How to enhance the ability of mesenchymal stem cells to alleviate intervertebral disc degeneration

Intervertebral disc(ID)degeneration(IDD)is one of the main causes of chronic low back pain,and degenerative lesions are usually caused by an imbalance between catabolic and anabolic processes in the ID.The environment in which the ID is located is harsh,with almost no vascular distribution within the disc,and the nutrient supply relies mainly on the diffusion of oxygen and nutrients from the blood vessels located under the endplate.The stability of its internal environment also plays an important role in preventing IDD.The main feature of disc degeneration is a decrease in the number of cells.Mesenchymal stem cells have been used in the treatment of disc lesions due to their ability to differentiate into nucleus pulposus cells in a nonspecific anti-inflammatory manner.The main purpose is to promote their regeneration.The current aim of stem cell therapy is to replace the aged and metamorphosed cells in the ID and to increase the content of the extracellular matrix.The treatment of disc degeneration with stem cells has achieved good efficacy,and the current challenge is how to improve this efficacy.Here,we reviewed current treatments for disc degeneration and summarize studies on stem cell vesicles,enhancement of therapeutic effects when stem cells are mixed with related substances,and improvements in the efficacy of stem cell therapy by adjuvants under adverse conditions.We reviewed the new approaches and ideas for stem cell treatment of disc degeneration in order to contribute to the development of new therapeutic approaches to meet current challenges.

Exosomes derived from stem cells as an emerging therapeutic strategy for intervertebral disc degeneration

Intervertebral disc(IVD)degenerative diseases are a common problem in the world,and they cause substantial social and economic burdens for people.The current methods for treating IVD degenerative diseases mainly include surgery and conservative treatment,which cannot fundamentally restore the normal structure of the disc.With continuous research on the mechanism of degeneration and the development of regenerative medicine,rapid progress has been made in the field of regenerative medicine regarding the use of stem cell-derived exosomes,which are active biological substances used in intercellular communication,because they show a strong effect in promoting tissue regeneration.The study of exosomes in the field of IVD degeneration has just begun,and many surprising achievements have been made.This paper mainly reviews the biological characteristics of exosomes and highlights the current status of exosomes in the field of IVD degeneration,as well as future developments regarding exosomes.

Karacoline,identified by network pharmacology, reduces degradation of the extracellular matrix in intervertebral disc degeneration via the NF-κB signaling pathway

Karacoline is a compound found in the plant Aconitum kusnezoffii Reichb.Although Aconitum kusnezoffii Reichb is widely used for the treatment of pain,very few studies have been carried out on the use of karacoline due to its potential toxicity.In this study,we selected key matrix metalloproteinases(MMPs),collagen II,and aggrecan as targets due to their association with intervertebral disc degeneration(IDD).Using these targets,we then used network pharmacology to predict a series of molecules that might exert therapeutic effects on IDD.Of these molecules,karacoline was predicted to have the best effect.Tumor necrosis factor(TNF)-a is known to promote the degeneration of the extracellular matrix in IDD.We therefore applied different concentrations of karacoline(0,1.25,or 12.88 mM)along with 100 ng/mL TNF-a to rat nucleus pulposus cells and found that karacoline reduced the expression of MMP-14 in IDD by inhibiting the nuclear factor(NF)-κB pathway,while collagen II and aggrecan expression was increased.This suggested that extracellular matrix degradation was inhibited by karacoline(P<0.05).Our data therefore reveal a new clinical application of karacoline and provide support for the use of network pharmacology in predicting novel drugs.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human

ObjectiveTo investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. MethodsThe PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. ResultsAmong the 4096 targets, there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases, comprising 298 up-regulated and 358 down-regulated ones. ConclusionDNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

Transcription regulators differentiate mesenchymal stem cells into chondroprogenitors,and their in vivo implantation regenerated the intervertebral disc degeneration

