| Allotransplant rejection remains to be a main cause for the failure of the allograft. Transplant rejection is a complex process, accompanied by large changes in gene expression and the interactions between so many genes form a complex system, especially lowly expressed genes, organ transplant rejection response is still a threat to long-term graft survival. The long-term acceptance of allograft still requires life-long administration of immunosuppressive drugs. Besides their ability to prevent acute rejection episodes, conventional immunosuppressive drugs are less successful with many side effects, such as the high expence of immunosuppressive agents, increasing toxicity to liver, kidney, nervous and other systems. These limitations have driven researchers to in-depth study the mechanisms of allograft rejection, identify reliable, new biomarkers and search for a more effective and less toxic immunosuppressive agent to avoid organ rejection.Transplant rejection is a complex process, T cells and macrophages play important role. The body contains various kinds of T cells, although CD8+ T cells alone seem to be able to initiate allorejection, numeral studies have shown that CD4+ T cells are necessary and sufficient to induce allograft rejection in the absence of other types of lymphocytes and depletion of peripheral CD4+ T cells can prolong the survival of skin allograft. The gene expression of CD4+ T cells in allotransplantation got more attention recently.Gene expression profiling in different tissues and organs provides the molecular basis for the differentiation, development, aging and other phenomena of organism, and it provides important information for function studies. At present, a large number of researchers have found that there are significant differences for gene expression in kidney, liver, heart, corneal and skin transplantation using RT-PCR and microarray methods. There are so many genes differently expressed in CD4+ T cells in the process of rejection, even though studies have shown that these genes are very important, using gene knockout mice or specific antibodies cannot completely block transplant rejection, and even the survival of the graft had no significant improvement. This phenomenon shows that in addition to the highly expressed genes, there must be some lowly expressed genes which play more important role. Thus the sensitive and powerful laboratory method is needed to assess gene expression, find lowly expressed genes and prevent transplant rejection. Another problem is most of the research used transplant graft to study the gene profiling, as so many kinds of cells and gene expression information, there must be a lot of useful information merge in the background of gene expression. So it is needed to establish a model which can avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the researched cell, together with the sensitive and powerful laboratory method, we can identify more differently expressed genes and in-depth study the molecular mechanism of transplant rejection.Serial Analysis of Gene Expression (SAGE) is a powerful technique that can quickly, exactly reflect expression profile of genes in a given sample in different situations. SAGE does not require prior knowledge of the genes of interest and it provides qualitative and quantitative data of potentially every transcribed sequence in a particular cell or tissue type. It can find out novel genes. Now SAGE has been widely applied in life sciences for understanding the mechanisms of tumor pathogenesis, signal transduction pathway, and inflammation, but it has not been wildly used in the field of organ transplantation. Application of SAGE technology is meaningful for in-depth study of the molecular mechanism of transplant rejection to find special biomarkers, develop effective immunosuppressant and avoid organ rejection.In the first part of current study, we used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells such as B cells and other T cells to the gene expression of the target cell, together with the sensitive and powerful SAGE method, we find lowly expressed genes important for allograft rejection and we determined their functional categories. This study identified the underlying genetic mechanisms regulate the process using SAGE method. Reactive oxygen species (ROS) and reactive nitrogen clusters (RNS) participates in the oxidative injury to the tissue. It has been proven that oxidative stress in transplantation induced graft injury. Recent studies have shown that antioxidant treatment can reduce the organ toxicity caused by cyclosporine A (CSA). Finding an effective immunosuppressive agent which can also prevent or attenuate the toxicity of grafts has meaningful clinical value. Recently, pygeum africanum extract (PAE) been shown to have anti-inflammatory, anti-tumor, inhibiting cell proliferation, inhibiting cell migration functions. We evaluate the effect of PAE on oxidative stress and found that early treatment with PAE could effectively suppress the oxidative stress status in diabetic bladder. According to these findings, we speculate that PAE has potential value to prevent transplant rejection.Nitric oxide (NO) is one of RNS and it plays an important role in the pathophysiology of immune system. NO is an important molecule involved in transplant rejection. Studies have shown that in the process of human, mouse and rat organ or tissue transplantation, and plasma NO level are related to post-transplant graft rejection closely and specifically inhibit the NO production can effectively prolong graft survival. Researchers found significantly lower NO levels when application of immunosuppressive agents such as CsA to effectively induce immune transplant tolerance. There are many factors regulate NO