| Background and objectiveLower back pain(low back PAIN,LBP)is more common in clinic,epidemiological survey shows that more than 80% of people,have one or more times in their life experience of lower back pain,and lower back pain is one of the common causes of outpatient visits.Low back pain seriously affects the quality of life of patients,and to society and individuals caused huge economic losses.At present,such as lumbar intervertebral disc protrusion,lumbar spinal stenosis and degenerative lateral convex,etc.,is recognized to cause the lower back pain main disease.A large number of studies have confirmed that these diseases are related to intervertebral disc degeneration(intervertebral Disc degeneration,IDD)and its secondary pathological changes.The incidence of lower back pain in our country is increasing year by year,with the aging of society,the patients with degenerative disease of lumbar spine are increasing trend.At present,in clinical treatment of intervertebral disc diseases,the most important therapy is still surgical treatment.Surgical treatment can effectively relieve clinical symptoms,but surgery does not fundamentally repair the degenerative intervertebral discs,and may cause serious complications,causing greater pain to the patient.Therefore,it is of great practical significance to study the mechanism of intervertebral disc degeneration and the biological treatment.The intervertebral disc is composed of the central nucleus,the outer fiber ring and the end plate of the upper and lower ends;The main function of the intervertebral disc is to maintain the normal spine structure and bear the biomechanical stress of the spine.The normal water content of the nucleus pulposus is the prerequisite for the physiological function and stress of the intervertebral disc,and the imaging manifestations of the early intervertebral disc degeneration are the decrease of the water content in the nucleus pulposus of the T2 weighted image,which is gradually converted from high signal to low signal or even no signal.The decrease of water content is believed to be caused by the broken ring of extracellular matrix.The decrease of water content leads to the decrease of intervertebral height,the instability of local motion segment,and the pathological changes such as nucleus pulposus,stenosis of nerve root canal and so on.At present,many clinical studies on intervertebral disc degeneration are based on the changes of the content of thenucleus pulposus in the diagnosis of intervertebral disc degeneration,which is one of the important indexes for the diagnosis of intervertebral disc degeneration in clinical work.A large number of studies on intervertebral disc degeneration show that the metabolic disorder of extracellular matrix(ECM)in Nucleus pulposus,the invasion of inflammatory factors and the increase of apoptosis of disc nucleus cells are the main causes of degeneration.The most important mechanism for intervertebral disc degeneration is ECM metabolic disorder.The large amount of protein-chitosan molecule formed in the nucleus pulposus provides an efficient water retention system for intervertebral disc tissue,and is a necessary prerequisite for normal water content of intervertebral discs.In IDD,ECM synthesis decreased in nucleus pulposus,such as proteoglycan(mainly aggrecan),?collagen and other ECM molecular synthesis decreased,and the extracellular matrix of the nucleus pulposus increased,such as matrix(MMPs)and aggregation of glycoprotein(AGGRECANASE,ADAMTS)expression increased.These two factors contribute to the further reduction of ECM in the nucleus pulposus in IDD.Tian ye et il-1? the key role of inflammatory factors in ECM decomposition through NF-?b pathway ADAMT-4,there are other scholars to prove the role of MMPs and ADAMTS-5 and other enzymes in IDD and its regulation mechanism.In the recent years,with the revelation of the biological characteristics of stem cells,regenerative medicine and biological therapy have aroused wide concern of scholars.The research on the treatment of intervertebral disc degeneration and regenerative medicine is also focused on the stem cells with the potential of repair and multi-directional differentiation.By means of injection,this kind of treatment can directly effect the stem cells with repair ability to the degenerative intervertebral disc tissue,promote the recovery of the cell number and the regeneration of the Matrix,and finally improve the degeneration and even reverse the treatment effect of the degenerative process.At present,mesenchymal stem cells are the main methods for regenerative treatment of intervertebral disc degeneration.Mesenchymal stem cells have the advantages of multi-directional differentiation potential,tissue substitution,regulatory microenvironment,local immunity and inflammatory response,and the different sources of mesenchymal stem cells,and the differences of the multiple energy between sources.The study on regenerative treatment of mesenchymal stem cells is very extensive,and many positive results have been obtained,however,the treatment of mesenchymal stem cells has some disadvantages,such as the difficulty of obtaining the source,the aging of cells and the potential of tumor-causing,which greatly limits the regenerative medicine