【作者】 王鹏；

【导师】 阚全程；

【作者基本信息】 郑州大学 ， 内科学， 2011， 博士

【摘要】 研究背景：器官移植是治疗终末期器官疾病的重要方法之一,但供源缺乏和排斥反应是困扰器官移植的两大难题。肝细胞移植(hepatocyte transplantation, HT)最大的优点是可以一肝多用,使更多的人得到移植机会,降低等待肝源时的死亡率,因此有可能取代原位肝移植(orthotopic liver tranplantation, OLT)用于终末期肝脏疾病患者,但免疫排斥仍是HT需要解决的一个重要问题。随着现代基因工程的发展,具有免疫逃逸功能的转基因肝细胞成为HT的研究热点。腺病毒载体能够将外源基因安全而高效地转染入多种细胞,因此被广泛地应用于基因治疗的各个领域,但宿主对基因表达产物的排斥反应严重地限制了基因治疗的发展,临床现有的免疫抑制剂并不总是有效,而且毒副作用大。因此,降低或消除机体对外源基因表达产物及其转染细胞的免疫反应,成为长期基因治疗中的研究热点。主要组织相容性复合体(major histocompatibility complex, MHC)抗原呈递途径在免疫排斥方面的研究中越来越引起人们的关注,阻断MHCⅠ-抗原肽复合物的表达,成为诱导免疫耐受的研究热点。MHCⅠ限制性细胞毒性T淋巴细胞(cytotoxic T lymphocytes, CTL)在病毒感染中发挥着重要作用,干扰感染细胞表面MHCⅠ类分子抗原呈递途径来逃避宿主免疫清除的策略被许多病毒所利用。单纯疱疹病毒Ⅰ型(herpes simplex virus I, HSV-1)基因编码的立即早期感染性细胞蛋白47(infected cell protein 47,ICP47)能与抗原多肽竞争抗原加工相关转运子(transporter associated with antigen presentation,TAP)的肽结合位点,有效地降低CTL的识别和杀伤活性,逃逸宿主的免疫清除。因此,本研究构建了一种表达His-tag-ICP47融合基因的腺病毒载体,并研究其免疫活性,以期能够为研究机体免疫耐受机制和基因治疗提供重要的探索方向。此外,这些研究可能为拓展病毒免疫学领域,探索病毒和宿主免疫系统之间的相互作用提供了新的研究方向,并为临床疾病的治疗和预防拓展了更广阔的空间。第一部分表达His-tag-ICP47融合基因腺病毒载体的构建目的：构建表达His-tag-ICP47融合基因的重组腺病毒,并且进行扩增和滴度测定,以便用于其免疫活性的实验研究。方法：应用AdEasy-1月泉病毒表达载体系统构建表达His-tag-ICP47融合基因的重组腺病毒r-H-ICP47和对照空载重组腺病毒r-Track。His-tag-ICP47融合基因首先克隆到pAdTrack-CMV载体上,用PmeⅠ酶切后与腺病毒骨架载体pAdEasy-1在BJ5183菌内同源重组构建重组腺病毒载体pAdEasy-H-ICP47,PacⅠ酶切线性化后,重组腺病毒载体pAdEasy-H-ICP47在293细胞内包装为重组腺病毒r-H-ICP47。以同样的方法构建对照空载重组腺病毒r-Track。最终,扩增并检测病毒滴度。结果：重组腺病毒r-H-ICP47和r-Track构建成功并且具有良好的感染性,可以在293细胞中包装成病毒颗粒。重组腺病毒r-H-ICP47和r-Track的滴度测定结果分别为3.7×1010efu/mL和4.4x 1010efu/mL结论：复制缺陷型重组腺病毒r-H-ICP47和r-Track成功构建,病毒滴度高,能够在细胞内有效地转染和表达,能够满足下一步免疫活性研究的要求。第二部分表达His-tag-ICP47融合基因重组腺病毒的免疫活性研究目的：观察表达His-tag-ICP47融合基因重组腺病毒的免疫活性。方法：1.重组腺病毒r-H-ICP47或r-Track转染HL-7702肝细胞,测定对HL-7702细胞的转染效率,并用MTT法测定CTL对转染HL-7702细胞的杀伤活性。2.以健康人外周血单核细胞(peripheral blood mononuclear cells, PBMCs)为来源,体外诱导培养树突状细胞(dendritic cells, DCs),测定重组腺病毒r-H-ICP47或r-Track对转染DCs的转染效率,并且采用MTT法测定第24h、48h和72h时重组腺病毒r-H-ICP47或r-Track对转染DCs刺激淋巴细胞增殖活性的影响。3.重组腺病毒r-H-ICP47或r-Track转染淋巴细胞,测定转染效率,并进行双向混合淋巴细胞反应(Two-way mixed lymphocyte reaction, Two-way MLR),采用ELISA法分别于第2d、第4d和第6d测定各组MLR上清液中白细胞介素-2 (Interleukin-2, IL-2)和穿孔素(perforin, PF)的浓度。结果：1.重组腺病毒r-H-ICP47和r-Track均能高效转染HL-7702肝细胞株,感染效率随着感染复数(multiplicity of infection, MOI)升高而升高。当MOI为100时,重组腺病毒r-H-ICP47和r-Track对HL-7702细胞的转染效率(86.87±3.14%和90.32±2.25%)均明显高于MOI为50的转染效率(29.52±5.22%和32.12±2.27%)(P<0.05),而与MOI为200的转染效率(88.53±3.69%和90.64±3.65%)无显著性差异(P>0.05)。当MOI为100的重组腺病毒r-H-ICP47或r-Track转染HL-7702肝细胞后,进行CTL杀伤活性实验,结果显示,与r-track组和正常对照组相比,r-H-ICP47组能明显降低CTL对转染HL-7702细胞的杀伤活性(P<0.05)。2.重组腺病毒r-H-ICP47和r-Track对DCs的感染效率随着MOI升高而升高,当MOI为100时,重组腺病毒r-H-ICP47和r-Track对DCs的转染效率(86.92±2.76%和84.08±2.62%)均明显高于MOI为50的转染效率(30.32±3.08%和23.70±3.23%)(P<0.05),而与MOI为200的转染效率(91.26±2.59%和87.51士2.41%)无显著性差异(P>0.05)。MTT检测结果显示,与重组腺病毒r-Track组和正常对照组相比,在培养的第48h和第72h,重组腺病毒r-H-ICP47能够较明显地降低转染DCs刺激淋巴细胞增殖的能力(P<0.05),而在第24h时,DCs刺激淋巴细胞的增殖活性在3组之间均无明显差异(P>0.05)。3.重组腺病毒r-H-ICP47和r-Track对淋巴细胞的感染效率随着MOI升高而升高,当MOI为100时,重组腺病毒r-H-ICP47和r-Track对淋巴细胞的转染效率(87.11±3.29%和82.21±4.01%)均明显高于MOI为50的转染效率(25.54±4.07%和27.67±2.31%)(P<0.05),而与MOI为200的转染效率(89.75±2.92%和85.99±3.02%)无显著性差异(P>0.05)。Two-way MLR分为5组,D-R-ICP47、D-ICP47组、D-R-Track组、D-Track组和正常对照组,分别用MOI为100的重组腺病毒r-H-ICP47或r-Track转染供体(D)或受体(R)的淋巴细胞后进行Two-way MLR培养,采用ELISA法分别在第2d、第4d和第6d检测MLR上清液中IL-2和PF的浓度。结果显示在各个时间段D-R-ICP47组IL-2和PF的浓度均明显低于其余4组(P<0.05),并且D-ICP47组IL-2和PF的浓度也低于D-R-Track组、D-Track组和正常对照组(P<0.05)。结论：1.表达His-tag-ICP47的重组腺病毒能够安全、高效地转染HL-7702细胞、DCs和淋巴细胞,并达到理想的表达水平。2.表达His-tag-ICP47融合基因的重组腺病毒能够有效地降低CTL对转染HL-7702肝细胞的特异性杀伤活性,降低转染DCs刺激淋巴细胞增殖的能力和Two-way MLR上清液中IL-2和PF的表达程度。这些结果在一定程度上可预测His-tag-ICP47融合基因能够降低机体对转染细胞和腺病毒载体排斥反应发生的程度,提示表达His-tag-ICP47融合基因的腺病毒载体及其转染细胞能够介导免疫耐受,达到外源基因长期表达的目的。更多还原

