Ua tsaug rau koj tuaj xyuas Nature.com. Qhov browser version koj siv tau txwv CSS kev txhawb nqa. Txhawm rau kom tau txais txiaj ntsig zoo tshaj plaws, peb pom zoo kom siv qhov browser tshiab (lossis disabling compatibility hom hauv Internet Explorer). Nyob rau lub sijhawm no, txhawm rau ua kom muaj kev txhawb nqa txuas ntxiv, peb yuav tso saib lub vev xaib yam tsis muaj qauv thiab JavaScript.
Kev tsim tsiaj cov qauv ntawm kev hloov pauv (MC) yog lub hauv paus tseem ceeb rau kev kawm MC. Tsib caug-plaub New Zealand Dawb luav tau muab faib ua pawg sham-kev ua haujlwm, pawg leeg nqaij (ME pawg) thiab pawg nucleus pulposus implantation group (NPE pawg). Nyob rau hauv pawg NPE, lub intervertebral disc tau nthuav tawm los ntawm anterolateral lumbar phais mus kom ze thiab ib rab koob tau siv los txhawm rau L5 vertebral lub cev ze ntawm lub phaj kawg. NP tau muab rho tawm los ntawm L1/2 intervertebral disc los ntawm ib tug syringe thiab txhaj rau hauv nws. Drilling ib qhov nyob rau hauv cov pob txha subchondral. Cov txheej txheem phais thiab cov txheej txheem drilling hauv cov leeg nqaij implantation thiab pawg sham-kev ua haujlwm tau zoo ib yam li cov hauv pawg NP implantation. Hauv pab pawg ME, ib daim ntawm cov leeg tau muab tso rau hauv lub qhov, thaum nyob hauv pawg neeg ua haujlwm sham, tsis muaj dab tsi tau muab tso rau hauv lub qhov. Tom qab ua haujlwm, MRI scanning thiab kuaj molecular lom tau ua. Lub teeb liab hauv pawg NPE tau hloov pauv, tab sis tsis muaj qhov pom tseeb hloov pauv hauv pawg sham-kev ua haujlwm thiab pawg ME. Kev soj ntsuam histological tau pom tias qhov txawv txav ntawm cov nqaij mos proliferation tau pom nyob rau ntawm qhov chaw cog, thiab kev qhia ntawm IL-4, IL-17 thiab IFN-γ tau nce hauv pawg NPE. Kev cog qoob loo ntawm NP rau hauv cov pob txha subchondral tuaj yeem tsim tus qauv tsiaj ntawm MC.
Cov kev hloov pauv Modic (MC) yog cov kab mob ntawm cov kab mob vertebral endplates thiab cov pob txha pob txha nyob ib sab pom ntawm magnetic resonance imaging (MRI). Lawv feem ntau tshwm sim hauv cov tib neeg uas muaj cov tsos mob sib xws 1. Ntau qhov kev tshawb fawb tau hais txog qhov tseem ceeb ntawm MC vim nws txoj kev koom tes nrog mob nraub qaum (LBP) 2,3. de Roos li al.4 thiab Modic et al.5 ntawm nws tus kheej ua ntej piav txog peb yam sib txawv ntawm cov teeb liab subchondral txawv txav hauv cov pob txha pob txha. Modic hom I hloov pauv yog hypointense ntawm T1-weighted (T1W) sequences thiab hyperintense ntawm T2-weighted (T2W) sequences. Cov kab mob no nthuav tawm fissure endplates thiab cov ntaub so ntswg vascular granulation nyob ib sab hauv cov pob txha pob txha. Modic hom II hloov pauv qhia cov teeb liab siab ntawm ob qho tib si T1W thiab T2W ib ntus. Nyob rau hauv hom kab mob no, endplate kev puas tsuaj tuaj yeem pom, nrog rau kev hloov pauv ntawm cov pob txha uas nyob ib sab. Modic hom III kev hloov pauv qhia cov teeb liab qis hauv T1W thiab T2W ib ntus. Cov kab mob Sclerotic sib thooj rau cov phaj kawg tau pom 6. MC suav hais tias yog kab mob pathological ntawm tus txha nqaj qaum thiab muaj kev cuam tshuam nrog ntau yam kab mob degenerative ntawm tus txha caj qaum 7,8,9.
