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Ukusungulwa kwamamodeli ezilwane okushintsha kwe-modic (MC) kuyisisekelo esibalulekile sokutadisha i-MC. Onogwaja abamhlophe abangamashumi amahlanu nane baseNew Zealand bahlukaniswe ngeqembu lokuhlinzwa kwe-sham, iqembu lokufakelwa kwemisipha (iqembu le-ME) kanye neqembu lokufakelwa kwe-nucleus pulposus (iqembu le-NPE). Eqenjini le-NPE, i-disc ye-intervertebral yavezwa ngendlela yokuhlinzwa ye-anterolateral lumbar futhi kwasetshenziswa inaliti ukubhoboza umzimba we-vertebral we-L5 eduze kwepuleti lokugcina. I-NP yakhishwa ku-disc ye-intervertebral ye-L1/2 ngesirinji futhi yafakwa kuyo. Ukubhoboza imbobo ethanjeni le-subchondral. Izinqubo zokuhlinzwa kanye nezindlela zokubhoboza eqenjini lokufakelwa kwemisipha kanye neqembu lokufakelwa kwe-sham zazifana nalezo eziseqenjini lokufakelwa kwe-NP. Eqenjini le-ME, ucezu lwemisipha lwafakwa emgodini, kanti eqenjini lokufakelwa kwe-sham, akukho lutho olwafakwa emgodini. Ngemva kokuhlinzwa, kwenziwa ukuskena kwe-MRI kanye nokuhlolwa kwe-molecular biological. Isignali eqenjini le-NPE yashintsha, kodwa akukho ushintsho olucacile lwesignali eqenjini lokufakelwa kwe-sham kanye neqembu le-ME. Ukuhlolwa kwe-histological kubonise ukuthi ukwanda kwezicubu okungajwayelekile kwabonwa endaweni yokufakelwa, futhi ukuvezwa kwe-IL-4, IL-17 kanye ne-IFN-γ kwanda eqenjini le-NPE. Ukufakwa kwe-NP ethanjeni le-subchondral kungakha imodeli yesilwane ye-MC.
Izinguquko ze-Modic (MC) ziyizilonda zama-vertebral endplates kanye nomnkantsha oseduze obonakala ku-magnetic resonance imaging (MRI). Zivame kakhulu kubantu abanezimpawu ezihlobene1. Izifundo eziningi zigcizelele ukubaluleka kwe-MC ngenxa yokuhlangana kwayo nobuhlungu obuphansi emhlane (LBP)2,3. u-de Roos et al.4 kanye no-Modic et al.5 baqale bachaza ngokuzimela izinhlobo ezintathu ezahlukene zokuphazamiseka kwesignali ye-subchondral emnkantsha wethambo lomgogodla. Izinguquko ze-Modic type I ziyi-hypointense kuma-T1-weighted (T1W) sequences kanye ne-hyperintense kuma-T2-weighted (T2W) sequences. Lesi silonda sembula ama-fissure endplates kanye nezicubu ze-vascular granulation eziseduze emnkantsha wethambo. Izinguquko ze-Modic type II zibonisa isignali ephezulu kokubili kuma-T1W kanye nama-T2W sequences. Kulolu hlobo lwesilonda, kungatholakala ukubhujiswa kwe-endplate, kanye nokufakwa esikhundleni kwamafutha e-histological komnkantsha wethambo oseduze. Izinguquko ze-Modic type III zibonisa isignali ephansi kuma-T1W kanye nama-T2W sequences. Izilonda ze-sclerotic ezihambisana nama-endplates ziye zabonwa6. I-MC ibhekwa njengesifo esibangela izifo emgogodleni futhi ihlotshaniswa eduze nezifo eziningi eziwohlokayo zomgogodla7,8,9.
Uma sibheka idatha etholakalayo, izifundo eziningana zinikeze ukuqonda okuningiliziwe ngezimbangela kanye nezindlela ze-MC. U-Albert nabanye basikisela ukuthi i-MC ingase ibangelwe yi-disc herniation8. U-Hu nabanye basikisela ukuthi i-MC ibangelwa ukuwohloka okukhulu kwe-disc10. UKroc uphakamise umqondo "wokuqhekeka kwe-disc yangaphakathi," othi ukulimala kwe-disc okuphindaphindiwe kungaholela ekuqhekekeni kwe-microplate. Ngemva kokwakheka kwe-cleft, ukubhujiswa kwe-endplate yi-nucleus pulposus (NP) kungabangela impendulo ye-autoimmune, okuholela ekuthuthukisweni kwe-MC11. UMa nabanye babe nombono ofanayo futhi babika ukuthi i-NP-induced autoimmune idlala indima ebalulekile ekuthuthukisweni kwe-MC12.
