| The role and mechanism of TBCB in the process of astrocytes protrusion growth and developmentObjective: To investigate the role,mechanism and regulatory mechanism of tubulin folding cofactors B in astrocytes.Starting with the perspective of TBCB on microtubule biosynthesis and dynamic balance regulation of astrocyte in the nervous system,it reveals the role and mechanism of TBCB in the process of astrocytes and their protrusion growth and development,which provides a theoretical basis for clinical practice..Methods: The experiment is composed of three parts.Part 1: To study the role of TBCB in the growth and development of astrocytes protrusion.Primary cultured mouse astrocytes exposed to acute and chronic alcohol,and TBCB was silenced by siRNA.The effect of chronic alcohol exposure on the activity of astrocytes was examined by CCK8.After immobilizing the astrocytes with polyoxymethylene,which can keep the cell membrane intact.The proteins dissolved in the cytoplasm and combined with the skeleton protein were tested by immunofluorescence.The morphological changes of microtubules(MT)were labeled with ?-tubulin(?-T).The expression of TBCB and MT and the morphological changes of astrocytes were observed under different stimulation.The effect of TBCB on microtubules and the effect of this mechanism on the growth and development of astrocytes were studied.Changes of TBCB and ?-T protein and mRNA content were detected with western blotting and quantitative reverse transcription polymerase chain reaction.Part2: To study the mechanism of TBCB affecting the growth of astrocytes protrusion.EBs are MT-positive tracer proteins.EB1 can be bound to TBCB,while EB3 has the same domain as EB1 that can bind to TBCB.It was proved that EB3 can also be combined with TBCB in our group.This part of the experiment intends to reveal whether TBCB binds and regulates the MT positive end through EB1 and 3,thereby affecting the growth of astrocytes.In this part of the IF test,methanol and acetone(1: 1)were used to fix the cells,so that the protein dissolved in the cytoplasm flowed out,and only the intracellular MT and the protein bound to it were displayed.After silencing the expression of TBCB and EB1 with siRNA and overexpression of TBCB with lentivirus,the expression of TBCB and EB1 at the positive end of microtubule were examined by immunofluorescence.The changes of TBCB and EB1 protein and mRNA content were detected with western blotting and quantitative reverse transcription polymerase chain reaction.In addition,the astrocytes were treated with chronic alcohol exposure,TBCB or EB3 was treated with siRNA silence and over expression with lentivirus respectively.The relationship between TBCB and EB3 protein and mRNA were studied by western blotting and quantitative reverse transcription polymerase chain reaction;The expression location of the MT positive end changed was detected with immunofluorescence.Part 3: To study the regulatory mechanism of TBCB expression.After astrocytes were exposed to acute and chronic alcohol,TBCB was siRNA silenced or lentivirus overexpressed,the p-PAK1 and MAPK(ERK,p38,JNK)protein phosphorylation changes were tested by western blotting.Then,agonists and inhibitors were used to interfere with PAK1 and MAPK signal pathways respectively.The expression of TBCB was examined by western blotting and the regulatory mechanism of TBCB expression was studied.Results: Part 1: CCK8 results showed that chronic alcohol exposure had a dose-dependent effect on the activity of astrocytes.The higher the alcohol concentration,the weaker the activity.In normal control astral and negative control astral,IF results showed that TBCB co-expressed with ?-T,which was extremely rich in MT tissue center around astrocytes protuberance and nucleus;MT was radiated from MT tissue center,in a straight line,dense and regular arrangement.After acute and chronic alcohol interference and TBCB silencing,the expression of TBCB and ?-T decreased synchronously,which was dose-dependent on alcohol;the expression of TBCB in the center of MT tissue around the nucleus did not change significantly,but decreased significantly in the cell protuberance;the number of MT decreased significantly,especially in the cell protuberance,and the positive end was arranged disorderly and curly.WB and qRT-PCR results showed that after acute and chronic alcohol interference and TBCB silencing,the expression of TBCB and ?