表达His-tag-ICP47融合基因腺病毒载体的构建及其免疫活性的研究
|
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。
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 gene
Objective:
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 gene
Objective:
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.
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。
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 gene
Objective:
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 gene
Objective:
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.
引文
[1]H Ma, Y Liu, S Liu, et al. Oral adeno-associated virus-sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice [J]. Hepatology,2005,42 (6):1355-1363.
[2]Peter Johannes Hoist, Cathrine (?)rskov, Allan Randrup Thomsen, et al. Quality of the Transgene-Specific CD8+ T Cell Response Induced by Adenoviral Vector Immunization Is Critically Influenced by Virus Dose and Route of Vaccination [J]. J. Immunol.,2010,184 (8): 4431-4439.
[3]陆华中,薛京伦.克服腺病毒载体介导免疫应答的新策略：关闭淋巴细胞反应[J].生物工程进展,2000,20(3)：62-66.
[4]J Bagley, K Aboody-Guterman, X Breakefield, et al. Long-term expression of the gene encoding green fluorescent protein in murine hematopoietic cells using retroviral gene transfer [J]. Transplantation,1998,65 (9):1233-1240.
[5]陈宗祐.阻断信号传导对延长小鼠同种异体移植心脏存活时间的影响[D].[博士学位论文].上海：复旦大学,2006.
[6]刘海忠.阻断ICOS共刺激通路抗肝移植急性排斥的研究[D].[硕士学位论文].重庆：重庆医科大学,2005.
[7]Nathan J. Felix, W. June Brickey, Robert Griffiths, et al. H2-DMα-/- Mice Show the Importance of Major Histocompatibility Complex-bound Peptide in Cardiac Allograft Rejection [J]. J. Exp. Med.,2000.192(1):192:31-40
[8]范荣.反义CⅡTA基因转移抑制MHC-Ⅱ类分子表达的实验研究[D].[硕士学位论文].上海：第二军医大学,2002
[9]Martina Sester, Katja Koebernick, Douglas Owen, et al. Conserved Amino Acids within the Adenovirus 2 E3/19K Protein Differentially Affect Downregulation of MHC Class Ⅰ and MICA/B Proteins [J]. J. Immunol.,2010,184 (1):255-267.
[10]艾伦·P·埃舍尔,李逢春.预防与治疗自身免疫性疾病的物质[P].中国专利,CN200710097957.2.2007-10-24
[11]洪敏,李卫中,李琦涵.单纯疱疹病毒Ⅰ型免疫逃避机制-ICP47抑制抗原加工相关转运蛋白[J].病毒学报,2007,23(1)：72-75.
[12]Margit H. Lampen, Marieke C. Verweij, Bianca Querido, et al. CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population [J]. J. Immunol., 2010,185(11):6508-6517.
[13]Christopher Aisenbrey, Christina Sizun, Joachim Koch, et al. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery [J]. J. Biol. Chem.,2006,281 (41):30365-30372.
[14]许学文.重组腺病毒介导的反义MMP-2cDNA基因治疗人表皮癌的实验研究[D].[博士学位论文].成都：四川大学,2006.
[15]汪圣毅,王玉琪,符伟国,等.白细胞介素-10腺病毒载体制备及其在血管平滑肌细胞的表达[J].中国普外基础与临床杂志,2007,14(6)：660-664.
[16]JC Lorenzi, AP Trombone, CD Rocha, et al. Intranasal vaccination with messenger RNA as a new approach in gene therapy:use against tuberculosis [J]. BMC Biotechnol,2010,10(1):77.
[17]S Pascolo. Vaccination with messenger RNA (mRNA) [J]. Handb Exp Pharmacol,2008, 183(1):221-235.
[18]OM Tepper. BJ Mehrara. Gene therapy in plastic surgery [J]. Plast Reconstr Surg,2002,109 (2):716-734.
[19]Jeffrey M. Isner, Peter R. Vale, James F. Symes, et al. Assessment of Risks Associated With Cardiovascular Gene Therapy in Human Subject [J]. Circ. Res.,2001,89 (5):389-400.
[20]FY Lim, BG Martin, M Sena-Esteves, et al. Adeno-associated virus (AAV)-mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture [J]. J Pediatr Surg,2002,37 (7):1051-1057.
[21]Y Kojima, S Sasaki, Y Umemoto, et al. Effects of adenovirus mediated gene transfer to mouse testis in vivo on spermatogenesis and next generation [J]. J Urol,2003,170 (5):2109-2114.
[22]Sneha Sundaram, Ruchit Trivedi, Chandrasekar Durairaj, et al. Targeted Drug and Gene Delivery Systems for Lung Cancer Therapy [J]. Clin. Cancer Res.,2009,15(10):7299-7308.
[23]刘思景,郭伟韬,王辉.腺病毒载体的研究新进展[J].现代中西医结合杂志.2010,19(3)：1669-1670.
[24]ME Csete, KE Drazan, M van Bree, et al. Adenovirus-mediated gene transfer in the transplant setting. I. Conditions for expression of transferred genes in cold-preserved hepatocytes [J]. Transplantation,1994,57 (10):1502-1507.
[25]毛立军,郑骏年.辅助腺病毒载体及其介导的基因治疗研究进展[J].国际病毒性杂志,2006,13(5)：151-154.
[26]刘根梅,陈瑞爱,罗满林.腺病毒载体的研究进展[J].广东农业科学,2010,37(4)：188-190.
[27]A Benham, A Tulp, J Neefjes. Synthesis and assembly of MHC-peptide complexes [J]. Immunol Today,1995,16 (7):359-362.
[28]A Alcami, UH Koszinowski. Viral mechanisms of immune evasion [J]. Immunol Today,2000, 21 (9):447-455.
[29]Padma Billam, Kathryn L. Bonaparte, Jie Liu, et al. T Cell Receptor Clonotype Influences Epitope Hierarchy in the CD8+ T Cell Response to Respiratory Syncytial Virus Infection [J]. J. Biol. Chem.,2011,286 (6):4829-4841.
[30]Palanee Ammaranond, David J. van Bockel, Kathy Petoumenos, et al. HIV Immune Escape at an Immunodominant Epitope in HLA-B*27-Positive Individuals Predicts Viral Load Outcome [J]. J. Immunol.,2011,186(1):479-488.
[31]H Pircher, D Moskophidis, U Rohrer, et al. Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo [J]. Nature,1990,346 (6285):629-633.
[32]L Lybarger, X Wang, M Harris, et al. Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways [J]. Curr Opin Immunol, 2005,17(1):71-78.
[33]J Koch, R Tampe. The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation [J]. Cell Mol Life Sci,2006,63 (6):653-662.
[34]T Elliott, A Williams. The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex [J]. Immunol Rev,2005,207 (10):89-99.
[35]Maaike E. Ressing, Sinead E. Keating, Daphne van Leeuwen, et al. Impaired Transporter Associated with Antigen Processing-Dependent Peptide Transport during Productive EBV Infection [J]. J. Immunol.,2005,174 (11):6829-6838.
[36]Christian Brander, Todd Suscovich, Yun Lee, et al. Impaired CTL Recognition of Cells Latently Infected with Kaposi's Sarcoma-Associated Herpes Virus [J]. J. Immunol.,2000,165 (4):2077-2083.
[37]S Koba, F Tsunoda, T Hirano, et al. Postprandial changes in LDL phenotypes in patients with myocardial infarction [J]. Eur J Clin Invest,2005,35 (3):171-179.
[38]Danijela Koppers-Lalic, Eric A. J. Reits, Maaike E. Ressing, et al. Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing [J]. PNAS,2005,102 (14):5144-5149.
[39]N Nozawa, N Inoue. Mechanisms of congenital CMV infection [J]. Nippon Rinsho,2006,64 (Suppl 3):446-450
[40]Nupur T. Pande, Colin Powers, Kwangseog Ahn, et al. Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11 [J].J. Virol,2005,79 (9):5786-5798.
[41]郑秀峰,韩金祥.单纯疱疹病毒研究现状[J].中国麻风病杂志,2008,24(5)：370-373.
[42]吴建兵,郑家润.单纯疱疹病毒潜伏感染的分子基础[J].国际皮肤性病学杂志,2006,32(3)：194-196.
[43]M Hong, WZ Li, QH Li. The immune escape mechanism of herpes simplex virus type 1 suppression of transporter associated with antigen processing (TAP) by ICP47 [J]. Bing Du Xue Bao,2007,23(1):72-75.
[44]Liesbeth E. M. Oosten, Danijela Koppers-Lalic, Els Blokland, et al. TAP-inhibiting proteins US6, ICP47 and UL49.5 differentially affect minor and major histocompatibility antigen-specific recognition by cytotoxic T lymphocytes [J]. Int. Immunol.,2007,19 (9): 1115-1122.
[45]Christopher Aisenbrey, Christina Sizun, Joachim Koch, et al. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery [J]. J. Biol. Chem.,2006,281 (41):30365-30372.
[46]范凌云,谢庆军.腺病毒载体的研究进展[J].中国生物制品学杂志,2008,21(2)：153-157.
[47]X Danthinne, MJ Imperiale. Production of first generation adenovirus vectors:a review [J]. Gene Ther,2000,7 (20):1707-1714.
[48]RW Wilmott, RS Amin, CR Perez, et al. Safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lungs of nonhuman primates [J]. Hum Gene Ther,1996,7 (3):301-318.
[49]Robin J. Parks, Liane Chen, Martina Anton, et al. A helper-dependent adenovirus vector system:Removal of helper virus by Cre-mediated excision of the viral packaging signa [J]. PNAS,1996,93 (24):13565-13570.
[50]范明齐,冯嘉瑜,黄赤兵,等.人ICOS胞外区腺病毒载体的构建与鉴定[J].重庆医学,2006,35(16)：1460-1462.
[51]金天明,武迎红.腺病毒及腺病毒载体的研究进展[J].天津农学院学报,2007,14(2)：48-51.
[52]方针强,叶钢,欧阳一辛,等.人BMP-7基因重组腺病毒的构建与鉴定[J].第三军医大学学报,2008,30(2)：153-156.
[53]JA St George. Gene therapy progress and prospects:adenoviral vectors [J]. Gene Ther, 2003,10(14):1135-1141.
[54]M Zeng, SK Smith, F Siegel, et al. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination [J]. Biotechniques,2001,31 (2): 260-262.