【Abstract】 Background:Organ transplantation is one of the most important treatments of end-stage organ failure, yet graft rejection and lack of donation are the two major problems persisting in the treatments. Hepatocyte transplantation (HT) may serve as an alternative to orthotopic liver transplantation (OLT) for patients with end-stage liver disease because the most important advantage of it is decreasing mortality in the waiting list and allowing more patients to be treated, but immune rejection of HT is still an important problem to be solved.With recent advances in transgenic technology, the availability of transgenic HT evading the clearance of host immune systems could be a critical subject of HT. Adenovirus vector is considered a safe and efficient way to introduce foreign genes into several kinds of cells and is widely used in the various fields of gene therapy.But the response of host immune systems against gene products expressed by genetically modified cells is a major obstacle to successful gene therapy. Immunosuppressants are not always effective and associated with well-known toxic effects. Therefore, prevention of host immune response against transplanted cells and products of introduced genes could be a critical subject for the long-term success of gene therapy.In particular, major histocompatibility complex (MHC) classⅠantigen presenting pathway is very important in acute allograft rejection and has been an attractive target for immune rejection, and blocking MHCⅠantigen expression is becoming a research hotspot of inducting immune tolerance. MHCⅠrestricted cytotoxic T lymphocytes (CTL) may be an important role in immunity to viral infection, many viruses have evolved mechanisms to evade clearance of host immune systems by blocking MHCⅠantigen presentation pathway. Infected cell protein 47 (ICP47), an immediate-early protein expressesd by herpes simplex virus type 1 (HSV-1), inhibits MHCⅠantigen presentation pathway by binding to host transporter associated with antigen presentation (TAP), and thereby reduces the rate of cytolysis of infected cells by sensitized CTL and evades the host immune clearance.Based on these studies, this subject was designed to construct a recombinant adenovirus vector expressing His-tag-ICP47 fusion gene and identify its immunological activity. We expect this finding should have important implications for analyzing the mechanisms of immune tolerance as well as human gene therapy. Moreover, these studies should open up new horizons for expanding the fields of viral immunology, exploring the interactions between host immune systems and viruses, and enable us to explore more effective preventions and treatments for clinical diseases.PartⅠConstruction of adenovirus vector expressing His-tag-ICP47 fusion geneObjective:To construct and amplify