-T protein and mRNA decreased simultaneously(p<0.05).The above results demonstrate that the change of TBCB in astralaffects the expression of MT,especially in the MT positive end;And TBCB also affects the growth of astral,especially the most active cell protrusions on the MT positive end.It is proposed that the change of TBCB may affect the growth of the MT positive end and thus affect the formation and growth of astrocyte protrusions.Part 2: In Con and NC astral gel,IF showed that TBCB was expressed along MT,especially at the MT positive end located at the periphery of the cell,and the expression was abnormally rich.Its expression pattern was similar to "tailed comet","comet head" toward the cell periphery,The gradually weakened "comet tail" was toward the center of the cell;EB1 and 3 were co-expressed with TBCB at the MT positive end,and were also "comet-shaped".After chronic alcohol exposure and silencing of TBCB,EB1,or EB3(siTBCB,siEB1,or siEB3),IF showed that the above interference made an opening significant decrease in the expression density and fluorescence intensity of the three or two other proteins.WB and qRT-PCR results also confirmed that after siTBCB,siEB1 or siEB3,TBCB,EB1,and EB3 protein and mRNA levels were reduced,and the changes were the same(p<0.05);After lentivirus overexpressed TBCB or EB3 that TBCB and EB1,EB3 protein and mRNA levels increased simultaneously(p<0.05).These results indicate that TBCB can be bound to EB3;TBCB,EB1,and 3 can all bind to the MT positive end;after interference,the changes of the three affect each other.It is suggested that TBCB can be bind to EB1 and EB3 to regulate the growth of the positive end of MT,thereby affecting the formation and growth of astrocyte protrusions.Part 3: WB results showed that the proteins of PAK1,ERK,and p38 signaling pathways were changed after astrocytes were exposed to acute and chronic alcohol,and silenced or overexpressed TBCB.Among them,ERK pathway had the most obvious changes,and JNK signaling pathway had no obvious changes.Agents and inhibitors interfered with the ERK pathway,causing synchronous changes in TBCB expression(p<0.05).These results indicate that TBCB expression is regulated by the ERK signaling pathway,and PAK1 and p38-MAPK signaling pathways are also involved in the regulation of TBCB expression.Conclusion: In mouse astrocytes,TBCB can affect the formation and growth of astrocyte protrusions through binds to EB1 and EB3,binds to the MT positive end and regulates its growth;ERK-MAPK signaling pathway can regulate the expression of TBCB,PAK1 and p38-MAPK signaling pathways may also participate in the regulation of TBCB expression,thereby affecting the growth of astrocytes.The mechanism of aquaporin-4(AQP4)polar expression change in scratch-injured astrocytes in acute phaseObjective: To investigate the possible pathogenesis of traumatic brain edema by exploring the reason for AQP4 polar expression change in scratch-injured astrocytes.Methods: Primary astrocytes were cultured with SD rat brain tissue,and the in vitro scratch model was used to simulate brain damage in vivo.To study the astrocyte reactivity in the acute stage of scratch and the polar expression Change of AQP4,and the relationship between this polarity change and the change of its anchor protein;The expression changes of AQP4,GFAP and its anchor protein-dystrophin(?-DG)were examined by IF.To clarify after brain trauma,Whether DG is related to the change of AQP4 polarity expression,so as to reveal the possible mechanism of traumatic brain edema formation.Changes of protein and m RNA expression of AQP4,?-DG and ?-DG were detected with IF,Western blot(Western Blot,WB)and quantitative reverse transcription PCR(Quantitative Reverse Transcription PCR,q RT-PCR).Results: IF results showed that the co-expression of AQP4 and GFAP in the astrocytes of the control group was dispersed or dotted.After 6h of scratching,GFAP and AQP4 still maintained co-expression mode,and their expression increased synchronously around the scratches,showing a patchy distribution of fusion,indicating that the astrocytes in the area were significantly more responsive,and the polar expression mode of AQP4 was changed;Similarly,AQP4 and DG were also co-expressed in the control group,showing a diffuse or punctate distribution.After scratching,the expression of the two was simultaneously enhanced to a plaque-like co-expression pattern.It is suggested that the change of AQP4 polarity expression after scratching is related to the change of anchor protein expression.Conclusion: After 6h of scratching,the astrocytes in the scratched area were in an activated state;the change in the expression of AQP4 polarity in this area may be related to the change in the expression of its anchoring protein,thereby affecting the formation of traumatic brain edema. |
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