[55]郭浩,彭毅志,辛海明.小鼠CCR7基因重组腺病毒的构建与鉴定[J].现代生物医学进展,2007,7(11)：1643-1645.
[56]徐竹蔚,刘莹,户义.抗3种标签蛋白单克隆抗体的制备及特性鉴定[J].细胞与分子免疫学杂志,2005,21(5)：613-614.
[57]M Hiraoka, N Nitta, M Nagai, et al. MCP-1-induced enhancement of THP-1 adhesion to vascular endothelium was modulated by HMG-CoA reductase inhibitor through RhoA GTPase-, but not ERK1/2-dependent pathway [J]. Life Sci,2004,75 (11):1333-1341.
[58]闵玉涛,王云龙,李晨阳,等.抗FLAG标签单克隆抗体的制备、鉴定及初步应用[J].中国生物工程杂志,2006,27(1)：93-97
[59]王文礼,王云龙,李晨阳,等.His标签单克隆抗体的制备、鉴定及初步应用[J].细胞与分子免疫学杂志,2008,24(4)：399-400.
[60]崔正军.腺病毒载体介导的反义VEGF基因治疗人皮肤恶性黑色素瘤的实验研究[D].[博士学位论文].成都：四川大学,2004.
[1]Jessamyn Bagley, Chaorui Tian, David H. Sachs, et al. Induction of T-cell tolerance to an MHC class Ⅰ alloantigen by gene therapy [J]. Blood,2002,99 (12):4394-4399.
[2]郭峰,卢永刚,何建平,肝细胞移植研究进展[J].中国普通外科杂志,2007,16(8)：803-805
[3]刘世呈,李靖.肝细胞移植的研究进展[J].中国普外基础与临床杂志,2008,15(2)：109-111.
[4]S Murasawa, T Asahara. Gene modified cell transplantation for vascular regeneration [J]. Curr Gene Ther,2007,7(1):1-6.
[5]Martina Sester, Katja Koebernick, Douglas Owen, et al. Conserved Amino Acids within the Adenovirus 2 E3/19K Protein Differentially Affect Downregulation of MHC Class Ⅰ and MICA/B Proteins [J]. J. Immunol.,2010,184 (1):255-267.
[6]艾伦·P·埃舍尔,李逢春.预防与治疗自身免疫性疾病的物质[P].中国专利,CN200710097957.2.2007-10-24
[7]洪敏,李卫中,李琦涵.单纯疱疹病毒Ⅰ型免疫逃避机制-ICP47抑制抗原加工相关转运蛋白[J].病毒学报,2007,23(1)：72-75.
[8]Margit H. Lampen, Marieke C. Verweij, Bianca Querido, et al. CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population [J]. J. Immunol., 2010,185 (11):6508-6517.
[9]Christopher Aisenbrey, Christina Sizun, Joachim Koch, et al. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery [J]. J. Biol. Chem.,2006,281 (41):30365-30372.
[10]孙祖玥,赵勇.基因工程在克服移植免疫排斥反应及诱导免疫耐受中的应用[J].中国生物工程杂志,2005,25(1)：6-9.
[11]陈宗祐.阻断信号传导对延长小鼠同种异体移植心脏存活时间的影响[D].[博士学位论文].上海：复口.大学,2006
[12]刘海忠.阻断ICOS共刺激通路抗肝移植急性排斥的研究[D].[硕士学位论文].重庆：重庆医科大学,2005.
[13]EM Mazaris, CT Roussos, VE Papalois. Hepatocyte transplantation:a review of worldwide clinical developments and experiences [J]. Exp Clin Transplant,2005,3 (1):306-315.
[14]T Kawahara, H Yagita, K Okumura, et al. T cell-mediated effector mechanisms in the rejection of allogeneically transplanted hepatocytes [J]. Transplant Proc,1999,31 (2): 830-831.
[15]王绯,杨永平.肝细胞移植基础与临床应用研究进展[J].肝脏,2007,12(2)：143-144.
[16]崔正军.腺病毒载体介导的反义VEGF基因治疗人皮肤恶性黑色素瘤的实验研究[D]. [博士学位论文].成都：四川大学,2004.
[17]Wang Y, Huang S, Sah VP, et al. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family [J]. J Biol Chem.1998, 273 (4):2161-2168.
[18]张文敏,陈裕庆,张萌,等.NY-ESO-1致敏树突状细胞诱导的CTL对肝癌细胞株的特异性杀伤作用[J].中国肿瘤生物治疗杂志,2009,16(1)：18-23.
[19]S Hao, O Bai, J Yuan, et al. Dendritic cell-derived exosomes stimulate stronger CD8+CTL responses and antitumor immunity than tumor cell-derived exosomes [J]. Cell Mol Immunol, 2006,3 (3):205-211.
[20]杨静悦,曹大勇,刘文超,等.IL-18基因增强肿瘤抗原致敏DC诱导的CTL特异诱导杀伤肝癌细胞[J].中国肿瘤生物治疗杂志,2009,16(1)：55-58
[21]付必莽.IL-2、TNF-β在混合淋巴细胞培养中的表达与肝移植急排反应的关系[D].[硕士学位论文].昆明：昆明医学院,2005.
[22]B Lindton, L Markling, O Ringden, et al. Mixed lymphocyte culture of human fetal liver cells [J]. Fetal Diagn Ther,2000,15 (2):71-78.
[23]JE Visentainer, SR Lieber, LB Persoli, et al. Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation [J]. Braz J Med Biol Res,2002,35 (5):567-572.
[24]黄建钊,夏穗生.对肝移植免疫耐受机理研究的思索[J].中国普外基础与临床杂志,2000,4(7)：261-262.
[25]刘利,张玉海.混合淋巴细胞培养上清液中细胞因子水平与移植肝急性排斥反应的联系[J].肝脏病与透析肝移植杂志,2000,1(9)：43-47.
[26]谢晋,李宁丽,陈福祥,等.异种淋巴细胞混合培养时T淋巴细胞表达细胞因子的格局[J].中华器官移植杂志,2001,3(2)：172-173.
[27]周光炎.免疫学原理.第一版[M].上海：上海科学技术文献出版社,2000,300-304.
[28]张儒有,郑永同,胡韶山,等.神经干细胞移植治疗脑卒中后遗症50例临床效果分析[J].中国临床康复,2006,10(9)：138-139.
[29]CB Johansson, S Momma, DL Clarke, et al. Identification of a neural stem cell in the adult mammalian central nervous system [J]. Cell,1999,96 (1):25-34.
[30]Y Li, M Chopp. Temporal profile of nestin expression after focal cerebral ischemia in adult rat [J]. Brain Res,1999,838 (2):1-10.
[31]王宁生,雷燕,刘平,等.关于血清药理学的若于思考[J].中国中西医结合杂志,1999,19(5)：263-266.
[32]赵军,沈振亚,徐五音,等.血清IL-2水平在大鼠心脏移植术后早期急性排斥反应诊断中的意义[J].苏州医学院学报,1999,19(9)：973-975.
[33]OM Martinez, JC Villanueva, J Lake, et al. IL-2 and IL-5 gene expression in response to alloantigen in liver allograft recipients and in vitro [J]. Transplantation,1993,55 (5): 1159-1166.
[34]H Egawa, OM Martinez, MB Quinn, et al. Acute liver allograft rejection in the rat. An analysis of the immune response [J]. Transplantation,1995,59 (1):97-102.
[35]MH Andersen, D Schrama, P Thor Straten, et al. Cytotoxic T cells [J]. J Invest Dermatol, 2006,126(1):32-41.
[36]朱鹏,陈义发,李丹,等.小鼠心脏移植后穿孔素及颗粒酶B表达水平与急性排斥反应的关系[J].世界华人消化杂志,2007,15(31)：3289-3293
[37]M Yannaraki, JM Rebibou, D Ducloux, et al. Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection [J]. Transpl Int,2006,19 (9): 759-768.
[38]邓薇薇,雷建平.穿孔素的研究进展[J].实用临床医学,2008,9(2)：128-129.
[39]Angela Kuhla, Christian Eipel, Kerstin Abshagen, et al. Role of the perforin/granzyme cell death pathway in D-Gal/LPS-induced inflammatory liver injury [J]. Am J Physiol Gastrointest Liver Physiol,2009,296 (5):G1069-G1076.
[40]Mark D McKenzie, Nadine L Dudek, Lina Mariana, et al. Perforin and Fas induced by IFN-γ and TNF-α mediate beta cell death by OT-I CTL [J]. Int. Immunol.,2006,18(6):837-846.
[1]A Alcami, UH Koszinowski, Viral mechanisms of immune evasion [J]. Immunol Today, 2000,21 (9):447-55.
[2]Eva Szomolanyi-Tsuda, Xueya Liang, Raymond M. Welsh, et al. Role for TLR2 in NK Cell-Mediated Control of Murine Cytomegalovirus In Vivo [J]. J. Virol.,2006,80 (9):4286-4291.
[3]Elizabeth K. Day, Andrew J. Carmichael, Ineke J. M. ten Berge, et al. Rapid CD8+ T Cell Repertoire Focusing and Selection of High-Affinity Clones into Memory Following Primary Infection with a Persistent Human Virus:Human Cytomegalovirus [J]. J. Immunol.,2007, 179 (5):3203-3213.
[4]JA Hamerman, K Ogasawara, LL Lanier. NK cells in innate immunity [J]. Curr Opin Immunol, 2005,17(1):29-35.
[5]SH Robbins, G Bessou, A Cornillon, et al. Natural killer cells promote early CD8 T cell responses against cytomegalovirus [J]. PLoS Pathog,2007,3 (8):e123.
[6]Yu Mori, Sukenao Tsuji, et al. Inhibitory Immunoglobulin-Like Receptors LILRB and PIR-B Negatively Regulate Osteoclast Development [J]. J. Immunol.,2008,181(7):4742-4751.
[7]Michael A. Caligiuri. Human natural killer cells [J]. Blood,2008,112(3):461-469.
[8]孙蕾,常雅萍.病毒逃逸免疫细胞杀伤机制[J].国外医学免疫学分册,2002,25(5)：265-269.
[9]M Vales-Gomez, H Browne, HT Reyburn. Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells [J]. BMC Immunol,2003; 4(1):4.