research and application of mesenchymal stem cells.So,based on the limitations of stem cell therapy,more researchers are looking at a substance secreted by mesenchymal stem cells,the exosome.It has been found that MSC derived exosomes can significantly promote the synthesis of nucleus pulposus ECM.The exocrine body is a nano-scale vesicle secreted by cells containing encoded RNA,non coded RNA,protein,antigen-presenting factor,and DNA.Because of its double layer membrane structure,it is easy to penetrate the cell membrane to achieve the effect of intercellular transduction of the contents,through the transfer of protein and RNA,the secretion can regulate the function of cells and other organs,activities.JCI also in 2016 to do a special issue on the discovery of the body,and the role of the future application of the outlook made a systematic review.At present,the regeneration effect of the external secretion has been verified in the study of other tissue and organ damage repair,including heart injury treatment,lung injury treatment,liver regeneration therapy,fracture healing,post stroke regenerative therapy,etc.However,the extracellular secretion of mesenchymal stem cells can promote the repair of intervertebral disc degeneration,and the effect of stem cell therapy is still in the research stage with only a few reports.Therefore,aiming at the above situation,the role of mesenchymal stem cells in the degeneration of nucleus pulposus cells was studied systematically,and a new method for the treatment of intervertebral disc degeneration was found in this study,which lay a foundation for future regenerative Medicine related treatment research.Part ? The isolation and identification of MSC derived exosomesMethods: In order to collect the fresh bone marrow specimens in the femur of the patients with non open femoral fracture trauma,the cells were separated and collected by Percoll density gradient centrifugation,and in 5% CO2 and 37 ? conditions,the dmen medium with 15% fetal bovine serum was still cultured,and the cell reached 90% fusion.The expression of the surface markers of mesenchymal stem cells was detected by flow cytometry,and the cells were induced by commercial osteogenesis and lipid differentiation induction medium,and their differentiation potentials were identified.After the cell passage to P2,the mesenchymal stem cells were inoculated into the T75 and above the cultured bottles for amplification,when the cell fusion degree reaches 90%,replace the low secretion serum culture base,every 2 days of liquid-collecting medium,continuous collection,the collected medium using a speeding centrifuge centrifuge,extraction ofexternal secretion.For the collected secretion,the properties were verified by the following methods,the expression of specific protein in the secretion was detected by using Western blot,the size,morphology and purity of the secretion were analyzed by electron microscope scanning and nanosight,and the fusion ability of the secretion was clarified by using the PKH67 experiment.Results:(1)bone marrows were collected and successful cultured in good condition.(2)Flow cytometry MSC markers CD105,CD34,CD44,CD14,CD90,CD45,CD19 showed primary culture MSC in line with internationally defined MSC indicators.Osteogenic and adipogenic induction,the primary culture of MSC showed significant calcium nodules and fat droplets.(3)Ultrafine centrifugation was carried out by culturing the MSC in large quantities and the supernatant was collected to obtain significant precipitation and to be centrifuged again.(4)Western Blot was used to detect the expression of exosomes-specific CD81 and CD63,and Nanosight and electron microscopy showed that the collected precipitate was exosomes.(5)The epithelium was stained with the PKH67 dye and added to the MSC.The results showed that the MSC could be stained with exosomes,suggesting that the collected exosomes had membrane fusion ability.Conclusions: We obtained bone marrow MSC cells that conformed to international standards through primary culture of bone marrow MSC cells.After the primary MSC cells were amplified and the supernatant was collected,the extracorporeal bodies were obtained by ultracentrifugation.At the same time the use of exoskeletal markers and functional experiments to confirm the collection of exosomes.Part ? The effect of MSC-Exo on IDDMethods: The primary culture of human nucleus pulposus(30-50 age group)with normal and no obvious degeneration was collected.The CCK-8 multiplication test was used to verify the cytotoxicity of the derived secretion and the ability of promoting the proliferation of the nucleus pulposus cells.Test group: The supernatant after speeding centrifugation as a group of outward secretion,as a control group,the original culture medium was not cultured after the Overspeed centrifugation,as the blank control group;HEK293 cells cultured and collected supernatant for speeding centrifugation collected by the above method as negative control group The mesenchymal stem cells were derived from exogenous secretions as experimental groups.After the protein quantification of each sample,the above samples with differentconcentrations were added in the nucleus pulposus cells and 24 h was cultured,and Western blot and PCR were used