| Intervertebral disc degeneration (IDD) is the major reason that cause the disc degenerated disease (DDD) such as lumbar intervertebral disc protrusion (LIDP). The pathogenesis mechanisms of the IDD are complex. IDD is influenced by multiple factors like age, cytokines, nutrition, metabolism, genetic, biomechanics. However, there is increasing evidence implicating cytokines, growth factors and inflammatory mediators in its pathogenesis. The aetiology of IDD is associated with an abnormal response at a cellular and molecular level due to genetic malignancies and/or environmental factors. At the cellular level, the degeneration process is characterised by a decline in the cell population caused by a decrease in pH, nutrient and oxygen supply. At a molecular level, a change of the extracellular matrix (ECM) composition is noted with a decrease in type II collagen and aggrecan content that are the major and uniformly distributed component of the ECM of the NP tissue. This phenomenon leads to a loss of water content that compromises the swelling properties of the disc. With the changes of the cellular and molecular level in the IDD, the structure of the disc becomes more fibrotic with an increase in type I collagen promoting the vascularisation and innervation of the disc. A structural disorganisation of the nucleus pulposus (NP) and the annulus fibrosus (AF) is induced leading to a loss of disc height. This leads to an overall effect on the biomechanics of the spinal column.Since mature intervertebral disc is largely avascular, survival of cells and extracellular matrix entirely relies on the ability of nutrients, and waste products, to move through the vertebral endplates into and out of the disc. As a result, any impairment in the diffusion of nutrients through the disc, vertebral endplate, and subchondral bone may jeopardize the integrity of the disc tissue. In particular, vertebral endplates and subchondral bone are known to act as a barrier that prevents the indiscriminate flow of substances from the vertebral bodies to the disc. If the function of this barrier is compromised, substances that usually are not allowed to penetrate within the disc may reach the disc tissue and cause damaging of extracellular matrix. For instance, if fractures of vertebral endplates allow an indiscriminate penetration of matrixdestroying enzymes within the disc, a damage of extracellular matrix eventually leading to disc degeneration may occur, and if sclerosis of vertebral endplates prevent the waste products to move through the vertebral endplates out of the disc, that ultimately results in the decrease in pH. The acid environment is fit for proteinase to produce a marked effect.The ECM is constantly synthesized by cells of disc or degraded by proteinases produced by disc cells. Normally rates of synthesis and breakdown are in balance, but in disc degeneration the balance is shifted towards degradation with consequent loss of matrix components, disturbances of tissue architecture, and of biomechanical function leading possibly to clinical consequences. The major enzymes responsible for this destruction are metalloproteinases, including the family of matrix metalloproteinases (MMPs), and the family of proteases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS). Tissue inhibitors of metalloproteinases (TIMPs) are also made by disc cells; these are endogenous inhibitors of metalloproteinase activities and thus also play an important role in regulating tissue turnover. The MMPs can degrade all the components of the ECM which exist within the intervertebral disc. The ADAMTS family have highly selective proteolytic activities and have a particular avidity for aggrecan and thus have been termed aggrecanases. The TIMPs (TIMP-1,-2,-3,-4) show little inhibitory specificity although TIMP-3 reportedly selectively inhibits members of the ADAMTS family.With aging and degeneration, intervertebral discs (IVD) undergo substantially morphological and cellular changes, including altered cellularity in the annulus fibrosus (AF) and the nucleus pulposus (NP), increased cell density but decreased cell viability, increased cell senescence and apoptosis, elevated matrix metal loproteinase and aggrecanase activity, and increased expression of catabolic cytokines. These changes lead to progressive loss of proteoglycans and water content in the NP, filling of the NP space with fibrocartilage, derangement/delamination of the AF, narrowing of the disc space, and mechanical failure of the disc. Susceptible gene-mediated familial predisposition, acute or chronic biomechanical injuries, inflamatory factors involved in unbalanced anabolic and catabolic metabolism, a reduced nutrient supply after chondrocyte endplate trauma or