| Intervertebral disc degeneration(IDD),with high morbidity and mortality,is the main cause of spinal disease associated with low back pain which is influenced by a variety of factors,including machinery,age,and genetics.As a mechanical unit contains,the intervertebral disc(IVD)is consisted by the central glial nucleus pulposus(NP),the surrounding annulus fibrosus(AF),and the cartilaginous endplate(CEP)connected to the adjacent vertebral body.The IVD is rich in moisture,type Ⅱ collagen and proteoglycan to maintain the elastic state of the disc so that the IVD can withstand normal activity,axial compression and tensile stress,protecting the vertebral body from injury under extreme physical load.In a healthy disc,anabolism and catabolism of the extracellular matrix(ECM)are in balance,IVD degeneration is characterized by a cascade of changes in cells,composition,structure,and function,primarily manifested in the progressive loss of ECM components and increased intervertebral acidosis.The IVD is the largest avascular structure in the body and receives oxygen from the surrounding vertebra via CEP diffusion.The NP cells in the center of the intervertebral disc are hypoxic and produce adenosine triphosphate(ATP)through anaerobic glycolysis,so their activity and vitality are first affected.Hypoxia is an important cellular stress mechanism and has important pathological significance in intervertebral disc degeneration.There are many ways of treatment low back pain caused by intervertebral disc degeneration,mainly including surgical and non-surgical treatment.However,there is fundamentally no satisfactory solution to reconstruct and restore the natural properties of the IVD.Hypoxia-inducible factor 1α(HIF-1α)is a transcription factor that initiates a synergistic cellular cascade under hypoxic stress,including the regulation of multiple enzymes in response to hypoxia.It is reported that HIF-1αexpression in intervertebral disc cells is critical for intervertebral disc development and balance as well as IVD degeneration,therefore,HIF-1α is associated with susceptibility and severity of intervertebral disc degeneration.HIF-1α is highly correlated with matrix synthesis and energy metabolism of cells in a hypoxic environment,since matrix remodeling and cell metabolism and the regulation of a large number of hypoxic response signaling pathways are disordered during the course of intervertebral disc degeneration.Therefore,is it possible to regulate cellular metabolism to restore disc homeostasis via HIF-1αif so,what are the mechanisms?In what form does HIF enter the intervertebral disc?How to avoid the HIF-1α metabolized rapidly so as to maintain its continued function in degenerative intervertebral discs?Stem cell transplantation has always been a hot topic in the field of degenerative disc therapy.Adipose tissue-derived stem cells(ADSCs)are widely used because they can differentiate into different types of cells in specific environments,including nucleus pulposus cells.However,for tissue regeneration,it is important for MSCs to maintain their vitality to induce in situ regeneration.The microenvironment of damaged tissues is often hypoxia and malnutrition,leading to cell apoptosis and death.Especially for degenerative IVD,the survival time of stem cells after transplantation is limited due to poor microenvironment such as hypoxia and low PH,and most mesenchymal stem cells die due to aging or apoptosis within a few days after transplantation.Therefore,how to keep stem cells alive and active in intervertebral disc has been an urgent problem to be solved.Studies have shown that hypoxic preconditioning of stem cells can significantly improve the expression of HIF-1α,and improve the survival rate of stem cells at the transplant site and cell activity to achieve tissue repair.Therefore,we wondered whether the intervention of stem cells could achieve precise regulation of HIF-1α and maintain the survival of transplanted stem cells.Meanwhile,the high expression of HIF-1α could continue to play a role in the intervertebral disc,so as to achieve the purpose of tissue repair of degenerative intervertebral disc.So in our study:1.We isolated and cultured ADSCs from SD rats and identified their stem cell properties.2.We established a hypoxic culture model of ADSCs in vitro,and observed the proliferation and apoptosis of ADSCs in the hypoxic environment,as well as the differentiation of ADSCs and the expression of HIF-1α.3.The overexpression of HIIF-1α and deletion of HIF-1α ADSCs were constructed by plasmid transduction containing HIIF-1α and Si-RNA technology,respectively.The cell proliferation,apoptosis,differentiation,collagen