Research Of Bone Marrow Hematopoietic Cells Inhibiting The Differentiation Of Mesenchymal Stem Cells Into Adipocytes

The first partAims:Two ways were used to injure hematopoietic cells in mouse bone marrow, whichincluded non-myeloablative total body irradiation and chemicals. The pathologicalchanges of bone marrow in mice were observed, and compared among differenttypes of myeloablative drugs. Its aims are to explore the effects of different lines ofhematopoietic cells on regulating the differentiation of bone marrow mesenchymalstem cells and their related mechanisms.Methods:1.8-week-old female specific pathogen free （SPF） Balb/c mice were dividedrandomly into normal group, radiation group and transplantation group. Radiationgroup and transplantion group mice received6.0Gy⁶⁰Coγ radiation; after radiation,transplantion group mice were injected with bone marrow mononuclear cells throughtail vein. At day3,6,9,12, and15, general condition, peripheral blood cells, bonemarrow histological changes, and the amount of the bone marrow adipose, PPARγ、CEBPα and other signal molecules expression in bone marrow were observed.2. The chemical experiment included control group （I）, busulfan （Bu） group （II）,fluorine fludarabine （Flu） group （III）, cyclosporine （CsA） group （IV）, combinedmyeloablative group （V）, transplantation group （VI）, granulocyte colony-stimulatingfactor （G-CSF） group （VII）, and PPARγ inhibitor group （VIII）. At day3,6,9,12,15,18,24, and36, related indexes were analysed.Results:1. Compared with normal mice, the general state of radiation group mice were poor,the hematopoiesis decreased to a very low level for2weeks, and the amount ofadipose tissue increased significantly in bone marrow; Compared with radiationgroup, the hematopoiesis in transplantion group mice was more active, the amount offat tissue was lower. These results suggest that hematopoietic cells improve the bone marrow microenvironment.2. The general condition of group III and IV mice was fine, peripheral blood cellscount fell slightly, and recovered rapidly, and had little fatty tissue in bone marrowcavity, indicating that fludarabine and cyclosporine which suppressed lymphocytesdo little effect on the bone marrow microenvironment. The general condition ofgroup II, V, and VI mice were bad, mice were suffered from severe bone marrowsuppression, peripheral blood cells indicators increased slowly, and a large numberof fat cells appeared in the bone marrow, indicating that busulfan increased theamount of bone marrow fat cells through suppressing granulocytes. Combinedmyeloablative treatment cause great damage to the hematopoiesis. Group VII andVIII mice were in poor condition during the first week, peripheral blood cellsindicators were low. Group VII mice began to recover after6days, group VIII after9days. The adipose tissue in bone marrow cavity in group VII and VIII were morethan group III and IV, but less than group II, V, and VI. GCSF can increase thenumber of granulocytes, which improved the bone marrow microenvironment.PPARγ inhibitors inhibit the formation of fat cells, which are beneficial to thehematopoietic reconstruction.Immunohistochemistry results showed that there were more PPARγ positivesignalings in radiation group and group V than Group VII and VIII. Similarly, therewere less PPARγ positive signalings in normal group mice bone marrow cells thanradiation group and myeloablative group.Conclusion:Hematopoietic cells in the bone marrow can regulate the differentiation ofmesenchymal stem cells, which means hematopoietic cells; especially granulocytescan inhibit MSCs differentiating into adipocytes. The second partAims:To observe whether myeloid precursor cells （32D） inhibit the differentiation ofpreadipocytes （3T3-L1） into a mature adipocyte through3T3-L1and32D cellsco-culture, and to explore related mechanisms.Methods:The experiment included five groups: the control group （3T3-L1）（A）, directco-culture group （3T3-L1+32D）（B）, indirect co-culture group （3T3-L1+32D）（C）,conditioned media group （3T3-L1）（D）, and positive control group （3T3-L1）（E）.Adipogenic differentiation experiments were carried among group B, C, D, and E. Atday8, mature adipocytes were observed by oil red O staining, RQ-PCR and Westernblot were uesd to analyze the expression of PPARγ, CEBPα, DLK1, FABP4, andWNT signal pathway molecules.Results:Experimental results showed that co-culture of32D cells and3T3-L1couldinhibit3T3-L1cells differentiating into mature adipocytes, especially in directcontact co-culture system. Indirect contact could also inhibit the differentiation andmaturation of preadipocytes, but the effect was weaker. RQ-PCR results showed thatFABP4and FASN mRNA expression levels in group B and C were significantlylower than in group E. CEBPα and PPARγ mRNA expression levels were also lower,but DLK1and RUNX2mRNA expression levels were higher. β-catenin and AxinmRNA expression levels in group B and C were higher than Group E, Western blotresults were in accordance with RQ-PCR results.Conclusion:Myeloid precursor cells inhibit the preadipocyte differentiating into a matureadipocyte by cell-cell direct contact or indirect contact, certain genes and signalpathways may be involved in the regulation.