ossification, loss of the immune privilege stage in the NP space, cell senescence with aging, and IVD apoptosis are considered to be the main causes or a secondary response of the triggering element in the process of IVD degeneration.Current treatments for disc degeneration such as bed rest, anti-inflammatories, analgesia, and physical therapy as well as surgical measures do not provide a uniformly good outcome. In clinical practice, anterior interbody fusion is performed to relieve discogenic pain and degenerative instability. However, such fusion may accelerate premature degeneration at the junctional level of the fused spine. Evidence from studies investigating the pathogenesis of IVD degeneration shows that it originates from the NP. To halt progressive spinal degeneration, the ultimate therapy is restoration of normal function of the discs. An ideal solution to managing disc degeneration would be to repair the IVDs, producing a matrix with similar or improved biological and biomechanical properties compared with the original. Recent approaches to biological repair and regeneration of the disc function are under investigation, including gene therapy, growth factor injections, cell therapy, and cell-based tissue engineering. With recent advancements in molecular biology (including recombinant DNA technology, the cloning of genes, and gene transfer technology), it becomes possible to contemplate treating the intervertebral disc itself at the molecular level to prevent or delay the progression of disc degeneration.The potential to treat DDD as well as other musculoskeletal disorders on a molecular level evolved with the recognition of potentially therapeutic genes, and has progressed to the successful clinical use of growth factors to improve spinal fusion rates. With regard to intervertebral disc degeneration, the ability to increase proteoglycan synthesis by intervertebral disc cells was first demonstrated by Thompson et al, who showed that the exogenous application of recombinant human transforming growth factor (TGF)-β1 to canine disc tissue in culture stimulated in vitro proteoglycan synthesis. These authors suggested that growth factors may be useful for the treatment of disc degeneration. Gruber et al also demonstrated that TGF-β1 can induce changes in proliferative and extracellular matrix cellular activities in human disc cells in three-dimensioned culture. Subsequent studies with other growth factors such as insulin-like growth factor 1 (IGF-1) and bone morphogenic protein 2 (BMP-2) have also exhibited the ability to upregulate proteoglycan synthesis activity in disc cells.Telomeres are specialized protein-DNA structures at the end of eukaryotic chromosomes. They protect chromosomes from end-to-end fusion and nuclease Amplify hTERT gene with designing special primers and PCR technology, Double enzyme cut the amplified hTERT gene with BglⅡ/Sal I, then link to the PIRES2-EGFP plasmid. Design primers with attb at the two ends, amplify hTERT-PIRES2-EGFP gene. Reclamate the gene in electrophoresis gel and purify it, then recombinate gene to pDONR222 plasmid. Transform the recombinated plasmid into DH5a, wave bacteria, extract plasmid, then recombinate gene to adenovirus vector and prepared for adenovirus packaging.1.2. Packaging, amplification, purification of adenovirusLinearizate the recombinated plasmid pAd-hTERT Pac I, transfect the linearizated plasmid into 293AD cells, cytopathic effect (CPE) achieve to 80%, collect cells, obtain virus with the method of repeated freezing and thawing. Amplify virus in 293AD cells, measure virus titre with end dilution assay and purify the amplified virus with continuous gradient centrifugation in the CsCl cation column and the titre achieve to 2.1×1012 pfu/ml.2. Establish degenerative disc model2.1. Construct degenerative disc model guided by CTIdentify purpose disc with CT and stab purpose disc percutaneously with 18G needle guided by CT after the New Zealand rabbits were anesthetized, with minimally invasive method to construct degenerative disc model.2.2. Identify the degree of the degenerative discIdentify success of construction of degenerative disc model with methods of X-ray and MRI imaging examination, general view and histopathological changes of the discs consistent with the character of degenerative disc, collagen type II reduced significantly in degenerative disc compared to the control group after being detected with immunohistochemical and Western Blot, digestive dyeing method to detect to degenerative discs proteoglycan content than the control group were obviously reduced.3. hTERT gene inhibits cell senescence of the nucleus pulposus3.1. Primary culture of the nucleus pulposus cellsCulture the nucleus pulposus cells with