Amaseli esistimu yomzimba, ikakhulukazi ama-lymphocyte asizayo e-CD4+ T, adlala indima ebalulekile ekuqaliseni kwe-autoimmune13. I-Th17 subset esanda kutholakala ikhiqiza i-cytokine IL-17 evuvukalayo, ikhuthaza ukuvezwa kwe-chemokine, futhi ivuselela amaseli e-T ezithweni ezonakele ukuthi akhiqize i-IFN-γ14. Amaseli e-Th2 nawo adlala indima eyingqayizivele ekuqaliseni kwempendulo yomzimba. Ukuvezwa kwe-IL-4 njengeseli elimele i-Th2 kungaholela emiphumeleni emibi ye-immunopathological15.
Nakuba kwenziwe izifundo eziningi zezokwelapha ku-MC16,17,18,19,20,21,22,23,24, kusenokuntuleka kwamamodeli okuhlola ezilwane afanele angalingisa inqubo ye-MC evame ukwenzeka kubantu futhi angasetshenziswa ukuphenya imbangela noma ukwelashwa okusha njengokwelashwa okuqondisiwe. Kuze kube manje, kubikwa ukuthi kuncane kakhulu amamodeli ezilwane e-MC afunda izindlela eziyisisekelo ze-pathological.
Ngokusekelwe kumbono wokuzivikela komzimba ohlongozwe ngu-Albert no-Ma, lolu cwaningo lusungule imodeli ye-MC yonogwaja elula nephindaphindwayo ngokufaka i-NP ngokuzenzakalela eduze kwepuleti lokugcina lomgogodla elibhoboziwe. Ezinye izinjongo ukubheka izici ze-histological zamamodeli ezilwane nokuhlola izindlela ezithile ze-NP ekuthuthukisweni kwe-MC. Ukuze sifeze lokhu, sisebenzisa amasu anjenge-molecular biology, i-MRI, kanye nezifundo ze-histological ukutadisha inqubekela phambili ye-MC.
Onogwaja ababili bafa ngenxa yokopha ngesikhathi sokuhlinzwa, kanti onogwaja abane bafa ngesikhathi sokuhlinzwa ngesikhathi se-MRI. Onogwaja abangu-48 abasele basinda futhi ababonisanga zimpawu zokuziphatha noma zezinzwa ngemva kokuhlinzwa.
I-MRI ikhombisa ukuthi ukuqina kwesignali yezicubu ezifakwe emigodini ehlukene kuhlukile. Ukuqina kwesignali yomzimba womgogodla we-L5 eqenjini le-NPE kwashintsha kancane kancane emavikini ayi-12, 16 kanye nama-20 ngemva kokufakwa (ukulandelana kwe-T1W kubonise isignali ephansi, kanti ukulandelana kwe-T2W kubonise isignali exubile kanye nesignali ephansi) (Umfanekiso 1C), kuyilapho ukubonakala kwe-MRI kwamanye amaqembu amabili ezingxenye ezifakiwe kwahlala kuzinzile phakathi nenkathi efanayo (Umfanekiso 1A, B).
(A) Ama-MRI alandelanayo amele umgogodla we-lumbar onogwaja ngezikhathi ezi-3. Akukho ukuphazamiseka kwesignali okutholakale ezithombeni zeqembu le-sham-operation. (B) Izici zesignali zomzimba we-vertebral eqenjini le-ME zifana nalezo eziseqenjini le-sham-operation, futhi akukho ushintsho olukhulu lwesignali olubonakalayo endaweni yokushumeka ngokuhamba kwesikhathi. (C) Eqenjini le-NPE, isignali ephansi ibonakala ngokucacile ochungechungeni lwe-T1W, kanti isignali exubile kanye nesignali ephansi kubonakala ngokucacile ochungechungeni lwe-T2W. Kusukela esikhathini samaviki ayi-12 kuya esikhathini samaviki angama-20, izimpawu eziphakeme ezingahleliwe ezizungeze izimpawu eziphansi ochungechungeni lwe-T2W ziyancipha.
