Promoting Proliferation And Mechanism Of CXCL12 On Rat Oligodendrocyte Precursor Cells

Background:Spinal cord injury(SCI)is a trauma that difficult to be treated with high incidence in the central nervous system(CNS).It used be results in severe impairment of sensory and motor below the level of injury,urination and defecation dysfunction.With the development of social economy and transportation industry,the incidence of SCI is increasing year by year,which seriously affects the life of patients.It also brings a lot of serious social and economic burden to the patient,family,and even the country.At present,the research of SCI has made some encouraging results,but in the actual clinical work,the treatment effect of SCI is still not very good.The treatment of spinal cord injury is still a problem to be overcome in the medical field.Visible,to carry out the treatment of SCI research is an urgent event which has a scientific and social significance.SCI is a long-term complex damage process: including the primary injury that direct mechanical violence itself on nerve cells,glial cells,blood vessels and the secondary cascade damage that cased by the inflammatory response,tissue edema,hypoxia,ischemia,lipid over oxidation,glutamate receptor excessive activation,activation of calcium overload pathway.SCI not only directly led to the death of nerve cells,but also cause the death of oligodendrocytes in the lesion areas,this may lead to the demyelination of axonal,which seriously affect the body’s various neural functions.Oligodendrocyte precursor cells(OPCs)are the initial cell of the glial cell,it is an immature cell type.In one hand,OPCs with strong proliferation ability and can rapidly proliferatein vivo;in other hand,OPCs can directly migrate and eventually differentiate into mature oligodendrocytes in certain environment.Related research results show that: through OPCs cell transplantation,OPCs can survive in vivo,promote the formation of myelin sheath,and ultimately improve the motor and nervous function of SCI rats.In addition to the traditional chemotactic role to inflammatory cells and bone marrow cells,it is also found that chemokines have different degrees of effect in the repair process of the central nervous system after nerve injury.Under normal physiological conditions,central nervous system widely expressed CXCL12/CXCR4,which plays a role in guiding the migration of neuronal precursor cells to the final functional area.Under pathological conditions,CXCL12/CXCR4 molecules are closely related to the damage and repair process of the nervous system.It has been reported in the literatures that CXCL12 has certain influence on proliferation,differentiation,growth of choroidal retinal vascular endothelial cells,tumor cells,glioma cells and their precursor cells,primary microglial cells,nerve anterior somatic cells and so on.Further researchs find that CXCL12 can through effect express of matrix metalloproteinase3(MMP3)and matrix metalloproteinase9(MMP9)to influence the proliferation and differentiation of neural precursor cells.So,we have reason to speculate that CXCL12 may also be involved in the regulation of proliferation and differentiation of OPCs by a similar mechanism.Objective:Therefore,in this study,the OPCs was purified,the in vitro culture model was established.We observe the growth and proliferation of the OPCs in vitro,explore the effect of CXCL12 on the proliferation of OPCs,detect the expression of CXCR4 and CXCR7 and MMP9 protein.So we can better understand the proliferation and differentiation characteristic of OPCs.Above all,this lays some experimental basis for later study of OPCs transplantation to treat spinal cord injury.Method:1.SD rats born within 48 hours(h)were selected,and the cerebral cortex tissue was taken from SD rats.OPCs were efficiently dissociated by the shaking process and differential adhesion,purified and culturedin the primary culture.OPCs identification was obtained by using immunocytochemistry technique according to the specific expression of PDGFR-α on the membrane of early OPCs cells.After the OPCs was transferred into the directional differentiation medium,the expression of O4 and MBP was induced in the middle and late stage,also according to the immunocytochemistry technique the maturation of the cells was identified.2.Under in vitro culture conditions,proliferation of OPCs under different concentrations of CXCL12(0ng/mL,5ng/mL,10ng/mL,20ng/mL)at different time points(0h,24 h,48h,72h)was assayed using CCK-8,Western blotting was used to detect the expression of CXCR4,CXCR7 and MMP9 protein of OPCs at different concentrations of CXCL12 condition for 72 h.3.In 20ng/mL CXCL12 culture conditions,CXCR7 si RNA and CXCR4 si RNA were used to interfere the expression of CXCR4 and CXCR7 protein respectively in rat OPCs.Proliferation of OPCs under different experiment conditions(CXCL12,CXCL12+con siRNA,CXCL12+CXCR4 si RNA,CXCL12+CXCR7 si RNA)at different time points(0h,24 h,48h,72h)was assayed using CCK-8;Western blotting was used to detect the expression of CXCR4,CXCR7 and MMP9 protein of OPCs different experiment conditions(CXCL12,CXCL12+con si RNA,CXCL12+CXCR4 si RNA,CXCL12+CXCR7 siRNA)after 72 h.4.With MMP9 si RNA interference MMP9 expressionin rat OPCs.And then,proliferation of OPCs under different experiment conditions(CXCL12,CXCL12+con siRNA,CXCL12+MMP9 siRNA)at different time points(0h,24 h,48h,72h)was assayed using CCK-8.Result:1.In primary culture of the ninth-tenth days,cells showed obvious layering growth.A large number of OPCs were isolated through the shaking separation and differential adhesion.At the beginning,the cell body was small,diameter of 6~10μm,approximate circular,strong refraction of small bright spot,some cell body began with small bipolar or tripolar cell processes.The results of immunocytochemistry showed that OPCs was able to express PDGFR-α.After directional differentiation of culture,OPCs tends to be mature,the slender processes around cells were slightly increased,with larger cell body.Induce metaphase O4 expression was positive.In induced advanced the cell body mesh slender processes were more obvious,even like “ramificated” or “cobweb-like” processes,and the expression of MBP was positive,they were mature myelin forming cells.2.In a certain concentration range,with the culture medium CXCL12 concentration increased,the proliferation of OPCs gradually increased,the expression of CXCR4 and CXCR7 and MMP9 protein increased gradually;in a certain range of time,proliferation of OPCS gradually increased,the expression of CXCR4 and CXCR7 and MMP9 protein gradually increased.Compared with 0ng/mL,at the condition of 20ng/m L CXCL12 for 72 h,the proliferation of OPCs and the expression of CXCR4,CXCR7,MMP9 protein increased most obvious(P<0.05).3.With CXCR4 siRNA,CXCR7 si RNA respectively interference CXCR4 and CXCR7 protein expression of OPCs,the proliferation in each time point were gradually suppressed.After cultured of 72 h,the most significant difference(P<0.05,P<0.05).Also,the expression of CXCR4 and CXCR7 and MMP9 protein gradually decrease,72 hours later the most significant difference(P<0.05,P<0.05).4.With MMP9 siRNA interference the expression of MMP9 protein,the proliferation of OPCs also inhibited(P<0.05).Conclusion:1.In this experiment,OPCs were efficiently dissociated by the shaking process and differential adhesion in the primary culture.2.In a certain concentration and time range,CXCL12 can significantly promote the proliferation of OPCs,this promoting effect is closely related with up-express of CXCR4,CXCR7,MMP9 protein.