digest method of typeⅡcollagenase, extract discs organization of rats, digest with trypsin for 30 min, then digest disc tissue with typeⅡcollagenase, obtain round like nucleus pulposus cells.3.2. Transfect hTERT-Ad into the nucleus pulposus cellsThe primary generation of nucleus pulposus cells were cultured to the 3rd-5th generation, the constructed hTERT-Ad were transfected to the nucleus pulposus cells, the no-transfected cells as blank group, the transfected EGFP-Ad as virus vector control group. Identify the success of transfection by detecting the fluorescence in the cells that have been transfected with hTERT-Ad.3.3. hTERT-Ad inhibits cell senescence of nucleus pulposusPerform serial passage of the primary culture cells after being transfected hTERT-Ad, the shape of the nucleus pulposus cells become hypertrophy and more particle appear and increase in the cellular cytoplasm when observed the no-transfected hTERT-Ad cells with light microscopy in the senescent cells, fluorescence reduced also; typeⅡcollagen and hTERT gene expression of the transfected hTERT-Ad nucleus pulposus cells that were detected with Polymerase chain reaction (PCR) are higher than that in control and EGFP-Ad group; typeⅡcollagen and hTERT protein expression of the transfected hTERT-Ad nucleus pulposus cells detected by Western Blot are also higher than that in control and EGFP-Ad group. Proteoglycan of the transfected hTERT-Ad nucleus pulposus cells detected by Alcian blue staining is significantly higher than that in the in control and EGFP-Ad group. The senescent cells percentage that detected and calculated by activity ofβ-galactosidase staining (SA-β-gal) assay in the transfected hTERT-AD degradation. In humans, telomeres consists of approximately 4 14 kb of TTAGGG duplex repeats and 150-200 bases of singlestranded DNA overhang running 5’to 3’ toward the end of chromosome. Because of the end replication problem, telomeres in human cells erode by approximately 100 bp with each cell division. Telomerase is the key enzyme for the stabilization of telomere by adding TTAGGG repeats to telomere ends. It is a ribonucleoprotein that utilizes its RNA component as the template to synthesis telomere repeats. Telomeres are terminal protein-DNA complexes forming capping structures that function to stabilize chromosomal ends and prevent them from being recognized by the cell as DNA double strand breaks. Functional telomeres require sufficient numbers of telomeric DNA repeats, as well as the proper repertoire and amounts of telomere associated proteins—mutation or loss of either can lead to "uncapping" and telomere dysfunction. Importantly, in most normal somatic cells, telomeric DNA shortens during cell division due to incomplete replication by standard DNA polymerases. In humans, telomerase activity is not detected in most somatic cells. Progressive telomere shortening after each cell division leads to cellular senescence after 60-80 population doublings. Escaping senescence leads to further shortening of telomeres and can eventually cause cells to enter crisis and cell death. Cells at this stage appear aneuploidy because telomere loss induces chromosome instability through the breakage/fusion/bridge cycles. Few cells stabilize telomere length through activating telomerase activity.Telomerase, an enzyme synthesizing TTAGGG telomere repeats, is pertinent to self-renewal potential of stem cells and is required for long-term cellular proliferation and survival. Telomerase is tightly regulated throughout development. In early embryonic tissues and stem cells, telomerase is highly expressed and telomeres are stably maintained. Although telomerase is detected in many adult stem and progenitor cells, its level in these cells is not sufficient to maintain telomere length. Furthermore, telomerase is undetectable in the majority of human adult somatic cells, which are programmed for a limited number of cell divisions. These cells undergo senescence and/or apoptosis after their telomeres are depleted, which is likely a molecular basis for the replicative senescence checkpoint in human cells, known as the Hayflick limit.Telomerase consists of three core subunits:hTR, the human telomerase RNA component, TP, telomerase-associated proteins, and hTERT, the human telomerase reverse transcriptase. In humans, telomerase is active in early fetal development, but is repressed in essentially all somatic tissues before birth. In postnatal somatic tissues, telomerase is repressed, or is present transiently or at very low levels, and telomeres gradually erode with time and cell division. The hTERT subunit is a catalytic subunit homologue protein of human telomerase and its gene transcription correlates with telomerase activity in most