| Degenerative lumbar disc herniation,a common encountered spine-related disorder,is a syndrome caused by degeneration of the intervertebral disc,disruption of the annulus fibrosus,irritation of the nucleus pulposus herniation and compression of nerves root or ponytail.LDH patients always accompany with severe sciatica,which has severely affected their daily activities and work,what's more,a few severe patients cannot take care of themselves in daily life.Clinical treatment strategies include conservative treatments and surgeries,but there is no consensus on the therapeutic methods at home and arboad so far.So it's necessary to scientifically analyze pathogenic factors in the process of LDH,so that we can have deeper and more comprehensive understanding of disease formation mechanism to provide more efficient options for clinical treatments.The medical imaging examinations on degenerative lumbar disc herniation patients have shown the disruption of the vertebral body or endplate in clinic,which changes the biomechanical structure adjacent to the spine and causes several disorders,among which low back pain is the most common symptom.However whether there is a correlation between degenerative lumbar disc herniation and disruption of vertebral body or endplate,we have no related researches so far.As shown in the research,the pain caused by the degenerative LDH has no correlation with the nervous root compression,while it's closely related to the inflammatory reactions.The animal experiments also demonstrate that the nucleus pulposus has strong inflammatory effects.In the vertebral body inflammatory environment,the blood sinus in the marrow cavity generates vascular bud,which decreases the blood into the cartilage endplate,cuts off the main pathway to get the nutrients for the intervertebral disc and leads to the vertebral disc degeneration.With the continuous existing inflammatory environment,the increase of osteoclast' number and the hyperfunction of osteoclast break out the dynamic balance and lose bone quality severely,which causes a serious of clinical symptoms including osteoporosis,disorder of the mineral and bone metabolism and the disruption of vertebral body or endplate.Osteoclast activation in the vertebral body,increasing number,the tiny shape of the bone trabecular,the overburden of the last bone trabecular and the decreased of the intensity are similar with the osteoporosis.In that case,a single bone trabecular will form microscopic fracture due to the too large stress,so that the liquid exchanging state will be influenced between vertebral spongy body,annulus fibrosus and nucleus pulposus without vessels to destroy the pathway of providing nutrition to the nucleus pulposus,as a result the degeneration of the intervertebral disc will be accelerated.The diseases and the fracture due to themselves will have significant influence on patients' life quality and survival time.To chase for the breakthrough on the pathogenetic mechanism and the treatments of the disorder of mineral and bone metabolism,osteoclast has become the research priority.However,how the osteoclast is activated and the mechanism of how osteoclast plays a pathological role in degenerative LDH are not clear so far.With the influence of inflammatory environments,the number and the activity of osteoclast have been on the rise,it's necessary for osteoclast,derived from mononuclear phagocyte system,to differentiate into multinuclear mature osteoclast via gene regulating in the body.The activated signal pathway is mediated through M-CFS,RANKL,]TAM,as well as non-classical pathway consisted of some cytokines,growth factor and so on.At present,the RANKL is regarded as the most immediate endogenous molecule activating osteoclast.The RANKL forms the complex including TRAF6 and c.Src and activates downstream signal pathway including NF-?J3?MAPK and Ca2+ pathway via combing and collecting the RANK,which is a receptor on the surface of the osteoclast.In addition,it activates the nuclear factor including NF-kB,AP.1 and NFATcl and activates the expression of osteoclast related genes activating,such as TRACP,MMP-9,integrin133 and cathepsin k.,via intracellular signal transformation pathway.The function above accelerate osteoclast differentiation,strengthen bone absorption activity,inhibits apoptosis and result in osteoclast proliferation and hyperfunction.MicroRNA is a naturally non-coding endogenous RNA which includes 20-22 nucleotide.MicroRNA affects the post-transcriptional expression level via recognizing and combining the 3'UTR of the target gene mRNA.With more and more miRNA being discovered,its biological effects have been paid more and more attention to.MiRNA plays an important role in regulating various bone diseases,such as bone tumor,osteoarthritis,necrosis of femoral head,bone metabolism related diseases and so on.However there are few researches focused on the function of the micro-RNA.Micro-RNA was firstly detected in 2007 and the researches were mainly about the changes of the pathological expression in pancreatic cancer.At present although a series of researches have been demonstrated that the osteoclast plays an important role in degeneration intervertebral disc herniation,we know little about the changes of expression and function of microRNA-216 in osteoclast differentiation.The degenerative operated lumbar disc herniations were classified based on CT and MRI.We found that the duration of pain was longer with the more severity of disrupture in the lumbar or endplate.And the expression of miR-216 was decreased in the intervertebral discs with vertebral or endplate disruption.by detecting the expression level of miR-216 in the nucleus pulposus.We also found that the expression of miR-216 was decreased in the process of osteoclast differentiation in vitro.In the experiment,it was found that MiR-216 can regulate the differentiation of osteoclasts by targeting the RANKL-RNAK-NF-kappa B signaling pathway.In a word,miR-216 as the central link,it was found that the expression of miR-216 was decreased,and the inhibition of osteoclast was relieved in the process of intervertebral disc