recombinant adenovirus vector expressing His-tag-ICP47 fusion gene, and determine virus particle titres for the studies of its immunological activities.Methods: The adenovirus vector system of AdEasy-1 was used to prepare the recombinant adenovirus expressing His-tag-ICP47 fusion gene or the control empty recombinant adenovirus r-Track. His-tag-ICP47 fusion gene was firstly cloned into the pAdTrack-CMV vector. The gene fragments digested by PmeⅠwere co-transformed with adenoviral backbone vector pAdEasy-1 in E.coli BJ5183 cells to produce recombinant adenovirus vector pAdEasy-H-ICP47. Linearized with PacⅠrecombinant adenovirus vector was subsequently transfected into 293 cells to product r-H-ICP47. Meanwhile, the control empty recombinant adenovirus r-Track was built in the same way. Finally, The viruses were amplified and virus particle titres were determined for the studies of its immunological activities.Results:The recombinant adenoviruses of r-H-ICP47 and r-Track were successfully constructed and successfully transfected into 293 cells and virus granules appeard. The virus particle titres of r-H-ICP47 and r-Track were determined with the resulting of 3.7×1010 efu/mL and 4.4×1010 efu/mL.Conclusion:The replication-defective recombinant adenoviruses of r-H-ICP47 and r-Track are constructed successfully. For the following studies of its immunological aetivities. the virus particle titres are highly enough to infect and express relevant genes in cells at a desired level, respectively.PartⅡImmunological activities of the recombinant adenovirus expressing His-tag-ICP47 fusion geneObjective:To investigate immunological activities of the recombinant adenovirus expressing His-tag-ICP47 fusion gene.Methods:1. HL-7702 cells were transfected with either r-H-ICP47 or r-Track, and efficiencies of transfection in HL-7702 with the recombinant adenoviruses at various multiplicity of infection (MOI) were analyzed. The cytotoxic activity of CTL was determined by using MTT assay.2. Dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMCs) derived from healthy donors in vitro and transfected with the recombinant adenoviruses at various MOI. Efficiencies of transfection and the ability to stimulate lymphocytes proliferation of DCs transfected by the recombinant adenoviruses were analyzed.3. Lymphocytes were transfected with either r-H-ICP47 or r-Track at various MOI, and efficiencies of transfection in lymphocytes with the recombinant adenoviruses were analyzed. Concentrations of Interleukin-2 (IL-2) and perforin (PF) were assayed in supernates of Two-way mixed lymphocyte reaction (Two-way MLR) by an ELISA procedure at the 2nd,4th and 6th day, respectively.Results:1. Recombinant adenovirus r-H-ICP47 and r-Track had efficiently and safely transferred genes into HL-7702 cells, and no cytotoxicity was detected. As MOI increased, efficiencies of transfection were increasing. Efficiencies of transfection with r-H-ICP47 and r-Track in HL-7702 at MOI of 100 (86.87±3.14% and 90.32±2.25%) were significantly higher than those at MOI of 50 (29.52±5.22% and 32.12±2.27%) (P<0.05), and there was no difference in the efficiencies of transfection between the groups of MOI 100 and MOI 200 (88.53±3.69% and 