Xav txog cov ntaub ntawv muaj, ntau qhov kev tshawb fawb tau muab cov ncauj lus kom ntxaws rau hauv etiology thiab pathological mechanisms ntawm MC. Albert et al. qhia tias MC tej zaum yuav tshwm sim los ntawm disc herniation8. Hu et al. ntaus nqi MC rau cov disc degeneration loj heev 10. Kroc tau tshaj tawm lub tswv yim ntawm "sab hauv disc rupture," uas hais tias rov ua dua disc raug mob tuaj yeem ua rau microtears hauv lub phaj kawg. Tom qab cleft tsim, endplate kev puas tsuaj los ntawm lub nucleus pulposus (NP) yuav ua rau ib tug autoimmune teb, uas ntxiv mus rau txoj kev loj hlob ntawm MC11. Ma et al. sib koom ib qho kev pom zoo sib xws thiab tshaj tawm tias NP-induced autoimmunity plays lub luag haujlwm tseem ceeb hauv pathogenesis ntawm MC12.
Cov kab mob tiv thaiv kab mob, tshwj xeeb tshaj yog CD4 + T pab lymphocytes, ua lub luag haujlwm tseem ceeb hauv cov kab mob autoimmunity13. Qhov tsis ntev los no nrhiav pom Th17 subset tsim cov proinflammatory cytokine IL-17, txhawb chemokine qhia, thiab stimulates T hlwb nyob rau hauv lub cev puas tsuaj los tsim IFN-γ14. Th2 hlwb kuj ua lub luag haujlwm tshwj xeeb hauv cov kab mob ntawm lub cev tiv thaiv kab mob. Kev nthuav qhia ntawm IL-4 ua tus neeg sawv cev Th2 ntawm tes tuaj yeem ua rau muaj kev tiv thaiv kab mob hnyav heev15.
Txawm hais tias muaj ntau qhov kev tshawb fawb soj ntsuam tau ua tiav ntawm MC16,17,18,19,20,21,22,23,24, tseem tsis muaj cov qauv kev sim tsiaj tsim nyog uas tuaj yeem ua raws li cov txheej txheem MC uas nquag tshwm sim hauv tib neeg thiab tuaj yeem ua tau. siv los soj ntsuam cov etiology lossis kev kho tshiab xws li kev kho mob. Txog niaj hnub no, tsuas yog ob peb tus tsiaj qauv ntawm MC tau tshaj tawm los kawm txog cov txheej txheem pathological.
Raws li autoimmune txoj kev xav tau los ntawm Albert thiab Ma, qhov kev tshawb fawb no tau tsim ib qho yooj yim thiab rov tsim dua luav MC qauv los ntawm autotransplanting NP ze ntawm qhov kawg ntawm qhov kawg phaj. Lwm lub hom phiaj yog los soj ntsuam cov yam ntxwv histological ntawm cov qauv tsiaj thiab tshuaj xyuas cov txheej txheem tshwj xeeb ntawm NP hauv kev txhim kho MC. Txog qhov kawg, peb siv cov tswv yim xws li molecular biology, MRI, thiab cov kev tshawb fawb histological los kawm txog kev nce qib ntawm MC.
Ob tug luav tuag los ntshav thaum lub sijhawm phais, thiab plaub tus luav tuag thaum lub sijhawm tshuaj loog thaum MRI. Lwm 48 tus luav tau muaj sia nyob thiab tsis pom tus cwj pwm lossis cov tsos mob ntawm lub paj hlwb tom qab kev phais.
MRI qhia tau hais tias lub teeb liab siv ntawm cov ntaub so ntswg embedded nyob rau hauv txawv qhov sib txawv. Lub teeb liab siv ntawm L5 vertebral lub cev nyob rau hauv pawg NPE maj mam hloov ntawm 12, 16 thiab 20 lub lis piam tom qab tso (T1W ib ntus pom cov teeb liab qis, thiab T2W ib ntus pom cov teeb liab sib xyaw ntxiv rau cov teeb liab qis) (Daim duab 1C), thaum MRI tshwm sim. ntawm ob pab pawg ntawm cov khoom embedded tseem nyob ruaj khov nyob rau tib lub sijhawm (Fig. 1A, B).
