较高滴度的重组腺病毒载体和较长的转染时间,而较高的滴度长时间作用存在负作用,同时当滴度达到一个阈值后,其转染效率不再发生明显的变化,因此在控制腺病毒滴度的前提下提高腺病毒载体对猪皮肤组织的转染效率不仅对于研制基因转染猪皮具有重要的意义,而且对皮肤基因治疗也具有重要的借鉴和参考作用。腺病毒对皮肤组织的转染过程大致可分为三个阶段,每个阶段都会有居多因素影响腺病毒的转染效率。第一阶段:腺病毒通过表皮屏障到达皮肤细胞周围。在这一阶段,由于表皮组织十分致密,影响腺病毒载体进入到皮肤组织内部,到达皮肤细胞的周围,从而影响转染效率;第二阶段:腺病毒载体与皮肤细胞互相接触。在此阶段,由于细胞表面和腺病毒载体的表面均携带负电荷,存在静电排斥作用,影响腺病毒与皮肤细胞互相接触,从而影响转染效率;第三阶段:腺病毒载体通过与皮肤细胞表面的CAR受体相互作用,以胞吞的形式进入细胞内,完成对细胞的转染。在转染的第三阶段,由于猪皮肤细胞表面的CAR受体较少,影响了腺病毒载体对猪皮肤组织的转染效率。本课题针对影响腺病毒载体转染猪皮肤组织的以上三个限制性原因,分三个部分对提高腺病毒载体转染猪皮肤组织的效率进行研究。通过定量PCR、ELISA、皮肤活性检测、体外淋巴刺激实验、移植试验等方法从基因、蛋白和功能三个层次进行转染效果的评估。以降低基因转染猪皮的腺病毒用量,缩短转染时间,提高腺病毒载体对猪皮肤组织的转染效率。第一部分:针对猪皮肤细胞表面的CAR受体较少的问题,构建利用在各类细胞表面均有广泛表达的CD46分子为受体进行感染的新型嵌合型腺病毒载体Ad5F35-CTLA4Ig,与传统的腺病毒载体Ad5-CTLA4Ig进行转染效果的比较。结果表明:Ad5F35的最佳转染条件是在滴度为5×108条件下转染60分钟,其次是在3×108条件下转染120分钟,在此条件下,Ad5F35的转染效率分别是Ad5的1.49倍和1.73倍,表明Ad5F35-CTLA4Ig对猪皮肤组织的转染效率要优于Ad5-CTLA4Ig。但较高的病毒滴度长时间对猪皮肤组织进行转染存在负作用,因此我们选择Ad5F35-CTLA4Ig作为基因转染猪皮的病毒载体进行后续研究。第二部分:针对皮肤细胞表面负电荷与腺病毒载体表面负电荷产生的静电排斥作用,分别将不同的阳离子物质(DEAE-dextran,protamine,Hexadimethrine bromide)在体外对腺病毒载体进行处理,在腺病毒载体衣壳外形成一层正电荷层,然后进行转染效果的研究,以降低腺病毒载体的转染用量,减少高的病毒滴度对猪皮肤组织进行长时间转染造成的负作用。结果表明:三种阳离子均能有效提高腺病毒转染效率,其中500μg/mL的DEAE-dextran效果最为明显,可将108PFU/mL Ad5F35-CTLA4Ig转染2小时的效率提高19.3倍。第三部分:针对腺病毒难以通过表皮屏障达到皮肤细胞周围的问题,在病毒载体转染液中添加皮肤促渗剂氮酮,同时结合活性皮肤转染后需要抗冻处理,然后进行深低温冻存以长期保持皮肤活性的情况,将转染和抗冻两步合二为一,以缩短转染时间。结果表明:1%的氮酮作用60分钟时效果最好,可将转染效率提高2.9倍。在此条件下加入500μg/mL的DEAE-dextran至转染抗冻液中,可将转染效率提高10.2倍,同时皮肤活力与对照组无明显差异,且效果要优于对照组。结论:使用Ad5F35-CTLA4Ig作为病毒载体,将转染和抗冻合二为一,在转染液中添加1%的氮酮和500μg/mL的DEAE-dextran,可减少处理时间1.5小时,提高转染效率10.2倍,且用此方法生产的基因转染猪皮的效果要优于常规生产的基因转染猪皮。创新点: 1)通过综合处理,在不提高腺病毒滴度的情况下,将腺病毒对猪皮肤组织的转染效率提高了10.2倍,同时减少了处理时间90分钟。 2)首次将Ad5F35病毒载体用于猪皮肤组织的转染,并与传统腺病毒载体进行详细的比较。 3)发明了基因转染猪皮用转染抗冻液,其配方为氮酮(1%)、DEAE(500μg/mL)和DMSO(10%)。将基因转染猪皮生产的转染和抗冻合二为一,缩短了处理时间90分钟。
[Abstract]:Gene transfected pigskin is transfected with adenovirus vector mediated CTLA4Ig gene transfection to active pig skin tissue. After transplantation to the recipient wound, CTLA4Ig protein can be secreted locally and effectively block the B7-CD28 co stimulation signal channel needed for T cell activation, thus inhibiting the activation of T cells, forming local immune tolerance and prolonging graft survival. Time effectively postpones the immune rejection of xenotransplantation, and avoids the adverse effects of conventional immunosuppressants on large area burned patients with weak immune function. It is a clinically ideal bioactive dressing. However, the transfection efficiency of the commonly used type 5 adenovirus vector (Ad5) to pig skin tissue is relatively low. One step to improve the efficiency of transfection in order to improve the therapeutic effect.
