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Kev tsim cov qauv tsiaj ntawm kev hloov pauv modic (MC) yog lub hauv paus tseem ceeb rau kev kawm MC. Tsib caug plaub tus luav New Zealand Dawb tau muab faib ua pawg phais sham, pawg cog cov leeg (ME pawg) thiab pawg cog nucleus pulposus (NPE pawg). Hauv pawg NPE, lub disc intervertebral tau raug nthuav tawm los ntawm kev phais anterolateral lumbar thiab siv rab koob los tho lub cev L5 vertebral ze ntawm lub phaj kawg. NP tau rho tawm ntawm lub disc L1/2 intervertebral los ntawm lub koob txhaj tshuaj thiab txhaj rau hauv nws. Khawb ib lub qhov hauv pob txha subchondral. Cov txheej txheem phais thiab cov txheej txheem khawb hauv pawg cog cov leeg thiab pawg phais sham zoo ib yam li cov hauv pawg NP cog. Hauv pawg ME, ib daim leeg tau muab tso rau hauv lub qhov, thaum nyob hauv pawg phais sham, tsis muaj dab tsi tau muab tso rau hauv lub qhov. Tom qab kev phais, MRI scanning thiab molecular biological kev kuaj mob tau ua tiav. Lub teeb liab hauv pawg NPE tau hloov pauv, tab sis tsis muaj kev hloov pauv teeb liab pom tseeb hauv pawg phais sham thiab pawg ME. Kev soj ntsuam histological qhia tau hais tias cov nqaij ntshiv tsis zoo li qub tau pom ntawm qhov chaw cog, thiab kev qhia tawm ntawm IL-4, IL-17 thiab IFN-γ tau nce ntxiv hauv pawg NPE. Kev cog NP rau hauv pob txha subchondral tuaj yeem tsim tus qauv tsiaj ntawm MC.
Kev hloov pauv Modic (MC) yog cov qhov txhab ntawm cov pob txha kawg thiab cov pob txha uas nyob ib sab uas pom tau ntawm cov duab magnetic resonance imaging (MRI). Lawv feem ntau tshwm sim rau cov tib neeg uas muaj cov tsos mob cuam tshuam1. Ntau txoj kev tshawb fawb tau hais txog qhov tseem ceeb ntawm MC vim nws cuam tshuam nrog mob nraub qaum qis (LBP)2,3. de Roos et al.4 thiab Modic et al.5 tau piav qhia txog peb hom teeb meem subchondral hauv cov pob txha vertebral. Kev hloov pauv Modic hom I yog hypointense ntawm T1-weighted (T1W) thiab hyperintense ntawm T2-weighted (T2W) sequences. Qhov txhab no qhia txog cov fissure endplates thiab cov leeg nqaij granulation nyob ib sab hauv cov pob txha. Kev hloov pauv Modic hom II qhia txog cov teeb meem siab ntawm ob qho tib si T1W thiab T2W sequences. Hauv hom kev mob no, kev puas tsuaj ntawm cov pob txha kawg tuaj yeem pom, nrog rau kev hloov pauv rog histological ntawm cov pob txha uas nyob ib sab. Kev hloov pauv Modic hom III qhia txog cov teeb meem qis hauv T1W thiab T2W sequences. Cov qhov txhab sclerotic uas sib raug rau cov endplates tau pom6. MC raug suav hais tias yog ib qho kab mob pathological ntawm tus txha nqaj qaum thiab nws muaj feem cuam tshuam nrog ntau yam kab mob degenerative ntawm tus txha nqaj qaum7,8,9.
Xav txog cov ntaub ntawv muaj, ntau txoj kev tshawb fawb tau muab cov kev nkag siab ntxaws ntxaws txog etiology thiab pathological mechanisms ntawm MC. Albert et al. tau hais tias MC tej zaum yuav yog los ntawm disc herniation8. Hu et al. tau hais tias MC yog vim muaj kev puas tsuaj loj heev ntawm disc10. Kroc tau tawm tswv yim txog lub tswv yim ntawm "kev tawg ntawm lub disc sab hauv," uas hais tias kev raug mob ntawm lub disc rov ua dua yuav ua rau muaj microtears hauv lub endplate. Tom qab kev tsim cleft, kev puas tsuaj ntawm lub endplate los ntawm nucleus pulposus (NP) yuav ua rau muaj kev teb autoimmune, uas ua rau muaj kev loj hlob ntawm MC11. Ma et al. tau qhia txog kev xav zoo sib xws thiab tau tshaj tawm tias NP-induced autoimmune ua lub luag haujlwm tseem ceeb hauv pathogenesis ntawm MC12.