production and studies have shown that macrophages produce NO is totally dependent on activated CD4+ T cells, NO production was significantly inhibited when treat the graft recipients with mAb anti-Thy 1.2 or anti-CD4, while the treatment with mAb anti-CD8 did not have significant effect. It has been shown that the balance of Thl/Th2 cytokines can alter the level of NO, Thl cytokines have been shown to promote the expression of iNOS in several systems, while Th2 cytokines may be responsible for down-regulation of NO production by regulating L-arginine metabolism.Recently, interferon regulatory factors (IRFs) get more attention in immune response. IRF-1 activates the transcription of many IFN-y-inducible genes which includes iNOS, IL12, P40. In addition, it is an important mediator for Thl, CD8, NK cell responses. IRF-1 has received attention in the field of organ transplantation. IRF-2 has originally been described as an antagonist of IRF-1-mediated transcription, however, more recent studies indicate that IRF-2 may act as a transcriptional activator for some genes, such as those encoding histone H4, Bcl-2 and FasL. It also has been shown that IRF-2 can cooperate with other transcription factors to activate the promoter. For example, IRF-2 activates the expression of TAP1 via IRF-2/STAT1complex on TAP1 promoter; in addition, IRF-2 cooperates with IRF-1to activate the CIITA and GBP1. Using gene knockout mice, researchers found that IRF-2 gene knockout mice, similar with IRF-1knockout mice, have defects in Thl and natural killer cell development and function, cytokine production. Bernd Elser et al. have found that IFN-y attenuates IL-4 expression via IRF-land IRF-2 pathways. These findings indicated that IRF-land IRF-2 play Key role in the mechanism of IFN-y represses Th2 response, but the role of IRF-2 in the process of transplant rejection has not been well studied. In the present results, there are some oxidative stress-related genes participated in IFN-gamma signal transduction pathway differently expressed, interferon regulatory factor 2 (IRF-2, allo:iso,8:3, p= 0.041) was over-expressed in the allograft rejection group. Thus, we speculated that IRF-2 is up-regulated in CD4+T cells after antigen stimulation, and this gene inhibits Thl immune response, thereby activating macrophages to produce NO, which is an effect molecule in transplant rejection.Thus, the second part of the current study was to further identify the mechanism of IRF-2 participate in transplant rejection and the function and regulation of PAE on allotransplantation.Part 1:Alternative Expressed Genes Identified in CD4+T Cells-Mediated AllorejectionPurpose:Using CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, we avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, together with the sensitive and powerful SAGE method, we find lowly expressed genes important for allograft rejection and identify the underlying genetic mechanisms regulating the process using SAGE method.Materials and methods: We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplanted SCID mice were used as the control. When the allotransplant skin were fully rejected, CD4+ T cells were harvested from the spleens by using the mouse CD4+ T cells enrichment columns, the cell purities were measured by flow cytometry. SAGE libraries were constructed using the total RNA of CD4+ T cells from allo-and iso-recipients. Our study identified hundreds of genes that significantly altered their express levels in the CD4+ T cells and Expression Analysis Systematic Explorer Program (EASE) version 2.0 was used to determine their functional categories.Results:1. The skin from C57BL/6 mouse was allotransplanted to SCID mouse and the skin from BABL/c mouse was isotransplanted to SCID mouse. After the transplant wound healed (21 days), we used BABL/c mouse naive CD4+T cell adoptive transfer to induce skin acute allograft rejection. Upon 14 days after adoptive transfer of CD4+T cells, the skin graft in the allotransplanted mouse was rejected but the skin graft in the isotransplanted mouse remained normal. Using SAGE method, we identified 185 differently expressed genes. Functional classification of these genes shows that these genes belongs to antigen presentation, apoptosis, cell activation, defense response, phosphorylation, transcription regulation, cell growth and maintenance, and signal transduction. This study provides a rich resource for the mechanism and function study in allograft rejection.2. There are some oxidative stress-related genes participated in IFN-gamma signal transduction pathway differently expressed, such as Statl (10:1,p = 0.003), Jak2 (1:5, p = 0.047), Irf2 (8:3, p=0.041), these results suggest that this signal transduction pathway play a crucial role in the process of allograft rejection.Conclusions:1. We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, this study provide a rich resource to the true gene expression information in allorejection.2. The SAGE libraries we constructed for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries. Functional classification of these genes shows that apoptosis, transcription regulation, and cell growth and maintenance, signal transduction, oxide stress regulation frequently changed. 