I-hyperplasia yamathambo esobala ingabonakala endaweni yokufakelwa komzimba womgogodla eqenjini le-NPE, futhi i-hyperplasia yamathambo yenzeka ngokushesha kusukela emavikini ayi-12 kuya kwangu-20 (Isithombe 2C) uma kuqhathaniswa neqembu le-NPE, akukho shintsho oluphawulekayo olubonakalayo emizimbeni yomgogodla efanisiwe; iqembu le-Sham kanye neqembu le-ME (Isithombe 2C) 2A,B).
(A) Ubuso bomzimba womgogodla engxenyeni efakwe bubushelelezi kakhulu, imbobo iphola kahle, futhi akukho hyperplasia emzimbeni womgogodla. (B) Ukuma kwendawo efakwe eqenjini le-ME kufana nalokho okuseqenjini lokuhlinzwa okungamanga, futhi akukho ushintsho olucacile ekubukekeni kwendawo efakwe ngokuhamba kwesikhathi. (C) I-Bone hyperplasia yenzeka endaweni efakwe eqenjini le-NPE. I-bone hyperplasia yanda ngokushesha futhi yaze yanwebeka nge-intervertebral disc yaya emzimbeni womgogodla ongaphandle.
Ukuhlaziywa kwe-Histological kunikeza ulwazi oluningiliziwe mayelana nokwakheka kwamathambo. Isithombe 3 sibonisa izithombe zezingxenye zangemuva kokuhlinzwa ezifakwe i-H&E. Eqenjini lokuhlinzwa kwe-sham, ama-chondrocyte ayehlelwe kahle futhi akukho ukwanda kwamaseli okutholiwe (Isithombe 3A). Isimo eqenjini le-ME sasifana neseqembu lokuhlinzwa kwe-sham (Isithombe 3B). Kodwa-ke, eqenjini le-NPE, inani elikhulu lama-chondrocyte kanye nokwanda kwamaseli afana ne-NP kwabonwa endaweni yokufakelwa (Isithombe 3C);
(A) Ama-Trabeculae angabonakala eduze kwepuleti lokugcina, ama-chondrocyte ahlelwe kahle ngobukhulu kanye nesimo samaseli esifanayo futhi akukho ukwanda (izikhathi ezingu-40). (B) Isimo sendawo yokufakelwa eqenjini le-ME sifana neseqembu lama-fake. Ama-Trabeculae nama-chondrocyte angabonakala, kodwa akukho ukwanda okusobala endaweni yokufakelwa (izikhathi ezingu-40). (B) Kungabonakala ukuthi ama-chondrocyte namaseli afana ne-NP ayanda kakhulu, futhi ukuma nobukhulu bama-chondrocyte akungalingani (izikhathi ezingu-40).
Ukuvezwa kwe-interleukin 4 (IL-4) mRNA, i-interleukin 17 (IL-17) mRNA, kanye ne-interferon γ (IFN-γ) mRNA kwabonwa kokubili emaqenjini e-NPE kanye ne-ME. Lapho amazinga okuvezwa kwezakhi zofuzo eziqondiwe eqhathaniswa, ukuvezwa kwezakhi zofuzo kwe-IL-4, IL-17, kanye ne-IFN-γ kwanda kakhulu eqenjini le-NPE uma kuqhathaniswa nalawo eqembu le-ME kanye neqembu lokusebenza kwe-sham (Isithombe 4) (P < 0.05). Uma kuqhathaniswa neqembu lokusebenza kwe-sham, amazinga okuvezwa kwe-IL-4, IL-17, kanye ne-IFN-γ eqenjini le-ME anda kancane futhi awazange afinyelele ushintsho lwezibalo (P > 0.05).
Ukuvezwa kwe-mRNA kwe-IL-4, IL-17 kanye ne-IFN-γ eqenjini le-NPE kubonise ukuthambekela okuphezulu kakhulu kunalabo abaseqenjini lokusebenza okungamanga kanye neqembu le-ME (P < 0.05).
Ngokuphambene nalokho, amazinga okubonakaliswa eqenjini le-ME awazange abonise mehluko obalulekile (P>0.05).
