Study Of The Effectivenesss Of The Leptin On The Hematopoiesis By Leptin Gene Knockout And Its Mechanism In Aplastic Anemia Mouse Model

Aplastic anemia(AA)patients are characterized by excessive adiposity in bone marrow,increased lipogenesis of mesenchymal stem cells and accumulation of leptin(LEP)in the microenvironment of bone marrow.LEP is a kind of cytokine like hormone encoded by the obesity gene,synthesized and secreted by fat cells.By binding to leptin receptors(LEP-R)it plays a variety of biological functions,such as regulating the body’s food intake,energy expenditure and fat storage.LEP can also be secreted by adipocytes in the microenvironment of bone marrow and can act on MSC to induce osteogenic differentiation and inhibit adipocyte differentiation as well.However,LEP does not play the roles mentioned above in AA hematopoietic microenvironment.It was concluded that MSC failed to use LEP in the microenvironment of AA bone marrow.That’s probably because that LEP cannot be used normally due to the destruction of LEP-R on the MSC,leading to increased adipogenic differentiation of MSC and decreased osteogenic differentiation,and excessive adipogenesis of bone marrow as well.LEP has immunomodulatory function of activating lymphocytes.The continuous accumulation of LEP in the bone marrow microenvironment of AA patients can activate T lymphocytes and induce their differentiation to Th1,which is consistent with the pathogenesis of immune-mediated AA.The activation of lymphocytes by LEP will expand the immune abnormality of AA,and then aggravate the immune injury of MSC,leading to a vicious cycle.It is concluded that LEP plays an important role in the immune abnormality of AA and the process of lipogenesis as well as osteogenesis disorder,which may be one of the reasons for the immune pathogenesis of AA,and may also be one of the reasons for the poor therapeutic effect of immunosuppressants.The production of LEP and its activation of T lymphocytes are important steps in the pathogenesis of AA.If the synthesis of LEP can be blocked or its immunomodulatory effect can be antagonized,it is expected to break the vicious cycle of immune disorder,so as to reduce the occurrence and development of AA.In this study,in vivo and in vitro experiments were conducted on mice with immune mediated LEP gene deficiency and normal mice AA model.The differences between the two mouse models and between their respective control groups are measured and compared at the cellular,molecular,and genetic levels to verify the hypothesis of failure using LEP and amplification of immune signal by LEP.We hope to block the immune injury process of AA through LEP intervention,so as to lay a foundation for seeking a new method of AA treatment from the hematopoietic microenvironment metabolism level.Part One:Effect of leptin gene knockout on aplastic anemia mouse model hematopoiesisObjectives:To determine the general state of mice,bone marrow failure and the degree of bone marrow adipogenesis at different time points during the AA process between the LEP-deficient mouse AA model and the normal mouse AA model.Methods:1.Immune-mediated methods were used to establish AA models of LEP gene-deficient ob/ob mice(C57BL/6 mice are used as modeling basis,hereinafter referred to as LEP-deficient mice)and AA models of normal C57BL/6 mice(hereinafter referred to as normal mice),respectively.LEP-deficient C57BL/6 mice and normal C57BL/6 mice were used as controls model mice,respectively.2.The general status of mice were observed on 0,3,6,9,12,15 and 18 days after modeling,respectively.3.The blood routine of mice were detected on 0,3,6,9,12,15 and 18 days after modeling,respectively.4.The pathological morphological changes of bone marrow were observed on 0,3,6,9,12,15 and 18 days after modeling,respectively.The above indexes of LEP-deficient mice model and normal mice model were compared at different time points by statistical method.Results:1.After modeling,normal mice gradually showed reduced food intake and activity,wrinkles,depilation,arched back and weight loss.The LEP gene deficient mice also showed the above phenomena after modeling,but to a lesser extent.2.Quantity of white blood cell(WBC),red blood cell(RBC)and platelet(PLT)were decreased rapidly in both LEP gene deficient and normal mice after modeling and the difference was statistically significant(P<0.05).However,the decline of WBC,RBC,and PLT of LEP gene deficient mice model were significantly less than those of normal mice model,with a higher minimum blood count than those of normal mice model.There were statistically significant differences between the two groups(P<0.05).3.After modeling,normal hematopoietic tissue structure in bone marrow of normal mouse model group was gradually destroyed,hematopoietic cells were gradually reduced,and was replaced by adipose tissue.The bone marrow was almost filled with fat cells on+18d.The hematopoietic structure of LEP defect model group mice were also gradually destroyed.From+6 days bone marrow hyperplasia were reduced,with a small amount of fat cells,and hematopoietic area reduced.Subsequently bone marrow hematopoietic cell decreases gradually,adipose aggravates gradually.Up to+18d hematopoietic cells were replaced by fat cells.But it is less destructive and fatty than normal mice model.Conclusions:In comparison with the normal mice AA model,LEP gene deficient mice AA model possessed less bone marrow adiposis and lesser bone marrow failure,which suggested that blocking LEP gene may improve hematopoiesis of AA bone morrow by reducing bone marrow adiposis.Part Two:Mechanism Investigation of leptin gene knockout on aplastic anemia mouse model hematopoiesisObjectives:To detect the expression