Cov hlwb ntawm lub cev tiv thaiv kab mob, tshwj xeeb tshaj yog CD4+ T helper lymphocytes, ua lub luag haujlwm tseem ceeb hauv kev mob autoimmunity13. Cov Th17 subset uas nyuam qhuav pom tsim cov proinflammatory cytokine IL-17, txhawb kev qhia chemokine, thiab txhawb cov T cell hauv cov kabmob puas tsuaj kom tsim IFN-γ14. Th2 cell kuj ua lub luag haujlwm tshwj xeeb hauv kev mob ntawm cov lus teb tiv thaiv kab mob. Kev qhia ntawm IL-4 ua tus sawv cev Th2 cell tuaj yeem ua rau muaj kev cuam tshuam loj heev rau kev tiv thaiv kab mob15.
Txawm hais tias muaj ntau txoj kev tshawb fawb txog MC16,17,18,19,20,21,22,23,24, los tseem tsis tau muaj cov qauv sim tsiaj uas tsim nyog uas tuaj yeem ua raws li cov txheej txheem MC uas feem ntau tshwm sim hauv tib neeg thiab tuaj yeem siv los tshawb nrhiav qhov ua rau lossis kev kho mob tshiab xws li kev kho mob tsom mus rau. Txog niaj hnub no, tsuas yog ob peb tus qauv tsiaj ntawm MC tau tshaj tawm tias kawm txog cov txheej txheem pathological hauv qab.
Raws li txoj kev xav txog tus kab mob autoimmune uas Albert thiab Ma tau hais, txoj kev tshawb fawb no tau tsim ib qho qauv MC luav yooj yim thiab rov ua dua los ntawm kev hloov NP ze ntawm lub phaj kawg ntawm lub vertebral. Lwm lub hom phiaj yog los soj ntsuam cov yam ntxwv ntawm cov qauv tsiaj thiab soj ntsuam cov txheej txheem tshwj xeeb ntawm NP hauv kev txhim kho MC. Txog qhov kawg no, peb siv cov txheej txheem xws li molecular biology, MRI, thiab kev tshawb fawb histological los kawm txog kev nce qib ntawm MC.
Ob tug luav tuag vim los ntshav thaum lub sijhawm phais, thiab plaub tug luav tuag thaum lub sijhawm siv tshuaj loog thaum lub sijhawm MRI. Cov luav seem 48 tus tseem muaj sia nyob thiab tsis muaj tsos mob lossis mob hlwb tom qab phais.
MRI qhia tau hais tias qhov muaj zog ntawm cov ntaub so ntswg uas tau muab tso rau hauv cov qhov sib txawv yog txawv. Qhov muaj zog ntawm lub cev L5 vertebral hauv pawg NPE maj mam hloov pauv ntawm 12, 16 thiab 20 lub lis piam tom qab muab tso rau hauv (T1W kab ke qhia tau tias muaj teeb liab qis, thiab T2W kab ke qhia tau tias muaj teeb liab sib xyaw ntxiv rau teeb liab qis) (Daim Duab 1C), thaum MRI tshwm sim ntawm ob pawg ntawm cov khoom uas tau muab tso rau hauv tseem ruaj khov thaum lub sijhawm tib yam (Daim Duab 1A, B).
(A) Cov duab MRI sawv cev ntawm tus luav lub duav ntawm 3 lub sijhawm. Tsis pom muaj qhov tsis zoo ntawm cov cim qhia hauv cov duab ntawm pawg ua haujlwm sham. (B) Cov yam ntxwv ntawm lub cim qhia ntawm lub cev vertebral hauv pawg ME zoo ib yam li cov hauv pawg ua haujlwm sham, thiab tsis muaj kev hloov pauv tseem ceeb ntawm qhov chaw embedding dhau sijhawm. (C) Hauv pawg NPE, lub cim qis pom tseeb hauv T1W kab ke, thiab lub cim sib xyaw thiab lub cim qis pom tseeb hauv T2W kab ke. Txij li 12-lub lis piam mus rau 20-lub lis piam, cov cim siab tsis tu ncua nyob ib puag ncig cov cim qis hauv T2W kab ke txo qis.