Ukuhlaziywa kwe-Western blot kwenziwa kusetshenziswa ama-antibodies atholakala kwezentengiselwano ngokumelene ne-IL-4 kanye ne-IL-17 ukuqinisekisa iphethini yokubonakaliswa kwe-mRNA eshintshiwe. Njengoba kuboniswe kuZithombe 5A, B, uma kuqhathaniswa neqembu le-ME kanye neqembu lokusebenza kwe-sham, amazinga e-protein e-IL-4 kanye ne-IL-17 eqenjini le-NPE akhuliswe kakhulu (P < 0.05). Uma kuqhathaniswa neqembu lokusebenza kwe-sham, amazinga e-protein e-IL-4 kanye ne-IL-17 eqenjini le-ME nawo ahlulekile ukufinyelela izinguquko ezibalulekile ngokwezibalo (P> 0.05).
(A) Amazinga amaprotheni e-IL-4 kanye ne-IL-17 eqenjini le-NPE ayephezulu kakhulu kunalawo eqenjini le-ME kanye neqembu le-placebo (P < 0.05). (B) I-Western blot histogram.
Ngenxa yenani elilinganiselwe lamasampula abantu atholwe ngesikhathi sokuhlinzwa, izifundo ezicacile nezinemininingwane nge-pathogenesis ye-MC zinzima kancane. Sizame ukusungula imodeli yesilwane ye-MC ukuze sifunde izindlela zayo ezingaba khona ze-pathological. Ngesikhathi esifanayo, ukuhlolwa kwe-radiological, ukuhlolwa kwe-histological kanye nokuhlolwa kwe-molecular biological kwasetshenziswa ukulandela inkambo ye-MC ebangelwa yi-NP autograft. Ngenxa yalokho, imodeli yokufakelwa kwe-NP yaholela ekushintsheni kancane kancane kokuqina kwesignali kusukela emavikini ayi-12 kuya kwamasonto angama-20 (isignali ephansi exubile ku-T1W sequences kanye nesignali ephansi ku-T2W sequences), okubonisa izinguquko zezicubu, kanye nokuhlolwa kwe-histological kanye ne-molecular biological kwaqinisekisa imiphumela yocwaningo lwe-radiological.
Imiphumela yalolu cwaningo ikhombisa ukuthi izinguquko ezibonakalayo neze-histological zenzeke endaweni yokwephulwa komzimba we-vertebral eqenjini le-NPE. Ngesikhathi esifanayo, ukuvezwa kwezakhi zofuzo ze-IL-4, IL-17 kanye ne-IFN-γ, kanye ne-IL-4, IL-17 kanye ne-IFN-γ kwabonwa, okubonisa ukuthi ukwephulwa kwezicubu ze-nucleus pulposus ze-autologous emzimbeni we-vertebral kungabangela uchungechunge lwezinguquko zesignali kanye ne-morphological. Kulula ukuthola ukuthi izici zesignali zemizimba ye-vertebral yemodeli yesilwane (isignali ephansi ku-T1W sequence, isignali exubile kanye nesignali ephansi ku-T2W sequence) zifana kakhulu nezeseli zomgogodla womuntu, futhi izici ze-MRI nazo ziqinisekisa ukubonwa kwe-histology kanye ne-gross anatomy, okungukuthi, izinguquko kumaseli omzimba we-vertebral ziyathuthuka. Nakuba impendulo yokuvuvukala ebangelwa ukulimala okukhulu ingavela ngokushesha ngemva kokubhoboza, imiphumela ye-MRI ibonise ukuthi izinguquko zesignali ezikhula kancane kancane zavela emavikini ayi-12 ngemva kokubhoboza futhi zaqhubeka kuze kube amasonto angama-20 ngaphandle kwezimpawu zokululama noma ukuguqulwa kwezinguquko ze-MRI. Le miphumela isikisela ukuthi i-autologous vertebral NP iyindlela ethembekile yokusungula i-MV eqhubekayo konogwaja.