BACKGROUND Intervertebral disc degeneration(IVDD)is the leading cause of lower back pain.Disc degeneration is characterized by reduced cellularity and decreased production of extracellular matrix(ECM).Mesenchymal stem cells(MSCs)have been envisioned as a promising treatment for degenerative illnesses.Cell-based therapy using ECM-producing chondrogenic derivatives of MSCs has the potential to restore the functionality of the intervertebral disc(IVD).AIM To investigate the potential of chondrogenic transcription factors to promote differentiation of human umbilical cord MSCs into chondrocytes,and to assess their therapeutic potential in IVD regeneration.METHODS MSCs were isolated and characterized morphologically and immunologically by the expression of specific markers.MSCs were then transfected with Sox-9 and Six-1 transcription factors to direct differentiation and were assessed for chondrogenic lineage based on the expression of specific markers.These differentiated MSCs were implanted in the rat model of IVDD.The regenerative potential of transplanted cells was investigated using histochemical and molecular analyses of IVDs.RESULTS Isolated cells showed fibroblast-like morphology and expressed CD105,CD90,CD73,CD29,and Vimentin but not CD45 antigens.Overexpression of Sox-9 and Six-1 greatly enhanced the gene expression of transforming growth factor beta-1 gene,BMP,Sox-9,Six-1,and Aggrecan,and protein expression of Sox-9 and Six-1.The implanted cells integrated,survived,and homed in the degenerated intervertebral disc.Histological grading showed that the transfected MSCs regenerated the IVD and restored normal architecture.CONCLUSION Genetically modified MSCs accelerate cartilage regeneration,providing a unique opportunity and impetus for stem cell-based therapeutic approach for degenerative disc diseases.

Cervical intervertebral disc degeneration and dizziness

Clinical studies have found that patients withcervical degenerative disease are usually accompanied by dizziness.Anterior cervical surgery can eliminate not only chronic neck pain,cervical radiculopathy or myelopathy,but also dizziness.Immunohistochemical studies show that a large number of mechanoreceptors,especially Ruffini corpuscles,are present in degenerated cervical discs.The available evidence suggests a key role of Ruffini corpuscles in the pathogenesis of dizziness caused by cervical degenerative disease(i.e.cervical discogenic dizziness).Disc degeneration is characterized by an elevation of inflammatory cytokines,which stimulates the mechanoreceptors in degenerated discs and results in peripheral sensitization.Abnormal cervical proprioceptive inputs from the mechanoreceptors are transmitted to the central nervous system,resulting in sensory mismatches with vestibular and visual information and leads to dizziness.In addition,neck pain caused by cervical disc degeneration can play a key role in cervical discogenic dizziness by increasing the sensitivity of muscle spindles.Like cervical discogenic pain,the diagnosis of cervical discogenic dizziness can be challenging and can be made only after other potential causes of dizziness have been ruled out.Conservative treatment is effective for the majority of patients.Existing basic and clinical studies have shown that cervical intervertebral disc degeneration can lead to dizziness.

Crosstalk between Autophagy and Apoptosis in Intervertebral Disc Degeneration

Objective: To describe the relationship between autophagy and apoptosis and the possible signaling pathways involved in degenerative lumbar intervertebral disc. Summary of Background Data: Autophagy and apoptosis are regulatory cellular mechanisms that determine many pathologies, including degenerative intervertebral disc disease. The interactions between these events in the damage or protection of intervertebral disc cells and in cellular homeostasis remain controversial. Methods: The sample size was twenty patients who underwent lumbar spine surgery for symptomatic disc herniation or spondylolisthesis. Intervertebral discs were classified by magnetic resonance as Pfirrmann grade IV and grade V. Six patients were operated on two levels, resulting in twenty-six intervertebral discs that were submitted to immunohistochemistry to verify the protein expression of autophagy and apoptosis markers. Results: The autophagic markers had greater protein expression in the human intervertebral disc (Pfirrmann Grades IV and V). Under these conditions, autophagy and apoptosis showed a negative correlation. Regarding apoptosis, caspase 8 presented the highest protein expression, which allows inferring the preference for the extrinsic pathway in cell death. Conclusions: Autophagy had the greatest protein expression negative profile compared to apoptosis. Caspase 8 had the highest protein expression in apoptosis.