Pom tseeb tias pob txha hyperplasia tuaj yeem pom ntawm qhov chaw cog rau ntawm lub cev vertebral hauv pawg NPE, thiab pob txha hyperplasia tshwm sim sai dua ntawm 12 txog 20 lub lis piam (Daim duab 2C) piv rau pawg NPE, tsis muaj kev hloov pauv tseem ceeb pom hauv cov qauv vertebral lub cev; pawg Sham thiab pawg ME (Daim duab 2C) 2A, B).
(A) Qhov chaw ntawm lub cev pob txha ntawm qhov chaw cog rau hauv lub cev yog du heev, qhov kho tau zoo, thiab tsis muaj hyperplasia hauv lub cev pob txha. (B) Cov duab ntawm qhov chaw cog rau hauv pawg ME zoo ib yam li cov hauv pawg phais sham, thiab tsis muaj kev hloov pauv pom tseeb hauv qhov tsos ntawm qhov chaw cog rau hauv lub sijhawm. (C) Pob txha hyperplasia tshwm sim ntawm qhov chaw cog rau hauv pawg NPE. Cov pob txha hyperplasia nce sai thiab txawm tias nthuav dav los ntawm lub disc intervertebral mus rau lub cev pob txha contralateral.
Kev tshuaj xyuas keeb kwm muab cov ntaub ntawv ntxaws ntxiv txog kev tsim pob txha. Daim duab 3 qhia cov duab ntawm cov seem tom qab phais uas tau pleev xim nrog H&E. Hauv pawg phais sham, cov chondrocytes tau teeb tsa zoo thiab tsis muaj kev loj hlob ntawm cov hlwb (Daim duab 3A). Qhov xwm txheej hauv pawg ME zoo ib yam li hauv pawg phais sham (Daim duab 3B). Txawm li cas los xij, hauv pawg NPE, muaj ntau cov chondrocytes thiab kev loj hlob ntawm cov hlwb zoo li NP tau pom ntawm qhov chaw cog (Daim duab 3C);
(A) Trabeculae tuaj yeem pom ze ntawm lub phaj kawg, cov chondrocytes tau teeb tsa zoo nkauj nrog cov cell loj thiab cov duab sib xws thiab tsis muaj kev loj hlob (40 zaug). (B) Qhov xwm txheej ntawm qhov chaw cog rau hauv pawg ME zoo ib yam li pawg sham. Trabeculae thiab chondrocytes tuaj yeem pom, tab sis tsis muaj kev loj hlob pom tseeb ntawm qhov chaw cog (40 zaug). (B) Nws tuaj yeem pom tias chondrocytes thiab NP-zoo li cell loj hlob ntau heev, thiab cov duab thiab qhov loj ntawm chondrocytes tsis sib xws (40 zaug).
Cov kev qhia tawm 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 qib kev qhia tawm ntawm cov noob caj noob ces tau muab piv rau, cov kev qhia tawm ntawm IL-4, IL-17, thiab IFN-γ tau nce ntxiv ntau heev hauv pawg NPE piv rau cov ntawm pawg ME thiab pawg ua haujlwm sham (Daim Duab 4) (P < 0.05). Piv nrog pawg ua haujlwm sham, cov qib kev qhia tawm ntawm IL-4, IL-17, thiab IFN-γ hauv pawg ME tsuas yog nce me ntsis xwb thiab tsis tau mus txog qhov kev hloov pauv ntawm cov lej (P > 0.05).
Qhov kev qhia tawm mRNA ntawm IL-4, IL-17 thiab IFN-γ hauv pawg NPE tau qhia txog qhov sib txawv siab dua li cov neeg hauv pawg ua haujlwm sham thiab pawg ME (P < 0.05).
