The Study On The Mechanism Of Endosomal Transportation Related Protein On Degeneration Of The Notochord

Part ?:Isolation of the notochord cellsOBJECTIVE:To isolate and culture the rat notochord cells that derived from the caudal intervertebral disc by percoll density gradient centrifugation and cell filter,and to observe the notochord cells by morphological phenotype.METHODS:SD rats aged 4 weeks were randomly divided into a non-treatment group,a percoll density gradient centrifugation group and a cell filter group.The notochord cells of the rat caudal intervertebral discs were taken,and the notochord were isolated by no treatment,percoll density gradient centrifugation and cell filter methods.The morphological markers of each group were observed under microscope,classified and counted.The morphology phenotypes of the notochord cells were observed under transmission electron microscope.RESULTS:The total number of cells extracted from the non-treatment group was higher(p<0.05),and the proportion of cells in the "bubble" structure cells/all cells in the field of view was lower than that of the two treatment methods(p<0.05).The cells with "bubble" structure extracted by percoll density gradient centrifugation were more than those of cell filter group(p<0.05),and the total number of cells extracted by percoll density gradient centrifugation was also higher than that cell filter group(p<0.05).There was no statistically significant difference between the two percoll density gradient centrifugation group and the filter group when compare the total number of cells in the "bubble" structure cells/field of view(p>0.05).Electron microscopy showed that the notochord cells were mostly 20-30?m in size The cytoplasm was rich in various types of vacuoles,which were subcellular structures encapsulated by incomplete cytoplasmic monolayers.The cytoplasmic component of the membrane is inconsistent into the vacuole CONCLUSION:The notochord cells can be extracted more efficiently by percoll density gradient centrifugationPart ? Effect of endosome inhibitors and culture time on vacuolar morphology in notochord and nucleus pulposus cellsOBJECTIVE:To observe the effect of endosome inhibitor-Brefeldin A(BFA)on intracellular vacuolar structure of notochord cells,and to compare the expression of endosome-associated proteins in notochord cells and nucleus pulposus cells at different culture time.METHODS:Three groups of NC were inoculated for 4 days.The BFA group,the blank group and the control group were given 5 ?g/?l BFA,equal volume of DMSO and culture medium.After 4 hours of culture,the cell morphology was observed.The NC cultured for 4 days and 7 days,and NP under normal conditions.The protein expression of Rab5,Rab32,Lampl and Lamp2 related to endosome transport was detected by western blot.RESULTS:Compared with the blank group and the control group,the intracellular vacuole area of the BFA group decreased(p<0.05).The expression of Rab32 in the NC cultured for 4 days was more than that of 7d in NC and the NP(P<0.05,).The expression of Lampl in NC cultured for 4 days and 7 days was more than that of cartilage-like NP(P<0.05).The expression of lamp2 and Rab5 in NC cultured for 4 was less than that of the other groups(P<0.05).CONCLUSION:The endosomal system is involved in the expression of vacuoles in the notochord cells.Th notochord cells in the long culture period tend to show de-vacuolation changes.Part ? Changes in expression of endosome transport-associated proteins innotochord cells under different conditionsOBJECTIVE:To investigate the effects of RNA interference on RAB32 gene,weak acidity,nutrient deprivation and mechanical damage on the expression of endosome transport-related proteins in NC,and to explore the relationship between notochord cells degeneration and endosomal transport system.METHODS:RNA interference was used to interfere with the expression of Rab32 in notochord cells.Transfection efficiency was observed by fluorescence microscopy.By using western blot analysis,detecting the expression of endosome transport-related protein Rab5,Rab32,Lamp1,Lamp2,in siRab32 transfection,weak acidity,nutrient deprivation mechanical damages and longtime culture in NC and cartilage-like NP.The morphological changes of each group were observed under microscope.RESULTS:Real-time PCR showed that the mRNA expression in the si-rab32 group was significantly lower than that in the blank group and the control group(P<0.05),but there was no significant difference between the control group and theblank group(P>0.05).Immunofluorescence showed that the proportion of positive cells after Si-rab32 transfection was greater than that of the blank control group(P<0.05).The results of Western Blot assay showed that the expression of Rab32 and Lampl in nucleus pulposus cells was significantly lower than that in normal control group(p<0.05),while Lamp2 and Rab5 protein expression were significantlyincreased(p<0.05)compared with normal treatment group in mechanical injury,acid environment and interference with Rab32.The expression of Rab32 and Lamp1 protein in NP cells was significantly decreased compared with the normal treatment group(p<0.05),while Lamp2 was significantly increased(p<0.05).There was no significant difference in Rab5 protein expression(p>0.05)CONCLUSION:The expression of endosome transport-related proteins in NC and NP cells changes under different environmental conditions and interfere expression of Rab32.The regulation of endosomal transport system may be related to the degeneration of nucleus pulposus cells.