Roles Of Astrocytes In Bone Marrow Stromal Cell Proliferation And Differentiation

In recent decades,the incidence of diseases of central nervous system (CNS) such as traumatic brain or spinal cord injury and various neurodegenerative diseases has increased greatly,while the CNS of adult mammals has poor regenerative abilities.At present,it is obliged to adopt the symptomatic treatment for various CNS diseases and there is no effective treatment in clinic.In recent years,with the presention of the concept of stem cells,transplantation of stem cells to repair various CNS injuries or neurodegenerative diseases becomes a promising treatment.The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types.Bone marrow stromal stem cells(BMSCs) are a population of stem cells derived from bone marrow. BMSCs have abundant sources,with easy operation and when autologous transplanted,there exist no immunorejection and thical issues.So the BMSCs become one of the most advantage seed cellsRecently,it has been found that BMSCs are not only capable of differentiating into a wide variety of cell types derived from mesenchym(for example,osteoblast,fibroblast,adipocyte),but also can transdifferentiate into neural cells under proper conditions in vitro.Furthermore,they can differentiate into various types of neural cells in different regions (microenvironment).So the BMSCs become one of the most advantage seed cells for studying the nerve tissue engeneering.Astrocytes,as the most plentiful residential cells in the CNS,are also the major component of local microenvironment in the CNS.Recent work found that in addition to the traditional accepted functions that providing nutrition, support,protection,isolation to the neurons,astrocytes can actively participate and play an important role in the development of neuron,synapsegenesis and neurogenesis.Lots of studies showed that once the CNS was injuried or damaged,the inflammation of the CNS was very obvious,while the astrocytes were prominently activated and expressed a serial of factors,which played key roles in the CNS.However,it has remained to be ascertained if inflammation-activated astrocytes can affect the behavior of BMSCs in the inflammatory microenvironment of CNS.In view of the complicated changes of cellular microenvironment and various impact factors under in vivo conditions,so it is important to simulate the in vivo microenvironment and further study the functions of astrocytes and the survival,proliferation and differentiation of BMSCs under inflammatory conditions using the in vitro model.一,Isolation,culture and expanding of BMSCsThe number of BMSCs is estimated to be too few in bone marrow(2-5 BMSCs per 10⁵ mononuclear cells),so it is important to expand and purify the BMSCs for the following experiments.BMSCs were isolated from adult rats using density centrifugation and anchoring culture,then cultured in low-glucose DMEM supplement with 10% fetal bovine serum(FBS) for expanding.Colonies of BMSCs were formed at 7^th day at primary culture,at about 10^th day,BMSCs got together and were subcultured by 0.25%trypsine-0.02%EDTA.After three passages,BMSCs became relatively homogeneous in appearance,presenting a more spindle-shaped,fibroblastic morphology.Because the antigenic profile displayed by BMSCs is not unique,thus it has been accepted that several antigens were detected to identify BMSCs.The results of fluorescent cell sorting at passage 3 demonstrated that the majority of the cells were positive for CD29 and CD90.二,Effects of ACM at different inflammatory states on the proliferation and neural differentiation of BMSCsTo understand the effects of astrocytes on the BMSCs, astrocyte-conditioned medium(ACM) was prepared in this study for treatment of BMSCs.The ACM derived from astrocytes stimulated by lipopolysaccharide for 12,36 or 72h,respectively,served as inflammatory ACM(12h ACM,36h ACM and 72h ACM),while that from unstimulated astrocytes was used as normal control ACM(N-ACM).Then BMSCs were treated with different ACM and their proliferation and differentiation were observed.First,the effects of cell proliferation showed that the inflammatory ACM(12h ACM,36h ACM and 72h ACM) obviously promoted the proliferation of BMSCs in comparison with N-ACM,and the effects of 36h ACM were significantly stronger than those of 12h or 72h ACM.Secondly,the effects of neural differentiation by immunocytochemistry showed that compared with N-ACM, the proportion of neural differentiation(both neuron and astrocytes) in BMSCs treated with inflammatory ACM was markedly elevated(p＜0.01).Besides,the effects of 36h ACM on the neuronal differentiation of BMSCs were evidently greater than that of 12h or 72h ACM(p＜0.05).However,there was no obvious expression change in astrocytic differentiation among 12h,36h and 72h ACM (p＞0.05).The results of Western blotting showed that the expression change of NSE protein was consistent with the immunostaining results.三,The mechanism study of ACM' roles in the proliferation and neural differentiation of BMSCsAlthough the studies above demonstrated that the factors produced by astrocytes play key roles in modulating the fate specification of BMSCs,the detailed molecules involved in the process are still unknown.In this connection, attention has been drawn to interleukin-6(IL-6).Previous studies showed that IL-6,as a pleiotropic inflammatory factor,its expression was significantly increased in various CNS disorders.Furthermore,some studies have extended that IL-6 can affect the survival of neuron,axonal regenenation and neurogenesis.However,it has remained to be elucidated whether IL-6 participates and plays an important role in astrocyte-regulated proliferation and neural differentiation of BMSCs.To explore the functions of IL-6,the content of IL-6 in the ACM was detected by Elisa assay firstly,It showed that the IL-6 produced by astrocytes obviously increased once the astrocytes were stimulated by LPS,and the content of IL-6 in 36h ACM was significantly higher than that of 12 or 72h ACM.The results of expression of IL-6 mRNA of astrocytes by RT-PCR were consistent with that of Elisa.Moreover,following neutralization of interleukin-6 IL-6 of the ACM, both the proliferation and astrocytic differentiation of BMSCs were decreased; on the other hand,the neuronal differentiation was significantly increased. In conclusion,the present findings suggest that inflammation-activated astrocytes can facilitate the proliferation and neural differentiation of BMSCs and the functions have been closely associated with different phases after CNS injuries.Moreover,astrocyte-derived IL-6 participates in the proliferation and neural differentiation of BMSCs.