Gene transfection efficiency is the core and key of gene therapy. It is a hot spot in the research of gene therapy. Because the tissue structure of the skin is very compact, the transfection efficiency of the virus carrier is low. In order to achieve the sufficient transfection effect, the recombinant adenovirus vector with high titer and the longer transfection time are needed, and the high titer is high. When the titer reaches a threshold, the transfection efficiency is no longer obvious. Therefore, it is of great significance not only to improve the transfection efficiency of adenovirus vector to pig skin, but also to gene therapy for skin gene therapy. It has important reference and reference function.
The transfection of adenovirus to skin tissue can be roughly divided into three stages. There are many factors affecting the transfection efficiency of adenovirus at each stage. First stage: adenovirus reaches the skin cells through the epidermal barrier. In this stage, the epidermal tissue is very dense, and the adenovirus vector enters the skin tissue, reaching the skin tissue. The second phase: the adenovirus carrier and the skin cells contact each other. In this stage, the cell surface and the surface of the adenovirus carrier are negatively charged, and there is electrostatic repulsion, which affects the contact between the adenovirus and the skin cells, thus affecting the transfection efficiency; the third stage: adenosis virus By interacting with the CAR receptor on the surface of the skin, the body enters the cell in the form of endocytosis and completes the transfection of the cells. In the third phase of the transfection, the transfection efficiency of the adenovirus vector to the pig skin tissue is affected by the less CAR receptor on the surface of the pig skin cells.
Aiming at the above three restrictive causes affecting the transfection of pig skin tissue by adenovirus vector, the efficiency of transfection of pig skin tissue by adenovirus vector was studied in three parts. The methods of quantitative PCR, ELISA, skin activity detection, in vitro lymphatic stimulation experiment, and transplantation test were carried out from three levels of gene, protein and function. The transfection effect was evaluated to reduce the dosage of adenovirus transfected into pig skin, shorten the time of transfection, and increase the transfection efficiency of adenovirus vector to porcine skin tissue.
The first part: a new type of chimeric adenovirus vector Ad5F35-CTLA4Ig, a new type of chimeric adenovirus vector, which is infected by CD46 molecules widely expressed on the surface of various cells, is constructed, aiming at the low CAR receptor on the surface of the pig skin. The result shows that the best transfer of Ad5F35 is compared with that of the traditional adenovirus vector Ad5-CTLA4Ig. The transfection condition was 60 minutes transfected under the condition of 5 x 108, followed by 3 x 108 transfection for 120 minutes. Under this condition, the transfection efficiency of Ad5F35 was 1.49 times and 1.73 times of that of Ad5 respectively. It showed that the transfection efficiency of Ad5F35-CTLA4Ig to pig skin tissue was better than that of Ad5-CTLA4Ig., but the higher virus titer for pig skin tissue for a long time. Transfection has negative effects. Therefore, we chose Ad5F35-CTLA4Ig as a viral vector for gene transfection in pigskin for further study.
The second part: in view of the electrostatic exclusion of the negative charges on the surface of the skin cells and the negative charge on the surface of the adenovirus vector, the different cationic substances (DEAE-dextran, protamine, Hexadimethrine bromide) are treated with the adenovirus vector in vitro, and a positive charge layer is formed outside the capsid of the adenovirus vector and then transfected. The results showed that three kinds of cations could effectively improve the transfection efficiency of adenovirus. The results showed that the three kinds of cations could effectively improve the transfection efficiency of adenovirus, of which the effect of 500 g/mL DEAE-dextran was the most obvious, and 108PFU/mL Ad5F35-CTLA4Ig could be transfected for 2 hours. The efficiency is 19.3 times higher.
The third part: in view of the problem that the adenovirus can not reach the skin cells through the epidermal barrier, the skin infiltration agent azone is added to the transfection fluid of the virus carrier, and the antifreeze treatment is needed after the transfection of the active skin, then the deep cryopreservation is carried out to keep the skin activity for a long time, and the two steps of transfection and antifreeze are combined into one. The transfection time was shortened. The results showed that the effect of 1% azone was best for 60 minutes, and the transfection efficiency was increased by 2.9 times. The transfection efficiency could be increased by 10.2 times of the transfection efficiency under the condition of adding 500 mu g/mL to the transfection antifreeze. At the same time, the skin vitality was not significantly different from the control group, and the effect was better than that of the control group.
Conclusion: using Ad5F35-CTLA4Ig as a virus carrier, the transfection and antifreeze are combined to one. Adding 1% azone and 500 g/mL DEAE-dextran in the transfection solution can reduce the treatment time for 1.5 hours and increase the transfection efficiency by 10.2 times.
Innovation point:
1) through comprehensive treatment, the transfection efficiency of adenovirus to pig skin tissue was increased by 10.2 times without increasing the titer of adenovirus, and the treatment time was reduced by 90 minutes.
2) for the first time, Ad5F35 virus vector was used to transfect pig skin tissue, and compared with traditional adenovirus vector.
3) the transfection antifreeze solution for gene transfection of pigskin was invented. The formula was azone (1%), DEAE (500 mu g/mL) and DMSO (10%). The transfection of gene transfected pigskin and the combination of antifreeze, shortened the treatment time for 90 minutes.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

【参考文献】