Qhov sib piv, cov theem kev qhia tawm hauv pawg ME tsis qhia qhov sib txawv tseem ceeb (P> 0.05).
Kev tshuaj ntsuam xyuas Western blot tau ua tiav los ntawm kev siv cov tshuaj tiv thaiv kab mob tiv thaiv IL-4 thiab IL-17 uas muaj nyob hauv khw los xyuas kom meej tias cov qauv kev hloov pauv ntawm mRNA. Raws li pom hauv Daim Duab 5A, B, piv nrog pawg ME thiab pawg ua haujlwm sham, cov protein ntawm IL-4 thiab IL-17 hauv pawg NPE tau nce ntxiv ntau heev (P < 0.05). Piv nrog pawg ua haujlwm sham, cov protein ntawm IL-4 thiab IL-17 hauv pawg ME kuj tsis tau hloov pauv ntau yam (P> 0.05).
(A) Cov protein theem ntawm IL-4 thiab IL-17 hauv pawg NPE siab dua li cov hauv pawg ME thiab pawg placebo (P < 0.05). (B) Western blot histogram.
Vim muaj tsawg tus neeg kuaj tau thaum lub sijhawm phais, kev tshawb fawb meej thiab ntxaws txog pathogenesis ntawm MC yog qhov nyuaj me ntsis. Peb tau sim tsim kom muaj tus qauv tsiaj ntawm MC los kawm txog nws cov txheej txheem pathological. Tib lub sijhawm, kev ntsuam xyuas radiological, kev ntsuam xyuas histological thiab kev ntsuam xyuas molecular biological tau siv los ua raws li txoj kev ntawm MC uas tshwm sim los ntawm NP autograft. Yog li ntawd, tus qauv NP implantation ua rau muaj kev hloov pauv maj mam ntawm lub zog teeb liab los ntawm 12-lub lis piam mus rau 20-lub lis piam lub sijhawm ntsiab lus (sib xyaw teeb liab qis hauv T1W kab ke thiab teeb liab qis hauv T2W kab ke), qhia txog kev hloov pauv ntawm cov ntaub so ntswg, thiab kev ntsuam xyuas histological thiab molecular biological tau lees paub cov txiaj ntsig ntawm kev tshawb fawb radiological.
Cov txiaj ntsig ntawm qhov kev sim no qhia tau tias kev hloov pauv pom thiab histological tau tshwm sim ntawm qhov chaw ntawm lub cev vertebral cuam tshuam hauv pawg NPE. Tib lub sijhawm, kev qhia tawm ntawm IL-4, IL-17 thiab IFN-γ noob, nrog rau IL-4, IL-17 thiab IFN-γ tau pom, qhia tias kev cuam tshuam ntawm autologous nucleus pulposus cov ntaub so ntswg hauv lub cev vertebral yuav ua rau muaj ntau yam teeb liab thiab morphological hloov pauv. Nws yooj yim nrhiav tau tias cov yam ntxwv teeb liab ntawm lub cev vertebral ntawm tus qauv tsiaj (cov teeb liab qis hauv T1W kab lus, cov teeb liab sib xyaw thiab cov teeb liab qis hauv T2W kab lus) zoo ib yam li cov tib neeg vertebral hlwb, thiab cov yam ntxwv MRI kuj lees paub qhov kev soj ntsuam ntawm histology thiab gross anatomy, uas yog, cov kev hloov pauv hauv cov hlwb vertebral yog nce zuj zus. Txawm hais tias qhov kev teb inflammatory los ntawm kev raug mob hnyav yuav tshwm sim sai sai tom qab puncture, cov txiaj ntsig MRI qhia tau tias cov kev hloov pauv teeb liab nce zuj zus tshwm sim 12 lub lis piam tom qab puncture thiab kav ntev txog 20 lub lis piam yam tsis muaj cov cim ntawm kev rov zoo lossis thim rov qab ntawm MRI hloov pauv. Cov txiaj ntsig no qhia tau hais tias autologous vertebral NP yog ib txoj hauv kev txhim khu kev qha rau kev tsim cov MV nce qib hauv cov luav.