cells and tissues that have been examined. Ectopic expression of the hTERT gene is sufficient for cellular immortalization; yet normal human cells rarely undergo spontaneous immortalization. The RNA component of human telomerase (hTR) provides the template for telomere repeat synthesis. Several studies have shown that disrupting the function of telomerase RNA leads to progressive shortening of telomeres, suggesting that this component plays an essential role in telomerase function. The functional significance of the telomerase-associated proteins remains unclear.Gene therapy that hTERT gene inhibit disc degeneration is not reported. In this research, the in vivo and in vitro study is to investigat the effect of hTERT gene on prevention of disc degeneration by transfecting the hTERT gene to the disc cells and to the NP.1. Construct the adenovirus plasmid of hTERT genes1.1. Construct process of the adenovirus plasmid of hTERT genes significant difference between CON, hTERT, PBS three groups after the first month of treatment, no significant sclerosis of endplate and bone formation of vegetation; the IDH percentage of hTERT group are significantly higher than PBS group and no significant difference with CON group at the third month and the sixth month time point. Began to appear sclerosis on endplate but no the bone formation of vegetation in hTERT group at the sixth month time point, but obvious sclerosis and hardening of vertebral endplate and the bone formation of vegetation were observed at the sixth month time point in PBS group. The signal changes are apparent decrease with the extension of time point gradually in PBS group detected by MRI images, but they decline slower in hTERT group and CON group, there is no significantly different.4.3. General observation of inhibition of disc degeneration by hTERT.Extract discs of the control, PBS and hTERT-Ad group of rabbits at the time points of 1,3 and 6 months, respectively. The apparent degenerative changes of the disc nucleus pulposus were observed in PBS group, but the changes of the hTERT group are not so significant after the injection at the sixth month time point. There are small amount of nucleus pulposus in nucleus pulposus central and just mild degeneration of anulus fibrosus in hTERT group as the changes of the first month that were observed at PBS group.4.4. Pathology changes of inhibition of disc degeneration by hTERT.Extract discs of the control, PBS and hTERT-Ad group of rabbits at the time points of 1,3 and 6 months, respectively. Fixed with 4% formalin, dehydrated conventionally, embeded with paraffin. The slices of control group, PBS and hTERT-Ad group were stained with HE and collagen II by immunohistochemistry methods. At the first month, the boundary of the anulus fibrosus and the nucleus pulposus is separated slightly in the hTERT group, the number of the nucleus pulposus notochord cells reduced, the gap between the nucleus pulposus cells also nucleus pulposus cells is significantly lower than that in the control group and EGFP-Ad group.4. hTERT inhibit disc degeneration in vivo4.1. Transfect hTERT-Ad into disc cells in vivo.18 New Zealand rabbit, divided into 1,3 and 6 months three group for observation,6 experimental rabbits in each of the three groups, in each rabbit, inject PBS into L5/6 for PBS control group; inject hTERT-Ad into 4/5 for treatment group; L3/4 as control group. All the rabbits were mitigated with SuMianXin and atropine quickly, then anesthetise with sodium pentobarbital by intravenous injection, take X-ray lateral projection of lumbar on the workbench with left-lateral position; take T1-weighted images and T2-weighted images of New Zealand rabbit lumbar spine by human knee detecting coil on MRI table. Perform percutaneous stab into intervertebral disc with 18G needles guided by CT, determine the needle tip achieve to the nucleus pulposus center with CT scan, repeatedly puncture three times, gel like samples of nucleus pulposus are visible in the tip of tube. Control group perform no stab, inject 20μL sterile PBS liquid into the nucleus pulposus after puncture with trace syringe in PBS group, inject 20μL hTERT-Ad liquid into the nucleus pulposus after puncture with trace syringe in hTERT-Ad treatment group, injects 0.2 million U/Kg penicillin sodium daily into the rabbits in postoperative three days.4.2. Imaging changes of inhibition of disc degeneration by hTERTTake 6 rabbits at the time points of 1,3 and 6 months, respectively. Take X-ray lateral projection of lumbar on the workbench with left-lateral position; take T1-weighted images and T2-weighted images of New Zealand rabbit lumbar spine by human knee detecting coil on MRI table after success of anesthesia. Intervertebral disc height (IDH) percentage of post/preoperation were calculated and compared between the CON, hTERT, PBS three groups. Found that the IDH percentage are no appeared separation; but in the PBS group, the disc degenerated apparently, the nucleus pulposus has transformed into class chondroid cells, cells arranged disorderly. The nucleus pulposus notochord cells further reduced in hTERT group at the third and sixth month, intercellular substance decreased, the nucleus pulposus and anulus fibrosus structures become more disorder, but still exist apparent normal tissue structure and component; in PBS group, nucleus pulposus further continue to differentiate into chondroid like cells, the boundary structure of the nucleus pulposus and anulus fibrosus become more disorder and the number of chondroid cells increased in nucleus pulposus, the degree of disc degeneration in PBS group is significantly serious than that in hTERT-Ad group and control group. Type II collagen within the cytoplasm of notochord cells is rich in the control group and hTERT group, positive expression of typeⅡcollagen are also strong at extracellular, but the expression of typeⅡcollagen at extracellular reduced significantly in PBS group at the first month. Though the chondroid like cells in the PBS group also express typeⅡcollagen, did the quantity of expression decrease obviously when compared with the control and hTERT-Ad group at the third and sixth month time point.4.5. Molecular biological changes of inhibition of disc degeneration by hTERT.4.5.1 TypeⅡcollagen expression of the discsExtract total protein of discs that taken from control, PBS and hTERT-Ad group rabbits at the 1 st,3rd,6th month with 2 x SDS lysis liquid, respectively. Detect the expression of typeⅡcollagen within the lytic protein with Western Blot test techniques. As time extend, typeⅡcollagen reduced significantly in the PBS group than that in hTERT and control group. The expression of typeⅡcollagen reduced slower in the hTERT group than that in PBS group.4.5.2 Proteoglycan expression of discs Extract discs of the control, PBS and hTERT-Ad group of rabbits at the time points of 1,3 and 6 months, respectively. Digest the discs with papain digestive method. Detect the optical density (OD) value of the digestive liquid by the absorption spectrum of the dye 1,9-dimethylmethylene blue (DMB) to calculate the proteoglycans concentration of the discs according to the OD value standard curve of chondroitin sulfate. Compare the proteoglycans concentration of the disc tissue of the control group, PBS group and hTERT-Ad treatment group. The results show that there is not significant difference between the hTERT and control group for the comparison of the proteoglycans concentration of the discs, but there is significant difference between the control group and PBS groups and hTERT group and PBS group in the first month. There is significant difference compared the hTERT group with PBS group, no difference between the hTERT group and the control group at the third month and the sixth month as well.Conclusions:In this study, we investigated the prevention effect of hTERT gene therapy on disc degeneration by in vivo and in vitro. Firstly, the recombinated plasmid pAd-hTERT was constructed with clon techniques, adenovirus recombinated with hTERT gene were amplified in HEK 293AD cells, the amplified adenovirus were purified with continuous gradient centrifugation in the CsCl cation column and virus titre was 2.1×1012 pfu/ml. Secondly, the degenerative disc model was established with percutaneous puncture method guided by CT. The typeⅡcollagen and proteoglycan content reduced significantly in degenerative disc compared with the control group. Thirdly, the hTERT gene prevents the disc cells senescence in primary cultured nucleus pulposus cells. The typeⅡcollagen and hTERT gene expression in the hTERT-Ad group are higher than that in CON and EGFP-Ad group; typeⅡcollagen and hTERT protein expression in the hTERT-Ad group are higher than that in CON and EGFP-Ad group as well. Proteoglycan of the hTERT-Ad group is significantly higher than that in the in CON and EGFP-Ad group. The senescent cells percentage in the hTERT-AD group is significantly lower than that in the CON group and EGFP-Ad group. Lastly, hTERT gene prevents the disc degeneration in vivo.when the hTERT gene was transfected into the rabbit discs. The IDH ratio of hTERT group is significantly higher than PBS group, but no significant difference compared with CON group at the third month and the sixth month time point. The type 11 collagen and proteoglycans concentration in the discs tissues reduced significantly in the PBS group than that in hTERT and CON group. |
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