Le modeli yokubhoboza idinga ikhono elanele, isikhathi, kanye nomzamo wokuhlinzwa. Ekuhlolweni kokuqala, ukuhlukaniswa noma ukukhuthazwa ngokweqile kwezakhiwo ze-paravertebral ligamentous kungabangela ukwakheka kwama-osteophyte e-vertebral. Kufanele kuqashelwe ukuthi kungalimazi noma kucasule ama-disc aseduze. Njengoba ukujula kokungena kumele kulawulwe ukuze kutholakale imiphumela ehambisanayo nephindaphindayo, senze ipulaki ngesandla ngokusika igobolondo lenaliti engu-3 mm ubude. Ukusebenzisa le pulaki kuqinisekisa ukujula okufanayo kokubhoboza emzimbeni we-vertebral. Ekuhlolweni kokuqala, odokotela abathathu bamathambo abahilelekile ekuhlinzeni bathole ukuthi kulula ukusebenza ngezinaliti ze-gauge ezingu-16 kunezinaliti ze-gauge ezingu-18 noma ezinye izindlela. Ukuze ugweme ukuphuma kwegazi ngokweqile ngesikhathi sokubhoboza, ukubamba inaliti inganyakazi isikhashana kuzohlinzeka ngembobo yokufaka efaneleke kakhulu, okuphakamisa ukuthi izinga elithile le-MC lingalawulwa ngale ndlela.
Nakuba izifundo eziningi zihlose i-MC, kuncane okwaziwayo nge-etiology kanye ne-pathogenesis ye-MC25,26,27. Ngokusekelwe ezifundweni zethu zangaphambilini, sithole ukuthi ukuzivikela komzimba kudlala indima ebalulekile ekuveleni nasekuthuthukisweni kwe-MC12. Lolu cwaningo luhlole ukubonakaliswa kwe-IL-4, IL-17, kanye ne-IFN-γ, okuyizindlela eziyinhloko zokuhlukanisa amaseli e-CD4+ ngemva kokukhuthazwa kwe-antigen. Esifundweni sethu, uma kuqhathaniswa neqembu elibi, iqembu le-NPE lalinokubonakaliswa okuphezulu kwe-IL-4, IL-17, kanye ne-IFN-γ, kanti amazinga amaprotheni e-IL-4 kanye ne-IL-17 nawo ayephezulu.
Ngokwezokwelapha, ukuvezwa kwe-mRNA ye-IL-17 kuyanda kumaseli e-NP avela ezigulini ezine-disc herniation28. Amazinga okwanda kokubonakaliswa kwe-IL-4 kanye ne-IFN-γ atholakale futhi kumodeli ye-disc herniation engacindezeli uma kuqhathaniswa nezilawuli ezinempilo29. I-IL-17 idlala indima ebalulekile ekuvuvukeni, ekulimaleni kwezicubu ezifweni zokuzivikela komzimba30 futhi ithuthukisa impendulo yomzimba ku-IFN-γ31. Ukulimala kwezicubu okuthuthukisiwe okubangelwa yi-IL-17 kubikwe kumagundane e-MRL/lpr32 kanye namagundane athinteka yi-autoimmune33. I-IL-4 ingavimbela ukuvezwa kwama-cytokines abangela ukuvuvukala (njenge-IL-1β kanye ne-TNFα) kanye nokusebenza kwe-macrophage34. Kubikwe ukuthi ukuvezwa kwe-mRNA kwe-IL-4 kwakuhlukile eqenjini le-NPE uma kuqhathaniswa ne-IL-17 kanye ne-IFN-γ ngesikhathi esifanayo; Ukuvezwa kwe-mRNA kwe-IFN-γ eqenjini le-NPE kwakuphakeme kakhulu kunakwamanye amaqembu. Ngakho-ke, ukukhiqizwa kwe-IFN-γ kungaba ngumlamuleli wempendulo yokuvuvukala ebangelwa ukuhlangana kwe-NP. Izifundo zibonise ukuthi i-IFN-γ ikhiqizwa izinhlobo eziningi zamaseli, okuhlanganisa amaseli e-T asebenzayo ohlobo 1, amaseli okubulala emvelo, nama-macrophages35,36, futhi iyi-cytokine eyinhloko ekhuthaza ukuvuvukala ekhuthaza izimpendulo zomzimba37.