synthesis and cell metabolism of the ADSCs were detected under normoxic conditions,and the mechanism was preliminarily studied.The degeneration model of tail imtervertebral disc of SD rats was established by acupuncture,and ADSCs with overexpression of HIF-1α were implanted.The repair effect of the degeneration intervertebral disc was determined by histological examination at different periods.Meanwhile,the transplanted cells were tracked to determine their distribution and survival.Our research results show that:1.The extracted and separated ADSCs of SD rat has good stem cell properties.The apoptosis rate of ADSCs in serum deprivation was decreased when cultured in hypoxic condition.When ADSCs were co-cultured with NP cells under hypoxia conditions,they promoted proliferation,increased type Ⅱ collagen synthesis,decreased catabolism,differentiated into nucleus posus cells,and increased HIF1α expression significantly.2.The overexpression and deletion of HIF-1α ADSCs were successfully constructed.When HIF-1α was overexpressed,the apoptosis rate decreased,proliferation increased,type Ⅱ ollagen synthesis increased,metabolism decreased,and promoted the differentiation of nucleus pulposus cells.When HIF-1α is knocked out,the apoptosis rate is increased,proliferation is decreased,type Ⅱ collagen synthesis is decreased,and metabolism is increased 3.1n vivo experiments confirmed that ADSCs with overexpression of HIF-1α can maintain long-term survival in degenerative intervertebral disc,effectively improve intervertebral height and degenerative grade of intervertebral disc,and increase ECM of degenerative intervertebral disc,suggesting that ADSCs with overexpression of HIF-1α can effectively repair and delay degeneration of intervertebral disc.Part Ⅰ:Effects of hypoxic pretreatment conditions on biological behavior of ADSCsObjective:To extract and identify ADSCs from SD rats and establish a culture system of ADSCs in low oxygen environment.The proliferation,apoptosis and differentiation of ADSCs were observed under the condition of environmental hypoxia.Methods:1.ADSCs were extracted from white adipose tissue of the inguinal region of SD rats,and identified by three-way induced differentiation and flow cytometry.2.ADSCs hypoxic culture system was constructed,and the apoptosis of ADSCs under the limit condition of serum-free culture was verified by Tunel assay.3.ADSCs and NP cells were co-cultured,and cell proliferation was tested by EdU technology.4.The expressions ofCol-Ⅱ,MMP-13 and HIF-αin ADSCs were detected by Western Blot to reflect the effect of environmental hypoxia on the differentiation and protein synthesis of ADSCs.Result:1.Flow cytometry results indicated that the extracted cells had the characteristics of surface antigen of stem cells.The cells were successfully induced to differentiate into osteogenic,adipogenic and chondrogenic cells,which proved that the cells had good stem cell properties;2.Tunel assay showed that the apoptosis rate of ADSCs in serum-free culture was significantly lower than that in normoxic culture when the environmental hypoxia concentration wasl%.3.EdU test showed that ADSCs proliferation rate was higher than other groups when co-cultured with NP cells under hypoxic culture conditions.4.Western Blot showed that the expression of HIF-1α,COLⅡ,SOX9,AC AN,HIF-1α were significantly increased when ADSCs and NP cells were co-cultured under hypoxic conditions.Conclusion:The hypoxic culture system of ADSCs was established in this study.When the ambient oxygen concentration is 1%,the expression of HIF-1α in ADSCs was significantly increased,the apoptosis rate decreased obviously in serum-free culture condition,ADSCs co-cultured with NP cells showed good proliferation efficiency,ADSCs can be better induced to differentiate into NP cells.Part Ⅱ:Molecular mechanism of hypoxia regulating ADSCs proliferation,migration and directed differentiation to nucleus pulposus cells through HIF-1α pathwayObjective:To study the specific mechanism of hypoxia regulating endoplasmic reticulum stress in ADSCs through HIF-1α in cell proliferation,migration and directional differentiation to nucleus pulposus cells.Methods:1.Transfect ADSCs with HIF-1α gene to determine the effect of HIF-1αgene overexpression on the proliferation and migration of ADSCs,and study its regulatory effect on the directional differentiation of nucleus pulposus cells by co-culture with nucleus pulposus cells;2.Silencing HIF-1α gene by siRNA technology,to clarify the effect of low expression of HIF-1α gene on the proliferation and migration of ADSCs,and to study its regulatory effect on the directional differentiation