| 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. |
