| ObjectiveThe injury of nervous system used to result in the irreversible neuron necrosis , the disorders of neural function from which has long been obsessing neurologists. For the past years mecenchymal stem cells has been applied in the repair and reconstruction of injured nervous system and then proposed a new solution. Packs of research showed that MSCs has the advantages of both easy access and quick isolation, in proper situation it can differentiate over embryonic layers and has less implant rejections, which makes it the most active one in the therapy of nervous diseases by transplation and the good result from animal experiment was already reported. While what roles the immune system takes in this process remains unknown, and what effect it exerts on the neurotrophic activity of MSCs keeps uncertain. Microglia is considered the representative of immune cells in central nervous system. It takes a dual role of repair or damage when activated during various nervous diseases. The in vitro study shows that;microglias secrets neurotrophic or neurotoxic factors depending on the circumstances, and directly promote the repair or death of neurons. In this study, from the view of microglia , we mainly evaluate its effect on the neurotrophic activities of MSCs during the circumstances of injured neurons and did a preliminary work on the signal transduction way in this circumstances.Methods1 Primary culture was used to culture marrow mecechymal stem cells, andPC 12 and BV2 were cultured and passed from cell line.2 The co - culture system was set up by 24 - well plate and the transwells with corresponding size, the pore size of transwell is 3 jxm which allows the communication of fluid and big - molecule protein in and outside transwell, but no cells were permitted to pass. The transwell and cells were removed after co -culture, and the cells left on 24 - well plate alone were detected.3 Immunofluorescence staining were adopted to identify marrow mechen-chymal stem cells, and so were the NSE and GFAP staining .4 Double - staining of FITC - PI were used to do a quantitive detection of PC 12 apoptosis with flowcytometry, morphology of apoptotic PC 12 were detected from the view of confocal and electric microscope.5 ELISA was used to detect the neurotrophic factors in supernatant quantitatively6 Western blot was used to do a semi - quantitative analysis in the expression of phosphotated - STAT, total - STAT in stimulated marrow mechechymal stem cells.Protocols1 PC 12 were injured with aged2 MSCs were incubated with the supernatant of injured PC12 with or without the presence of BV2, and then grouped as following;A MSCs incubated with supernatant of normal PC 12B MSCs incubated with supernatant of injured PC 12C MSCs incubated with supernatant of normal PC 12 in the presence of BV2D MSCs incubated with supernatant of injured PC 12 in the presence of BV2E MSCs incubated with plain 16403 Remove BV2 and the supernatant of PC 12, the MSCss and their supernatant were detected.4 The NSE positive cells were detected in MSCss5 Apoptosis of injured PC12was detected after incubated with MSCs supernatant6 Phosptatate STAT3 and total STAT3 in MSCss were detected.7 Neuroiivphic factors of bFGF, BDNF, NGF in MSCs supernatant were detected.8 Statistical analyses were finished with t - test or ANOVA.Results1 The culture and identification of marrow mechenchymal cells: on the second day of primary culture of MSCss, the cells took a round shining shape, and were adherent to the bottom dispursely, the cells are dense and fibroblasts -like one week later. MSCss were passaged after 80% of bottom were coverd, after 2 - 3 passages, MSCss were identified with CD44 with immunofluorescence, a light green fluorescence was visible under the microscope. MSCss were observed in the same field with both bright mode and fluorescence mode, over 95% cells were CD44 positive. PC 12 suspended in cluster and take polygone shape at the early stage, after 10 passages, some cells tend to differentiate with axons showing up and adhering. BV2 cells grows rapidly and the cells are adherent and spindle - like.2 We adopted immunofluorescence staining of NSE to identify the MSCss with neuron - differentiating tendency. NSE positive cells are bigger ones with various morphology, most shows 1-3 long dendrites, presenting with neurons looking. In group D ( MSCss co - cultured with BV2 and incubated with the supernatant from injured PC12) and group B(MSCs incubated with injured PC12 supernatant, no coculture) , NSE positive cells are apparently visible, while in other groups, all fields under microscope are mostly in darkness. All NSE positive cells are counted and normalized by MTT, the resulted were compared a-mong groups. Group D took the highest NSE positive cell number and is significantly different from that of control. While group B came second and is significantly higher than control, but no difference from group D.3 The apoptosis of injured PC 12 after incubation with MSCs supernatant was detected, the result demonstrated that there is significant difference among groups, in which group D ( cultured with supernatant from MSCs incubated withinjured PC 12 media and BV2 coculture) had the least apoptotic cells (P <0. 