The Effects Of Growth Hormone On The Formation Of Human Lympho-hematopoietic System In Humanized Mice

Objective:The differences between species resulted in the inability to accurately predict human physiology,pathology,and to evaluate the drug effectively,through the traditional animal model.A humanized mouse model that human embryonic thymus and fetal liver hHSC were transplanted to immunodeficient mice was internationally recognized as the effective tool to study human immunological problems and diseases.However,some of the hematopoietic and immune system development-related factors secreted by mice and human cells have no response or reaction with each other,and the human immune system composition and function in humanized mice are not perfect until now and needs to be further optimized.As an important component of the neuroendocrine system,growth hormone(GH)has been shown to be closely related to thymus development and lymphoid hematopoietic cell differentiation.Studies have shown that mouse growth hormone(mGH)and human growth hormone receptor(GHR)without cross-reaction,which may be the limitation of human immune system reconstruction in humanized mice.Therefore,it is important to study the effect of human growth hormone on the human immune system in humanized mice and its mechanism,and to optimize the humanized mouse model.This study was designed to optimize the human immune system in humanized mice by recombinant human growth hormone(rhGH).Meanwhile,the effect of rhGH on the proliferation and differentiation of human hematopoietic stem cells(hHSC)was investigated by means of humanized mouse model.Methods:(1)The HSC expansion system in vitro was constructed,and the effenciency of humanized mice as grafts into another group of immunodeficient mice.To investigate the effect of rhGH on the reconstitution of human hematopoietic system in humanized mice by secondary transplantation of hHSCs.Results:(1)HSC amplification efficiency in rhGH group was significantly weaker than SCF group.The effect of rhGH with different concentrations on hematopoietic stem cell proliferation efficiency was different: low concentration(10ng/ml)group could promote hematopoietic stem cell proliferation,moderate and high concentration of rhGH did not promte or inhibit the proliferation of hHSCs.Low concentration rhGH in combined with SCF could further enhance the expansion of hHSCs.(2)The state of mice and the hematopoietic tissue of bone marrow in rhGH group recovered faster after the construction.The proportion of leukocytes expressing CD45 in peripheral blood of humanized mice in rhGH group was significantly increased at 3rd weeks,6th weeks and 9th weeks after construction,which was about 2 times of that of control group.The number of cells in each subsets of immunocytes increased,and the proportion of them in the human WBC showed no significantly difference between rhGH group and control group.The results of those cells in spleen and bone marrow were consistent with peripheral blood at the 10 th week.(3)The percentage of T cells and their subgroups in the spleen,peripheral blood,thymus and bone marrow of humanized mice were measured at the 10 th week after the human immune system construction.Only the newly generated Th cells of the thymus in rhGH group were less than the control group.The proportion of the remaining T cells showed no significant difference between two groups.(4)The percentage of total B cells in the spleen,peripheral blood and bone marrow of the rhGH group was significantly higher than that in control group.The Pro-B cells in the bone marrow were higher than those in the control group.In peripheral blood and spleen,the ratios of Pro-B cells and Pre-B cells in rhGH group was higher and immature-B cells was lower comparing with control group,the tend of mature-B was opposite.(5)Total red blood cells and the erythrocytes of different stage in bone marrow were detect at the 10 th week after humanized mice construction,it was found that the RBC reconstruction level increased in rhGH group comparing with the control group.Also,the ratio of juvenile big-red blood cells increased.The ratio of proerythroblasts,intermediate or late erythroblast was higher than that of the control group,but the ratio of reticulocytes and mature red blood cells showed no difference between two groups.In addition,human erythrocytes were not detected in peripheral blood.(6)In vitro experiments confirmed that rhGH promoted hHSC proliferation,while the proportion of continuous multi-potential hHSCs compared with the blank control group did not change,in vivo experimental also confirmed that rhGH did not change the proporation of those hHSCs in bone marrow of humanized mice.Secondary transplantation humanized mice model also confirmed the above findings.Conclusions:The rhGH could increase the proliferation effeciency of hHSCs and promote the reconstruction of human lympho-hematopoietic system in humanized mice,without influencing the ablility of continuous multi-potential differention of h HSCs,and finally optimize the humanized mice model:(1)rhGH could promote the reconstruction of human leukocytes(mainly the B lymphocyte)in humanized mice bone marrow,spleen and peripheral blood,especially increased the ratio of Pro-B,Pre-B cells.(2)rhGH could promote the reconstruction of human erythrocytes,including the proerythroblasts,intermediate and late erythroblast cells.