[10]Alexandre Iannello, Olfa Debbeche, Elodie Martin, et al. Viral strategies for evading antiviral cellular immune responses of the host [J]. J. Leukoc. Biol.,2006,79 (1):16-35.
[11]M Kubin, L Cassiano, J Chalupny, et al. ULBP1,2,3:novel MHC class Ⅰ-related molecules that bind to human cytomegalovirus glycoprotein UL16, activate NK cells [J]. Eur J Immunol, 2001,31(5):1428-1437.
[12]D Cosman, J Mullberg, CL Sutherland, et al. ULBPs, novel MHC class Ⅰ-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor [J]. Immunity,2001,14 (2):123-133.
[13]Alessandro Poggi, Roberta Carosio, Grazia Maria Spaggiari. NK Cell Activation by Dendritic Cells Is Dependent on LFA-1-Mediated Induction of Calcium-Calmodulin Kinase Ⅱ:Inhibition by HIV-1 Tat C-Terminal Domain [J]. J. Immunol,2002,168 (1):95-101.
[14]程玉兴,胡云章.病毒免疫逃逸机制研究进展[J].国外医学病毒学分册,2005,12(5)：150-154.
[15]Collin R. Brooks, Tim Elliott, Peter Parham, et al. The Inhibitory Receptor NKG2A Determines Lysis of Vaccinia Virus-Infected Autologous Targets by NK Cells [J]. J. Immunol.,2006,176(2):1141-1147.
[16]Peter Tomasec, Veronique M. Braud, Carole Rickards, et al. Surface Expression of HLA-E, an Inhibitor of Natural Killer Cells, Enhanced by Human Cytomegalovirus gpUL40 [J]. Science,2000,287 (5455):1031.
[17]N Pizzato, B Garmy-Susini, P Le Bouteiller, et al. Differential down-modulation of HLA-G and HLA-A2 or-A3 cell surface expression following human cytomegalovirus infection [J]. J Reprod Immunol,2004,62 (1-2):3-15.
[18]JA Hamerman, K Ogasawara, LL Lanier. NK cells in innate immunity [J]. Curr Opin Immunol,2005.17(1):29-35.
[19]Katherine Belov, Claire E. Sanderson, Janine E. Deakin, et al. Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system [J].Genome Res.,2007,17 (7):982-991.
[20]赵朴,郑玉姝,刘兴友.病毒干扰MHCⅠ类抗原呈递策略的研究进展[J].生命科学,2008,20(2)：300-303.
[21]Karen A. O'Connell, Yefei Han, Thomas M, et al. Role of Natural Killer Cells in a Cohort of Elite Suppressors:Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type Ⅰ Replication In Vitro [J]. J. Virol.,2009,83 (10): 5028-5034.
[22]M Lopez-Botet, M Llano, M Ortega.Human cytomegalovirus and natural killer-mediated surveillance of HLA class Ⅰ expression:a paradigm of host-pathogen adaptation [J]. Immunol Rev,2001,181(12):193-202.
[23]W. L. William Chang, Nicole Baumgarth, Dong Yu. et al. Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality [J]. J. Virol.,2004,78 (16):8720-8731.
[24]C Cerboni, A Achour, A Warnmark, et al. Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR-1/ILT2/CD85j [J].Eur J Immunol,2006; 36(3):732-41.
[25]Elena G. Chiorean, Scott J. Dylla, Krista Olsen, et al. BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIRs) [J]. Blood,2003,101 (9):3527-3533.
[26]DM Andrews, CE Andoniou. F Granucci, et al. Infection of dendritic cells by murine cytomegalovirus induces functional paralysis [J]. Nat Immunol,2001,2(11):1077-84.
[27]Marie Mandron, Helene Martin, Beatrice Bonjean, et al. Dendritic cell-induced apoptosis of human cytomegalovirus-infected fibroblasts promotes cross-presentation of pp65 to CD8+ T cells [J]. J. Gen. Virol.,2008,89(1):78-86.
[28]赵宏,马伟杭.人巨细胞病毒感染中的细胞免疫机制[J].国际流行病学传染病学杂志[J].2008,35(3)：182-184.
[29]Sara Gredmark, Tamara Tilburgs, and Cecilia Soderberg-Naucler. Human Cytomegalovirus Inhibits Cytokine-Induced Macrophage Differentiation [J].J. Virol.,2004,78 (19):10378-10389.
[30]Franziska Schwartzkopff, Tobias A. Grimm, Carla S.R. Lankford, et al. Platelet factor 4 (CXCL4) facilitates human macrophage infection with HIV-1 and potentiates virus replication [J]. Innate Immunity,2009,15 (6):368-379.
[31]R Hewer, D Meyer. Peptide immunogens designed to enhance immune responses against human immunodeficiency virus (HIV) mutant strains:a plausible means of preventing viral persistence [J]. J Theor Biol,2005,233 (1):85-90.
[32]M. Kathryn Liszewski, Marilyn K. Leung, Richard Hauhart, et al. Structure and Regulatory Profile of the Monkeypox Inhibitor of Complement:Comparison to Homologs in Vaccinia and Variola and Evidence for Dimer Formation [J]. J. Immunol.,2006,176 (6):3725-3734.
[33]欧元祝,瞿涤.病毒逃逸补体的机制[J].微生物与感染,2006,1(2)：114-117.
[34]O. Brad Spiller, David J. Blackbourn, Linda Mark, et al. Functional Activity of the Complement Regulator Encoded by Kaposi's Sarcoma-associated Herpesvirus [J]. J. Biol. Chem.,2003,278 (11):9283-9289.
[35]J Bernet, J Mullick, AK Singh, et al. Viral mimicry of the complement system [J]. J Biosci, 2003,28 (3):249-264.
[36]Herman W. Favoreel, Gerlinde R. Van de Walle, Hans J. Nauwynck, et al. Virus complement evasion strategies [J].J. Gen. Virol.,2003,84 (1):1-15.
[37]沈小燕,丛国正,常惠芸,等.病毒感染与逃避免疫反应的研究进展[J].医学分子生物学杂杂志,2005,2(4)：284-287.
[38]C Speth, H Stoiber, MP Dierich. Complement in different stages of HIV infection and pathogenesis [J]. Int Arch Allergy Immunol,2003,130 (4):247-257.
[39]Patricia Devaux, Dale Christiansen, Sebastien Plumet, et al. Cell surface activation of the alternative complement pathway by the fusion protein of measles virus [J]. J. Gen. Virol., 2004,85(6):1665-1673.
[40]Mauricio Urquiza, Ramses Lopez, Helena Patino, et al. Identification of Three gp350/220 Regions Involved in Epstein-Barr Virus Invasion of Host Cells [J]. J. Biol. Chem.,2005,280 (42):35598-35605.
[41]DT Haydon, AR Samuel, NJ Knowles. The generation and persistence of genetic variation in foot-and-mouth disease virus [J]. Prev Vet Med,2001,51 (1-2):111-24.
[42]JP Moorman, M Joo, YS Hahn. Evasion of host immune surveillance by hepatitis C virus: potential roles in viral persistence [J]. Arch Immunol Ther Exp (Warsz),2001,49 (3): 189-194.
[43]Christine M. Posavad, Meei Li Huang, Serge Barcy,. Long Term Persistence of Herpes Simplex Virus-Specific CD8+ CTL in Persons with Frequently Recurring Genital Herpes [J]. J. Immunol.,2000,165(2):1146-1152.
[44]E Maidji, S McDonagh, O Genbacev, et al. Maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal Fc receptor-mediated transcytosis [J]. Am. J. Pathol.,2006,168 (4):1210-26.
[45]John M. Lubinski, Ming Jiang, Lauren Hook, et al. Herpes Simplex Virus Type 1 Evades the Effects of Antibody and Complement In Vivo [J]. J. Virol.,2002; 76 (18):9232-9241.
[46]Michael Basler, Maya Dajee, Carlo Moll, et al. Prevention of Experimental Colitis by a Selective Inhibitor of the Immunoproteasome [J]. J. Immunol.,2010,185 (10):634-641.
[47]Paul Schmid-Hempel. Immune defence, parasite evasion strategies and their relevance for 'macroscopic phenomena' such as virulence [J]. Phil Trans R Soc B,2009,364 (1513):85-98.
[48]Yili Yin, Benedicte Manoury, Robin Fahraeus, et al. Self-Inhibition of Synthesis and Antigen Presentation by Epstein-Barr Virus-Encoded EBNA1 [J]. Science,2003,301 (5638):1371.
[49]Christopher T. Cornell, William B. Kiosses, Stephanie Harkins, et al. Coxsackievirus B3 Proteins Directionally Complement Each Other To Downregulate Surface Major Histocompatibility Complex Class Ⅰ [J]. J. Virol.,2007,81 (13):6785-6797.
[50]NL Glosson, AW Hudson. Human herpesvirus-6A and- 6B encode viral immunoevasins that downregulate class Ⅰ MHC molecules [J]. Virology,2007,365 (1):125-135.
[51]Lydie Trautmann, Marie Rimbert, Klara Echasserieau, et al. Selection of T Cell Clones Expressing High-Affinity Public TCRs within Human Cytomegalovirus-Specific CD8 T Cell Responses [J]. J. Immunol.,2005,175 (9):6123-6132.
[52]J Koch, R Tampe.The macromolecular peptide-loading complex in MHC class Ⅰ-dependent antigen presentation [J]. Cell Mol Life Sci,2006,63 (6):653-662.
[53]洪敏,李卫中,李琦涵.单纯疱疹病毒Ⅰ型免疫逃避机制-ICP47抑制抗原加工相关转运蛋白[J].病毒学报,2007,23(1)：72-75.
[54]Maaike E. Ressing, Sinead E. Keating, Daphne van Leeuwen, et al. Impaired Transporter Associated with Antigen Processing-Dependent Peptide Transport during Productive EBV Infection [J]. J. Immunol.,2005,174 (11):6829-6838.
[55]Christian Brander, Todd Suscovich, Yun Lee, et al. Impaired CTL Recognition of Cells Latently Infected with Kaposi's Sarcoma-Associated Herpes Virus [J]. J. Immunol.,2000, 165(4):2077-2083.
[56]S Koba, F Tsunoda, T Hirano, et al. Postprandial changes in LDL phenotypes in patients with myocardial infarction [J]. Eur J Clin Invest,2005,35 (3):171-179.