05 ) , while group B ( cultured with supernatant from MSCs incubated with injured PC 12 without BV2 coculture) had the second least apopototic cells, but has no statistical significance with other group. While the apoptotic cells are significantly lower in group D than that in group B ( P <0.05)4 Detection of neurotrophic factors in MSCs culture media: bFGF and TGF - pcan be detected in all groups, bFGF is highest in group D and has statisticalsignificance compared with controls( P < 0.05 ). while no statistical significance was found in the expression of TGF - pin all groups ( P > 0. 05 ). BDNF and NGF were under detection level in all 5 groups.5 The detection of phosphotate STAT3 and total STAT3 shows that: phos-photate STAT3 in group D is significantly different from that in control (P <0. 05) , suggesting that when injured neurons and microglias are present , STAT3 is actively involved in the biological activity of MSCs ( neurotrophicity and differentiation) .DiscussionImmune reaction usually takes a wide participation in neurological diseases , and is also the starting cause of neurodegeneration or nervous injury, it inevitably has effect on the stem cells entering nervous system for the purpose of neurotrophicity or therapy. In Wong G's study he and colleagues found that IFN was able to promote the differentiation of neural stem cells and neurites growth. Microglias has long been considered to derive from the monocytes or pre - mono-cytes in blood, and do an important immune work in brain, playing dual roles of repairing and injuring in the disorders of nervous system. While no report was yet seen about its neurotrophic or supporting functions on stem cells. In our study we started from microglia and focus on the effect of microglia presence on MSCss biological activities in the microcircumstance of injured neurons.BV2 and PC 12 came from the microglioma and pheochromocytoma of mouse respectivley, and were widely used as substitute the microglias and neurons. PC 12 was demonstrated to secrete various injury -related factors after stimulatedwith aged Ap^^l -40, while the supernatant of injured PC12 and MSCs,BV2 compose the microcircumstance of injured neurons in brain. Our results showed that;the NSE positive MSCs incubated with supernatant of injured PC 12 is more than MSCss incubated with supernatant of normal PC12 or controls, suggesting that injured neurons can exert effect on MSCs by exocrine factors, inducing the differentiation of MSCs to neurons. While no difference was found in MSCs incubated by normal PC 12 supernatant with that in control, which indicated that injured neurons is essential to the start of oriented differentiation of MSCs. NSE positive MSCs is more with the presence of BV2 but no statistical significance could be found, which suggested that microglias had no bad effect on the differentiation of MSCs to neurons.We then detected the effect of microglia on MSCss' neurotrophic or neuro-toxicity in the microcircumstance of injured neurons. We injured PC 12 with aged A.3i-4ol -40, and injured PC 12 were incubated with MSCss' supernatant , the PC 12 apoptosis were detected 24 hours later to study the neurotrophic or neuro-toxicity of supernatant of MSCss. The results showed that: incubated with injured PC 12 supernatant, supernatant of MSCs cocultured with BV2 could reduce the PC12 apoptosis significantly, suggesting that: the presence of BV2 in the circumstance of injured neurons could promote the neurotrophicity of MSCs, and improve the viability of injured neurons, while BV2 take an indirect role of protecting neurons. We further assay the neurotrophic factors in the supernatant of MSCss, including bFGF,BDNF,TGF - p,NGF. The results showed that, TGF and bFGF can be detected in all groups, and reached the highest value in the group with BV2 coculture and PC12 injury, which is significantly different from all other groups (P <0. 05) , while BDNF and NGF is negative in all groups. The results indicates that;BV2 promote the neurotrophic function of MSCs by improving the bFGF secretion in MSCs.Whether MSCs would save or replace injured neurons depends on what it will do in such a microenviroment, while the signal transduction within the MSCs is the basis of its biological activites. We collect the cell lysate of MSCs and detect both total STAT3 and phosphotate - STAT3, the results shows that: there is no difference of total STAT3 expression among groups, while phosphotate -STAT3 is significantly improved in MSCss with BV2 coculture, indicating that;in the microenviroment of our experiment, the phosphotation of STAT3 is involved in the biological activities of MSCs, and it is plausible the STAT3 pathway is involved here.Our results showed that the microglia was able to affect the MSCs acitivites in the microenviroment of injured neurons, and promote its differentiation to neurons. What will mciroglias do to affect the differentiation of MSCs in such microenviroment and by what pathway remains a lot to be explored.Conclusions1 Under the microenviroment of injured neurons, the presence of microglia is favorable to the proliferation and neuron differentiation of MSCs2 Under the microenviroment of injured neurons, the presence of microglias could promote the neurotrophic activities of MSCs and promote the production of neurotrophic factors.3 The phosphotation of STAT3 increased in MSCs lysates, indicating the involvement of STAT3 pathway in the biological activities of MSCs.
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