本文選題：CTLA4Ig + 重組腺病毒載體��； 參考：《第三軍醫(yī)大學(xué)》2008年博士論文

【摘要】： 基因轉(zhuǎn)染豬皮是由腺病毒載體介導(dǎo)CTLA4Ig基因轉(zhuǎn)染活性豬皮膚組織而成,其移植于受體創(chuàng)面后,可在移植局部分泌CTLA4Ig蛋白,有效阻斷T細(xì)胞活化所需的B7-CD28共刺激信號通道,從而抑制T細(xì)胞活化,形成移植局部免疫耐受,延長移植物存活時間,有效延緩了異種皮膚移植免疫排斥發(fā)生,同時避免了使用常規(guī)免疫抑制劑對免疫功能脆弱的大面積燒傷病人造成的副作用,是臨床理想的活性生物敷料。但由于常用的5型腺病毒載體(Ad5)對豬皮膚組織的轉(zhuǎn)染效率相對較低,需要進(jìn)一步提高轉(zhuǎn)染效率以提高治療效果。 基因轉(zhuǎn)染效率是基因治療的核心和關(guān)鍵,是當(dāng)前基因治療研究的熱點。由于豬的皮膚組織結(jié)構(gòu)十分致密,因此病毒載體的轉(zhuǎn)染效率較低。為了達(dá)到足夠的轉(zhuǎn)染效果,需要使用較高滴度的重組腺病毒載體和較長的轉(zhuǎn)染時間,而較高的滴度長時間作用存在負(fù)作用,同時當(dāng)?shù)味冗_(dá)到一個閾值后,其轉(zhuǎn)染效率不再發(fā)生明顯的變化,因此在控制腺病毒滴度的前提下提高腺病毒載體對豬皮膚組織的轉(zhuǎn)染效率不僅對于研制基因轉(zhuǎn)染豬皮具有重要的意義,而且對皮膚基因治療也具有重要的借鑒和參考作用。 腺病毒對皮膚組織的轉(zhuǎn)染過程大致可分為三個階段,每個階段都會有居多因素影響腺病毒的轉(zhuǎn)染效率。第一階段:腺病毒通過表皮屏障到達(dá)皮膚細(xì)胞周圍。在這一階段,由于表皮組織十分致密,影響腺病毒載體進(jìn)入到皮膚組織內(nèi)部,到達(dá)皮膚細(xì)胞的周圍,從而影響轉(zhuǎn)染效率;第二階段:腺病毒載體與皮膚細(xì)胞互相接觸。在此階段,由于細(xì)胞表面和腺病毒載體的表面均攜帶負(fù)電荷,存在靜電排斥作用,影響腺病毒與皮膚細(xì)胞互相接觸,從而影響轉(zhuǎn)染效率;第三階段:腺病毒載體通過與皮膚細(xì)胞表面的CAR受體相互作用,以胞吞的形式進(jìn)入細(xì)胞內(nèi),完成對細(xì)胞的轉(zhuǎn)染。在轉(zhuǎn)染的第三階段,由于豬皮膚細(xì)胞表面的CAR受體較少,影響了腺病毒載體對豬皮膚組織的轉(zhuǎn)染效率。 本課題針對影響腺病毒載體轉(zhuǎn)染豬皮膚組織的以上三個限制性原因,分三個部分對提高腺病毒載體轉(zhuǎn)染豬皮膚組織的效率進(jìn)行研究。通過定量PCR、ELISA、皮膚活性檢測、體外淋巴刺激實驗、移植試驗等方法從基因、蛋白和功能三個層次進(jìn)行轉(zhuǎn)染效果的評估。以降低基因轉(zhuǎn)染豬皮的腺病毒用量,縮短轉(zhuǎn)染時間,提高腺病毒載體對豬皮膚組織的轉(zhuǎn)染效率。 第一部分:針對豬皮膚細(xì)胞表面的CAR受體較少的問題,構(gòu)建利用在各類細(xì)胞表面均有廣泛表達(dá)的CD46分子為受體進(jìn)行感染的新型嵌合型腺病毒載體Ad5F35-CTLA4Ig,與傳統(tǒng)的腺病毒載體Ad5-CTLA4Ig進(jìn)行轉(zhuǎn)染效果的比較。