Tus qauv tho qhov no xav tau kev txawj ntse, lub sijhawm, thiab kev siv zog phais. Hauv kev sim ua ntej, kev txiav lossis kev txhawb nqa ntau dhau ntawm cov qauv paravertebral ligamentous yuav ua rau muaj cov pob txha caj qaum. Yuav tsum ceev faj kom tsis txhob ua puas tsuaj lossis ua rau khaus cov discs uas nyob ib sab. Vim tias qhov tob ntawm kev nkag mus yuav tsum tau tswj kom tau txais cov txiaj ntsig zoo sib xws thiab rov ua dua, peb tau ua lub ntsaws los ntawm kev txiav lub hnab ntawm koob ntev 3 hli. Siv lub ntsaws no ua kom muaj qhov tob sib npaug hauv lub cev vertebral. Hauv kev sim ua ntej, peb tus kws phais pob txha uas koom nrog hauv kev phais pom tias koob 16-gauge yooj yim dua rau kev ua haujlwm nrog dua li koob 18-gauge lossis lwm txoj hauv kev. Txhawm rau kom tsis txhob los ntshav ntau dhau thaum lub sijhawm tho, tuav lub koob kom ruaj khov rau ib pliag yuav muab qhov ntxig zoo dua, qhia tias qee qib ntawm MC tuaj yeem tswj tau hauv txoj kev no.
Txawm hais tias muaj ntau txoj kev tshawb fawb tau tsom mus 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 ua lub luag haujlwm tseem ceeb hauv qhov tshwm sim thiab kev loj hlob ntawm MC12. Txoj kev tshawb fawb no tau tshuaj xyuas qhov kev qhia tawm ntawm IL-4, IL-17, thiab IFN-γ, uas yog cov kev sib txawv tseem ceeb ntawm CD4 + hlwb tom qab antigen stimulation. Hauv peb txoj kev tshawb fawb, piv nrog pawg tsis zoo, pawg NPE muaj kev qhia tawm ntau dua ntawm IL-4, IL-17, thiab IFN-γ, thiab cov protein theem ntawm IL-4 thiab IL-17 kuj siab dua.
Hauv kev kho mob, IL-17 mRNA kev qhia tawm tau nce ntxiv hauv cov hlwb NP los ntawm cov neeg mob uas muaj disc herniation28. Cov qib kev qhia tawm IL-4 thiab IFN-γ ntxiv kuj pom muaj nyob rau hauv tus qauv mob disc herniation tsis-compressive piv rau cov neeg noj qab haus huv tswj29. IL-17 ua lub luag haujlwm tseem ceeb hauv kev o, kev raug mob ntawm cov nqaij hauv cov kab mob autoimmune30 thiab txhim kho kev tiv thaiv kab mob rau IFN-γ31. Kev txhim kho IL-17-mediated kev raug mob ntawm cov nqaij tau tshaj tawm hauv MRL/lpr nas32 thiab autoimmunity-susceptible nas33. IL-4 tuaj yeem tiv thaiv kev qhia tawm ntawm proinflammatory cytokines (xws li IL-1β thiab TNFα) thiab macrophage activation34. Nws tau tshaj tawm tias mRNA kev qhia tawm ntawm IL-4 txawv hauv pawg NPE piv rau IL-17 thiab IFN-γ tib lub sijhawm; Kev qhia tawm mRNA ntawm IFN-γ hauv pawg NPE tau siab dua li ntawd hauv lwm pawg. Yog li ntawd, IFN-γ kev tsim khoom yuav yog tus neeg nruab nrab ntawm kev teb inflammatory uas tshwm sim los ntawm NP intercalation. Cov kev tshawb fawb tau qhia tias IFN-γ yog tsim los ntawm ntau hom cell, suav nrog cov activated type 1 helper T cell, natural killer cell, thiab macrophages35,36, thiab yog ib qho tseem ceeb pro-inflammatory cytokine uas txhawb nqa kev tiv thaiv kab mob37.