Lolu cwaningo luphakamisa ukuthi impendulo yokuzivikela komzimba ingase ihileleke ekwakhekeni nasekuthuthukisweni kwe-MC. ULuoma nabanye bathole ukuthi izici zesignali ze-MC kanye ne-NP evelele ziyafana ku-MRI, futhi zombili zibonisa isignali ephezulu ku-T2W sequence38. Amanye ama-cytokine aqinisekisiwe ukuthi ahlobene eduze nokuvela kwe-MC, njenge-IL-139. UMa nabanye baphakamise ukuthi ukuvela okuphezulu noma okuphansi kwe-NP kungaba nethonya elikhulu ekuveleni nasekuthuthukisweni kwe-MC12. UBobechko40 noHerzbein nabanye41 babike ukuthi i-NP iyizicubu ezingabekezeleli amasosha omzimba ezingakwazi ukungena ekujikelezeni kwegazi kusukela ekuzalweni. Ukuvela kwe-NP kungenisa imizimba yangaphandle egazini, ngaleyo ndlela kuqondise ukusabela kwe-autoimmune kwendawo42. Ukusabela kwe-autoimmune kungadala inani elikhulu lezinto zokuzivikela komzimba, futhi lapho lezi zinto zivezwa njalo ezicutshini, zingabangela izinguquko ekuboniseni izimpawu43. Kulolu cwaningo, ukuvezwa ngokweqile kwe-IL-4, IL-17 kanye ne-IFN-γ kuyizinto zokuzivikela zomzimba ezivamile, okufakazela ubudlelwano obuseduze phakathi kwe-NP kanye ne-MCs44. Le modeli yesilwane ilingisa kahle ukugqashuka kwe-NP kanye nokungena epuletini lokugcina. Le nqubo yembula futhi umthelela wokuzivikela komzimba ku-MC.
Njengoba bekulindelekile, le modeli yesilwane isinikeza ipulatifomu engaba khona yokufunda i-MC. Kodwa-ke, le modeli isenemikhawulo ethile: okokuqala, ngesikhathi sesigaba sokubuka izilwane, onogwaja abathile abasesigabeni esiphakathi badinga ukubulawa ukuze kuhlolwe i-histological kanye ne-molecular biology, ngakho-ke ezinye izilwane "ziyayeka ukusetshenziswa" ngokuhamba kwesikhathi. Okwesibili, yize kubekwe amaphuzu amathathu esikhathi kulolu cwaningo, ngeshwa, senze imodeli yohlobo olulodwa lwe-MC (ushintsho lohlobo lwe-Modic I), ngakho-ke akwanele ukumelela inqubo yokuthuthukiswa kwesifo somuntu, futhi kudingeka kubekwe amaphuzu esikhathi engeziwe ukuze kubonwe kangcono zonke izinguquko zesignali. Okwesithathu, izinguquko esakhiweni sezicubu zingabonakala ngokucacile ngokudaya kwe-histological, kodwa amanye amasu akhethekile angaveza kangcono izinguquko zesakhiwo kule modeli. Isibonelo, i-polarized light microscopy yasetshenziswa ukuhlaziya ukwakheka kwe-fibrocartilage kuma-disc e-intervertebral kanogwaja45. Imiphumela yesikhathi eside ye-NP ku-MC kanye ne-endplate idinga ukufundwa okwengeziwe.
Onogwaja abamhlophe abangamaduna abangamashumi amahlanu nane baseNew Zealand (abanesisindo esingaba ngu-2.5-3 kg, abaneminyaka engu-3-3.5 ubudala) bahlukaniswe ngokungahleliwe baba yiqembu lokuhlinzwa okungamanga, iqembu lokufakelwa kwemisipha (iqembu le-ME) kanye neqembu lokufakelwa kwezimpande zemizwa (iqembu le-NPE). Zonke izinqubo zokuhlola zavunyelwa yiKomidi Lokuziphatha laseTianjin Hospital, futhi izindlela zokuhlola zenziwa ngokuhambisana neziqondiso ezivunyiwe.