to detect the secretory expression of extracellular matrix.The expression of MMP and adamts related to the degradation of the nucleus pulposus extracellular matrix and the expression of the gene related to the extracellular matrix synthesis were validated.The effects of external secretion on the degeneration of nucleus pulposus cells in vitro were analyzed by high throughput sequencing technique and the target gene was found.Results: 5 strains of Nucleus pulposus cells were successfully established in vitro.CCK-8 proliferation test showed that mesenchymal stem cells had no cytotoxicity to nucleus pulposus cells and could improve the proliferation ability of nucleus pulposus cells.After treatment by Western blot and PCR,the expression of type 2 collagen and proteoglycan in the extracellular matrix of the experimental group were significantly higher than that in the control group and the negative control group.The degradation of extracellular matrix of nucleus pulposus cells in experimental group was related to MMP1,MMP2,The expression of MMP7 was significantly lower than that in control group and negative control group.The expression of ACAN,colii and chsy in the extracellular matrix of nucleus pulposus cells in experimental group were significantly higher than that in control group and negative control group.A high throughput transcription group sequencing technique was used to find that the level of transcription group gene expression was significantly different from that in the control group.Conclusions: 5 strains of human nucleus pulposus cells were obtained by tissue culture of nucleus pulposus.The extracellular secretion of mesenchymal stem cells has no cytotoxicity to the nucleus pulposus cells,and can improve the proliferation ability of nucleus pulposus cells.The cells were quantitatively treated by Western blot and PCR,and it was found that the exogenous secretion of mesenchymal stem cells could promote the synthesis of extracellular matrix,increase the extracellular matrix synthetase of nucleus pulposus cells and reduce the expression of extracellular matrix degrading enzymes.Part ? MSC-Exo upregulates HO-1 to protect NP cells from degenerationMethods:(1)Screening candidate target genes according to high throughput RNA sequencing results.(2)Combining with high throughput miRNA sequencing data of MSC external secretion,the effects of miRNA components on target genes were analyzed bybioinformatics.(3)Screening the miRNA in the extracellular secretion of MSC derived from the BACH1 associated with HO-1 and verifying the gene by double fluorescein enzyme.(4)To study the change of expression of HO-1 in BACH1 and nucleus pulposus cells by the candidate miRNA of expression prediction.(5)The effect of microRNA on the binding sites of mutant BACH1 in vitro was determined by the double fluorescein enzyme experiment,and the target mechanism was confirmed.Results:(1)Screening candidate target genes according to high throughput RNA sequencing results.(2)Combining with high throughput miRNA sequencing data of MSC external secretion,the effects of miRNA components on target genes were analyzed by bioinformatics.(3)Screening the miRNA in the extracellular secretion of MSC derived from the BACH1 associated with HO-1 and verifying the gene by double fluorescein enzyme.(4)To study the change of expression of HO-1 in BACH1 and nucleus pulposus cells by the candidate miRNA of expression prediction.(5)The effect of microRNA on the binding sites of mutant BACH1 in vitro was determined by the double fluorescein enzyme experiment,and the target mechanism was confirmed....Conclusions: Using High-throughput detection combined with bioinformatics analysis,we found that the extracellular secretion of MSC can significantly improve the expression of the regulatory factors after the transfer of Nucleus pulposus cells,which means that miRNA and other transcriptional regulatory factors are closely related to the synthesis of ECM in IDD.By using high throughput miRNA group to analyze the extracellular secretion of MSC and the technology of bioinformatics prediction,we found a batch of BACH1 inhibitory miRNA,and validated it by corresponding experiments,and found the mechanism of the increase of HO-1 in the secretion of the body.SummaryBy exploring the role of the secreted body from MSc in the nucleus pulposus cells,the first discovery of the related gene changes caused by the degeneration of nucleus pulposus can be recovered by the BACH1 targeted miRNA factors in the secretion by using high throughput Transcription group and miRNA assay.We found that the expression of miR-21,-23,-125 and-let7 were significantly increased in the nucleus pulposus cells treated by external secretion,and it was confirmed by the functional study that the miRNA can cause the change of BACH1 expression,thus causing the increase of HO-1 activity and promoting the repair of intervertebral disc degeneration.The above research has inspiredthe regulation mechanism of HO-1.In the early stage,the mechanism of HO-1 was enriched by the extracellular secretion of HO-1,and the mechanisms of the extracellular secretion of MSC to promote the repair of intervertebral discs were further proved by the study. |
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