Daim ntawv tshawb fawb no qhia tau hais tias kev teb autoimmune tej zaum yuav koom nrog rau qhov tshwm sim thiab kev loj hlob ntawm MC. Luoma et al. pom tias cov yam ntxwv ntawm lub teeb liab ntawm MC thiab NP tseem ceeb zoo sib xws ntawm MRI, thiab ob qho tib si qhia cov teeb liab siab hauv T2W sequence38. Qee cov cytokines tau 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 lossis nqis ntawm NP yuav muaj kev cuam tshuam loj 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 hauv cov hlab ntsha txij thaum yug los. NP protrusions coj cov khoom txawv teb chaws mus rau hauv cov ntshav, yog li ua rau cov tshuaj tiv thaiv kab mob hauv zos42. Cov tshuaj tiv thaiv kab mob tuaj yeem ua rau muaj ntau yam tshuaj tiv thaiv kab mob, thiab thaum cov yam ntxwv no raug rau cov ntaub so ntswg tas li, lawv tuaj yeem ua rau muaj kev hloov pauv hauv kev taw qhia43. Hauv daim ntawv tshawb fawb no, kev tshaj tawm ntau dhau ntawm IL-4, IL-17 thiab IFN-γ yog cov tshuaj tiv thaiv kab mob ib txwm muaj, ua pov thawj ntxiv txog kev sib raug zoo ntawm NP thiab MCs44. Tus qauv tsiaj no ua raws li NP qhov kev tawg thiab nkag mus rau hauv lub phaj kawg. Cov txheej txheem no qhia ntxiv txog qhov cuam tshuam ntawm autoimmunity rau MC.
Raws li qhov xav tau, tus qauv tsiaj no muab rau 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 xyuas tsiaj, qee tus luav theem nrab yuav tsum tau euthanized rau kev kuaj mob histological thiab molecular biology, yog li qee tus tsiaj "poob ntawm kev siv" dhau sijhawm. Qhov thib ob, txawm hais tias peb lub sijhawm tau teeb tsa hauv kev tshawb fawb no, hmoov tsis zoo, peb tsuas yog ua qauv ib hom MC (Modic hom I hloov), yog li nws tsis txaus los sawv cev rau cov txheej txheem kev loj hlob ntawm tib neeg, thiab ntau lub sijhawm yuav tsum tau teeb tsa kom zoo dua saib txhua qhov kev hloov pauv teeb liab. Qhov thib peb, cov kev hloov pauv hauv cov qauv ntaub so ntswg tuaj yeem pom tseeb los ntawm kev pleev xim histological, tab sis qee cov txheej txheem tshwj xeeb tuaj yeem qhia tau zoo dua cov kev hloov pauv microstructural hauv tus qauv no. Piv txwv li, polarized lub teeb microscopy tau siv los tshuaj xyuas qhov tsim ntawm fibrocartilage hauv luav intervertebral discs45. Cov teebmeem ntev ntawm NP ntawm MC thiab endplate xav tau kev kawm ntxiv.
Tsib caug plaub tug luav dawb New Zealand txiv neej (hnyav li 2.5-3 kg, hnub nyoog 3-3.5 lub hlis) tau raug faib ua pawg phais sham, pawg cog cov leeg (ME) thiab pawg cog cov hauv paus paj hlwb (NPE). Txhua txoj kev sim tau txais kev pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsev Kho Mob Tianjin, thiab cov txheej txheem sim tau ua raws li cov lus qhia pom zoo.
Muaj qee qhov kev txhim kho tau ua rau txoj kev phais mob ntawm S. Sobajima 46. Txhua tus luav tau muab tso rau hauv txoj hauj lwm pw sab nraud thiab qhov chaw pem hauv ntej ntawm tsib lub discs intervertebral lumbar (IVDs) tau raug nthuav tawm siv txoj kev posterolateral retroperitoneal. Txhua tus luav tau muab tshuaj loog dav dav (20% urethane, 5 ml/kg ntawm cov hlab ntsha pob ntseg). Ib qho kev phais mob ntev tau ua los ntawm ntug qis ntawm cov tav mus rau lub pelvic brim, 2 cm ventral mus rau cov leeg paravertebral. Lub pob txha caj qaum sab xis ntawm L1 txog L6 tau raug nthuav tawm los ntawm kev txiav cov ntaub so ntswg subcutaneous, cov ntaub so ntswg retroperitoneal, thiab cov leeg (Daim Duab 6A). Qib disc tau txiav txim siab siv lub pelvic brim ua lub cim anatomical rau qib disc L5-L6. Siv rab koob puncture 16-gauge los tho qhov ze ntawm lub phaj kawg ntawm L5 vertebra mus rau qhov tob ntawm 3 hli (Daim Duab 6B). Siv lub koob txhaj tshuaj 5-ml los nqus cov nucleus pulposus hauv L1-L2 intervertebral disc (Daim Duab 6C). Tshem tawm cov nucleus pulposus lossis cov leeg raws li qhov yuav tsum tau ua ntawm txhua pawg. Tom qab lub qhov tho tau tob zuj zus, cov xov uas nqus tau yuav muab tso rau ntawm cov fascia tob, superficial fascia thiab daim tawv nqaij, ceev faj kom tsis txhob ua rau cov ntaub so ntswg periosteal ntawm lub cev vertebral puas tsuaj thaum phais.