[57]Danijela Koppers-Lalic, Eric A. J. Reits, Maaike E. Ressing, et al. Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing [J]. PNAS,2005,102 (14):5144-5149.
[58]N Nozawa, N Inoue. Mechanisms of congenital CMV infection [J]. Nippon Rinsho,2006,64 (Suppl 3):446-50
[59]Nupur T. Pande, Colin Powers, Kwangseog Ahn, et al. Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11 [J]. J. Virol.,2005,79 (9):5786-5798.
[60]MJ Bevan, et al. Antigen presentation to cytotoxic T lymphocytes in vivo [J]. J. Exp. Med., 1995,182(1):639.
[61]Naeem Khan, Rachel Bruton, Graham S. et al. Identification of Cytomegalovirus-Specific Cytotoxic T Lymphocytes In Vitro Is Greatly Enhanced by the Use of Recombinant Virus Lacking the US2 to US11 Region or Modified Vaccinia Virus Ankara Expressing Individual Viral Genes [J] J. Virol.,2005,79 (5):2869-2879.
[62]Dimitrios Lagos, Matthew W. B. Trotter, Richard J. Vart, et al. Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells [J]. Blood,2007,109 (4):1550-1558.
[63]GH Ashrafi, MR Haghshenas, B Marchetti, et al. E5 protein of human papillomavirus type 16 selectively downregulates surface HLA class Ⅰ [J]. Int J Cancer,2005,113 (2):276-283.
[64]Shihe Hou, Hancheng Guan, Robert P. Ricciardi. In Adenovirus Type 12 Tumorigenic Cells, Major Histocompatibility Complex Class Ⅰ Transcription Shutoff Is Overcome by Induction of NF-κB and Relief of COUP-TFII Repression [J]. J. Virol.,2002,76 (7):3212-3220.
[65]MT Barel, GC Hassink, S van Voorden, et al. Human cytomegalovirus-encoded US2 and US11 target unassembled MHC class Ⅰ heavy chains for degradation [J]. Mol Immunol,2006, 43(8):1258-1266.
[66]Xiaoli Wang, Yihong Ye, Wayne Lencer, et al. The Viral E3 Ubiquitin Ligase mK3 Uses the Derlin/p97 Endoplasmic Reticulum-associated Degradation Pathway to Mediate Down regulation of Major Histocompatibility Complex Class Ⅰ Proteins [J]. J. Biol. Chem.,2006, 281 (13):8636-8644.
[67]Vanessa M. Noriega,Domenico Tortorella. Human Cytomegalovirus-Encoded Immune Modulators Partner To Downregulate Major Histocompatibility Complex Class Ⅰ Molecules [J]. J Virol.2009,83 (3):1359-1367.
[68]Scott G. Hansen, Colin J. Powers, Rebecca Richards, et al. Evasion of CD8+ T Cells Is Critical for Superinfection by Cytomegalovirus [J]. Science,2010,328 (5974):102-106.
[69]Zheng W. Chen, Abie Craiu, Ling Shen, et al. Simian Immunodeficiency Virus Evades a Dominant Epitope-Specific Cytotoxic T Lymphocyte Response Through a Mutation Resulting in the Accelerated Dissociation of Viral Peptide and MHC Class Ⅰ [J]. J. Immunol., 2000,164 (12):6474-6479.
[70]Laurent Coscoy, David Jesse Sanchez, Don Ganem. A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition [J]. J. Cell Biol.,2001,155 (7):1265.
[71]Yves Chalandon, Sylvie Degermann, Jean Villard, et al. Pretransplantation CMV-specific T cells protect recipients of T-cell-depleted grafts against CMV-related complications [J]. Blood,2006,107(1):389-396.
[72]Yujuan Yue, Amitinder Kaur, Meghan K. Eberhardt, et al. Immunogenicity and Protective Efficacy of DNA Vaccines Expressing Rhesus Cytomegalovirus Glycoprotein B, Phosphoprotein 65-2, and Viral Interleukin-10 in Rhesus Macaques [J]. J. Virol.,2007,81 (3): 1095-1109.
[73]Z Wang, C La Rosa, Z Li, et al. Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1.Vaccine [J],2007,25 (6):1132-1141.
[74]Graeme E. Price, Rong Ou, Hong Jiang, et al. Viral Escape by Selection of Cytotoxic T Cell-Resistant Variants in Influenza a Virus Pneumonia [J]. J. Exp. Med.,2000,191 (11): 1853-1868.
[75]Massimo Maffei, Fabio Ghiotto, Marzia Occhino, et al. Human Cytomegalovirus Regulates Surface Expression of the Viral Protein UL18 by Means of Two Motifs Present in the Cytoplasmic Tail [J]. J. Immunol.,2008,180 (2):969-979.
[76]Susan McDonagh, Ekaterina Maidji, Wenge Ma, et al. Viral and bacterial pathogens at the maternal-fetal interface.The Journal of Infectious Disease [J],2004,190 (4):826-834
[77]Todd A. Fehniger, Megan A. Cooper, Gerard J. Nuovo, et al. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2:a potential new link between adaptive and innate immunity [J]. Blood,2003,101 (8):3052-3057.
[78]DM Andrews, AA Scalzo, WM Yokoyama, et al. Functional interactions between dendritic cells and NK cells during viral infection [J]. Nat Immunol,2003,4 (2):175-181.
[79]Fan Xiu Zhu, Sonya M. King, Eric J. Smith, et al. A Kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type Ⅰ interferon by blocking IRF-7 phosphorylation and nuclear accumulation [J]. PNAS,2002,99 (8):5573-5578.
[80]A Arena, D Iannello, D Gazzara. et al. Role of interleukin-18 in peripheral blood mononuclear cells infected with human herpes virus type 6 [J]. Intervirology,2001,44 (4): 250-254.
[81]Ikka Helantera, Raisa Loginov, Petri Koskinen, et al. Persistent cytomegalovirus infection is associated with increased expression of TGF-β1, PDGF-AA and ICAM-1 and arterial intimal thickening in kidney allografts.Nephrol [J]. Dial. Transplant.,2005,20 (4):790-796.
[82]Selina Khan, Albert Zimmermann, Michael Basler, et al. A Cytomegalovirus Inhibitor of Gamma Interferon Signaling Controls Immunoproteasome Induction [J]. J. Virol.,2004,78 (4):1831-1842.
[83]Martin Savard, Carole Belanger, Michel J. Tremblay, et al. EBV Suppresses Prostaglandin E2 Biosynthesis in Human Monocytes [J]. J. Immunol.,2000,164 (12):6467-6473.
[84]Martin J. Raftery, Dorte Wieland, Stefanie Gronewald, Shaping Phenotype, Function, and Survival of Dendritic Cells by Cytomegalovirus-Encoded IL-10 [J]. J. Immunol.,2004,173 (5):3383-3391.
[85]Christina Jenkins, Allison Abendroth, Barry Slobedman. A Novel Viral Transcript with Homology to Human Interleukin-10 Is Expressed during Latent Human Cytomegalovirus Infection [J]. J. Virol.,2004,78 (3):1440-1447.
[86]Juliet V. Spencer, Kristen M. Lockridge, Peter A. Barry, et al. Potent Immunosuppressive Activities of Cytomegalovirus- Encoded Interleukin-10 [J]. J. Virol.,2002,76 (3):1285-1292.
[87]Christina Jenkins, Allison Abendroth, Barry Slobedman. A Novel Viral Transcript with Homology to Human Interleukin-10 Is Expressed during Latent Human Cytomegalovirus Infection [J]. J. Virol.,2004,78 (3):1440-1447.
[88]TE Sparer, J Gosling, TJ Schall, et al. Expression of human CXCR2 in murine neutrophils as a model for assessing cytomegalovirus chemokine vCXCL-1 function in vivo [J]. J Interferon Cytokine Res,2004,24 (10):611-620.
[89]K Iwatsuki, T Yamamoto, K Tsuji, et al. A spectrum of clinical manifestations caused by host immune responses against Epstein-Barr virus infections [J]. Acta Med Okayama,2004, 58(4):169-180.
[90]Rhiannon N. Silk, Gavin C. Bowick, Charles C. Abrams, et al. African swine fever virus A238L inhibitor of NF-KB and of calcineurin phosphatase is imported actively into the nucleus and exported by a CRM1-mediated pathway [J]. J Gen Virol,2007,88 (2):411-419.
[91]BT Seet, JB Johnston, CR Brunetti, et al. Poxviruses and immune evasion [J]. Annu Rev Immunol,2003,21(5):377-423.
[92]NL Nakhoul and LL Hamm. Non-erythroid Rh glycoproteins:a putative new family of mammalian ammonium transporters [J]. Pflugers Arch,2004,447 (5):807-812.
[93]Robert Y. Suruki,Nancy Mueller,K.atsuhiro Hayashi,et al. Host Immune Status and Incidence of Hepatocellular Carcinoma among Subjects Infected with Hepatitis C Virus:A Nested Case-Control Study in Japan [J]. Cancer Epidemiol. Biomarkers Prev,2006,15 (12): 2521-2525.
[94]Sebastian Voigt, Gordon R. Sandford, Gary S. Hayward, et al. The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region:further evidence for a separate evolutionary lineage from that of rat CMV Maastricht [J]. J Gen Virol.,2005,86(2):263-274.
[95]张欣文,韩蕾,李芬.母胎界面人巨细胞病毒免疫逃逸机制研究进展[J].中国妇幼健康研究,2006,17(4)：302-304.
[96]CE Andoniou, MA Degli-Esposti. Insights into the mechanisms of CMV-mediated interference with cellular apoptosis [J]. Immunol Cell Biol,2006,84 (1):99-106.
[97]Dustin T. Petrik, Kimberly P. Schmitt, Mark F. Stinski. Inhibition of Cellular DNA Synthesis by the Human Cytomegalovirus IE86 Protein Is Necessary for Efficient Virus Replication [J]. J. Virol.,2006,80 (8):3872-3883.
[98]M Irmler, M Thome, M Hahne, et al. Inhibition of death receptor signals by cellular FLIP [J]. Nature,1997,388(6638):190-195.
[99]Joseph DeMasi, Michael C. Chao, Ashu S. Kumar, et al. Bovine Papillomavirus E7 Oncoprotein Inhibits Anoikis [J]. J.Virol.,2007,81 (17):9419-9425.
