| Backgrornd:Astrocytes(AST),which is one of principal cell populations in CNS,play a considerabe role in the development and regeneration of Central Nervous System(CNS). Astrocytes can secrete numerous cell factors up to more than 100 kinds, compose of neurotrophic factors, such as Nerve Growth Factor(NGF), Brain-derived Neurotrophic Factor(BDNF). Clinical research has impled that the effect of single neurotrophic factor was different between patients even though their disease condition is similar. It is believed that one of the most important reasons is that some neruotrophic factor may need cooperation each other when they exerted their effection on neurons. To implement the cooperation of numerous neurotrophic factors, bioreactor could be effective instrument. Astrocyes culturing in bioreactor can secrete multiple neurotrophic factors into medium, which named as the Astrocyte-conditional -medium (ACM). In past years, bioreactor with polyethersulfone (PES) semipermeable membrane had successfully been used to culture hepatic cells, and play a good role in treatment liver disease. However, whether the astrocytes culturing in this bioreactor can normally growth and secrete cytokines, as hepatic cell? Whether PES membrane would effect their morphous, survival, proliferation and function? All of these are unclear at present.It was reported that astrocyte-conditioned medium (ACM) is a nutritious liquid for neuronal growth containing various soluble cytokines, such as NGF, BDNF and so on, released from astrocytes. Like a layer of astrocytes wherein neurons are cultured, ACM constructs and keeps an environment of certain glial–neuron interaction, which implys that it could have the role of promoting injuried neurons'survival and functional recovery. Zhu and Colleagues reported that ACM can effectively protect hippocampal neurons in primary cultures against corticosterone-induced damages. Eriksin reported ACM support developmental serotonin neuron against ethanol and so on damage. However, whether the ACM can protect the neuron from mechanical or hypoxia injury or not was unclear. So, this work will evaluate the protective effect of ACM on cultured rat neurons against hypoxia and mechanical injury.As all known, the secondary brain injury would occur as pathophysiologic process after ischemia and hypoxia or traumatic injury of CNS. These damage factors will be switching neurovirulence, a progressive and cascading injury reaction. In the process, Nitrogen monoxidum (NO) play a significant role, generous NO can mediate glumatic acid to activate NMDA receptor, then aggravate the damage of neurons; excess productive NO can also damage the electron transfer system of chondriosome, inhibit the energy metabolism and DNA synthesis. The releasing quantity of Lactate Dehydrogenase (LDH), one of intracellular marker enzymes, will obviously increase after cells were damaged and the cellular membrane permeability happened to be changed. These changes will give us a chance to check the contents of these factors in culture medium as an index to evaluate damage degree of cells. So, with this work, we will investigate the protective effect of ACM on cultured mouse neurons against hypoxia and mechanical injury, moreover to study relative mechanism by test the contect of NO, LDH and activity of ATPase changes.Objective:To research the regularity of mouse AST proliferation, secreting NGF and BDNF in vitro, and the effect of PES membrane on AST's growth, survival/proliferation and secreting neurotrophic factors; by cell model in vitro, to investigate the protective effect and correlated mechanism of mouse ACM on hypoxia and mechanical damaged neurons in vitro.Methods:1. Cortical ASTs were isolated and purified from neonatal KM mouse. In primary culture, the ASTs were divided into 2 groups: Group A, common plate culture; Group B, PES membrane culture, the cell numbers of ASTs at the 1st , 3rd , 5th , 7th , 9th day were counted with CCK8 kit, and compared, and then, the numbers of ASTs of serial subcultivation in the 1st era were counted and compared in the same way separately at 5 periods, the 1st , 3rd , 7th, 14th, 21st day. Moreover, the numbers of the 5th day of every era (from 1st to 6th era) in conventional serial subcultivation, were counted and compared. The ACMs of every period were harvested and the concentration of NGF and BDNF was detected by ELISA, and the variation tendency of both factors was invested.2. After serial subcultivation, the purified AST were divided into three groups: Group A, common plate cultures; Group B, PES membrane cultures; Group C, PES membrane coated with laminin cultures. The changes of morphology, survival/proliferation capability, the concentration of NGF and BDNF in medium were analyzed and compared between three groups in above-mentioned ways.3. Cortical neurons from prenatal KM mouse were isolated and cultured by conventional way. The different concentration of ACMs harvested from 5th day, 3rd era were added to medium of culturing neurons, the numbers of neurons were counted 3 days or 6 days later with CCK8 kit, the effect of different concentration of ACM on neurons'survival was invested by morphology observation and immunochemistry staining of NF.4. Hypoxia injuried neurons model was made through put into dish with neurons into 3%O2 in 3-gas incubator, and the mechanical injured model of neurons was made by scratching neurons culturing