degeneration.So it is one of the reasons for the disrupture of the vertebral body or endplate.So the study of mechanism of targeting gene miR-216 and its biological functions are of clinical value for the treatment and prevention of degenerative lumbar disc herniation patients with great potential;we hope that with further research,we can discover more and more targets with new breakthrough in clinical treatment.PART1Objective:To discuss the relation between the medical maging examinations and clinical characteristics and the correlation between whether the intervertebral disc is degeneration and the expression level of the miR-216 in nucleus pulposus in degeneration intervertebral disc herniation.To illuminate the correlation between the degeneration degree of vertebral disc and the disruption of vertebral body or endplate,iin the meantime to illuminate the role of miR-216 in degeneration lumbar intervertebral herniation.Method:In this research,we recruited 192 patients who had received surgical therapy.According to AAOS&ISSLS classify and Pfirnnann grading these patients were divided into the following groups based on CT and MRI:the bulging lumbar disc herniation group,the herniated lumbar disc herniation group and the prolapse lumbar disc herniation goup.The research needed to record the essential feature of every cases,such as:gender,age,occupation,the progression of the disease,the blood glucose and so on.Meanwhile we observed the disruption of the vertebral body or endplate based on the imaging examination results and the change of clinical characteristics between different patients.The control group comprised of 29 cases,all of which were young,suffered acute burst trauma and had no degeneration in intervertebral disc on MR1 examination.Then we cut off the nucleus pulposus of all patients and detected miR-216 expression in the nucleus pulposus in the normal and degeneration LDH patients by such means including qRT-PCR,Western blot,luciferase experiment and so onResult:According to AAOS&ISSLS classify and Pfirrmann grading and combined with CT and MRI characteristics:intervertebral herniation degree,signal change of disc,structure change of disc,whether the boundary between nucleus pulposus and annulus fibrosus is clear and intervertebral compression signs,such as compression of dural sac or nerve root,transformation,transposition or disappearing of epidural fat,these patients were divide into following groups:bulging LDH in 36 patients,herniated LDH in 117 patients,prolapse LDH in 39 patients and the control group in 27patients.It was discovered that the percentages of vertebral body or endplate disruption were 2.8%in bulging LDH group,84%in herniated LDH group and 95%in prolapse LDH group respectively(P<0.01)based on observing CT and MRI imaging examination results.There was statistically significant difference between the bulging group and the other two groups on average duration of low back pain from the onset of symptoms to surgery(P<0.05);the ratio of patients who were undertaking physical labor was higher than the herniated group and the prolapse group compared to the bulging group(P<0.01).The change of miR-216 expression level was detected in the samples of nucleus pulposus of two group:the degeneration LDH patients and other traumatic patients.The result showed that miR-216 expression level decreased in nucleus pulposus in degeneration LDH patients compared to the normal control groupConclusion:1.The degeneration of LDH patients is heavier,the disruption of vertebral body or endplate will be heavier.2.The average duration of low back pain has a positive correlation with the degree of degeneration LDH.3.If people undertake higher physical labor intensity,the degeneration will be heavier.4.The rate of disruption of vertebral body or endplate in degenerative disc herniation is higher than that of normal.5.The miR-216 expression level downregulates in the intervertebral disc tissues of degeneration LDH patients.PART2Objective:During the process of invitro monocyte being induced into osteoclast,we observed the correlation between the miR-216 and intracellular key protein and confirmed the regulating function and molecule biology mechanism in the osteoclastogenesis process to provide new strategy for clinical treatmentsMethods:Osteoclast differentiation was induced from human peripheral blood mononuclear cells via colony stimulating factor in vitro,the expression level of proteins and nucleic acid including miR-216,CALCR,CTSK,TRAP and so on was detected via using these means such as TRAP staining,Western blot,qRT-PCR,and luciferase report system.Results:1.Osteoclast differentiation was induced from human peripheral blood mononuclear cells in vitro.The proteins expression including CALCR,CTSK and TRAP increased,and the degeneration of osteoclast formation was confirmed through TRAP staining too.2.It was shown that miR-216 expression decreased during the osteoclast differentiation process by qRT-PCR detection,which suggested that miR-216 may play a role in the osteoclast differentiation.3.It was confirmed that the expression level of proteins and nucleic acid including miR-216,CALCR and CTSK,TRAP decreased via transfecting miR-216,Western blot and qRT-PCR,and TRAP staining also verified that the osteoclast differentiation was inhibited in the process of osteoclast differentiation in vitro.4.It was verified that miR-216 could target RANK directly via Western blot,qRT-PCR,and luciferase report system transfection.5.The Western blot defection result showed that p-NF-kB p65 expression decreased,which suggested that miR-216 downregulated osteoclast differentiation via regulating RANKL-RNAK-NF-Kb signal pathway.Conclusion:1.MiR-216 expression will decrease during the osteoclast differentiation process.2.MiR-216 regulates the osteoclast differentiation via targeting RANKL-RNAK-NF-Kb signal pathway.Experience summary:During the process of degeneration LDH,low expression miR-216 in nucleus pulposus may release the inhibition of osteoclast,which is one of the causes leading to vertebral bone or endplate disruption. |
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