(A) Cov neeg sawv cev ua ntu zus MRIs ntawm tus luav lumbar qaum ntawm 3 lub sij hawm cov ntsiab lus. Tsis muaj teeb meem txawv txav tau pom nyob rau hauv cov duab ntawm pawg sham-kev ua haujlwm. (B) Cov yam ntxwv ntawm lub cev vertebral hauv pawg ME zoo ib yam li cov hauv pawg sham-kev ua haujlwm, thiab tsis pom qhov hloov pauv tseem ceeb ntawm qhov chaw embedding lub sijhawm. (C) Hauv pab pawg NPE, cov teeb liab qis tau pom meej meej hauv T1W ib ntus, thiab cov teeb liab sib xyaw thiab cov teeb liab qis tau pom meej meej hauv T2W ntu. Los ntawm 12-lub lim tiam mus rau 20-lub lim tiam, cov teeb meem tsis sib haum xeeb nyob ib puag ncig cov teeb liab qis hauv T2W ib ntus txo.
Pom tseeb pob txha hyperplasia tuaj yeem pom ntawm qhov chaw cog qoob loo ntawm lub cev vertebral hauv pawg NPE, thiab cov pob txha hyperplasia tshwm sim sai ntawm 12 mus rau 20 lub lis piam (Daim duab 2C) piv nrog rau pawg NPE, tsis muaj kev hloov pauv tseem ceeb hauv cov qauv vertebral. lub cev; Sham pawg thiab ME pawg (Fig. 2C) 2A,B).
(A) Qhov saum npoo ntawm lub cev vertebral ntawm qhov implanted feem yog du heev, lub qhov zoo zoo, thiab tsis muaj hyperplasia nyob rau hauv lub cev vertebral. (B) Cov duab ntawm qhov chaw cog rau hauv pawg ME zoo ib yam li hauv pawg sham lag luam, thiab tsis muaj qhov pom tseeb hloov pauv ntawm qhov pom ntawm qhov chaw cog rau lub sijhawm. (C) Pob txha hyperplasia tshwm sim ntawm qhov chaw cog hauv NPE pawg. Cov pob txha hyperplasia tau nce sai heev thiab txawm txuas ntxiv los ntawm intervertebral disc mus rau contralateral vertebral lub cev.
Kev tshuaj xyuas histological muab cov ncauj lus kom ntxaws ntxiv txog kev tsim cov pob txha. Daim duab 3 qhia cov duab ntawm cov seem tom qab ua haujlwm nrog H&E. Hauv pab pawg sham-kev ua haujlwm, cov chondrocytes tau npaj tau zoo thiab tsis pom cov cell proliferation (Fig. 3A). Qhov xwm txheej hauv pab pawg ME zoo ib yam li hauv pawg sham-kev ua haujlwm (Fig. 3B). Txawm li cas los xij, hauv pawg NPE, ntau tus chondrocytes thiab kev loj hlob ntawm NP-zoo li cov hlwb raug pom ntawm qhov chaw cog qoob loo (Fig. 3C);
(A) Trabeculae tuaj yeem pom nyob ze ntawm lub phaj kawg, cov chondrocytes tau ua kom zoo zoo nrog cov cell loj thiab cov duab thiab tsis muaj kev loj hlob (40 zaug). (B) Cov xwm txheej ntawm qhov chaw cog qoob loo hauv pawg ME zoo ib yam li ntawm pawg sham. Trabeculae thiab chondrocytes tuaj yeem pom, tab sis tsis muaj qhov pom tseeb tshwm sim ntawm qhov chaw cog qoob loo (40 zaug). (B) Nws tuaj yeem pom tias chondrocytes thiab NP-zoo li cov hlwb proliferate loj, thiab cov duab thiab qhov loj ntawm chondrocytes tsis sib npaug (40 zaug).
Kev qhia ntawm interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, thiab interferon γ (IFN-γ) mRNA tau pom nyob rau hauv ob pawg NPE thiab ME. Thaum cov kev qhia theem ntawm cov hom phiaj tau muab piv, cov kab lus ntawm IL-4, IL-17, thiab IFN-γ tau nce ntau hauv pawg NPE piv nrog cov pab pawg ME thiab pawg ua haujlwm sham (Fig. 4) (P <0.05). Piv nrog rau pawg neeg ua haujlwm sham, cov lus qhia theem ntawm IL-4, IL-17, thiab IFN-γ hauv ME pab pawg tau nce me ntsis thiab tsis ncav cuag kev hloov pauv (P> 0.05).
Qhov kev qhia mRNA ntawm IL-4, IL-17 thiab IFN-γ hauv pawg NPE tau pom muaj qhov sib txawv ntau dua li cov hauv pawg neeg ua haujlwm sham thiab pawg ME (P <0.05).