3. Oxidative stress-related genes participated in IFN-y signal transduction pathway play a crucial role in the process of allograft rejection.Part 2:Study of the Anti-allorejection Effect of PAEAntioxidant treatment can reduce the renal toxicity caused by cyclosporine A. The first part of this study identifies that oxidative stress-related genes participated in IFN-y signal transduction pathway play a crucial role in the process of allograft rejection. Pygeum africanum extract (PAE) has been shown to have anti-inflammatory, anti-tumor, inhibiting cell proliferation, inhibiting cell migration effects. Thus we speculate that PAE has potential application value to prevent transplant rejection. PAE has not been concerned in the field of transplantation. In this study, we first evaluate the effect of PAE skin on allograft survival, and further identify the effect of PAE on oxidative stress status in diabetic and transplant recipients, oxidative stress-related genes IFN-y, IRF-2, iNOS expression and Thl cytokine production. The purpose of the current study was to further identify the mechanism of IRF-2 participated in transplant rejection and the regulation and mechanism of PAE.Materials and methods:1. We used mice spleen cells adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplantated SCID mice were used as control. The next day after cell adoptive transferring, PAE was given intraperitoneally to transplant recipients at various doses (12.5 mg/kg,25 mg/kg,50 mg/kg, and 100 mg/kg) in sterile saline solution; control group was given sterile saline solution. The survival conditions of the skin grafts were observed daily.2. Wistar rats received an i.v. injection with STZ to induce diabetes oxide stress medol. After diabetes induction, PAE groups were fed with PAE (100 mg/kg, orally) in peanut oil. And control groups were fed with same dose peanut oil. The levels of iNOS expression, CAT, SOD activity were measured.3. We used mice spleen cells adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplantated SCID mice were used as control. The next day after cell adoptive transferring, PAE was given intraperitoneally to transplant recipients at various doses (12.5 mg/kg,25 mg/kg,50 mg/kg, and 100 mg/kg) in sterile saline solution; control group was given sterile saline solution. Serum NOS activity was assessed using a NOS activity assay kit, the expression of iNOS in the grafted skin and spleen was detected by immunohistochemistry, the expression of IRF-2 and IFN-y mRNA was measured by RT-PCR, serum levels of TNF-a were measured with specific radioimmunoassay.The splenocytes from control and PAE+allotransplantat group were cultured in vitro for 24h, and the supernatant were used to culture macrophage stimulated with LPS for 24h, the NOS activity was assessed using a NOS activity assay kit.Results:1. Application of 25mg/kg PAE after transplantation can effectively prolong allograft survival (p< 0.05).2. Compared with diabetic group, PAE decreased iNOS expression significantly and increased CAT and SOD (p<0.05).3. Compared with the control group, serum NOS activity was decreased, expression of iNOS protein, IFN-y and IRF-2 mRNA in the spleen tissue was significantly decreased, serum TNF-a level significantly decreased after application of 25 mg/kg PAE (p< 0.05). And 50 mg/kg Pygeum africanum extract also can improve the oxidative stress status in some extent. The splenocytes supernatant from 25 mg/kg PAE group can downregulate NOS level of macrophage (p< 0.05).Conclusions:Application of 25mg/kg/day PAE in vivo can effectively prolong allograft survival. And in vivo and vitro study indicate the mechanism is suppress the oxidative stress status, through down-regulating the expression of IRF-2,suppressing Thl-type cytokines levels, shifting Thl/Th2 cytokines balance, then decrease NOS activity and expression, it will be a new effective immunosuppressive agents to prevent allograft rejection. This research provides the foundation for further illustrate the mechanism of NO production dependent on CD4+ T cells, also provides important experimental basis for application of PAE on transplantation. Conclusions and Significances1. We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, this study provide a rich resource to the true gene expression information in allorejection. The SAGE libraries we constructed for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries. Functional classification of these genes shows that apoptosis, transcription regulation, and cell growth and maintenance, signal transduction, oxide stress regulation frequently changed. This study provides a rich resource for the mechanism and function study in allograft rejection.2. Through functional classification, we found that several oxidative stress-related genes participated in IFN-y signal transduction pathway were differently expressed, these results suggest that IFN-y signal transduction pathway play a crucial role in the process of allotransplantat rejection.3. We evaluated the regulation and mechanism of PAE on skin allograft survival conditions first time, and application of PAE after transplantation can effectively prolong allograft survival, through down-regulating the expression of oxidative stress-related gene and protein, shifting Th1/Th2 cytokines balance. This research provides the foundation for further illustrate the mechanism of NO production dependent on CD4+ T cells, also provides important experimental basis for application of PAEon transplantation.Points of Innovation1. We used CD4+T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection to construct SAGE libraries. The SAGE libraries for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries.2. Most of the 185 genes we identified in SAGE libraries have not been studied in previous transplantation studies. The SAGE method is a sensitive and powerful tool for gene expression study. And the SAGE libraries have potential research value in transplantation. It is useful for us to find the effective way to prevent allorejection.3. We evaluated the regulation and mechanism of PAE on skin allograft survival conditions for the first time, and this research provides important experimental basis for application of PAE on transplantation. |
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