[100]Eli Boritz, Brent E. Palmer, Cara C. Wilson. Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD4+T Cells That Proliferate In Vitro Detected in Samples from Most Viremic Subjects and Inversely Associated with Plasma HIV-1 Levels [J]. J.Virol.,2004,78 (22):12638-12646
[2]Peter Johannes Hoist, Cathrine (?)rskov, Allan Randrup Thomsen, et al. Quality of the Transgene-Specific CD8+ T Cell Response Induced by Adenoviral Vector Immunization Is Critically Influenced by Virus Dose and Route of Vaccination [J]. J. Immunol.,2010,184 (8): 4431-4439.
[3]陆华中,薛京伦.克服腺病毒载体介导免疫应答的新策略：关闭淋巴细胞反应[J].生物工程进展,2000,20(3)：62-66.
[4]J Bagley, K Aboody-Guterman, X Breakefield, et al. Long-term expression of the gene encoding green fluorescent protein in murine hematopoietic cells using retroviral gene transfer [J]. Transplantation,1998,65 (9):1233-1240.
[5]陈宗祐.阻断信号传导对延长小鼠同种异体移植心脏存活时间的影响[D].[博士学位论文].上海：复旦大学,2006.
[6]刘海忠.阻断ICOS共刺激通路抗肝移植急性排斥的研究[D].[硕士学位论文].重庆：重庆医科大学,2005.
[7]Nathan J. Felix, W. June Brickey, Robert Griffiths, et al. H2-DMα-/- Mice Show the Importance of Major Histocompatibility Complex-bound Peptide in Cardiac Allograft Rejection [J]. J. Exp. Med.,2000.192(1):192:31-40
[8]范荣.反义CⅡTA基因转移抑制MHC-Ⅱ类分子表达的实验研究[D].[硕士学位论文].上海：第二军医大学,2002
[9]Martina Sester, Katja Koebernick, Douglas Owen, et al. Conserved Amino Acids within the Adenovirus 2 E3/19K Protein Differentially Affect Downregulation of MHC Class Ⅰ and MICA/B Proteins [J]. J. Immunol.,2010,184 (1):255-267.
[10]艾伦·P·埃舍尔,李逢春.预防与治疗自身免疫性疾病的物质[P].中国专利,CN200710097957.2.2007-10-24
[11]洪敏,李卫中,李琦涵.单纯疱疹病毒Ⅰ型免疫逃避机制-ICP47抑制抗原加工相关转运蛋白[J].病毒学报,2007,23(1)：72-75.
[12]Margit H. Lampen, Marieke C. Verweij, Bianca Querido, et al. CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population [J]. J. Immunol., 2010,185(11):6508-6517.
[13]Christopher Aisenbrey, Christina Sizun, Joachim Koch, et al. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery [J]. J. Biol. Chem.,2006,281 (41):30365-30372.
[14]许学文.重组腺病毒介导的反义MMP-2cDNA基因治疗人表皮癌的实验研究[D].[博士学位论文].成都：四川大学,2006.
[15]汪圣毅,王玉琪,符伟国,等.白细胞介素-10腺病毒载体制备及其在血管平滑肌细胞的表达[J].中国普外基础与临床杂志,2007,14(6)：660-664.
[16]JC Lorenzi, AP Trombone, CD Rocha, et al. Intranasal vaccination with messenger RNA as a new approach in gene therapy:use against tuberculosis [J]. BMC Biotechnol,2010,10(1):77.
[17]S Pascolo. Vaccination with messenger RNA (mRNA) [J]. Handb Exp Pharmacol,2008, 183(1):221-235.
[18]OM Tepper. BJ Mehrara. Gene therapy in plastic surgery [J]. Plast Reconstr Surg,2002,109 (2):716-734.
[19]Jeffrey M. Isner, Peter R. Vale, James F. Symes, et al. Assessment of Risks Associated With Cardiovascular Gene Therapy in Human Subject [J]. Circ. Res.,2001,89 (5):389-400.
[20]FY Lim, BG Martin, M Sena-Esteves, et al. Adeno-associated virus (AAV)-mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture [J]. J Pediatr Surg,2002,37 (7):1051-1057.
[21]Y Kojima, S Sasaki, Y Umemoto, et al. Effects of adenovirus mediated gene transfer to mouse testis in vivo on spermatogenesis and next generation [J]. J Urol,2003,170 (5):2109-2114.
[22]Sneha Sundaram, Ruchit Trivedi, Chandrasekar Durairaj, et al. Targeted Drug and Gene Delivery Systems for Lung Cancer Therapy [J]. Clin. Cancer Res.,2009,15(10):7299-7308.
[23]刘思景,郭伟韬,王辉.腺病毒载体的研究新进展[J].现代中西医结合杂志.2010,19(3)：1669-1670.
[24]ME Csete, KE Drazan, M van Bree, et al. Adenovirus-mediated gene transfer in the transplant setting. I. Conditions for expression of transferred genes in cold-preserved hepatocytes [J]. Transplantation,1994,57 (10):1502-1507.
[25]毛立军,郑骏年.辅助腺病毒载体及其介导的基因治疗研究进展[J].国际病毒性杂志,2006,13(5)：151-154.
[26]刘根梅,陈瑞爱,罗满林.腺病毒载体的研究进展[J].广东农业科学,2010,37(4)：188-190.
[27]A Benham, A Tulp, J Neefjes. Synthesis and assembly of MHC-peptide complexes [J]. Immunol Today,1995,16 (7):359-362.
[28]A Alcami, UH Koszinowski. Viral mechanisms of immune evasion [J]. Immunol Today,2000, 21 (9):447-455.
[29]Padma Billam, Kathryn L. Bonaparte, Jie Liu, et al. T Cell Receptor Clonotype Influences Epitope Hierarchy in the CD8+ T Cell Response to Respiratory Syncytial Virus Infection [J]. J. Biol. Chem.,2011,286 (6):4829-4841.
[30]Palanee Ammaranond, David J. van Bockel, Kathy Petoumenos, et al. HIV Immune Escape at an Immunodominant Epitope in HLA-B*27-Positive Individuals Predicts Viral Load Outcome [J]. J. Immunol.,2011,186(1):479-488.
[31]H Pircher, D Moskophidis, U Rohrer, et al. Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo [J]. Nature,1990,346 (6285):629-633.
[32]L Lybarger, X Wang, M Harris, et al. Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways [J]. Curr Opin Immunol, 2005,17(1):71-78.
[33]J Koch, R Tampe. The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation [J]. Cell Mol Life Sci,2006,63 (6):653-662.
[34]T Elliott, A Williams. The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex [J]. Immunol Rev,2005,207 (10):89-99.
[35]Maaike E. Ressing, Sinead E. Keating, Daphne van Leeuwen, et al. Impaired Transporter Associated with Antigen Processing-Dependent Peptide Transport during Productive EBV Infection [J]. J. Immunol.,2005,174 (11):6829-6838.
[36]Christian Brander, Todd Suscovich, Yun Lee, et al. Impaired CTL Recognition of Cells Latently Infected with Kaposi's Sarcoma-Associated Herpes Virus [J]. J. Immunol.,2000,165 (4):2077-2083.
[37]S Koba, F Tsunoda, T Hirano, et al. Postprandial changes in LDL phenotypes in patients with myocardial infarction [J]. Eur J Clin Invest,2005,35 (3):171-179.
[38]Danijela Koppers-Lalic, Eric A. J. Reits, Maaike E. Ressing, et al. Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing [J]. PNAS,2005,102 (14):5144-5149.
[39]N Nozawa, N Inoue. Mechanisms of congenital CMV infection [J]. Nippon Rinsho,2006,64 (Suppl 3):446-450
[40]Nupur T. Pande, Colin Powers, Kwangseog Ahn, et al. Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11 [J].J. Virol,2005,79 (9):5786-5798.
[41]郑秀峰,韩金祥.单纯疱疹病毒研究现状[J].中国麻风病杂志,2008,24(5)：370-373.
[42]吴建兵,郑家润.单纯疱疹病毒潜伏感染的分子基础[J].国际皮肤性病学杂志,2006,32(3)：194-196.
[43]M Hong, WZ Li, QH Li. The immune escape mechanism of herpes simplex virus type 1 suppression of transporter associated with antigen processing (TAP) by ICP47 [J]. Bing Du Xue Bao,2007,23(1):72-75.
[44]Liesbeth E. M. Oosten, Danijela Koppers-Lalic, Els Blokland, et al. TAP-inhibiting proteins US6, ICP47 and UL49.5 differentially affect minor and major histocompatibility antigen-specific recognition by cytotoxic T lymphocytes [J]. Int. Immunol.,2007,19 (9): 1115-1122.
[45]Christopher Aisenbrey, Christina Sizun, Joachim Koch, et al. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery [J]. J. Biol. Chem.,2006,281 (41):30365-30372.
[46]范凌云,谢庆军.腺病毒载体的研究进展[J].中国生物制品学杂志,2008,21(2)：153-157.
[47]X Danthinne, MJ Imperiale. Production of first generation adenovirus vectors:a review [J]. Gene Ther,2000,7 (20):1707-1714.
[48]RW Wilmott, RS Amin, CR Perez, et al. Safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lungs of nonhuman primates [J]. Hum Gene Ther,1996,7 (3):301-318.
[49]Robin J. Parks, Liane Chen, Martina Anton, et al. A helper-dependent adenovirus vector system:Removal of helper virus by Cre-mediated excision of the viral packaging signa [J]. PNAS,1996,93 (24):13565-13570.
[50]范明齐,冯嘉瑜,黄赤兵,等.人ICOS胞外区腺病毒载体的构建与鉴定[J].重庆医学,2006,35(16)：1460-1462.
[51]金天明,武迎红.腺病毒及腺病毒载体的研究进展[J].天津农学院学报,2007,14(2)：48-51.
[52]方针强,叶钢,欧阳一辛,等.人BMP-7基因重组腺病毒的构建与鉴定[J].第三军医大学学报,2008,30(2)：153-156.
[53]JA St George. Gene therapy progress and prospects:adenoviral vectors [J]. Gene Ther, 2003,10(14):1135-1141.
[54]M Zeng, SK Smith, F Siegel, et al. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination [J]. Biotechniques,2001,31 (2): 260-262.
[55]郭浩,彭毅志,辛海明.小鼠CCR7基因重组腺病毒的构建与鉴定[J].现代生物医学进展,2007,7(11)：1643-1645.