of adipogenic genes(C/EBPα、C/EBPβ、PPARγ)and osteogenic genes(RUNx2)in bone marrow tissues at different time during the AA process between the AA model of LEP deficient mice and the AA model of normal mice;To detect changes and differences of LEP-R expression in serum,bone marrow and MSC at different time during the AA process between the AA model of LEP deficient mice and the AA model of normal mice;To detect the immune signal amplification conditions such as inversion of CD4+T/CD8+T ratio and the increase of Th1/Th2 at different time during the AA process between the AA model of LEP deficient mice and the AA model of normal mice.Methods:1.Immune-mediated methods were used to establish AA models of LEP gene-deficient ob/ob mice(C57BL/6 mice are used as modeling basis,hereinafter referred to as LEP-deficient mice)and AA models of normal C57BL/6 mice(hereinafter referred to as normal mice),respectively.LEP-deficient C57BL/6 mice and normal C57BL/6 mice were used as controls model mice,respectively.2.The proportion of subsets of CD4~+and CD8~+cells were determined by flow cytometry on 0,3,6,9,12,15 and 18 days after modeling,respectively.3 The changes of soluble leptin receptor(sLEP-R),cytokines of Th1 such as IFN-γ,IL-2 and cytokines of Th2 such as IL-4,IL-5 in mice serum were detected by enzyme-linked immunosobent assay(ELISA)on 0,3,6,9,12,15 and 18 days after modeling,respectively.4.LEP-R in mice bone marrow was measured by immunohistochemistry on 0,3,6,9,12,15 and 18 days after modeling,respectively.5.Real-time fluorescence quantitative PCR were used to detect the expression levels of LEP-R on the MSC of mice bone marrow,as well as the expression levels of adipogenic genes(C/EBPα,C/EBPβ,PPARγ)and osteogenic genes(RUNx2)in mice bone marrow on 0,3,6,9,12,15 and 18 days after modeling,respectively.6.MSCs were isolated and cultured,and the LEP-R expression levels of MSCs were detected by real-time quantitative PCR on 0,3,6,9,12,15,18 days after modeling.The above indexes of LEP-deficient model mice and normal model mice at different time points were compared by statistical method.Results:1.The expressions of the adipogenic genes C/EBPα,C/EBPβ,PPARγshowed a gradually increasing tendency in bone marrow of both the LEP gene deficient mice model and the normal mice model.The difference was statistically significant(P<0.05).However,the LEP gene deficient mice model had a low expression level of adipogenic genes than the normal mice model.There were statistically significant differences between the two groups(P<0.05).The expressions of the osteogenic genes RUNx2 showed a gradually decreasing trend in bone marrow of both the LEP gene deficient mice model and the normal mice model.The difference was statistically significant(P<0.05).However,the LEP gene deficient mice model had a high osteogenic genes than the normal mice model,and the difference was statistically significant(P<0.05)..2.The levels of sLEP-R in serum of showed a gradually decreasing trend in bone marrow of both the LEP gene deficient mice model and the normal mice model.The difference was statistically significant(P<0.05).But the level of the LEP-deficient mice model was higher than that of the normal mice model,and the difference was statistically significant(P<0.05).The expression of LEP-R in bone marrow presented a decreasing trend after modeling in both LEP gene deficient mice model and normal mice model,but the decreasing trend of the normal mice model was more serious than that of the LEP gene deficient mice model.The LEP-R expression of MSC showed a gradual downtrend in both LEP gene deficient mice model and normal mice model,respectively.The difference was statistically significant(P<0.05).While the degree of LEP-R downtrend of the LEP gene deficient mice model is less than those of the normal mice model,and the difference was statistically significant(P<0.05).3.After modeling,in both LEP gene deficient and normal mice,the value of CD4~+T cell presented a gradual downtrend while the value of CD8~+T cell presented a gradual uptrend,so the ratio of CD4~+/CD8~+in both model mice were inverted.The difference was statistically significant(P<0.05).But the CD4~+/CD8~+ratio inversion of the normal mice model was less than that of the LEP gene deficient mice model,and the difference was statistically significant(P<0.05).4.Both the LEP gene deficient mice model and the normal mice model showed a situation that Th1 associated cytokines(IFN-γ,IL-2)showed a gradually increasing trend and Th2 associated cytokines(IL-4,IL-5)showed a gradually decreasing trend.The difference was statistically significant(P<0.05).But the degree of improvement of IFN-γand IL-2 and decline of IL-4 and IL-5 of the LEP gene deficient mice model were less than those of the normal mice model,and the difference was statistically significant(P<0.05).Conclusions:1.In comparison with the normal AA mice model,LEP gene deficient mice AA model showed less bone marrow adiposis,increased osteogenic gene expression,decreased adipogenic gene expression and decreased LEP-R,which suggested that disuse of LEP in the bone marrow microenvironment led to excessive adipose of the bone marrow2.The increase of Th1/Th2 ratio of LEP gene deficient mice AA model was less than that of the normal AA model mice,which suggested that blocking LEP gene may hamper the immune signal amplification effect and inhibit the occurrence and development of AA.3.This study proposed that LEP metabolism disorder caused AA bone marrow fat and immune signal amplification for the first time.So reducing the expression of LEP gene,increasing the level of LEP-R,or increasing the sensitivity of the combination of LEP-R and LEP may be new ideas and methods for AA treatment.