Application of molybdenum target X-ray photography in imaging analysis of caudal intervertebral disc degeneration in rats

BACKGROUND Conventional plain X-ray images of rats,the most common animals used as degeneration models,exhibit unclear vertebral structure and blurry intervertebral disc spaces due to their small size,slender vertebral bodies.AIM To apply molybdenum target X-ray photography in the evaluation of caudal intervertebral disc(IVD)degeneration in rat models.METHODS Two types of rat caudal IVD degeneration models(needle-punctured model and endplate-destructed model)were established,and their effectiveness was verified using nuclear magnetic resonance imaging.Molybdenum target inspection and routine plain X-ray were then performed on these models.Additionally,four observers were assigned to measure the intervertebral height of degenerated segments on molybdenum target plain X-ray images and routine plain X-ray images,respectively.The degeneration was evaluated and statistical analysis was subsequently conducted.RESULTS Nine rats in the needle-punctured model and 10 rats in the endplate-destructed model were effective.Compared with routine plain X-ray images,molybdenum target plain X-ray images showed higher clarity,stronger contrast,as well as clearer and more accurate structural development.The McNemar test confirmed that the difference was statistically significant(P=0.031).In the two models,the reliability of the intervertebral height measured by the four observers on routine plain X-ray images was poor(ICC<0.4),while the data obtained from the molybdenum target plain X-ray images were more reliable.CONCLUSIONMolybdenum target inspection can obtain clearer images and display fine calcification in the imaging evaluation of caudal IVD degeneration in rats,thus ensuring a more accurate evaluation of degeneration.

Genotoxicity Clues to Predict Intervertebral Disc Degeneration: A Systematic Review

Objective: To characterize the association between DNA damage and Intervertebral disc degeneration (IDD). Summary of Background Data: IDD is the main disorder causing low back pain and is the most promising target for intervention. Many factors can contribute to the etiology, such as genetics, environment and lifestyle, but it is not yet fully understood. DNA damage can influence this process and needs to be studied, as well as the agents that can determine these damages. Methods: A systematic literature search of PubMed, Web of Science and Scopus was performed to identify studies related to DNA damage to the intervertebral disc. Results: After screening 61 records, 7 articles were included according to the selection criteria. All studies showed some relation between DNA damage and IDD. However, DNA damage was always considered a secondary issue to be investigated. Conclusions: Many factors can influence DNA damage induced by different genotoxic agents on the degenerative cascade of IVD. However, the correlation between IDD severity and DNA damage, as well as the factual role of DNA damage in disc degeneration could not be defined.

An animal model to create intervertebral disc degeneration characterized by radiography and molecular biology

Objectives： To develop a rabbit model of intervertebral disc degeneration that more exactly simulates the pathological changes of human intervertebral disc degeneration. Methods： Twelve New Zealand white rabbits were utilized to establish three different disc injury models according to the following protocol; group A： anulus punctures were done with a 18-gauge needle at L2-L3 and L5-L6; Group B： intradiscal injection of interleukin-1 IL-1β with a 23-gauge needle at L3-L4; and Group C： intradiscal injection of phosphate buffer saline（PBS） with a 23-gauge needle at L4-LS. The L1-L2 level was used as a control. Rabbits were killed after 24 weeks. The intervertebral disc height was measured by lateral plain radiographs. After the radiographic measurements were obtained, the intervertebral discs were removed and analyzed for DNA, sulfated glycosaminoglycan（s-GAG） and water contents of nucleus pulposus. Results： The intervertebral disc height, s-GAG, and water contents in anulus needle punctures were significantly decreased in Group A, but the DNA content in the nucleus pulposus was significantly increased when compared to the control. The significant decrease of disc height and water contents were demonstrated, only the s-GAG and DNA contents did not show a significant difference in Group B when compared to the control. The significant decrease of disc height, s-GAG, water, and DNA contents did not show in Group C when compared to the control. Conclusion： The 18-gauge puncture models produced the most consistent disc degeneration in the rabbit lumbar spine.