Hauv qhov sib piv, cov theem qhia hauv pawg ME tsis pom qhov txawv txav (P> 0.05).
Western blot tsom xam tau ua tiav siv cov khoom lag luam muaj cov tshuaj tiv thaiv kab mob tiv thaiv IL-4 thiab IL-17 kom paub meej tias qhov hloov pauv mRNA tus qauv qhia. Raws li pom hauv daim duab 5A,B, piv nrog ME pab pawg thiab pawg neeg ua haujlwm sham, cov protein ntau ntawm IL-4 thiab IL-17 hauv pawg NPE tau nce ntau (P <0.05). Piv nrog rau pawg neeg ua haujlwm sham, cov qib protein ntawm IL-4 thiab IL-17 hauv ME pab pawg kuj ua tsis tau raws li kev hloov pauv tseem ceeb (P> 0.05).
(A) Cov qib protein ntawm IL-4 thiab IL-17 hauv pawg NPE tau nce siab dua li cov hauv pawg ME thiab pab pawg placebo (P <0.05). (B) Western blot histogram.
Vim muaj tsawg tus tib neeg cov qauv tau txais thaum lub sij hawm phais, cov kev tshawb fawb meej thiab cov ncauj lus kom ntxaws ntawm cov kab mob ntawm MC yog qhov nyuaj me ntsis. Peb tau sim tsim tus qauv tsiaj ntawm MC los kawm txog nws cov txheej txheem pathological. Nyob rau tib lub sijhawm, kev ntsuam xyuas hluav taws xob, kev tshuaj xyuas histological thiab kev ntsuam xyuas molecular biological tau siv los ua raws li cov chav kawm ntawm MC induced los ntawm NP autograft. Raws li qhov tshwm sim, NP implantation qauv ua rau maj mam hloov ntawm cov teeb liab siv los ntawm 12-lub lis piam mus rau 20-lub lim tiam cov ntsiab lus (sib xyaw cov teeb liab qis hauv T1W ib ntus thiab qis teeb liab hauv T2W ib ntus), qhia cov ntaub so ntswg hloov, thiab cov kab mob thiab cov molecular. Kev soj ntsuam lom neeg tau lees paub cov txiaj ntsig ntawm kev tshawb fawb hluav taws xob.
Cov txiaj ntsig ntawm qhov kev sim no qhia tau hais tias kev pom thiab kev hloov pauv histological tshwm sim ntawm qhov chaw ntawm kev ua txhaum ntawm lub cev vertebral hauv pawg NPE. Nyob rau tib lub sijhawm, kev qhia ntawm IL-4, IL-17 thiab IFN-γ cov noob, nrog rau IL-4, IL-17 thiab IFN-γ tau pom, qhia tias kev ua txhaum ntawm autologous nucleus pulposus cov ntaub so ntswg hauv vertebral. Lub cev tuaj yeem ua rau muaj kev hloov pauv ntawm cov teeb liab thiab morphological. Nws yog ib qho yooj yim kom pom tias cov yam ntxwv ntawm lub cev vertebral ntawm tus tsiaj qauv (tso teeb liab hauv T1W ib ntus, cov teeb liab sib xyaw thiab cov teeb liab qis hauv T2W sib lawv liag) zoo ib yam li cov tib neeg vertebral hlwb, thiab MRI cov yam ntxwv kuj. paub meej tias qhov kev soj ntsuam ntawm histology thiab tag nrho lub cev nqaij daim tawv, uas yog, cov kev hloov hauv lub cev vertebral hlwb tau nce zuj zus. Txawm hais tias cov lus teb inflammatory tshwm sim los ntawm kev raug mob hnyav tuaj yeem tshwm sim sai tom qab kev sib tsoo, MRI cov txiaj ntsig tau pom tias kev hloov pauv ntawm cov teeb liab tshwm sim 12 lub lis piam tom qab puncture thiab txuas ntxiv mus txog 20 lub lis piam yam tsis muaj cov tsos mob ntawm kev rov qab los lossis kev hloov pauv ntawm MRI. Cov txiaj ntsig no qhia tias autologous vertebral NP yog ib txoj hauv kev txhim khu kev qha rau kev tsim kom muaj kev vam meej MV hauv luav.
