| Background and objectiveLow back Pain and neck pain(Neck pain)as one of the most common diseases in modern society,seriously affecting people’s quality of life.Epidemiological survey shows that more than 80% of people have had low back pain or shoulder and neck pain in their lives,of which more than 15% of the people need to be able to achieve the severity of hospitalization to ease.As a hospital outpatient treatment of one of the common causes of each year due to the above symptoms caused by direct or indirect socio-economic losses.At present,more than 40% of low back pain and 20% of patients with neck and shoulder pain are mainly due to their intervertebral disc degeneration(IDD)and its secondary a series of lesions caused.For IDD patients,the current effective treatment can not be separated from surgical resection of degenerative disc treatment.However,in the removal of degeneration of the disc at the same time need to implant the fusion device and screw fixation,at the expense of the patient’s spine activity at the same time to the patient’s economic burden.Therefore,the search for new disc degeneration or intervertebral disc regeneration has been one of the hotspots in this field.With the development of stem cell research,stem cell-based regenerative medicine and biotherapy have attracted wide attention.Through the use of stem cell transplantation,tissue engineering transplantation and change the local micro-environment induced stem cell regeneration and other innovative medical means to help repair damaged tissue,reconstruction of lesions and other purposes.In the cells that have more tissue regeneration and repair capacity,the most widely used and reliable seed cells are the most widely used cells in the cells,which are relatively easy to obtain,the ability to differentiate and the deepening of mesenchymal stem cells.At present,one of the research hotspots of regenerative medicine.MSC-based regimen is expected to provide another treatment option for patients with intervertebral disc degeneration.However,almost all MSCs and other stem cells are found in invasive acquisition,the number of separation is small,age-dependent and in vitro expansion capacity is limited and other limitations greatly limit its clinical application.As a pluripotent stem cell capable of acting as a therapeutic agent,the number of pluripotent stem cells in the body can be reduced and the ability to repair is diminishing.Second,because of its rare,in the collection and in vitro amplification often mixed with non-stem cell adult cells,and ultimately in vitro amplification and treatment of stem cells affect the purity and efficacy.In addition,MSC-based adult pluripotent stem cells,in the process of in vitro expansion will continue to occur cell senescence(Senescence),and in the process of loss of its multi-directional differentiation potential.In the intervertebral disc degeneration,due to the special structure of the nucleus of its internal environment in the hypoxia and lack of nutrients in the state,the traditional MSC transplant survival efficiency is low.In addition,due to the small volume of nucleus pulposus,surrounded by a solid fiber ring wrapped,water and other characteristics,so that it can only accommodate a small amount of MSC into the nucleus pulposus,greatly limiting the effect of its treatment.In summary,for the re-treatment of intervertebral disc degeneration,the urgent need to find a easy to obtain,shape stability,low immunogenicity,good repair stem cell substitutes to make up for the above deficiencies.In order to find suitable stem cell substitutes,scholars from the stem cells repair damaged tissue or promote the mechanism of tissue regeneration in-depth study,and found that MSC not only through its own proliferation and differentiation of damaged tissue,but also by paracrine important micro Environmental regulation substances-exosomes(Exosomes)to change the surrounding microenvironment of damaged tissue,so as to regulate cell proliferation,differentiation,apoptosis and other biological functions,and indirectly achieve the role of repair and treatment.Exosomes are cystic vesicles of nanoscale(40-120 nm)bilayer membrane cells secreted by budding.Because of its cell membrane-like double-layer phospholipid membrane structure,it has good permeability and stability,can be combined with the target cells;it is rich in cell-derived mRNA,miRNA and protein components,and the above ingredients can participate in The target cells play an important role in the regulation of the function and signal of the target cells.Since almost all cells will analyze the exosomes,and the different components of the cells are different,so they are considered to be important local microenvironment regulating the media,but also stem cells on the repair of many organs play an important role.There have been many reports that MSC derived exosomes have similar functions to MSC,such as their ability to repair damaged tissue,inhibit inflammatory responses,and regulate immune responses.Compared with the simple MSC application,MSC exosomes have many advantages,such as stable nature,easy to save,no immunogenicity,easy access and easy to transform and many other advantages,for the treatment of a variety of diseases provides a new means.And the MSC-derived exosomes with the above-mentioned characteristics may be an ideal substitute for deficiencies in the treatment of intervertebral disc degeneration with traditional MSC treatment.In the previous study,it has been found that MSC derived exosomes can significantly promote the synthesis of nucleus pulposus ECM.As an important synthase of nucleus pulposus ECM,CHSY is involved in the development of various diseases.The abnormal function of CHSY leads to a decrease in the synthesis of chondroitin sulfate(CS),which is also closely related to the occurrence of IDD.Based on the above,we hypothesize that exosomes derived from MSC may promote the repair of intervertebral discs by activating the expression of related enzymes,CHSY,in ECM synthesis of nucleus pulposus cells.Part Ⅰ The isolation and identification of MSC derived exosomesMethods:(1)The rinsed liquid was made into a single cell suspension,and the nucleated cells were isolated and collected by Percoll density gradient centrifugation.The bone marrow was resuspended in DMEN medium containing 15% fetal bovine serum.Substance stem cells for primary culture.