[56]徐竹蔚,刘莹,户义.抗3种标签蛋白单克隆抗体的制备及特性鉴定[J].细胞与分子免疫学杂志,2005,21(5)：613-614.
[57]M Hiraoka, N Nitta, M Nagai, et al. MCP-1-induced enhancement of THP-1 adhesion to vascular endothelium was modulated by HMG-CoA reductase inhibitor through RhoA GTPase-, but not ERK1/2-dependent pathway [J]. Life Sci,2004,75 (11):1333-1341.
[58]闵玉涛,王云龙,李晨阳,等.抗FLAG标签单克隆抗体的制备、鉴定及初步应用[J].中国生物工程杂志,2006,27(1)：93-97
[59]王文礼,王云龙,李晨阳,等.His标签单克隆抗体的制备、鉴定及初步应用[J].细胞与分子免疫学杂志,2008,24(4)：399-400.
[60]崔正军.腺病毒载体介导的反义VEGF基因治疗人皮肤恶性黑色素瘤的实验研究[D].[博士学位论文].成都：四川大学,2004.
[1]Jessamyn Bagley, Chaorui Tian, David H. Sachs, et al. Induction of T-cell tolerance to an MHC class Ⅰ alloantigen by gene therapy [J]. Blood,2002,99 (12):4394-4399.
[2]郭峰,卢永刚,何建平,肝细胞移植研究进展[J].中国普通外科杂志,2007,16(8)：803-805
[3]刘世呈,李靖.肝细胞移植的研究进展[J].中国普外基础与临床杂志,2008,15(2)：109-111.
[4]S Murasawa, T Asahara. Gene modified cell transplantation for vascular regeneration [J]. Curr Gene Ther,2007,7(1):1-6.
[5]Martina Sester, Katja Koebernick, Douglas Owen, et al. Conserved Amino Acids within the Adenovirus 2 E3/19K Protein Differentially Affect Downregulation of MHC Class Ⅰ and MICA/B Proteins [J]. J. Immunol.,2010,184 (1):255-267.
[6]艾伦·P·埃舍尔,李逢春.预防与治疗自身免疫性疾病的物质[P].中国专利,CN200710097957.2.2007-10-24
[7]洪敏,李卫中,李琦涵.单纯疱疹病毒Ⅰ型免疫逃避机制-ICP47抑制抗原加工相关转运蛋白[J].病毒学报,2007,23(1)：72-75.
[8]Margit H. Lampen, Marieke C. Verweij, Bianca Querido, et al. CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population [J]. J. Immunol., 2010,185 (11):6508-6517.
[9]Christopher Aisenbrey, Christina Sizun, Joachim Koch, et al. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery [J]. J. Biol. Chem.,2006,281 (41):30365-30372.
[10]孙祖玥,赵勇.基因工程在克服移植免疫排斥反应及诱导免疫耐受中的应用[J].中国生物工程杂志,2005,25(1)：6-9.
[11]陈宗祐.阻断信号传导对延长小鼠同种异体移植心脏存活时间的影响[D].[博士学位论文].上海：复口.大学,2006
[12]刘海忠.阻断ICOS共刺激通路抗肝移植急性排斥的研究[D].[硕士学位论文].重庆：重庆医科大学,2005.
[13]EM Mazaris, CT Roussos, VE Papalois. Hepatocyte transplantation:a review of worldwide clinical developments and experiences [J]. Exp Clin Transplant,2005,3 (1):306-315.
[14]T Kawahara, H Yagita, K Okumura, et al. T cell-mediated effector mechanisms in the rejection of allogeneically transplanted hepatocytes [J]. Transplant Proc,1999,31 (2): 830-831.
[15]王绯,杨永平.肝细胞移植基础与临床应用研究进展[J].肝脏,2007,12(2)：143-144.
[16]崔正军.腺病毒载体介导的反义VEGF基因治疗人皮肤恶性黑色素瘤的实验研究[D]. [博士学位论文].成都：四川大学,2004.
[17]Wang Y, Huang S, Sah VP, et al. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family [J]. J Biol Chem.1998, 273 (4):2161-2168.
[18]张文敏,陈裕庆,张萌,等.NY-ESO-1致敏树突状细胞诱导的CTL对肝癌细胞株的特异性杀伤作用[J].中国肿瘤生物治疗杂志,2009,16(1)：18-23.
[19]S Hao, O Bai, J Yuan, et al. Dendritic cell-derived exosomes stimulate stronger CD8+CTL responses and antitumor immunity than tumor cell-derived exosomes [J]. Cell Mol Immunol, 2006,3 (3):205-211.
[20]杨静悦,曹大勇,刘文超,等.IL-18基因增强肿瘤抗原致敏DC诱导的CTL特异诱导杀伤肝癌细胞[J].中国肿瘤生物治疗杂志,2009,16(1)：55-58
[21]付必莽.IL-2、TNF-β在混合淋巴细胞培养中的表达与肝移植急排反应的关系[D].[硕士学位论文].昆明：昆明医学院,2005.
[22]B Lindton, L Markling, O Ringden, et al. Mixed lymphocyte culture of human fetal liver cells [J]. Fetal Diagn Ther,2000,15 (2):71-78.
[23]JE Visentainer, SR Lieber, LB Persoli, et al. Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation [J]. Braz J Med Biol Res,2002,35 (5):567-572.
[24]黄建钊,夏穗生.对肝移植免疫耐受机理研究的思索[J].中国普外基础与临床杂志,2000,4(7)：261-262.
[25]刘利,张玉海.混合淋巴细胞培养上清液中细胞因子水平与移植肝急性排斥反应的联系[J].肝脏病与透析肝移植杂志,2000,1(9)：43-47.
[26]谢晋,李宁丽,陈福祥,等.异种淋巴细胞混合培养时T淋巴细胞表达细胞因子的格局[J].中华器官移植杂志,2001,3(2)：172-173.
[27]周光炎.免疫学原理.第一版[M].上海：上海科学技术文献出版社,2000,300-304.
[28]张儒有,郑永同,胡韶山,等.神经干细胞移植治疗脑卒中后遗症50例临床效果分析[J].中国临床康复,2006,10(9)：138-139.
[29]CB Johansson, S Momma, DL Clarke, et al. Identification of a neural stem cell in the adult mammalian central nervous system [J]. Cell,1999,96 (1):25-34.
[30]Y Li, M Chopp. Temporal profile of nestin expression after focal cerebral ischemia in adult rat [J]. Brain Res,1999,838 (2):1-10.
[31]王宁生,雷燕,刘平,等.关于血清药理学的若于思考[J].中国中西医结合杂志,1999,19(5)：263-266.
[32]赵军,沈振亚,徐五音,等.血清IL-2水平在大鼠心脏移植术后早期急性排斥反应诊断中的意义[J].苏州医学院学报,1999,19(9)：973-975.
[33]OM Martinez, JC Villanueva, J Lake, et al. IL-2 and IL-5 gene expression in response to alloantigen in liver allograft recipients and in vitro [J]. Transplantation,1993,55 (5): 1159-1166.
[34]H Egawa, OM Martinez, MB Quinn, et al. Acute liver allograft rejection in the rat. An analysis of the immune response [J]. Transplantation,1995,59 (1):97-102.
[35]MH Andersen, D Schrama, P Thor Straten, et al. Cytotoxic T cells [J]. J Invest Dermatol, 2006,126(1):32-41.
[36]朱鹏,陈义发,李丹,等.小鼠心脏移植后穿孔素及颗粒酶B表达水平与急性排斥反应的关系[J].世界华人消化杂志,2007,15(31)：3289-3293
[37]M Yannaraki, JM Rebibou, D Ducloux, et al. Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection [J]. Transpl Int,2006,19 (9): 759-768.
[38]邓薇薇,雷建平.穿孔素的研究进展[J].实用临床医学,2008,9(2)：128-129.
[39]Angela Kuhla, Christian Eipel, Kerstin Abshagen, et al. Role of the perforin/granzyme cell death pathway in D-Gal/LPS-induced inflammatory liver injury [J]. Am J Physiol Gastrointest Liver Physiol,2009,296 (5):G1069-G1076.
[40]Mark D McKenzie, Nadine L Dudek, Lina Mariana, et al. Perforin and Fas induced by IFN-γ and TNF-α mediate beta cell death by OT-I CTL [J]. Int. Immunol.,2006,18(6):837-846.
[1]A Alcami, UH Koszinowski, Viral mechanisms of immune evasion [J]. Immunol Today, 2000,21 (9):447-55.
[2]Eva Szomolanyi-Tsuda, Xueya Liang, Raymond M. Welsh, et al. Role for TLR2 in NK Cell-Mediated Control of Murine Cytomegalovirus In Vivo [J]. J. Virol.,2006,80 (9):4286-4291.
[3]Elizabeth K. Day, Andrew J. Carmichael, Ineke J. M. ten Berge, et al. Rapid CD8+ T Cell Repertoire Focusing and Selection of High-Affinity Clones into Memory Following Primary Infection with a Persistent Human Virus:Human Cytomegalovirus [J]. J. Immunol.,2007, 179 (5):3203-3213.
[4]JA Hamerman, K Ogasawara, LL Lanier. NK cells in innate immunity [J]. Curr Opin Immunol, 2005,17(1):29-35.
[5]SH Robbins, G Bessou, A Cornillon, et al. Natural killer cells promote early CD8 T cell responses against cytomegalovirus [J]. PLoS Pathog,2007,3 (8):e123.
[6]Yu Mori, Sukenao Tsuji, et al. Inhibitory Immunoglobulin-Like Receptors LILRB and PIR-B Negatively Regulate Osteoclast Development [J]. J. Immunol.,2008,181(7):4742-4751.
[7]Michael A. Caligiuri. Human natural killer cells [J]. Blood,2008,112(3):461-469.
[8]孙蕾,常雅萍.病毒逃逸免疫细胞杀伤机制[J].国外医学免疫学分册,2002,25(5)：265-269.
[9]M Vales-Gomez, H Browne, HT Reyburn. Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells [J]. BMC Immunol,2003; 4(1):4.
[10]Alexandre Iannello, Olfa Debbeche, Elodie Martin, et al. Viral strategies for evading antiviral cellular immune responses of the host [J]. J. Leukoc. Biol.,2006,79 (1):16-35.
