The Effect Of Estrogen On Hematopoiesis And Its Mechanism

Background:Estrogen is one kind of18carbon steroid hormone, which is produced from ovary, testis, placenta and adrenal cortex of vertebrate. The main type of estrogen in most mammals is17β-estradiol and other important types include estriol and oestrone. Estrogen is a steroid compound with various biological activities, which not only induces and maintains the functions of female genital organs and secondary sex characters, but also makes some effects on endocrine system, cardiovascular system, hematopoietic system, the metabolism of the body and the growth and maturity of bone.Hematopoiesis in bone marrow is enforced by hematopoietic stem cell and hematopoietic microenvironment and their interactions. Hematopoietic stem cell is a multipotent somatic stem cell with two characteristics, self-renewal and multilineage differentiation. On the one hand, they finish the process of self-renewal by asymmetric cell division to maintain the relatively constant volume of hematopoietic stem cells. On the other hand, they're regulated by various stoma cells and cytokines secreted of stromal cells and then they differentiate into hematopoietic progenitor cells and finally to be the mature hematopoietic cells, which release into peripheral blood to meet the body's need. The key components of hematopoietic microenvironment which plays an important role on hematopoiesis are developed from mesenchymal stem cells in bone marrow. Mesenchymal stem cell is another kind of pluripotent stem cells, which owns the same two characteristics as hematopoietic stem cells. Mesenchymal stem cells which play a support and auxiliary role on hematopoiesis can differentiate into osteoblasts, adipocytes, chondroblasts and so on under different inductions. And osteoblast is one of the important components in HSC niche. It could secret some adhersion molecules to make the hematopoietic stem cells on the surface of endosteum and then other kinds of cytokines are produced through autocrine and paracrine to regulate the number and function of hematopoietic stem cells.Recently, some researches have discovered that estrogen receptors exist on the surface of hematopoietic stem cells and mesenchymal stem cells, which indicates that there may be some internal relationship between endocrine system and hematopoietic system. On the discovery, we put forward some basic ideas on this experimental research. We further investigate the bone marrow hematopoiesis due to estrogen-associated changes in vivo and vitro, including the effects on hematopoietic stem cells and mesenchymal stem cells, and then analyse the mechanism underlying. This research makes the deeper relationship between endocrine system and hematopoietic system, and offers the new diagnosis and therapy strategy and more sufficient theoretical basis on various hematological diseases caused by the change of hormone.Part one:The Effects of Estrogen on Hematopoiesis in Ovariectomized Rats and Its MechanismObjective:To discuss the effect of estrogen on hematopoiesis in ovariectomized rats and its mechanism underlying.Method:To establish an estrogen-deficiency rat model by ovariectomy and analyse the extent of hematopoiesis failure in bone marrow from the percentages of various components in bone marrow pathological sections, such as hematopoietic tissues, adipose tissues, bone trabecular, osteoblasts and osteoclasts, and from the extramedullary hematopoiesis and the changes in peripheral blood. To identify the effects of estrogen-associated changes on the number and function of hematopoietic stem cells by flow cytometry and colony-forming analysis, and then to detect the expression of hematopoietic growth factors, such as GM-CSF, IL-3, SCF, Epo and TPO in order to further explain the effects of estrogen-associated changes on hematopoietic microenvironment.Result:by comparison, the estrogen concentration in OVX group began to decrease remarkably from the12th week after operations, and the percentage of hematopoietic tissues/megakaryocytes, bone trabecular and osteoblasts decreased significantly in bone marrow pathological slices and the adipose tissues, osteoclasts accordingly increased significantly(p<0.05), compared with the Sham group, following by the appearance of extramedullary hematopoiesis since the16week. However, began from the24th week, the differences in all indexes above between the OVX and Sham group became narrowed(p>0.05). Hb and PLT in peripheral blood began to decrease significantly since the24th week after operations and WBC decreased significantly since the28th week(p<0.05). The number of CFUs-GM and HSCs in bone marrow began to decrease significantly since the16th and20th weeks respectively, following by the obviously increased number of the HSCs in liver and spleen(p<0.05). Compared with the Sham group, hematopoietic growth