on dish with micropipette tip. The 20% ACM of 5th day of 3rd era were added to the medium of anoxia/reoxygenation or mechanical injuried neurons. The survival of neurons was observed, and the concentration of NO, LDH of the neurons, and the activity of ATPase within cell membrane of hypoxia injuried neurons was examined with corresponding kits. The results was analyzed and compared with Control Group.Results:1. In primary culture, ASTs showed an trend of fast proliferation in two groups. However, the cells OD value culturing on PES membrane was obviously lower than the other group at the 3rd, 5th, 7thday. During the 1st era serial subcultivation, cell number was fast growing and reached peak by 7th day, then decreased; in continuous serial subcultivation, Comparing the change of cell number at 5th days of different era, the number of cells was increasing from 1st to 3rd era, and then declined, but no obvious difference occurred between 1st and 5th, however, the cell number of 6th era was obviously decreased than 5th era (P<0.05). The change of NGF and BDNF concentration of these cells was similar to their proliferation.2. Compared Group A with Group B during serial subcultivation under fluorescence microscope, the size of cell body of ASTs in Group B was smaller than group A, processus shorter, GFAP staining stronger; but Group C has similar changed with group A. From cell growth curve, after 3 days, the cell numbers of Group B were obviously lower than Group A and Group C (P<0.05), but no different was observed between Group A and Group C (P>0.05). The capacity of secreting NGF and BDNF by these cells has similar changes to cell proliferation. The concentration of NGF and BDNF reached peak up to 7th day, then decreased gradually. If using the ratio of concentration of NGF/BDNF to cell OD value as an representation of capacity of ASTs secreting NGF/BDNF, the result showed that the secreting capacity of NGF and BDNF by ASTs reached high peak when cells culturing to 7th day, and then gradually decreased, decreased obviously 14 day later, especially in Group B, this change was more obvious than Group A and C.3. After adding ACM into medium 3 days or 6 days later, neurons'OD value (0.145±0.079/0.206±0.083) in 10%ACM Group and 20% ACM Group (0.187±0.074/0.210±0.077) were more higher than control Group (0.125±0.067/0.150±0.066) and 50%ACM Group (0.134±0.034/0.145±0.034), 70%ACM Group (0.075±0.035/0.085±0.020), the difference was statistically remarkable (P<0.05), but when culturing these cells up to 6 days, neurons'OD value was no obviously different between 10%ACM Group and 20% ACM Group (P>0.05).4. Comparing the protect effect of ACM on damaged neuron, the morphology and number of neurons had obviously change between with and without ACM breeding group. In Conventional Medium Group (without ACM), the cell numbers of hypoxia injuried neurons gradually decreased as hypoxia time extending. The cells'OD values of hypoxia 24 hours without reoxygenation (H24RO) was 0.048±0.004, with reoxygenation 24 hours(H24R24) was: 0.039±0.010, it was more lower than that of hypoxia 8 hours without (H8RO) and with reoxygenation (H8R24), P<0.05. However, after adding 20%ACM, these changes was obviously improvement, the cells'OD values , no matter in hypoxia with or without reoxygenation group, were higher than that of Conventional Medium Group (without ACM) in every period (besides of H4R24 ) (P<0.05).Comparing with Control Group, in 20% ACM Group, NO concentration was lower at H4R24, H8R0, H8R24, H24R0, H24R24 ; the releasing ratio of LDH was smaller at H8R0, H8R24, H24R0, H24R24; the value of ATPase was higher at H4R24,H8R0,H8R24,H24R0,H24R24, the different was statistically remarkable ( P<0.05) .5. To mechanical damaged neurons, the cell number descending just occurred after post-injury 1 hour, and the change was more obvious as time extend or injuried degree was more severe, especially post-injury 6 hours and 24 hours. Comparing with Control Group, after adding 20% ACM, the cell number was higher, no matter in light injuried group, middle or severe injuried group. The change would be observed 1 hour later in severe injuried group, more obvious 24 hours later (P<0.05). After post-injury 1 hour, the concentrations of NO/LDH of injuried neurons were increasing gradually. In the middle injuried neurons, the NO concentrations was more lower after adding 20% ACM into medium for 6, 24 hours than Control Group, P<0.05. The concentrations of LDH had similar change in injuried groups after 1, 6, 24 hourswith NO, P<0.05.Conclusions:1. The AST numbers of serial subcultivation reached peak gradually by 7th day, then decreased; in continuous serial subcultivation, AST's proliferation obviously decreased after 6th era in conventional culture.2. PES membrane take an inhibiting role on the ASTs survival/proliferation and secreting NGF, BDNF in some extent, however, the laminin will lighten this effect of PES on AST and improve the capability secreting NGF and BDNF.3. ACM can promote normal and anoxia injuried neurons'survival, can protect anoxia/reoxygenation injuried neurons by reducing the production of NO and LDH, and raising the activity of ATPase.4. ACM has similar effects on mechanical damaged neurons to promote damaged neurons'survival. The protect mechanism of ACM may have some relations with reducing the production of NO and LDH.
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