Classification of Intervertebral Disc Degeneration in Low Back Pain Using Diffusional Kurtosis Imaging

Degenerative disc disease is the most common cause of low back pain. Intervertebral disc abnormalities are commonly evaluated by magnetic resonance imaging (MRI), and Pfirrmann’s system involves the use of T2-weighted images (T2WI) to classify disc degeneration. However, as this classification is based on visual evaluation, it is not possible to quantify degeneration using this method. The present study was performed to establish an MRI-based intervertebral disc classification system using diffusional kurtosis imaging (DKI), to quantify intervertebral disc water content according to the Pfirrmann classification. Sagittal mean diffusional kurtosis (MK) mapping was performed for the L3/4, L4/5, and L5/S1 intervertebral discs in 32 patients (15 female, 17 male;age range, 24 - 82 years;mean age, 57.7 years). The degree of disc degeneration was assessed in the midsagittal section on T2WI according to the Pfirrmann classification (grade I - V). The relationships between MK values, which are correlated with intervertebral disc composition changes, and grade of degeneration determined using the Pfirrmann classification were analyzed. The MK values tended to decrease with increasing grade of degeneration, and differed significantly between grades I and IV, but not between grade IV and V (P < 0.05, Mann-Whitney U test). DKI is an effective means of detecting the early stages of disc degeneration. Therefore, DKI may be a useful diagnostic tool for quantitative assessment of intervertebral disc degeneration.

Correlation Analysis of Intervertebral Disc Degeneration with HTRA1 and HAPLN1 Gene Polymorphisms

The lumbar spine,an important part of the body’s motor mechanism,is more susceptible to damage as it bears most of the body’s load.Age can cause clinical manifestations such as neurological impairment,back and leg pain in the lumbar spine.External forces result in nucleus pulposus out,destruction of the intervertebral disc fibrous ring,and gradual aging and damage.Lumbar degenerative change is a common middle-aged and old-aged disease,and its clinical symptoms on the initial stage are not obvious,but it becomes more and more serious as they get older.Patients with severe lumbar degenerative changes will appear symptoms such as urinary and fecal incontinence,lower extremity numbness,back pain,and sexual dysfunction.The main reason for back pain and leg pain is the degenerative changes in the lumbar intervertebral discs,at the same time which also leads to patients’lumbar instability.This study focuses on the correlation analysis of intervertebral disc degeneration with HTRA1 and HAPLN1 gene polymorphisms.

Multiple nano-drug delivery systems for intervertebral disc degeneration: Current status and future perspectives

Low back pain(LBP)is a common disease that imposes a huge social and economic burden on people.Intervertebral disc(IVD)degeneration(IVDD)is often considered to be the leading cause of LBP and further aggravate and cause serious spinal problems.The established treatment strategy for IVDD consists of physiotherapy,pain medication by drug therapy,and,if necessary,surgery,but none of them can be treated from the etiology;that is,it cannot fundamentally reverse IVD and reconstruct the mechanical function of the spine.With the development of nanotechnology and regenerative medicine,nano-drug delivery systems(NDDSs)have improved treatment results because of their good biodegradability,biocompatibility,precise targeted specific drug delivery,prolonged drug release time,and enhanced drug efficacy,and various new NDDSs for drugs,proteins,cells,and genes have brought light and hope for the treatment of IVDD.This review summarizes the research progress of NDDSs in the treatment of IVDD and provides prospects for using NDDSs to address the challenges of IVDD.We hope that the ideas generated in this review will provide insight into the precise treatment of IVDD.

Delivery of coenzyme Q10 loaded micelle targets mitochondrial ROS and enhances efficiency of mesenchymal stem cell therapy in intervertebral disc degeneration

Stem cell transplantation has been proved a promising therapeutic instrument in intervertebral disc degeneration(IVDD).However,the elevation of oxidative stress in the degenerated region impairs the efficiency of mesenchymal stem cells(BMSCs)transplantation treatment via exaggeration of mitochondrial ROS and promotion of BMSCs apoptosis.Herein,we applied an emulsion-confined assembly method to encapsulate Coenzyme Q10(Co-Q10),a promising hydrophobic antioxidant which targets mitochondria ROS,into the lecithin micelles,which renders the insoluble Co-Q10 dispersible in water as stable colloids.These micelles are injectable,which displayed efficient ability to facilitate Co-Q10 to get into BMSCs in vitro,and exhibited prolonged release of Co-Q10 in intervertebral disc tissue of animal models.Compared to mere use of Co-Q10,the Co-Q10 loaded micelle possessed better bioactivities,which elevated the viability,restored mitochondrial structure as well as function,and enhanced production of ECM components in rat BMSCs.Moreover,it is demonstrated that the injection of this micelle with BMSCs retained disc height and alleviated IVDD in a rat needle puncture model.Therefore,these Co-Q10 loaded micelles play a protective role in cell survival and differentiation through antagonizing mitochondrial ROS,and might be a potential therapeutic agent for IVDD.