90.64±3.65%) (P>0.05).After HL-7702 cells were transfected with either r-H-ICP47 or r-Track at MOI of 100, the cytotoxic activity of CTL was determined by using MTT assay. The results showed that percent cytolysis of HL-7702 cells transfected with r-H-ICP47 by CTL was reduced comparing with those of the r-track group and the control group (P<0.05).2. Recombinant adenovirus r-H-ICP47 and r-Track had efficiently and safely transferred genes into DCs, and as MOI increased, efficiencies of transfection were increasing. Efficiencies of transfection with r-H-ICP47 and r-Track at MOI of 100 (86.92±2.76% and 84.08±2.62%) were significantly higher than those at MOI of 50 (30.32±3.08% and 23.70±3.23%) (P<0.05), but there was no diffference in the efficiencies of transfection between the groups of MOI 100 and MOI 200 (91.26± 2.59% and 87.51±2.41%) (P>0.05).The results of MTT assay showed that the ability to stimulate lymphocytes proliferation of DCs transfected with r-H-ICP47 at MOI of 100 was decreased significantly in comparison with those of the r-Track group and the control group at the 48th h and 72th h (P<0.05), but no difference was found between the three groups at the 24th h (P>0.05).3. Recombinant adenovirus r-H-ICP47 and r-Track had efficiently and safely transferred genes into lymphocytes, and as MOI increased, efficiency of transfection was increasing. Efficiencies of transfection with r-H-ICP47 and r-Track at MOI of 100 (87.11±3.29% and 82.21±4.01%) were significantly higher than those at MOI of 50 (25.54±4.07% and 27.67±2.31%) (P<0.05), but there was no diffference in efficiencies of transfection between the groups of MOI 100 and MOI 200 (89.75±2.92 and 85.99±3.02%) (P>0.05).Experimental groups of Two-way MLR were divided into 5 groups, the D-R-ICP47 group, the D-ICP47 group, the D-R-Track group, the D-Track group and the control group. After lymphocytes of donor (D) and recipient (R) were transfected with either r-H-ICP47 or r-Track at MOI of 100, the supernates of Two-way MLR were removed and concentrations of IL-2 and PF were assayed by an ELISA procedure at the 2nd,4th and 6th day, respectively. The results showed that concentrations of IL-2 and PF in the D-R-ICP47 group were significantly lower than those of the other groups (P<0.05). Similarly, concentrations of IL-2 and PF in the D-H-ICP47 group were also lower than those of the D-R-Track group, the D-Track group and the control group (P<0.05).Conclusion:1. Recombinant adenovirus expressing His-tag-ICP47 fusion gene can efficiently and safely transfer genes into the HL-7702 cells, DCs and lymphocytes, and the expression of introduced genes is at a desired level.2. Recombinant adenovirus r-H-ICP47 have the abilities of reducing the percent cytolysis by CTL, suppressoring lymphocyte populations, and significantly decreasing the concentrations of IL-2 and PF in supernates of Two-way MLR. To some extents, these results can predict an absence of deleterious host immune responses against the transplanted cells or gene products and indicate that recombinant adenovirus expressing His-tag-ICP47 fusion gene and transplanted cells can confer immune tolerance and lead to long-term cell survival in recipients.更多还原