Tus qauv puncture no yuav tsum muaj kev txawj txaus, sijhawm, thiab kev siv zog phais. Hauv kev sim ua ntej, kev txiav tawm lossis kev txhawb nqa ntau dhau ntawm cov txheej txheem paravertebral ligamentous tuaj yeem ua rau tsim cov vertebral osteophytes. Kev saib xyuas yuav tsum tsis txhob ua kom puas lossis ua rau cov discs uas nyob ib sab. Txij li thaum qhov tob ntawm kev nkag mus yuav tsum tau tswj kom tau txais cov txiaj ntsig zoo ib yam thiab rov ua dua tshiab, peb tau ua ib lub ntsaws los ntawm kev txiav tawm lub plhaub ntawm 3 hli ntev koob. Kev siv lub ntsaws no ua kom zoo li qhov tob tob hauv lub cev vertebral. Hauv kev sim ua ntej, peb tus kws kho mob orthopedic koom nrog hauv kev ua haujlwm pom 16-gauge koob yooj yim ua haujlwm nrog ntau dua 18-gauge koob lossis lwm txoj hauv kev. Txhawm rau kom tsis txhob los ntshav ntau thaum lub sijhawm drilling, tuav lub koob tseem nyob rau ib ntus yuav muab lub qhov ntxig ntxig zoo dua, tawm tswv yim tias qee qib ntawm MC tuaj yeem tswj tau li no.
Txawm hais tias ntau cov kev tshawb fawb tau tsom rau MC, me ntsis paub txog etiology thiab pathogenesis ntawm MC25,26,27. Raws li peb cov kev tshawb fawb yav dhau los, peb pom tias autoimmunity plays lub luag haujlwm tseem ceeb hauv qhov tshwm sim thiab kev loj hlob ntawm MC12. Txoj kev tshawb no tau tshuaj xyuas qhov kev qhia ntau ntawm IL-4, IL-17, thiab IFN-γ, uas yog txoj hauv kev sib txawv tseem ceeb ntawm CD4 + hlwb tom qab antigen stimulation. Hauv peb txoj kev tshawb fawb, piv nrog cov pab pawg tsis zoo, pawg NPE tau qhia ntau dua ntawm IL-4, IL-17, thiab IFN-γ, thiab cov protein ntau ntawm IL-4 thiab IL-17 kuj siab dua.
Clinically, IL-17 mRNA qhia tau nce hauv NP hlwb los ntawm cov neeg mob uas muaj disc herniation28. Kev nce qib IL-4 thiab IFN-γ qhia tau kuj pom nyob rau hauv ib qho mob uas tsis yog-compressive disc herniation qauv piv nrog kev noj qab haus huv tswj29. IL-17 ua lub luag haujlwm tseem ceeb hauv kev mob, cov ntaub so ntswg raug mob hauv cov kab mob autoimmune30 thiab txhim kho lub cev tiv thaiv kab mob rau IFN-γ31. Txhim kho IL-17-mediated cov ntaub so ntswg raug mob tau tshaj tawm hauv MRL / lpr nas32 thiab autoimmunity-susceptible nas33. IL-4 tuaj yeem cuam tshuam qhov kev qhia ntawm proinflammatory cytokines (xws li IL-1β thiab TNFα) thiab macrophage activation34. Nws tau tshaj tawm tias mRNA qhia ntawm IL-4 yog qhov sib txawv hauv NPE pawg piv rau IL-17 thiab IFN-γ tib lub sijhawm; Qhov kev qhia mRNA ntawm IFN-γ hauv NPE pawg tau nce siab dua li ntawm lwm pab pawg. Yog li, IFN-γ ntau lawm yuav yog tus neeg nruab nrab ntawm cov lus teb inflammatory induced los ntawm NP intercalation. Cov kev tshawb fawb tau pom tias IFN-γ yog tsim los ntawm ntau hom ntawm tes, suav nrog hom 1 pab T hlwb, ntuj tua cov hlwb, thiab macrophages35,36, thiab yog ib qho tseem ceeb pro-inflammatory cytokine uas txhawb kev tiv thaiv kab mob.