[11]M Kubin, L Cassiano, J Chalupny, et al. ULBP1,2,3:novel MHC class Ⅰ-related molecules that bind to human cytomegalovirus glycoprotein UL16, activate NK cells [J]. Eur J Immunol, 2001,31(5):1428-1437.
[12]D Cosman, J Mullberg, CL Sutherland, et al. ULBPs, novel MHC class Ⅰ-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor [J]. Immunity,2001,14 (2):123-133.
[13]Alessandro Poggi, Roberta Carosio, Grazia Maria Spaggiari. NK Cell Activation by Dendritic Cells Is Dependent on LFA-1-Mediated Induction of Calcium-Calmodulin Kinase Ⅱ:Inhibition by HIV-1 Tat C-Terminal Domain [J]. J. Immunol,2002,168 (1):95-101.
[14]程玉兴,胡云章.病毒免疫逃逸机制研究进展[J].国外医学病毒学分册,2005,12(5)：150-154.
[15]Collin R. Brooks, Tim Elliott, Peter Parham, et al. The Inhibitory Receptor NKG2A Determines Lysis of Vaccinia Virus-Infected Autologous Targets by NK Cells [J]. J. Immunol.,2006,176(2):1141-1147.
[16]Peter Tomasec, Veronique M. Braud, Carole Rickards, et al. Surface Expression of HLA-E, an Inhibitor of Natural Killer Cells, Enhanced by Human Cytomegalovirus gpUL40 [J]. Science,2000,287 (5455):1031.
[17]N Pizzato, B Garmy-Susini, P Le Bouteiller, et al. Differential down-modulation of HLA-G and HLA-A2 or-A3 cell surface expression following human cytomegalovirus infection [J]. J Reprod Immunol,2004,62 (1-2):3-15.
[18]JA Hamerman, K Ogasawara, LL Lanier. NK cells in innate immunity [J]. Curr Opin Immunol,2005.17(1):29-35.
[19]Katherine Belov, Claire E. Sanderson, Janine E. Deakin, et al. Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system [J].Genome Res.,2007,17 (7):982-991.
[20]赵朴,郑玉姝,刘兴友.病毒干扰MHCⅠ类抗原呈递策略的研究进展[J].生命科学,2008,20(2)：300-303.
[21]Karen A. O'Connell, Yefei Han, Thomas M, et al. Role of Natural Killer Cells in a Cohort of Elite Suppressors:Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type Ⅰ Replication In Vitro [J]. J. Virol.,2009,83 (10): 5028-5034.
[22]M Lopez-Botet, M Llano, M Ortega.Human cytomegalovirus and natural killer-mediated surveillance of HLA class Ⅰ expression:a paradigm of host-pathogen adaptation [J]. Immunol Rev,2001,181(12):193-202.
[23]W. L. William Chang, Nicole Baumgarth, Dong Yu. et al. Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality [J]. J. Virol.,2004,78 (16):8720-8731.
[24]C Cerboni, A Achour, A Warnmark, et al. Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR-1/ILT2/CD85j [J].Eur J Immunol,2006; 36(3):732-41.
[25]Elena G. Chiorean, Scott J. Dylla, Krista Olsen, et al. BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIRs) [J]. Blood,2003,101 (9):3527-3533.
[26]DM Andrews, CE Andoniou. F Granucci, et al. Infection of dendritic cells by murine cytomegalovirus induces functional paralysis [J]. Nat Immunol,2001,2(11):1077-84.
[27]Marie Mandron, Helene Martin, Beatrice Bonjean, et al. Dendritic cell-induced apoptosis of human cytomegalovirus-infected fibroblasts promotes cross-presentation of pp65 to CD8+ T cells [J]. J. Gen. Virol.,2008,89(1):78-86.
[28]赵宏,马伟杭.人巨细胞病毒感染中的细胞免疫机制[J].国际流行病学传染病学杂志[J].2008,35(3)：182-184.
[29]Sara Gredmark, Tamara Tilburgs, and Cecilia Soderberg-Naucler. Human Cytomegalovirus Inhibits Cytokine-Induced Macrophage Differentiation [J].J. Virol.,2004,78 (19):10378-10389.
[30]Franziska Schwartzkopff, Tobias A. Grimm, Carla S.R. Lankford, et al. Platelet factor 4 (CXCL4) facilitates human macrophage infection with HIV-1 and potentiates virus replication [J]. Innate Immunity,2009,15 (6):368-379.
[31]R Hewer, D Meyer. Peptide immunogens designed to enhance immune responses against human immunodeficiency virus (HIV) mutant strains:a plausible means of preventing viral persistence [J]. J Theor Biol,2005,233 (1):85-90.
[32]M. Kathryn Liszewski, Marilyn K. Leung, Richard Hauhart, et al. Structure and Regulatory Profile of the Monkeypox Inhibitor of Complement:Comparison to Homologs in Vaccinia and Variola and Evidence for Dimer Formation [J]. J. Immunol.,2006,176 (6):3725-3734.
[33]欧元祝,瞿涤.病毒逃逸补体的机制[J].微生物与感染,2006,1(2)：114-117.
[34]O. Brad Spiller, David J. Blackbourn, Linda Mark, et al. Functional Activity of the Complement Regulator Encoded by Kaposi's Sarcoma-associated Herpesvirus [J]. J. Biol. Chem.,2003,278 (11):9283-9289.
[35]J Bernet, J Mullick, AK Singh, et al. Viral mimicry of the complement system [J]. J Biosci, 2003,28 (3):249-264.
[36]Herman W. Favoreel, Gerlinde R. Van de Walle, Hans J. Nauwynck, et al. Virus complement evasion strategies [J].J. Gen. Virol.,2003,84 (1):1-15.
[37]沈小燕,丛国正,常惠芸,等.病毒感染与逃避免疫反应的研究进展[J].医学分子生物学杂杂志,2005,2(4)：284-287.
[38]C Speth, H Stoiber, MP Dierich. Complement in different stages of HIV infection and pathogenesis [J]. Int Arch Allergy Immunol,2003,130 (4):247-257.
[39]Patricia Devaux, Dale Christiansen, Sebastien Plumet, et al. Cell surface activation of the alternative complement pathway by the fusion protein of measles virus [J]. J. Gen. Virol., 2004,85(6):1665-1673.
[40]Mauricio Urquiza, Ramses Lopez, Helena Patino, et al. Identification of Three gp350/220 Regions Involved in Epstein-Barr Virus Invasion of Host Cells [J]. J. Biol. Chem.,2005,280 (42):35598-35605.
[41]DT Haydon, AR Samuel, NJ Knowles. The generation and persistence of genetic variation in foot-and-mouth disease virus [J]. Prev Vet Med,2001,51 (1-2):111-24.
[42]JP Moorman, M Joo, YS Hahn. Evasion of host immune surveillance by hepatitis C virus: potential roles in viral persistence [J]. Arch Immunol Ther Exp (Warsz),2001,49 (3): 189-194.
[43]Christine M. Posavad, Meei Li Huang, Serge Barcy,. Long Term Persistence of Herpes Simplex Virus-Specific CD8+ CTL in Persons with Frequently Recurring Genital Herpes [J]. J. Immunol.,2000,165(2):1146-1152.
[44]E Maidji, S McDonagh, O Genbacev, et al. Maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal Fc receptor-mediated transcytosis [J]. Am. J. Pathol.,2006,168 (4):1210-26.
[45]John M. Lubinski, Ming Jiang, Lauren Hook, et al. Herpes Simplex Virus Type 1 Evades the Effects of Antibody and Complement In Vivo [J]. J. Virol.,2002; 76 (18):9232-9241.
[46]Michael Basler, Maya Dajee, Carlo Moll, et al. Prevention of Experimental Colitis by a Selective Inhibitor of the Immunoproteasome [J]. J. Immunol.,2010,185 (10):634-641.
[47]Paul Schmid-Hempel. Immune defence, parasite evasion strategies and their relevance for 'macroscopic phenomena' such as virulence [J]. Phil Trans R Soc B,2009,364 (1513):85-98.
[48]Yili Yin, Benedicte Manoury, Robin Fahraeus, et al. Self-Inhibition of Synthesis and Antigen Presentation by Epstein-Barr Virus-Encoded EBNA1 [J]. Science,2003,301 (5638):1371.
[49]Christopher T. Cornell, William B. Kiosses, Stephanie Harkins, et al. Coxsackievirus B3 Proteins Directionally Complement Each Other To Downregulate Surface Major Histocompatibility Complex Class Ⅰ [J]. J. Virol.,2007,81 (13):6785-6797.
[50]NL Glosson, AW Hudson. Human herpesvirus-6A and- 6B encode viral immunoevasins that downregulate class Ⅰ MHC molecules [J]. Virology,2007,365 (1):125-135.
[51]Lydie Trautmann, Marie Rimbert, Klara Echasserieau, et al. Selection of T Cell Clones Expressing High-Affinity Public TCRs within Human Cytomegalovirus-Specific CD8 T Cell Responses [J]. J. Immunol.,2005,175 (9):6123-6132.
[52]J Koch, R Tampe.The macromolecular peptide-loading complex in MHC class Ⅰ-dependent antigen presentation [J]. Cell Mol Life Sci,2006,63 (6):653-662.
[53]洪敏,李卫中,李琦涵.单纯疱疹病毒Ⅰ型免疫逃避机制-ICP47抑制抗原加工相关转运蛋白[J].病毒学报,2007,23(1)：72-75.
[54]Maaike E. Ressing, Sinead E. Keating, Daphne van Leeuwen, et al. Impaired Transporter Associated with Antigen Processing-Dependent Peptide Transport during Productive EBV Infection [J]. J. Immunol.,2005,174 (11):6829-6838.
[55]Christian Brander, Todd Suscovich, Yun Lee, et al. Impaired CTL Recognition of Cells Latently Infected with Kaposi's Sarcoma-Associated Herpes Virus [J]. J. Immunol.,2000, 165(4):2077-2083.
[56]S Koba, F Tsunoda, T Hirano, et al. Postprandial changes in LDL phenotypes in patients with myocardial infarction [J]. Eur J Clin Invest,2005,35 (3):171-179.
[57]Danijela Koppers-Lalic, Eric A. J. Reits, Maaike E. Ressing, et al. Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing [J]. PNAS,2005,102 (14):5144-5149.