結(jié)果表明:Ad5F35的最佳轉(zhuǎn)染條件是在滴度為5×108條件下轉(zhuǎn)染60分鐘,其次是在3×108條件下轉(zhuǎn)染120分鐘,在此條件下,Ad5F35的轉(zhuǎn)染效率分別是Ad5的1.49倍和1.73倍,表明Ad5F35-CTLA4Ig對豬皮膚組織的轉(zhuǎn)染效率要優(yōu)于Ad5-CTLA4Ig。但較高的病毒滴度長時間對豬皮膚組織進(jìn)行轉(zhuǎn)染存在負(fù)作用,因此我們選擇Ad5F35-CTLA4Ig作為基因轉(zhuǎn)染豬皮的病毒載體進(jìn)行后續(xù)研究。 第二部分:針對皮膚細(xì)胞表面負(fù)電荷與腺病毒載體表面負(fù)電荷產(chǎn)生的靜電排斥作用,分別將不同的陽離子物質(zhì)(DEAE-dextran,protamine,Hexadimethrine bromide)在體外對腺病毒載體進(jìn)行處理,在腺病毒載體衣殼外形成一層正電荷層,然后進(jìn)行轉(zhuǎn)染效果的研究,以降低腺病毒載體的轉(zhuǎn)染用量,減少高的病毒滴度對豬皮膚組織進(jìn)行長時間轉(zhuǎn)染造成的負(fù)作用。結(jié)果表明:三種陽離子均能有效提高腺病毒轉(zhuǎn)染效率,其中500μg/mL的DEAE-dextran效果最為明顯,可將108PFU/mL Ad5F35-CTLA4Ig轉(zhuǎn)染2小時的效率提高19.3倍。 第三部分:針對腺病毒難以通過表皮屏障達(dá)到皮膚細(xì)胞周圍的問題,在病毒載體轉(zhuǎn)染液中添加皮膚促滲劑氮酮,同時結(jié)合活性皮膚轉(zhuǎn)染后需要抗凍處理,然后進(jìn)行深低溫凍存以長期保持皮膚活性的情況,將轉(zhuǎn)染和抗凍兩步合二為一,以縮短轉(zhuǎn)染時間。結(jié)果表明:1%的氮酮作用60分鐘時效果最好,可將轉(zhuǎn)染效率提高2.9倍。在此條件下加入500μg/mL的DEAE-dextran至轉(zhuǎn)染抗凍液中,可將轉(zhuǎn)染效率提高10.2倍,同時皮膚活力與對照組無明顯差異,且效果要優(yōu)于對照組。 結(jié)論:使用Ad5F35-CTLA4Ig作為病毒載體,將轉(zhuǎn)染和抗凍合二為一,在轉(zhuǎn)染液中添加1%的氮酮和500μg/mL的DEAE-dextran,可減少處理時間1.5小時,提高轉(zhuǎn)染效率10.2倍,且用此方法生產(chǎn)的基因轉(zhuǎn)染豬皮的效果要優(yōu)于常規(guī)生產(chǎn)的基因轉(zhuǎn)染豬皮。 創(chuàng)新點: 1)通過綜合處理,在不提高腺病毒滴度的情況下,將腺病毒對豬皮膚組織的轉(zhuǎn)染效率提高了10.2倍,同時減少了處理時間90分鐘。 2)首次將Ad5F35病毒載體用于豬皮膚組織的轉(zhuǎn)染,并與傳統(tǒng)腺病毒載體進(jìn)行詳細(xì)的比較。 3)發(fā)明了基因轉(zhuǎn)染豬皮用轉(zhuǎn)染抗凍液,其配方為氮酮(1%)、DEAE(500μg/mL)和DMSO(10%)。將基因轉(zhuǎn)染豬皮生產(chǎn)的轉(zhuǎn)染和抗凍合二為一,縮短了處理時間90分鐘。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R346

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相關(guān)期刊論文 前4條

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本文編號：1823008