Txoj kev tshawb no qhia tias cov lus teb autoimmune tuaj yeem koom nrog qhov tshwm sim thiab kev loj hlob ntawm MC. Luoma et al. pom tias cov yam ntxwv ntawm cov teeb liab ntawm MC thiab cov NP tseem ceeb zoo ib yam ntawm MRI, thiab ob qho tib si qhia cov teeb liab siab hauv T2W ib ntus 38. Qee cov cytokines tau raug lees paub tias muaj feem cuam tshuam nrog qhov tshwm sim ntawm MC, xws li IL-139. Ma et al. tau hais tias qhov nce siab lossis qis qis ntawm NP tuaj yeem cuam tshuam zoo rau qhov tshwm sim thiab kev loj hlob ntawm MC12. Bobechko40 thiab Herzbein et al.41 tau tshaj tawm tias NP yog cov ntaub so ntswg tiv thaiv kab mob uas tsis tuaj yeem nkag mus rau vascular ncig thaum yug los. NP protrusions qhia lub cev txawv teb chaws rau hauv cov ntshav, yog li kho cov tshuaj autoimmune hauv zos42. Cov tshuaj tiv thaiv kab mob autoimmune tuaj yeem ua rau muaj ntau yam kev tiv thaiv kab mob, thiab thaum cov xwm txheej no txuas ntxiv mus rau cov ntaub so ntswg, lawv tuaj yeem ua rau muaj kev hloov pauv hauv signaling43. Nyob rau hauv txoj kev tshawb no, overexpression ntawm IL-4, IL-17 thiab IFN-γ yog ib qho kev tiv thaiv kab mob, ntxiv ua pov thawj ntawm kev sib raug zoo ntawm NP thiab MCs44. Tus qauv tsiaj no zoo ua raws li NP kev tawg thiab nkag mus rau hauv lub phaj kawg. Cov txheej txheem no tau qhia ntxiv txog qhov cuam tshuam ntawm autoimmunity ntawm MC.
Raws li qhov xav tau, tus qauv tsiaj no muab peb lub platform ua tau los kawm MC. Txawm li cas los xij, tus qauv no tseem muaj qee qhov kev txwv: ua ntej, thaum lub sijhawm saib tsiaj, qee qhov nruab nrab-theem luav yuav tsum tau euthanized rau kev kuaj histological thiab molecular biology, yog li qee cov tsiaj "poob siv" dhau sijhawm. Qhov thib ob, txawm hais tias peb lub sij hawm cov ntsiab lus tau teeb tsa hauv txoj kev tshawb no, hmoov tsis, peb tsuas yog ua qauv ib hom MC (Modic hom kuv hloov), yog li nws tsis txaus los sawv cev rau tib neeg txoj kev txhim kho kab mob, thiab ntau lub sij hawm cov ntsiab lus yuav tsum tau teem rau. zoo saib tag nrho cov teeb liab hloov. Thib peb, cov kev hloov pauv ntawm cov ntaub so ntswg tuaj yeem pom meej meej los ntawm histological staining, tab sis qee cov txheej txheem tshwj xeeb tuaj yeem nthuav tawm cov kev hloov pauv ntawm microstructural hauv cov qauv no. Piv txwv li, polarized lub teeb microscopy tau siv los txheeb xyuas qhov tsim ntawm fibrocartilage hauv luav intervertebral discs45. Qhov cuam tshuam mus sij hawm ntev ntawm NP ntawm MC thiab daim ntawv kawg yuav tsum tau kawm ntxiv.
Tsib caug plaub tus txiv neej New Zealand dawb luav (qhov hnyav txog 2.5-3 kg, hnub nyoog 3-3.5 lub hlis) tau muab faib ua pawg ua haujlwm sham, pawg leeg nqaij (ME pab pawg) thiab pawg paj hlwb cog (NPE pawg). Tag nrho cov txheej txheem kev sim tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tianjin Tsev Kho Mob, thiab cov txheej txheem kev sim tau ua tiav nruj raws li cov lus pom zoo.