(A) Lub disc L5–L6 raug nthuav tawm los ntawm txoj kev posterolateral retroperitoneal. (B) Siv rab koob 16-gauge los tho qhov ze ntawm L5 endplate. (C) Autologous MFs raug sau.
Cov tshuaj loog dav dav tau muab nrog 20% urethane (5 ml/kg) muab los ntawm cov hlab ntsha pob ntseg, thiab cov duab xoo hluav taws xob ntawm tus txha nqaj qaum tau rov ua dua ntawm 12, 16, thiab 20 lub lis piam tom qab phais.
Cov luav tau raug tua los ntawm kev txhaj tshuaj ketamine (25.0 mg / kg) thiab sodium pentobarbital (1.2 g / kg) rau hauv cov leeg ntawm 12, 16 thiab 20 lub lis piam tom qab phais. Tag nrho cov pob txha nqaj qaum tau raug tshem tawm rau kev tshuaj xyuas histological thiab kev tshuaj xyuas tiag tiag tau ua tiav. Quantitative reverse transcription (RT-qPCR) thiab Western blotting tau siv los ntes cov kev hloov pauv hauv cov yam ntxwv tiv thaiv kab mob.
Kev kuaj MRI tau ua rau cov luav siv lub hlau nplaum 3.0 T clinical magnet (GE Medical Systems, Florence, SC) uas muaj lub orthogonal limb coil receiver. Cov luav tau muab tshuaj loog nrog 20% urethane (5 mL/kg) los ntawm cov hlab ntsha pob ntseg thiab tom qab ntawd muab tso rau hauv lub hlau nplaum nrog thaj tsam lumbar nyob rau hauv nruab nrab ntawm lub voj voog 5-nti (GE Medical Systems). Cov duab Coronal T2-weighted localizer (TR, 1445 ms; TE, 37 ms) tau txais los txhais qhov chaw ntawm lub lumbar disc los ntawm L3–L4 mus rau L5–L6. Sagittal plane T2-weighted slices tau txais nrog cov chaw hauv qab no: fast spin-echo sequence nrog lub sijhawm rov ua dua (TR) ntawm 2200 ms thiab lub sijhawm echo (TE) ntawm 70 ms, matrix; thaj chaw pom ntawm 260 thiab yim stimuli; Lub thickness txiav yog 2 hli, qhov sib txawv yog 0.2 hli.
Tom qab thaij daim duab kawg thiab tua tus luav kawg lawm, daim sham, uas muaj cov leeg nqaij, thiab NP discs raug tshem tawm rau kev kuaj histological. Cov ntaub so ntswg raug kho rau hauv 10% neutral buffered formalin rau 1 lub lim tiam, decalcified nrog ethylenediaminetetraacetic acid, thiab paraffin sectioned. Cov blocks ntaub so ntswg raug muab tso rau hauv paraffin thiab txiav ua ntu sagittal (5 μm tuab) siv microtome. Cov ntu tau stained nrog hematoxylin thiab eosin (H&E).
Tom qab sau cov intervertebral discs ntawm cov luav hauv txhua pawg, tag nrho RNA tau rho tawm siv UNIQ-10 kem (Shanghai Sangon Biotechnology Co., Ltd., Tuam Tshoj) raws li cov lus qhia ntawm cov chaw tsim khoom thiab ImProm II reverse transcription system (Promega Inc., Madison, WI, USA). Reverse transcription tau ua tiav.