(2)The expression of CD105,CD34,CD44,CD14,CD90,CD45,CD19 and other mesenchymal stem cells were detected by flow cytometry.The cells were induced by commercialization of osteogenic and adipogenic differentiation induction medium.(3)The second generation of MSC cells in T75 above the culture flask to expand.When the cells reached 90% fusion,the low serum medium was replaced,and the culture medium was collected every two days.The supernatant was centrifuged and purified by ultracentrifuge.(4)The specific protein expression in the exosomes was detected by SDS-PAGE and Western-Blot.The size and morphology of the exosomes can be analyzed by transmission electron microscopy or Nanosight.(5)PKH67 experiments to determine the ability of fusion of exosomes.Results:(1)8 cases of bone marrow collection,the successful origin of bone marrow MSC original culture in 7 cases,both 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 Ⅱ The effect of MSC-Exo on IDDMethods:(1)Collect 30-50 age of normal nucleus pulposus for primary culture.(2)Supernatant after ultracentrifugation was used as the supernatant supernatant,that is,the control group;the supernatant was used as the blank control group by using the original culture medium of untreated cells;the culture supernatant of HEK293 cells was used Exosomes were isolated and the exosomes were obtained as negative control groups;and MSC derived exosomes as the experimental group to treat primary nucleus pulposus cells.(3)After the protein was added to the nucleus pulposus cells at different concentrations,the expression of extracellular matrix was detected by PCR and Western Blot.(4)The expression of MMP and ADAMTS in nucleus pulposus cells was detected by PCR and Western Blot after incubation for 24 hours after adding different concentrations of the above samples to nucleus pulposus cells.(5)The expression of CHSY and other enzymes related to ECM synthesis in nucleus pulposus cells was detected by PCR and Western Blot.Results:(1)6 strains of nucleus pulposus cell lines were successfully established by primary culture of nucleus pulposus.(2)The exosomes,exosomes supernatant,primitive culture medium and HEK293 exosomes were successfully collected from MSC culture supernatant.(3)The expression of type 2 collagen and proteoglycan in the nucleus pulposus cells of the experimental group was higher than that of the control group and the negative control group.(3)The expression of MMP1,MMP2 and MMP7 in the nucleus pulposus of the experimental group was significantly lower than that of the control group and the negative control group.(5)The expression of CHSY1,CHSY2 and CHSY3 in the experimental group was significantly higher than that in the control group and the negative control group.Conclusions: Five normal nucleus pulposus cell lines were obtained by primary culture of nucleus pulposus.Quantitative and postconditioned cells were identified by BCA method to detect the role of ECM synthetase and ECM synthesis in the nucleus of NK cells,and to reduce the expression of ECM-degrading enzyme.Part Ⅲ MSC-Exo upregulates CHSY to protect NP cells from degenerationMethods:(1)High-throughput RNA sequencing was used to detect the expression of transcriptional cells in nucleus pulposus cells treated with exosomes.(2)bioinformatics analysis of MSC derived exosomes to promote the nucleus pulposus ECM synthetase mechanism.(3)The microRNA molecules in the exosomes were detected by high-throughput sequencing and the mi RNAs associated with CHSY-associated intervertebral disc degeneration were screened and verified by double luciferase reporter assay.(4)overexpression of predicted candidate mi RNAs to study their changes in the secretion of ECM from CHSY and nucleus pulposus cells.(5)double luciferase experiments to determine the microsurgical microRNA on the target mechanism of CHSY.Results:(1)The high level transcription group sequencing technique showed that there was a significant difference in the level of gene expression in the nucleus pulposus of MSC after treatment with exosomes.(2)The use of GO analysis showed that exosomes of MSC could significantly affect the regulation of transcription factors in nucleus pulposus cells,suggesting that exosomes promoted CHSY expression may be related to post-transcriptional regulation.(3)The use of high-throughput miRNA sequencing technology found that MSC-derived exosomes had higher microRNA expression compared with control and HEK293 exosomes.(4)Predictive analysis of target genes found that MSC exosomes containing miRNA that could significantly inhibit CHSY negative regulatory factor,IL-1 and TNF-α pathway downstream factor NF-κB.(5)Targeted experiments confirmed that the candidate miRNAs in the exosomes of MSC could directly inhibit the expression of NF-κB and promote the expression of MMP in CHSY.Conclusions: Using high-throughput detection combined with bioinformatics analysis,we found that MSC-derived exosomes can significantly increase the expression of regulatory factors in the nucleus pulposus pathway,suggesting that miRNA and other post-transcriptional regulatory factors and exosomes promote IDM in ECM synthesis closely related.Using a high-throughput miRNA group to analyze MSC-derived exosomes and bioinformatics prediction techniques,we identified a number of targeted miRNAs of CHSY-inhibiting NF-κB and verified them with corresponding experiments.SummaryIn this study,we investigated the effect of exosomes derived from MSC on the nucleus pulposus cells,and found that the decrease of ECM synthase caused by degeneration of nucleus pulposus cells was mainly mediated by IL-TNF Such as proinflammatory cytokine-induced CHSY-targeted mi RNAs.We found that miR-194 and miR-515 were found to be significantly upregulated in IDD,and that the miRNAs were indeed able to cause changes in CHSY expression by functional verification,and that the miRNAs were also directly regulated by IL / TNF.Through the above research,we have inspired the regulation mechanism of CHSY.Based on the discovery of CHSY expression in exosomes of MS,we further studied the mi RNA-rich miRNAs in exosomes,and found that miR-199,-16,-195 Can bind NFκB,and NFκB as an important upstream regulatory element of IL / TNF,disc degeneration and ECM synthesis of the reduction has a crucial role.Subsequent studies have further confirmed that exosomes can affect the production of IL / TNF in nucleus pulposus cells by delivering miR-199,-16,-195,thereby reducing the decrease in CHSY expression and promoting the recovery of intervertebral disc degeneration. |
PDF Full Text Request