Qee qhov kev txhim kho tau ua rau cov txheej txheem phais ntawm S. Sobajima 46 . Txhua tus luav tau muab tso rau hauv txoj haujlwm rov qab sab nraud thiab sab xub ntiag ntawm tsib lub lumbar intervertebral discs (IVDs) tau nthuav tawm siv txoj hauv kev posterolateral retroperitoneal. Txhua tus luav tau muab tshuaj loog (20% urethane, 5 ml / kg ntawm pob ntseg leeg). Qhov ntev ntawm daim tawv nqaij incision yog tsim los ntawm sab ntug ntawm lub tav mus rau lub pelvic brim, 2 cm ventral mus rau paravertebral cov leeg. Txoj cai anterolateral qaum los ntawm L1 mus rau L6 tau nthuav tawm los ntawm cov ntse thiab blunt dissection ntawm overlying subcutaneous cov ntaub so ntswg, cov ntaub so ntswg retroperitoneal, thiab cov leeg nqaij (Fig. 6A). Cov qib disc tau txiav txim siab siv lub plab hnyuv siab raum ua ib qho chaw anatomical rau qib L5-L6 disc. Siv 16-gauge puncture koob los tho ib lub qhov ze ntawm lub phaj kawg ntawm L5 vertebra mus rau qhov tob ntawm 3 hli (Fig. 6B). Siv 5-ml syringe rau aspirate lub autologous nucleus pulposus hauv L1-L2 intervertebral disc (Fig. 6C). Tshem tawm cov nucleus pulposus lossis cov leeg nqaij raws li qhov xav tau ntawm txhua pab pawg. Tom qab lub qhov laum tob tob, cov sutures absorbable yog muab tso rau ntawm qhov tob fascia, fascia thiab daim tawv nqaij, saib xyuas kom tsis txhob ua puas cov ntaub so ntswg periosteal ntawm lub cev vertebral thaum phais.
(A) Lub L5-L6 disc raug nthuav tawm los ntawm txoj hauv kev posterolateral retroperitoneal. (B) Siv 16-gauge koob los tho ib lub qhov ze ntawm L5 endplate. (C) Autologous MFs raug sau.
Kev siv tshuaj loog dav dav tau muab nrog 20% urethane (5 ml / kg) siv los ntawm pob ntseg leeg, thiab lumbar spine radiographs tau rov ua dua ntawm 12, 16, thiab 20 lub lis piam tom qab ua haujlwm.
Cov luav tau muab xyeem los ntawm kev txhaj tshuaj intramuscular ntawm ketamine (25.0 mg / kg) thiab intravenous sodium pentobarbital (1.2 g / kg) ntawm 12, 16 thiab 20 lub lis piam tom qab kev phais. Tag nrho cov nqaj qaum raug tshem tawm rau kev soj ntsuam histological thiab kev soj ntsuam tiag tiag tau ua. Quantitative reverse transcription (RT-qPCR) thiab Western blotting tau siv los txheeb xyuas cov kev hloov pauv hauv kev tiv thaiv kab mob.
Kev kuaj MRI tau ua nyob rau hauv luav siv lub 3.0 T chaw kho mob hlau nplaum (GE Medical Systems, Florence, SC) nruab nrog ib qho orthogonal limb coil receiver. Cov luav tau muab tshuaj loog nrog 20% urethane (5 mL / kg) ntawm pob ntseg leeg thiab tom qab ntawd muab tso rau hauv cov hlau nplaum nrog lub lumbar cheeb tsam nyob rau ntawm 5-nti kab uas hla ntawm lub voj voog (GE Medical Systems). Coronal T2-weighted localizer dluab (TR, 1445 ms; TE, 37 ms) tau txais los txheeb xyuas qhov chaw ntawm lub lumbar disc los ntawm L3-L4 txog L5-L6. Sagittal dav hlau T2-hnyav hlais tau txais nrog cov kev teeb tsa hauv qab no: nrawm nrawm nrawm nrawm nrog lub sijhawm rov ua dua (TR) ntawm 2200 ms thiab lub sijhawm ncha (TE) ntawm 70 ms, matrix; pom kev ntawm 260 thiab yim stimuli; Txiav thickness yog 2 hli, qhov sib txawv yog 0.2 hli.
Tom qab thaij duab kawg thiab tus luav kawg raug tua, cov sham, cov leeg-pob, thiab NP discs raug tshem tawm rau kev kuaj mob. Cov ntaub so ntswg tau kho nyob rau hauv 10% nruab nrab buffered formalin rau 1 lub lis piam, decalcified nrog ethylenediaminetetraacetic acid, thiab paraffin sectioned. Cov ntaub so ntswg tau muab tso rau hauv paraffin thiab txiav rau hauv sagittal seem (5 μm tuab) siv microtome. Cov seem tau stained nrog hematoxylin thiab eosin (H&E).
Tom qab sau cov intervertebral discs los ntawm cov luav hauv txhua pab pawg, tag nrho RNA tau muab rho tawm siv UNIQ-10 kem (Shanghai Sangon Biotechnology Co., Ltd., Tuam Tshoj) raws li cov chaw tsim khoom cov lus qhia thiab ib qho ImProm II thim rov qab qhov system (Promega Inc. , Madison, WI, USA). Reverse transcription tau ua.
