Establishment Of Humanized Mouse Model And Its Preliminary Exploration In Gene Therapy And The Clinical Significance Of Interleukin-35 In Diagnosis Of Early-onset Neonatal Sepsis

PART Ⅰ ESTABLISHMENT OF HUMANIZED MOUSE MODEL AND ITS PRELIMINARY EXPLORATION IN GENE THERAPYBackground: Humanized mouse models are widely used in the study of human physiopathological mechanisms and evaluation of clinical efficacy of drugs.After nearly three decades’ development,humanized mouse models have become increasingly mature.New immunodeficient mice strains with IL2 rg and prkdc/rag mutations,which lack T,B,and NK cells are the most commonly used humanized mouse models.Gene therapy is the first choice for the patients with primary immunodeficiency disease who do not have suitable donor.Preclinical evaluation of safety and efficacy in vitro and in vivo of humanized mice is required prior to clinical application.Methods: Discarded bone marrow specimens of children with juvenile idiopathic arthritis were collected,and then CD34+ cells were sorted by CD34+ magnetic beads.8 to 9 weeks old NOD-Prkdc^scid Il2rg^null（NPG）female mice were irradiated and then transplanted with the 1-5x105 CD34+ cells via the tail vein for human immune system reconstruction.Four weeks after transplantation,Flow cytometry was used to detect the implantation of human CD45 cells in blood.The mice were sacrificed 12-14 weeks after transplantation.Flow cytometry was used to analyze the subpopulations of immune cells.Tissue sections were used to observe the morphology of spleen and thymus.CD34+ cells were transduced with GFP lentivirus.After the transduction,the efficiency was analyzed by flow cytometry.The colony formation assay was also performed after transduction.The cell differentiation was observed in NPG mice.Results: The purity of CD34+ cells was higher than 90%.The implantation of human CD45+ cells could be found in peripheral blood of NPG mice 4 weeks after transplantation,accounting for 2.81%±2.27%（n=9）of peripheral blood leukocytes.The chimeric rate of CD45+ in peripheral blood increased significantly after 6 weeks and maintained at a constant level.Twelve weeks after transplantation,in bone marrow（43.57%±16.52%,n=6）and thymus（98.12%±1.75%,n=6）,higher human CD45+ cells were implanted.Cells in blood,spleen,and bone marrow were mainly CD19+B cells,mostly Ig D+CD27-naive B cells.CD24int/hiCD38 hi immature B cells were accumulated in the spleen.Most of the B cells showed CD10+ immature phenotypes.CD19+Ig M-CD20-CD34+ pro-B,CD19+Ig M-CD20-CD34-pre-B cells,and CD19-CD10+CD38+CD34+ common lymphoid progenitors was detected in the spleen.Human CD4+ and CD8+ T cell subsets were detected in the thymus of mice.The TREC value of thymocytes was 4560±2907（n=3）.Plasma Ig M concentration was 932.9±596 ng/m L（n=9）at 12-14 weeks after transplantation and Ig G was not detected.After sorting,the CD34+ cells were transduced with GFP lentivirus.The GFP gene-labeled CD34+ cells reached 30%,and the vector copy number was 0.55.After transduction,63 colonies were formed per 1000 cells,which was comparable to the un-transduced group.After transduction,cells were transplanted into NPG mice and developed into all lineage cells.Conclusion: The use of bone marrow-derived CD34+ cells can reconstitute the human immune system in NPG mice.The B cells were partially differentiated and have abnormal functions.T cells can hardly migrate to the periphery in the thymus.The efficiency of stem cells transduced with GFP reached the requirement of gene therapy and the ability of colony formation was not compromised.The transduced stem cells’ differentiation was confirmed in vivo.Part Ⅱ THE CLINICAL SIGNIFICANCE OF INTERLEUKIN-35 IN DIAGNOSIS OF EARLY-ONSET NEONATAL SEPSISBackground: Early onset sepsis（EOS）remains a major cause of morbidity and mortality in newborns.However,current diagnostic tools are inadequate.We evaluated the accuracy of a novel cytokine,interleukin（IL）-35,for the diagnosis of EOS in comparison with other infection markers.Methods: One hundred fifty-seven neonates with suspected sepsis in the first 3 days of life were enrolled in this perspective study.All enrolled patients were divided into infected group and unlikely infected group according to clinical data.IL-35,C-reactive protein（CRP）,procalcitonin（PCT）,white blood cell（WBC）count,and blood culture were measured once the suspected EOS was documented.Results: Serum concentration of IL-35 was increased significantly in the infected group compared with the unlike-ly infected group（median 36.4 versus 27.1 pg/ml,respectively,p b 0.001）.The ROC curve value were 0.756 for IL-35,0.713 for PCT（age-adjusted）,0.670 for CRP,and 0.619 for WBC.With a cut-off value of 31.7 pg/ml,the diagnostic sensitivity and specificity of IL-35 were 78.48% and 66.67%,respectively. Moreover,unlike PCT concentration,IL-35 concentration did not fluctuate in neonates who were unlikely to be infected（p = 0.885）.Conclusion: The diagnostic performance of IL-35 was superior to that of PCT and other commonly used markers,suggesting that IL-35 may be a valuable tool for EOS diagnosis.