[58]N Nozawa, N Inoue. Mechanisms of congenital CMV infection [J]. Nippon Rinsho,2006,64 (Suppl 3):446-50
[59]Nupur T. Pande, Colin Powers, Kwangseog Ahn, et al. Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11 [J]. J. Virol.,2005,79 (9):5786-5798.
[60]MJ Bevan, et al. Antigen presentation to cytotoxic T lymphocytes in vivo [J]. J. Exp. Med., 1995,182(1):639.
[61]Naeem Khan, Rachel Bruton, Graham S. et al. Identification of Cytomegalovirus-Specific Cytotoxic T Lymphocytes In Vitro Is Greatly Enhanced by the Use of Recombinant Virus Lacking the US2 to US11 Region or Modified Vaccinia Virus Ankara Expressing Individual Viral Genes [J] J. Virol.,2005,79 (5):2869-2879.
[62]Dimitrios Lagos, Matthew W. B. Trotter, Richard J. Vart, et al. Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells [J]. Blood,2007,109 (4):1550-1558.
[63]GH Ashrafi, MR Haghshenas, B Marchetti, et al. E5 protein of human papillomavirus type 16 selectively downregulates surface HLA class Ⅰ [J]. Int J Cancer,2005,113 (2):276-283.
[64]Shihe Hou, Hancheng Guan, Robert P. Ricciardi. In Adenovirus Type 12 Tumorigenic Cells, Major Histocompatibility Complex Class Ⅰ Transcription Shutoff Is Overcome by Induction of NF-κB and Relief of COUP-TFII Repression [J]. J. Virol.,2002,76 (7):3212-3220.
[65]MT Barel, GC Hassink, S van Voorden, et al. Human cytomegalovirus-encoded US2 and US11 target unassembled MHC class Ⅰ heavy chains for degradation [J]. Mol Immunol,2006, 43(8):1258-1266.
[66]Xiaoli Wang, Yihong Ye, Wayne Lencer, et al. The Viral E3 Ubiquitin Ligase mK3 Uses the Derlin/p97 Endoplasmic Reticulum-associated Degradation Pathway to Mediate Down regulation of Major Histocompatibility Complex Class Ⅰ Proteins [J]. J. Biol. Chem.,2006, 281 (13):8636-8644.
[67]Vanessa M. Noriega,Domenico Tortorella. Human Cytomegalovirus-Encoded Immune Modulators Partner To Downregulate Major Histocompatibility Complex Class Ⅰ Molecules [J]. J Virol.2009,83 (3):1359-1367.
[68]Scott G. Hansen, Colin J. Powers, Rebecca Richards, et al. Evasion of CD8+ T Cells Is Critical for Superinfection by Cytomegalovirus [J]. Science,2010,328 (5974):102-106.
[69]Zheng W. Chen, Abie Craiu, Ling Shen, et al. Simian Immunodeficiency Virus Evades a Dominant Epitope-Specific Cytotoxic T Lymphocyte Response Through a Mutation Resulting in the Accelerated Dissociation of Viral Peptide and MHC Class Ⅰ [J]. J. Immunol., 2000,164 (12):6474-6479.
[70]Laurent Coscoy, David Jesse Sanchez, Don Ganem. A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition [J]. J. Cell Biol.,2001,155 (7):1265.
[71]Yves Chalandon, Sylvie Degermann, Jean Villard, et al. Pretransplantation CMV-specific T cells protect recipients of T-cell-depleted grafts against CMV-related complications [J]. Blood,2006,107(1):389-396.
[72]Yujuan Yue, Amitinder Kaur, Meghan K. Eberhardt, et al. Immunogenicity and Protective Efficacy of DNA Vaccines Expressing Rhesus Cytomegalovirus Glycoprotein B, Phosphoprotein 65-2, and Viral Interleukin-10 in Rhesus Macaques [J]. J. Virol.,2007,81 (3): 1095-1109.
[73]Z Wang, C La Rosa, Z Li, et al. Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1.Vaccine [J],2007,25 (6):1132-1141.
[74]Graeme E. Price, Rong Ou, Hong Jiang, et al. Viral Escape by Selection of Cytotoxic T Cell-Resistant Variants in Influenza a Virus Pneumonia [J]. J. Exp. Med.,2000,191 (11): 1853-1868.
[75]Massimo Maffei, Fabio Ghiotto, Marzia Occhino, et al. Human Cytomegalovirus Regulates Surface Expression of the Viral Protein UL18 by Means of Two Motifs Present in the Cytoplasmic Tail [J]. J. Immunol.,2008,180 (2):969-979.
[76]Susan McDonagh, Ekaterina Maidji, Wenge Ma, et al. Viral and bacterial pathogens at the maternal-fetal interface.The Journal of Infectious Disease [J],2004,190 (4):826-834
[77]Todd A. Fehniger, Megan A. Cooper, Gerard J. Nuovo, et al. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2:a potential new link between adaptive and innate immunity [J]. Blood,2003,101 (8):3052-3057.
[78]DM Andrews, AA Scalzo, WM Yokoyama, et al. Functional interactions between dendritic cells and NK cells during viral infection [J]. Nat Immunol,2003,4 (2):175-181.
[79]Fan Xiu Zhu, Sonya M. King, Eric J. Smith, et al. A Kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type Ⅰ interferon by blocking IRF-7 phosphorylation and nuclear accumulation [J]. PNAS,2002,99 (8):5573-5578.
[80]A Arena, D Iannello, D Gazzara. et al. Role of interleukin-18 in peripheral blood mononuclear cells infected with human herpes virus type 6 [J]. Intervirology,2001,44 (4): 250-254.
[81]Ikka Helantera, Raisa Loginov, Petri Koskinen, et al. Persistent cytomegalovirus infection is associated with increased expression of TGF-β1, PDGF-AA and ICAM-1 and arterial intimal thickening in kidney allografts.Nephrol [J]. Dial. Transplant.,2005,20 (4):790-796.
[82]Selina Khan, Albert Zimmermann, Michael Basler, et al. A Cytomegalovirus Inhibitor of Gamma Interferon Signaling Controls Immunoproteasome Induction [J]. J. Virol.,2004,78 (4):1831-1842.
[83]Martin Savard, Carole Belanger, Michel J. Tremblay, et al. EBV Suppresses Prostaglandin E2 Biosynthesis in Human Monocytes [J]. J. Immunol.,2000,164 (12):6467-6473.
[84]Martin J. Raftery, Dorte Wieland, Stefanie Gronewald, Shaping Phenotype, Function, and Survival of Dendritic Cells by Cytomegalovirus-Encoded IL-10 [J]. J. Immunol.,2004,173 (5):3383-3391.
[85]Christina Jenkins, Allison Abendroth, Barry Slobedman. A Novel Viral Transcript with Homology to Human Interleukin-10 Is Expressed during Latent Human Cytomegalovirus Infection [J]. J. Virol.,2004,78 (3):1440-1447.
[86]Juliet V. Spencer, Kristen M. Lockridge, Peter A. Barry, et al. Potent Immunosuppressive Activities of Cytomegalovirus- Encoded Interleukin-10 [J]. J. Virol.,2002,76 (3):1285-1292.
[87]Christina Jenkins, Allison Abendroth, Barry Slobedman. A Novel Viral Transcript with Homology to Human Interleukin-10 Is Expressed during Latent Human Cytomegalovirus Infection [J]. J. Virol.,2004,78 (3):1440-1447.
[88]TE Sparer, J Gosling, TJ Schall, et al. Expression of human CXCR2 in murine neutrophils as a model for assessing cytomegalovirus chemokine vCXCL-1 function in vivo [J]. J Interferon Cytokine Res,2004,24 (10):611-620.
[89]K Iwatsuki, T Yamamoto, K Tsuji, et al. A spectrum of clinical manifestations caused by host immune responses against Epstein-Barr virus infections [J]. Acta Med Okayama,2004, 58(4):169-180.
[90]Rhiannon N. Silk, Gavin C. Bowick, Charles C. Abrams, et al. African swine fever virus A238L inhibitor of NF-KB and of calcineurin phosphatase is imported actively into the nucleus and exported by a CRM1-mediated pathway [J]. J Gen Virol,2007,88 (2):411-419.
[91]BT Seet, JB Johnston, CR Brunetti, et al. Poxviruses and immune evasion [J]. Annu Rev Immunol,2003,21(5):377-423.
[92]NL Nakhoul and LL Hamm. Non-erythroid Rh glycoproteins:a putative new family of mammalian ammonium transporters [J]. Pflugers Arch,2004,447 (5):807-812.
[93]Robert Y. Suruki,Nancy Mueller,K.atsuhiro Hayashi,et al. Host Immune Status and Incidence of Hepatocellular Carcinoma among Subjects Infected with Hepatitis C Virus:A Nested Case-Control Study in Japan [J]. Cancer Epidemiol. Biomarkers Prev,2006,15 (12): 2521-2525.
[94]Sebastian Voigt, Gordon R. Sandford, Gary S. Hayward, et al. The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region:further evidence for a separate evolutionary lineage from that of rat CMV Maastricht [J]. J Gen Virol.,2005,86(2):263-274.
[95]张欣文,韩蕾,李芬.母胎界面人巨细胞病毒免疫逃逸机制研究进展[J].中国妇幼健康研究,2006,17(4)：302-304.
[96]CE Andoniou, MA Degli-Esposti. Insights into the mechanisms of CMV-mediated interference with cellular apoptosis [J]. Immunol Cell Biol,2006,84 (1):99-106.
[97]Dustin T. Petrik, Kimberly P. Schmitt, Mark F. Stinski. Inhibition of Cellular DNA Synthesis by the Human Cytomegalovirus IE86 Protein Is Necessary for Efficient Virus Replication [J]. J. Virol.,2006,80 (8):3872-3883.
[98]M Irmler, M Thome, M Hahne, et al. Inhibition of death receptor signals by cellular FLIP [J]. Nature,1997,388(6638):190-195.
[99]Joseph DeMasi, Michael C. Chao, Ashu S. Kumar, et al. Bovine Papillomavirus E7 Oncoprotein Inhibits Anoikis [J]. J.Virol.,2007,81 (17):9419-9425.
[100]Eli Boritz, Brent E. Palmer, Cara C. Wilson. Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD4+T Cells That Proliferate In Vitro Detected in Samples from Most Viremic Subjects and Inversely Associated with Plasma HIV-1 Levels [J]. J.Virol.,2004,78 (22):12638-12646