RT-qPCR tau ua tiav siv Prism 7300 (Applied Biosystems Inc., USA) thiab SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) raws li cov chaw tsim khoom cov lus qhia. PCR cov tshuaj tiv thaiv ntim yog 20 μl thiab muaj 1.5 μl ntawm diluted cDNA thiab 0.2 μM ntawm txhua tus primer. Primers tau tsim los ntawm OligoPerfect Designer (Invitrogen, Valencia, CA) thiab tsim los ntawm Nanjing Golden Stewart Biotechnology Co., Ltd. (Tuam Tshoj) (Table 1). Cov nram qab no thermal cycling tej yam kev mob: thawj zaug polymerase activation kauj ruam ntawm 94 ° C rau 2 min, ces 40 cycles ntawm 15 s txhua ntawm 94 ° C rau template denaturation, annealing rau 1 min ntawm 60 ° C, extension, thiab fluorescence. Kev ntsuas tau ua rau 1 min ntawm 72 ° C. Tag nrho cov qauv tau nthuav dav peb zaug thiab qhov nruab nrab tus nqi tau siv rau RT-qPCR tsom xam. Amplification cov ntaub ntawv raug soj ntsuam siv FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). IL-4, IL-17, thiab IFN-γ gene qhia tau normalized mus rau endogenous tswj (ACTB). Tus txheeb ze qhia theem ntawm lub hom phiaj mRNA tau suav nrog siv 2-ΔΔCT txoj kev.
Tag nrho cov protein tau muab rho tawm los ntawm cov ntaub so ntswg siv cov ntaub so ntswg homogenizer hauv RIPA lysis buffer (muaj cov tshuaj protease thiab phosphatase inhibitor cocktail) thiab tom qab ntawd centrifuged ntawm 13,000 rpm rau 20 min ntawm 4 ° C kom tshem tawm cov ntaub so ntswg. Tsib caug micrograms ntawm cov protein tau thauj khoom ib txoj kab, sib cais los ntawm 10% SDS-PAGE, thiab tom qab ntawd xa mus rau PVDF daim nyias nyias. Thaiv tau ua nyob rau hauv 5% nonfat qhuav mis nyuj nyob rau hauv Tris-buffered saline (TBS) uas muaj 0.1% Tween 20 rau 1 h ntawm chav tsev kub. Lub membrane yog incubated nrog luav anti-decorin thawj antibody (diluted 1:200; Boster, Wuhan, Tuam Tshoj) (diluted 1:200; Bioss, Beijing, Suav teb) thaum hmo ntuj ntawm 4 ° C thiab reacted nyob rau ob hnub; nrog cov tshuaj tiv thaiv kab mob thib ob (tshis los tiv thaiv luav immunoglobulin G ntawm 1: 40,000 dilution) ua ke nrog horseradish peroxidase (Boster, Wuhan, Tuam Tshoj) rau 1 teev ntawm chav tsev kub. Western blot signals tau kuaj pom los ntawm kev nce chemiluminescence ntawm chemiluminescent membrane tom qab X-ray irradiation. Rau kev tsom xam densitometric, blots tau soj ntsuam thiab ntsuas siv BandScan software thiab cov txiaj ntsig tau nthuav tawm raws li qhov piv ntawm lub hom phiaj gene immunoreactivity rau tubulin immunoreactivity.
Kev suav txheeb suav tau ua tiav siv SPSS16.0 software pob (SPSS, USA). Cov ntaub ntawv khaws tseg thaum txoj kev tshawb fawb tau nthuav tawm raws li qhov txhais tau tias ± tus qauv sib txawv (txhais tau tias ± SD) thiab tshuaj xyuas siv ib txoj kev rov ntsuas ntsuas qhov sib txawv (ANOVA) los txiav txim qhov sib txawv ntawm ob pawg. P <0.05 tau suav tias yog qhov tseem ceeb.
Yog li, kev tsim cov tsiaj qauv ntawm MC los ntawm kev cog autologous NPs rau hauv lub cev vertebral thiab ua kev soj ntsuam macroanatomical, MRI tsom xam, kev ntsuam xyuas histological thiab molecular biological tsom xam yuav dhau los ua ib qho tseem ceeb rau kev ntsuam xyuas thiab nkag siab txog cov txheej txheem ntawm tib neeg MC thiab tsim kho tshiab. kev cuam tshuam.
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Post lub sij hawm: Dec-13-2024