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 lus qhia ntawm cov chaw tsim khoom. Lub ntim PCR tshuaj tiv thaiv yog 20 μl thiab muaj 1.5 μl ntawm diluted cDNA thiab 0.2 μM ntawm txhua tus primer. Cov primers tau tsim los ntawm OligoPerfect Designer (Invitrogen, Valencia, CA) thiab tsim los ntawm Nanjing Golden Stewart Biotechnology Co., Ltd. (Tuam Tshoj) (Rooj 1). Cov xwm txheej thermal cycling hauv qab no tau siv: thawj kauj ruam polymerase activation ntawm 94 ° C rau 2 feeb, tom qab ntawd 40 lub voj voog ntawm 15 s txhua ntawm 94 ° C rau template denaturation, annealing rau 1 feeb ntawm 60 ° C, extension, thiab fluorescence. kev ntsuas tau ua rau 1 feeb ntawm 72 ° C. Tag nrho cov qauv tau raug nthuav dav peb zaug thiab tus nqi nruab nrab tau siv rau kev tshuaj xyuas RT-qPCR. Cov ntaub ntawv nthuav dav tau raug tshuaj xyuas siv FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). IL-4, IL-17, thiab IFN-γ gene expression tau normalized rau endogenous control (ACTB). Cov qib kev qhia sib piv ntawm lub hom phiaj mRNA tau suav siv txoj kev 2-ΔΔCT.
Tag nrho cov protein tau rho tawm ntawm cov ntaub so ntswg siv cov ntaub so ntswg homogenizer hauv RIPA lysis buffer (muaj protease thiab phosphatase inhibitor cocktail) thiab tom qab ntawd centrifuged ntawm 13,000 rpm rau 20 feeb ntawm 4 ° C kom tshem tawm cov khib nyiab 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 hloov mus rau PVDF membrane. Kev thaiv tau ua tiav hauv 5% cov mis nyuj qhuav tsis muaj roj hauv Tris-buffered saline (TBS) uas muaj 0.1% Tween 20 rau 1 teev ntawm chav tsev kub. Lub membrane tau incubated nrog luav anti-decorin thawj antibody (diluted 1: 200; Boster, Wuhan, Tuam Tshoj) (diluted 1: 200; Bioss, Beijing, Tuam Tshoj) hmo ntuj ntawm 4 ° C thiab reacted rau hnub thib ob; nrog cov tshuaj tiv thaiv kab mob theem ob (tshis 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 cov cim tau pom los ntawm kev nce chemiluminescence ntawm chemiluminescent membrane tom qab X-ray irradiation. Rau kev tshuaj xyuas densitometric, blots tau luam theej duab thiab ntsuas siv BandScan software thiab cov txiaj ntsig tau qhia ua qhov piv ntawm lub hom phiaj gene immunoreactivity rau tubulin immunoreactivity.
Cov kev suav lej suav tau ua tiav los ntawm kev siv SPSS16.0 software pob (SPSS, USA). Cov ntaub ntawv sau tau thaum lub sijhawm kawm tau qhia ua qhov nruab nrab ± tus qauv sib txawv (qhov nruab nrab ± SD) thiab tshuaj xyuas siv ib txoj kev ntsuas rov ua dua ntawm kev ntsuas qhov sib txawv (ANOVA) los txiav txim siab qhov sib txawv ntawm ob pawg. P <0.05 tau suav tias yog qhov tseem ceeb ntawm kev suav lej.
Yog li, kev tsim kom muaj tus qauv tsiaj ntawm MC los ntawm kev cog cov NPs autologous rau hauv lub cev vertebral thiab ua qhov kev soj ntsuam macroanatomical, MRI tsom xam, kev ntsuam xyuas histological thiab kev tshuaj xyuas molecular biological yuav dhau los ua ib qho cuab yeej tseem ceeb rau kev ntsuam xyuas thiab nkag siab txog cov txheej txheem ntawm tib neeg MC thiab tsim cov kev kho mob tshiab.
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Lub sijhawm tshaj tawm: Lub Kaum Ob Hlis-13-2024