factors such as GM-CSF, SCF and IL-3began to decrease in OVX group since the12th,16th and24th weeks, respectively, and Epo and TPO began to increase significantly since the12th week after operations(p<0.05).Conclusion:Bilateral ovariectomy could successfully make the establishment of the osteoporosis. Estrogen-deficiency due to ovariectomy could lead to the decrease in hematopoiesis in bone marrow, the increase in extramedullary hematopoiesis in liver and spleen and finally make the decline on peripheral blood. The hematopoiesis failure in bone marrow is concerned with the decrease in hematopoietic tissue/megakaryocytes, osteoblasts and the increase in adipose tissue, following by the decrease in the number and function of hematopoietic stem cells. At the same time, hematopoietic growth factors join in the regulation on hematopoiesis in bone marrow. SD rat with ovariectomy is a perfect model to investigate the hematopoiesis failure in bone marrow and the best age is3-9months.Part two:The Effects of Estrogen on Mesenchymal Stem Cells and Hematopoietic Stem Cells in Bone MarrowObjective:to investigate the effects of estrogen on the growth, proliferance and differentiation of mesenchymal stem cells and the ability of hematopoietic stem cells in bone marrow.Method:to obtain the mesenchymal stem cells in bone marrow by whole BM cells adhering plus density gradient centrifugation. To identify the cell surface antigens of mesenchymal stem cells by flow cytometry and to illustrate the adipogenic and osteogenic differentiation abilities of mesenchymal stem cells by Oil red O, Alizarin red dye, RT-PCR and ELISA analysis. To observe the grow curve and the effects of estrogen-associated changes on the adipogenic and osteogenic differentiation abilities of mesenchymal stem cells in vitro at the different estrogen concentrations (0,0.001nmol/L,0.01nmol/L,0.1nmol/L,1nmol/L). To detect the expressions of estrogen receptors α and β on the surface of mesenchymal stem cells under different inductions by RT-PCR. To acquire and identify the percentage of hematopoietic stem cells derived from the bone marrow by immunomagnetic microbeads and flow cytometry. To observe the hematopoietic stem cells by a microscope and analyse the ability of hematopoietic stem cells into CFUs-GM when at the different concentrations of estrogen (0,0.01nmol/L,1nmol/L).Result:Mesenchymal stem cells acquired are positive for CD29, CD44, CD90and negative for CD45and CD34. Mesenchymal stem cells are about in subround, triquetrous and irregular shapes during early culture, and in late culture they change into spindle shape or even grow in radiating. When differentiated into adipose tissues, oil red o can make the lipid droplet to be jacinth and the expression of LPL mRNA increase obviously by the detection of RT-PCR. When differentiated into osteoblasts, alizarin red dye can make the calcium nodules around the osteoblasts to be red. The decrease in adipocytes is followed by an increase in the concentration of estrogen and the number and function of osteoblasts, such as the extent of alizarin red dye and the expression of osteocalcin accordingly increases at the same condition. The expressions on estrogen receptors α and β have no significant difference between the groups with no induction and osteoblastic induction(p>0.05), and the estrogen receptor a under adipogenesis decreases significantly compared with the group under no induction and the receptor (3increases significantly correspondingly (p<0.05). Immunomagnetic microbeads could acquire the hematopoietic stem cells at the greatest extent. The shape of hematopoietic stem cells is small, subround or ellipitical and nucleolus at different size could also be seen by a microscope. The ability of hematopoietic stem cells proliferates and differentiates in to CFUs-GM has a positive correlation with the concentration of estrogen. That means, when estrogen increases, the number of CFUs-GM could increase accordingly.Conclusion:Mesenchymal stem cells derived from bone marrow in rats could be successfully separated, purified, cultured and passaged in vitro using whole BM cells adhering plus density gradient centrifugation. Estrogen could induce the mesenchymal stem cells to incline the osteogenesis and the adipogenesis would be reduced accordingly. The expressions of estrogen receptors a and (3make no difference between the groups with no induction and osteoblastic induction, and the estrogen receptor a under adipogenesis decreases significantly compared with the group under no induction and the receptor β increases significantly correspondingly. The purification of hematopoietic stem cells could be increased in vitro by immunomagnetic microbeads. Estrogen could make some effects on the proliferation and differentiation of hematopoietic stem cells. When estrogen increases, the number of CFUs-GM increases accordingly.