Engineered high-strength biohydrogel as a multifunctional platform to deliver nucleic acid for ameliorating intervertebral disc degeneration

Intervertebral disc degeneration(IVDD)is a leading cause of low back pain.The strategy of using functional materials to deliver nucleic acids provides a powerful tool for ameliorating IVDD.However,the immunogenicity of nucleic acid vectors and the poor mechanical properties of functional materials greatly limit their effects.Herein,antagomir-204-3p(AM)shows low immunogenicity and effectively inhibits the apoptosis of nucleus pulposus cells.Moreover,a high-strength biohydrogel based on zinc-oxidized sodium alginate-gelatin(ZOG)is designed as a multifunctional nucleic acid delivery platform.ZOG loaded with AM(ZOGA)exhibits great hygroscopicity,antibacterial activity,biocompatibility,and biodegradability.Moreover,ZOGA can be cross-linked with nucleus pulposus tissue to form a high-strength collagen network that improves the mechanical properties of the intervertebral disc(IVD).In addition,ZOGA provides an advantageous microenvironment for genetic expression in which AM can play an efficient role in maintaining the metabolic balance of the extracellular matrix.The results of the radiological and histological analyses demonstrate that ZOGA restores the height of the IVD,retains moisture in the IVD,and maintains the tissue structure.The ZOGA platform shows the sustained release of nucleic acids and has the potential for application to ameliorate IVDD,opening a path for future studies related to IVD.

Nimbolide targeting SIRT1 mitigates intervertebral disc degeneration by reprogramming cholesterol metabolism and inhibiting inflammatory signaling

Inflammation,abnormal cholesterol metabolism,and macrophage infiltration are involved in the destruction of the extracellular matrix of the nucleus pulposus(NP),culminating in intervertebral disc degeneration(IDD).Whether nimbolide(Nim),a natural extract,can alleviate IDD is unclear.In this study,we demonstrated that Nim promotes cholesterol efflux and inhibits the activation of the nuclear factor kappa B(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways by activating sirtuin 1(SIRT1)in nucleus pulposus cells(NPCs)during inflammation.Thus,Nim balanced matrix anabolism and catabolism of NPCs.However,the inhibition of SIRT1 significantly attenuated the effects of Nim.We also found that Nim promoted the expression of SIRT1 in RAW 264.7,which enhanced the proportion of M2 macrophages by facilitating cholesterol homeostasis reprogramming and impeded M1-like macrophages polarization by blocking the activation of inflammatory signaling.Based on these results,Nim can improve the microenvironment and facilitate matrix metabolism equilibrium in NPCs.Furthermore,in vivo treatment with Nim delayed IDD progression by boosting SIRT1 expression,modulating macrophage polarization and preserving the extracellular matrix.In conclusion,Nim may represent a novel therapeutic strategy for treating IDD.

An esterase-responsive ibuprofen nano-micelle pre-modified embryo derived nucleus pulposus progenitor cells promote the regeneration of intervertebral disc degeneration

Stem cell-based transplantation is a promising therapeutic approach for intervertebral disc degeneration(IDD).Current limitations of stem cells include with their insufficient cell source,poor proliferation capacity,low nucleus pulposus(NP)-specific differentiation potential,and inability to avoid pyroptosis caused by the acidic IDD microenvironment after transplantation.To address these challenges,embryo-derived long-term expandable nucleus pulposus progenitor cells(NPPCs)and esterase-responsive ibuprofen nano-micelles(PEG-PIB)were prepared for synergistic transplantation.In this study,we propose a biomaterial pre-modification cell strategy;the PEG-PIB were endocytosed to pre-modify the NPPCs with adaptability in harsh IDD microenvironment through inhibiting pyroptosis.The results indicated that the PEG-PIB pre-modified NPPCs exhibited inhibition of pyroptosis in vitro;their further synergistic transplantation yielded effective functional recovery,histological regeneration,and inhibition of pyroptosis during IDD regeneration.Herein,we offer a novel biomaterial pre-modification cell strategy for synergistic transplantation with promising therapeutic effects in IDD regeneration.