of nucleus pulposus cells by co-culture with nucleus pulposus cells.3.To clarify whether endoplasmic reticulum stress is regulated by hypoxia in ARDSs cells,and whether HIF-1α plays a role in this regulation,and preliminarily explore whether hypoxia regulates endoplasmic reticulum stress through HIF-1α in regulating the proliferation,migration and progression of ADSCs.Specific mechanisms in directed differentiation.Results:EdU results showed that HIF-1α(+)in ADSCs could significantly increase the proliferation of ADSCs,while the proliferation of HIF-1α(-)was significantly lower than that in the normal group.The results of Transwell migration chamber experiments showed that HIF-1α(+)could significantly improve the migration ability of ADSCs,while HIF-1α(-)could significantly reduce the migration ability of ADSCs.Western Blot results showed that the expressions of HIF-1α,Col-Ⅱ,Aggrecan and Sox9 in ADSCs were significantly increased under the co-culture condition of HIF-1α(+)ADSCs and NP cells.Under hypoxic co-culture of HIF-1α(-)ADSCs and NP cells,the expressions of HIF-1α,Col-Ⅱ,Aggrecan and Sox9 in ADSCs were significantly decreased.It is preliminarily demonstrated that hypoxia regulates the cell proliferation and migration of ADSCs caused by the radicalization of the endoplasmic reticulum through the HIF-1α pathway.Conclusion:HIF-1α plays an important role in the proliferation,migration and differentiation of ADSCs into nucleus pulposus cells.The high expression of HIF-1α can significantly increase the proliferation and migration ability of ADSCs,and can significantly enhance the ability of ADSCs to differentiate into nucleus pulposus cells.It is preliminarily demonstrated that in ADSCs cells,hypoxia reduces the level of cellular ER stress through the HIF-1α pathway,thereby enhancing cell proliferation and migration.HIF-1α may be an effective target for improving the treatment of intervertebral disc degeneration by ADSCs transplantation,and greatly improve the effect of stem cell transplantation in the treatment of intervertebral disc degeneration.Part Ⅲ:Evaluation of the effect of ADSCs transplantation with overexpression of HIF-1α in rat intervertebral disc degeneration modelObjective:To construct ADSCs with overexpression of HIF-1α in vitro and implant them into a rat acupunctured intervertebral disc degeneration model to evaluate the repair effect in vivo.The degeneration model of rat intervertebral disc acupuncture was established,Sham group,NC group,ADSCgroup and H-ADSC group.Samples were collected at 4 and 8 weeks postoperatively,and imaging(X-ray)and histological staining(HE,Safranin O-Fast green,and immunohistochemical staining)were performed respectively.Then,calculating Disc Height Index(DHI),observing the histological morphology of intervertebral disc,evaluating the intervertebral disc degeneration index.COL-Ⅱ and MMP-13 were detected by immunohistochemistry to reflect intervertebral disc matrix synthesis and catabolism.Similarly,GFP-ADSCs were implanted into degenerative intervertebral discs,and GFP-ADSCs were traced 4 weeks after surgery to observe cell survival and distribution.Result:In the H-ADSC group,the survival time of ADSCs in the degenerative intervertebral vertebrae was significantly prolonged and the distribution was wider.The intervertebral disc morphology and intervertebral disc degeneration index,as well as the DHI,in H-ADSC group were better than those in NC group and ADSC group,but still worse than the control group.Immunohistochemistry showed that COL-Ⅱ of H-ADSC group was significantly higher than that of NC group and ADSC group,while MMP-13 was significantly lower than that of NC group and ADSC group.All the results showed that the degenerative disc was repaired in the H-ADSC group,and the repair effect was better than that in the other groups.Conclusion:Injecting HIF-1α ADSCs into degenerative intervertebral discs can better resist the harsh environment of degenerative intervertebral discs,have a longer survival time,and have a wider distribution in intervertebral discs.Meanwhile,Intervertebral disc structure and shape can be restored to some extent,degeneration grade index can be improved,and intervertebral disc matrix synthesis increased accompanied with catabolism reduced.Therefore,ADSCs with overexpressed HIF-1α as seed cells implanted into degenerative intervertebral discs can better resist the harsh microenvironment of degenerative intervertebral discs to alleviate and repair the degeneration of intervertebral discs.It may be an ideal way to delay and treat intervertebral disc degeneration. |
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