A Research Of Effects On G-CSF To Function Of Microglia Activated By Hemoglobin

Objective: To establish a method of improving deficient isolation and purification of primary rats microglia.Methods: Primary microglia culture was modified based on the classic microglia culture method of MeCarthy's. The isolation of the microglia from mixed cultured cells was by hand. Microglial cells were identified by flow cytometric analysis.Results: Separation, cultivate microglia, strong sex of refractive index, round, have burr appearance cell swelled. Streaming cell detection instrument of cells isolated CDllb (+) MCHII (-) cell content in 95%.Conclusion: the method of cultivated by microglial cells for its function of high purity, establishes the foundation.Objective: Study the regulation and mechanisms of G-CSF to proinflammatory role of microglia activated byHb.Methods: Primary microglia were planted into 72 well plate (0.5 x 105/well) and incubated in DMEM with 10% fetal calf serum. Microglia were divided into three groups, the control group, Hb group and G-CSF groups, 24 well every group. Hb group was acubated with 1 ul hemoglobin, while the G-CSF group was acubated with rhG-CSF (10 ng/mL) except Hb. 6 well were tested of 3 groups at 2 h, 6 h, 12 h and 24 h each time. ELISA was used to determin IL-1, TNF alpha content in the liquid of the 3 groups, and IL-1 and TNF alpha mRNA expressions were determined by QRT-PCR, western blot was utilized to quantitative p-JNK and p-p38MAPK in microglia of 3 groups at 12 hResults: The IL-1 and TNF alpha content in Hb group and G-CSF group were higher than in the time control; Hb group and G-CSF group clear liquid on the IL-1 in 2 h when beginning to rise, 6 h peak when, when 12 h began to decline, G-CSF group IL-1 in 6 h and 12 h Hb group when below, and statistically significant (p < 0.05); Hb group and G-CSF group clear liquid on the TNF alpha 2 h in when beginning to rise, when 12 h peak, when 24 h began to decline, G-CSF group of TNF alpha in 6 h and 12 h Hb group when below, and statistically significant (p < 0.05), 24 h significantly lower than Hb group (p < 0.01).Hb group and G-CSF microglial cells of IL-1 and TNF alpha mRNA levels were higher than the time control; Hb group and G-CSF group clear liquid on the IL-1 mRNA in 2 h start when the highest, 6 h when began to fall, when 24 h minimum, G-CSF group IL-1 mRNA in 6 h and 12 h Hb group when below, and statistically significant (p < 0.05); Hb group and G-CSF microglial cells of TNF alpha mRNA 2 h in when beginning to rise, 6 h peak when, when 12 h began to decline, G-CSF group of TNF alpha mRNA in 6 h and 12 h Hb group when below, and statistically significant (p < 0.05), 24 h significantly lower than Hb group (p < 0.01).12 h 3 groups in microglial cells are p-JNK and p-p38MAPK expression; Compared with the control, Hb group in microglial cells p-JNK and p-p38MAPK are significantly increased (p < 0.01); G-CSF microglial cells of the p-JNK and p-p38MAPK was significantly lower than the Hb group (p < 0.05), but higher than those in the control group (p < 0.05).Conclusion: Hb can by stimulating microglial cells in IL-1, TNF alpha mRNA synthesis and the expression and cause the inflammation. G-CSF can reduce Hb activated by microglial cells synthesis and the release of inflammatory factor IL-1 and TNF alpha thus inhibiting microglial cells the role of inflammation, G-CSF the anti-inflammatory effects may inhibit JNK and p38MAPK phosphorylation to achieve thisObjective: Study the effects and mechanism of G-CSF to hemoglobin phagocytosis of activated microglia.Methods: Primary microglia were planted into 72 well plate (0.5 x 105/well) and incubated in DMEM with 10% fetal calf serum. Microglia were divided into three groups, the control group, Hb group and G-CSF groups, 24 well every group. Hb group was acubated with 1 ul hemoglobin, while the G-CSF group were acubated with rhG-CSF (10 ng/mL) except Hb. 6 well were tested of 3 groups at 2 h, 6 h, 12 h and 24 h each time. HiCN method was used to determine the content of Hb, QRT-PCR determined CD163 / HO-1 mRNA expressions in microglia, immunofluorescence stain was utilized to dectect the CD163 protein expression on microglial membranes, and western blot determined the HO-1 content.Results: Hb content in the fluid Hb group and G-CSF group gradually reduced with time; the Hb content of G-CSF group lower than those of the control group in 6 h (p < 0.05), and ignificantly lower than those of the control group in the 12 h, 24 h in s (p < 0.01).QRT-PCR was used to detect CD163 mRNA expression and found that CD163 mRNA expression are visible in each group. CD163 mRNA level was no change each time in the control group; After joining Hb, CD163 mRNA level change was no change in Hb group, compared with the control group, CD163 mRNA level had no obvious difference each time (p > 0.05); After joining Hb, change with time, G-CSF microglial cells group CD163 mRNA level gradually raised, when 12 h to reach the top, and 24 h falls, and G-CSF CD163 mRNA level 6 in group h, 12 h and 24 h are higher than those of the Hb group when, with 12 h most significant (p < 0.01).HO-1 mRNA in the control group was found no significant changes with time; HO-1 mRNA level in Hb group changes with time gradually raised, and reached its peak at 24 h, compared with the control, HO-1 mRNA levels are increased each time (p < 0.05); After joining Hb, HO-1 mRNA in G-CSF group level gradually raised with time, when 24 h peak, and G-CSF group HO-1 mRNA levels were higher than in the time of the Hb group (p < 0.05).Western blot was utilized to determine HO-1 content in the 3 groups and we found that there are HO-1 expressing in microglia of the three groups; In the control group, HO-1 expression level is low, there is no change in different time points; Hb group HO-1 level changes with time gradually raised, at 24 h to peak, compared with the control, HO-1 level are increased each time (p < 0.05); Changing over time, HO-1 level gradually raised in the G-CSF group, and reached peak at 24 h. HO-1 level of G-CSF group are higher than those of the Hb group in every time (p < 0.05). Conclusion: CD163 protein don't express and HO-1 expression was low in microglia at physiological state; When microglia stimulated by the Hb, CD163 mRNA level don't change obviously than before but the CD163 protein is higher than before. Presumably, there would be some post-transcription regulations; HO-1 mRNA level increased in activated microglia gradually over time, HO-1 protein also increased gradually; G-CSF can promot HO-1 and CD163 mRNA synthesis, making the CD163 / HO-1 high express and promoting the absorption of hemoglobin and metabolism.The results showed G-CSF may play an neuroprotective role after intracerebral hemorrhage, through adjusting inflammatory role of microglia one of the path.