Effect of Tuina along“bladder meridian”alleviating intervertebral disc degeneration by regulating the transforming growth factor-β1/Smad signaling pathway in a rabbit model

OBJECTIVE:The aim of this study was to investigate the protective effects of Tuina(a traditional Chinese massage therapy)on intervertebral disc(IVD)degeneration and the regulatory mechanisms of the transforming growth factor-β1(TGF-β1)/small mothers against decapentaplegic(Smad)signaling pathway.METHODS:Thirty New Zealand white rabbits were randomized into five groups:the control group,model group,model+Tuina group(Tuina group),model+TGF-β1 group(TGF-β1 group),and model+TGF-β1 inhibitor SB431542 group(SB431542 group).The model was established by posterolateral annulus fibrosus puncturing(AFP).Recombinant TGF-β1 and inhibitor SB431542 was injected into the TGF-β1 group and SB431542 group with a microsyringe,respectively.The rabbits in the Tuina group received Tuina treatment along the bladder meridian for 4 weeks.Magnetic resonance imaging(MRI)was performed on rabbits before AFP and after 4 weeks of intervention.Lumbar IVDs(L2-L3 to L4-L5)were harvested after intervention.Histopathological changes in the IVDs were measured by hematoxylin and eosin(HE)staining.Type I collagen was analyzed by immunohistochemistry detection.The expression level of matrix metalloproteinase-3(MMP3)was determined by enzyme-linked immunosorbent assay.Cell apoptosis was evaluated by terminal deoxynucleotidyl transferasemediated nick end labeling and Western blotting.Realtime polymerase chain reaction and Western blotting were used to analyze the expression of TGF-β1 and Smad2/3/4 and a disintegrin and metalloproteinase with thrombospondin motifs 5.RESULTS:Posterolateral AFP induced IVD degeneration in rabbits with histopathological damage and noticeable changes in MRI images.Tuina alleviated histopathological changes and reversed the expression of extracellular matrix degeneration-related molecules and apoptosis-related proteins.Furthermore,AFP induced the activation of TGF-β1 and Smad2/3/4,whereas Tuina therapy markedly reduced the protein expression of Smad2/3 and the gene expression of TGF-β1 and Smad2/3/4.Additionally,the TGF-β1/Smad signaling pathway was activated in the TGF-β1 group,while the TGF-β1/Smad signaling pathway was inhibited in the SB431542 group.CONCLUSION:Posterolateral AFP induced disc degeneration as determined by MRI assessment and histological analysis.Tuina alleviated disc degeneration,possibly by inhibiting the fibrotic response mediated by the TGF-β1/Smad pathway,thus alleviating extracellular matrix degeneration and reducing cell apoptosis.

Reabsorption of intervertebral disc prolapse after conservative treatment with traditional Chinese medicine:A case report

BACKGROUND Conservative treatments have been reported to diminish or resolve clinical symptoms of lumbar intervertebral disc herniation(LIDH)within a few weeks.CASE SUMMARY Computed tomography and magnetic resonance imaging(MRI)of the lumbar region of a 25-yearold male diagnosed with LIDH showed prolapse of the L5/S2 disc.The disc extended 1.0 cm beyond the vertebral edge and hung along the posterior vertebral edge.The patient elected a conservative treatment regimen that included traditional Chinese medicine(TCM),acupuncture,and massage.During a follow-up period of more than 12 mo,good improvement in pain was reported without complications.MRI of the lumbar region after 12 mo showed obvious reabsorption of the herniation.CONCLUSION A conservative treatment regimen of TCM,acupuncture,and massage promoted reabsorption of a prolapsed disc.

Construction of recombinant adenoviral vector Ad-CMV-hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer

To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.