| Background:Mesenchymal stem cells (MSCs) are multipotential stem cells with the capacity for self-renewal and multilineage differentiation, which can be isolated from various tissues, such as bone marrow, umbilical cord, umbilical cord blood, placental and so on. These cells have emerged as powerful tools in tissue engineering and regeneration, because they have strong capacity for expansion and differentiation into multiple cell types like osteoblasts, fat cells, chondrocytes, et. Different sources of MSCs have different biological characteristics. So they are usually used in different fields when applied to basic and clinical research. Bone marrow mesenchymal stem cells (BM-MSCs) are easily to differentiate into adipocytes, which makes they the best candidate for adipose tissue regeneration research. From a clinical standpoint, soft tissue damaged patients are afflicted with tissue resorption. For example, burn patients and HIV patients often suffer from soft tissue atrophy, which severely impact the quality of life. Depending on the resources and application, the culture methods to induce adipocyte differentiation of BM-MSCs differ from one another. This kind of situation greatly hinders the research of adipocyte differentiation of BM-MSCs, become of the lack of interpublication comparability. At the sometime, the current culture system take a lot of time and money, so it is necessary to optimize the adipocyte differentiation culture method. This research aim at comparing the adipogenic feature of BM-MSCs under different culture systems and the efficiency of three culture systems, which provide experimental evidence for optimizing the the induction methods.Object:(1) Comparing the adipogenic feature of BM-MSCs under different culture systems. (2) Comparing the efficiency of three culture methods and optimizing the method to induce adipocyte differentiation of BM-MSCs.Methods:(1) BM-MSCs were induced to differentiate into adipocytes by three culture methods separately. (2) PPARγ, FABP-4, adiponectin, leptin, C/EBPa, LPL mRNA were detected by Real-time PCR at different time point during adipocyte induction. (3) The secretion of adiponectin in supernatant were detected by ELISA. (4) To visualize lipid accumulation over the differentiation time course, cells were stained with Oil-Red O and examined using confocal fluorescent microscope.Results:(1) BM-MSCs expressed different level of PPARγ, FABP-4, adiponectin, leptin, C/EBPα, LPL mRNA under different culture systems. (2) Adiponectin section increased gradually during the adipocyte induction time course and the BM-MSCs cultured in DIMR produced more adiponectin than the others. (3) The efficiency of three induction system varied from each other. The DIMR treated BM-MSCs accumulated lipid droplets in only 7-8 days. (4) In early periods, DIMI treated BM-MSCs expressed more PPARγ protein than the others. And the PPARγ protein did not increased continuously during the induction.Conclusion:(1) Different culture methods treated BM-MSCs showed different features. (2) DIMR culture system showed higher induction efficiency than the others and the period of cultivation could be shortened to 7-8 days. (3) The PPARγ protein level could not be a good marker to evaluate the adipogenic differentiation of BM-MSCs under DIMR culture system.Background:Aplastic anemia (AA) is a bone marrow failure syndrome characterized by bone marrow aplasia and peripheral blood pancytopenia. It has a complex pathophysiology. A large amount of laboratory and clinical data suggest that immune-mediated suppression of haematopoiesis plays an essential role in the pathogenesis of acquired AA (a AA). However, recent studies provide evidence that bone marrow mesenchymal stem cells (BM-MSCs) from patients with aAA carry intrinsic deficits, such as poor proliferation potential, deficient support of hematopoietic colony-forming activity and poor immunosuppressive deficiency potential, which contribute to their vulnerability in developing bone marrow failure. In conclusion, BM-MSCs maybe play a role in the pathogenesis of AA.Leptin is a kind of active biological molecule, mainly produced by white adipocytes. It can regulate energy metabolism and immune function by acting on leptin receptor (Lep-R).Leptin seems to act as a proinflammatory factor to promote Thl response. Animal models show that leptin is associated with some autoimmune diseases, such as Rheumatoid Arthritis(RA), multiple sclerosis and so on. In haematological diseases, leptin is at high level in bone marrow of AML and ALL patients, and the high expression of Lep-R maybe enhance the engraftment of leukemia cells to bone marrow. Until now, there are no research about the relationship between leptin and aplastic anemia. Thus, in here, we investigate the leptin level in bone marrow plasma and BM-MSCs derived from AA. Further more, we explore the effect of pro-inflammatory (IL-1β and TNF-α) on leptin secretion during adipogenesis in vitro.Objective:To investigate the relationship between leptin and aplastic anemia, and the new mechanism of BM-MSCs in the pathogenesis of aplastic anemia.Methods:(1) Leptin level in bone marrow plasma was detected by ELISA. (2) BM-MSCs were isolated and purified from bone marrow by adherent culture and passsaging in the culture medium (DMEM/DF12 and 2% fetal bovine serum) in vitro. (3) The immunophenotype markers of BM-MSCs were detected using flow cytometry. (4) BM-MSCs were induced to differentiate into adipocytes. (5) The expression of leptin and leptin receptor mRNA in BM-MSCs were detected by Real-time PCR. (6) The leptin level in supernatant was detected by ELISA. (7) ELISA and Real-time PCR were used to detect the leptin expression when BM-MSCs were cultured with IL-1β and TNF-a.Results:(1) Leptin was higher in bone marrow plasma of aplastic anemia. (2) Leptin mRNA level and secreted leptin level were higher in BM-MSCs of aplastic anemia. (3) Proinflammatory factor, IL-1β and TNF-a, were able to prevent adipogenesis, but enhance the secretion of leptin. (4) BM-MSCs derived from AA were more easily to differentiate into adipocyte than normal control. (5) Leptin receptor mRNA level were higher in BM-MSCs of aplastic anemia.Conclusion:(1) Leptin in bone marrow plasma of aplastic anmia is higher than normal control. (2) High leptin level in BM-MSCs of AA maybe contribute to the high leptin level in plasma. (3) Proinflammatory factor maybe the reason that makes BM-MSCs express high level leptin. (4) BM-MSCs expressed higher level Lep-R maybe the mechanism that they are more easily to differentiate into adipocytes. |
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