Establishment Of Humanized Mouse Model And Monitoring Of Human Cytomegalovirus

Hematopoietic stem cell transplant(HSCT) is one of effective method in the treatment of blood diseases including leukemia, myelodysplastic syndromes, aplastic anemia, lymphoma. With decades of development, HSCT have been widely used in clinical settings, and techniques and methods have been improved greatly, but post-HSCT human cytomegalovirus(HCMV) infection is one of major factor to patients survival. HCMV infection is very common in the Chinese population(95%), and the incidence rate is higher with older age and is transmitted through saliva, urine, sexual contact, and blood products. HCMV infection consisted of primary infections, recurrent infections, and latent infections. Primary infection, also called initial infection, is defined by it or its product being found in serum-negative patients. Recurrent infection is categorized by re-activation of latent virus in serum-positive patients or re-infection with different exogenous strains of HCMV. Latent infection means HCMV DNA genome persists within the host cells and virus are unable to be detected by detection methods yet under immuno-suppression HCMV can be activated and produce new infectious virus particles. HCMV disease is defined by the detection of HCMV in specimens obtained through biopsy or other invasive(such as bronchoalveolar lavage) operation with detection method of sensitivity and specificity, and by the occurrence of symptoms and(or) physical sign so infections with a corresponding organ. HCMV disease includes interstitial pneumonia, retinitis, enteritidis and other rare diseases such as hepatitis and hemorrhagic cystitis.For people with normal immune systems, HCMV infection normally occurs without any shown symptoms, but the virus is latent in your body for your whole life, and is latent in monocytes, macrophages, dendritic cells and hematopoietic stem cells in blood systems. For immunosuppression individuals(such as infants, those with immunologicdeficiency syndrome,or those receiving hematopoietic stem cell transplantation or solid organ transplantation, etc.),regardless of primary/recurrent infection, obvious symptom sever serious HCMV diseases would occur. Post-HSCT human cytomegalovirus(HCMV) infection have been common especially for patients with acute graft-versus-host disease(GVHD).45%–85% of Allo-SCT recipients experience one or more episodes of active HCMV infection within the first 100 days after transplantation. In the absence of antiviral treatment, 25%–30% of these patients will progress to HCMV end-organ disease.HCMV pneumonia is mortality disease, and the morbidity and mortality(90%) are both high. The incidence of HCMV pneumonia ranges from 1% to 6% in autologous HSCT recipients and from 10% to 30% in allogeneic HSCT recipients. Specific risk factors for severe HCMV pneumonia include lymphocytopenia, male sex, and severe acute GVHD.HCMV infection is very common in the worldwide and has arouses attention. Each year more than 130,000 patients receipt solid organ transplantation and HSCT the whole world, According to statistics,8%–32% of Kidney transplants, 22%–29% of liver transplants, 39%–41% of lung transplants and/or heart transplantation, 50% of pancreas transplantation are suffer from HCMV infection. HCMV is one of the most important infectious factors that lead to birth defects in newborns.HCMV infection associated with many kinds of cancer, such as cervical carcinoma, colorectal cancer,etc. HCMV has been implicated as a possible cofactor in the development of vascular diseases.Currently, the infection mechanisms are still not entirely clear. Antiviral drug and adoptive immunotherapy are mainly treatment, but both have its limitations. CMV is highly species specific, that is to say, mouse cannot be infected with HCMV directly and mouse Cytomegalovirus(MCMV, mouse Cytomegalovirus) is impossible to infect people. This experiment is explore CD34~+ hematopoietic stem cells from G-CSF-mobilization peripheral blood of HCMV serum-positive donor to establish the Hu-HSC-NPG mouse model, and then to detect whether the mice are infected with HCMV, In order to investigate the infection mechanisms and new treatments.1. Blood test before irradiation. 4–24 hours after irradiation, mice were injected CD34~+ hematopoietic stem cells by magnetic bead separation through bone marrow cavity. On the 10 th day after transplantation executed one of irradiation mice and normal mice and then removed the mouse femur, after fixing and making pathological slices observed effect of myeloablative conditioning. Test hemogram at different time point. After transplantation 4th, 6th, 8th, 10 th, 12 th weeks by flow cytometry monitor peripheral blood human CD45+ cells in mice blood.2. After transplantation, by PCR methods to test HCMV in mouse blood; Experimental endpoint get out liver, spleen and bone marrow cells of mice to detect CD45+ cells, CD19+ cells and HCMV DNA copies in bone marrow, liver, blood cells; Removed the lung and intestinal tissue of mice and fixed by formalin. In order to detect HCMV by immunohistochemistry method; To extracted bone marrow cells DNA, and PCR method was used to detect human Alu gene expression.Experimental results:(1) Results: Human CD34~+ stem cell(in mice) purity reached up to 96.3% by bead selection.;(2) Nucleated cells and megakaryocytes in bone marrow cavity from post-irradiation mice were significantly reduced or even disappeared, achieving the effect of myeloablative conditioning;(3) Hemogram recovered to pre-radiation level in mice from transplantation group in week;(4) Human CD45+ cells have been detected in transplanted mice at week 4,6,8,10,and12;(5) There is no HCMV detected in peripheral blood, tissue, and histopathology slices from transplanted mice.(6) All experimental mouse bone marrow cells were detected of human Alu gene expression.Conclusions: human-mouse chimeric model was successfully created in NPG mouse receiving CD34~+ hematopoietic stem cells from peripheral blood of HCMV serum positive donors. HCMV infection was not detected in any blood/tissues from transplanted mice showing that HCMV serum-positive CD34~+ hematopoietic stem cells whether or not make mouse infected HCMV need further investigation.In the past, cord blood is mainly graft to establish humanized mouse model and mouse strains were mainly selected NOD/SCID(non-obese diabetic/severe combined immunodeficiency) mice. HCMV mouse model was intraperitoneal injected with HCMV infected fibroblast cells to mice. Although this approach was feasible, but the virus was not reactivated from latent, could not provide mouse models for cellular immunotherapy. At present, NSG mouse is the most severe immunodeficiency(domestic NSG mouse known as the NPG). The innovation of this experiment is exploratory research using HCMV serum positive donor CD34~+stem cell transplant to serious immune defects NPG mouse to observe its humanized level and detect whether mice are infected with HCMV. The model can provide platform for further exploration infection mechanism, and follow-up research anti-HCMV drug and the adoptive immune treatment.