Kuthuthukiswe okuthile enkambisweni yokuhlinzwa ye-S. Sobajima 46. Unogwaja ngamunye wabekwa endaweni yokulala eceleni kanti ubuso bangaphambili bama-disc amahlanu alandelanayo e-lumbar intervertebral (IVDs) bavezwa kusetshenziswa indlela ye-posterolateral retroperitoneal. Unogwaja ngamunye wanikezwa i-anesthesia ejwayelekile (20% i-urethane, 5 ml/kg nge-vein yendlebe). Kwenziwa ukusikwa kwesikhumba eside kusukela emaphethelweni angezansi ezimbambo kuya emphethweni we-pelvic, 2 cm ventral kuya emisipheni ye-paravertebral. Umgogodla we-anterolateral wesokudla kusukela ku-L1 kuya ku-L6 wavezwa ngokuhlukaniswa okubukhali nokungacacile kwezicubu ezingaphansi kwesikhumba ezimboze, izicubu ze-retroperitoneal, kanye nemisipha (Isithombe 6A). Izinga le-disc lanqunywa kusetshenziswa umngcele we-pelvic njengophawu olubonakalayo lwezinga le-disc le-L5-L6. Sebenzisa inaliti yokubhoboza engu-16-gauge ukubhoboza imbobo eduze kwepuleti lokugcina le-vertebra ye-L5 kuze kube ukujula okungu-3 mm (Isithombe 6B). Sebenzisa isirinji esingu-5-ml ukuze ukhiphe i-autologous nucleus pulposus ku-L1-L2 intervertebral disc (Isithombe 6C). Susa i-nucleus pulposus noma imisipha ngokwezidingo zeqembu ngalinye. Ngemva kokuba imbobo yokubhoboza isijulile, kufakwa ama-sutures angena emzimbeni ku-fascia ejulile, i-fascia engaphezulu kanye nesikhumba, kuqikelelwe ukuthi kungalimazi izicubu ze-periosteal zomzimba womgogodla ngesikhathi sokuhlinzwa.
(A) Idiski ye-L5–L6 ivezwa ngendlela ye-posterolateral retroperitoneal. (B) Sebenzisa inalithi engu-16-gauge ukubhoboza imbobo eduze kwe-endplate ye-L5. (C) Kuvunwa ama-MF e-Autologous.
Ukuhlinzwa okuvamile kwenziwa nge-urethane engu-20% (5 ml/kg) enikezwa ngemithambo yendlebe, kanti ama-radiograph omgogodla we-lumbar aphindaphindwa emavikini angu-12, 16, kanye nama-20 ngemva kokuhlinzwa.
Onogwaja bahlatshwa ngokujova nge-ketamine (25.0 mg/kg) kanye ne-sodium pentobarbital (1.2 g/kg) ngemithambo yegazi emavikini ayi-12, 16 kanye nama-20 ngemva kokuhlinzwa. Umgogodla wonke wasuswa ukuze kuhlaziywe nge-histological futhi kwenziwa ukuhlaziywa kwangempela. I-Quantitative reverse transcription (RT-qPCR) kanye ne-Western blotting kwasetshenziswa ukuthola izinguquko ezicini zomzimba.
Ukuhlolwa kwe-MRI kwenziwe konogwaja kusetshenziswa i-3.0 T clinical magnet (GE Medical Systems, Florence, SC) ehlonyiswe nge-orthogonal limb coil receiver. Onogwaja bahlanzwa nge-20% urethane (5 mL/kg) nge-ear vein base bebekwa e-sleep ngaphakathi kwe-magnet lapho i-lumbar region igxile kwi-circular surface coil engu-5 intshi ububanzi (GE Medical Systems). Izithombe ze-localizer ze-Coronal T2-weighted (TR, 1445 ms; TE, 37 ms) zatholakala ukuze kuchazwe indawo ye-lumbar disc kusukela ku-L3–L4 kuya ku-L5–L6. Izingcezu ze-Sagittal plane T2-weighted zitholwe ngezilungiselelo ezilandelayo: i-fast spin-echo sequence ene-repetition time (TR) engu-2200 ms kanye ne-echo time (TE) engu-70 ms, matrix; visual field engu-260 kanye ne-stimuli eziyisishiyagalombili; Ubukhulu bokusika babungu-2 mm, igebe lalingu-0.2 mm.
Ngemva kokuthathwa kwesithombe sokugcina kanye nonogwaja wokugcina ebulewe, ama-disc e-sham, afakwe imisipha, kanye ne-NP asusiwe ukuze kuhlolwe i-histological. Izicubu zaqiniswa ku-formalin engathathi hlangothi engu-10% isonto eli-1, zasuswa i-calcium nge-ethylenediaminetetraacetic acid, kwase kuhlukaniswa i-paraffin. Amabhulokhi ezicubu afakwa ku-paraffin futhi anqunywa abe yizingxenye ze-sagittal (ubukhulu obungu-5 μm) kusetshenziswa i-microtome. Izingxenye zafakwa i-hematoxylin kanye ne-eosin (H&E).
Ngemva kokuqoqa ama-disc e-intervertebral kusuka kunogwaja eqenjini ngalinye, i-RNA iyonke yakhishwa kusetshenziswa ikholomu ye-UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., eShayina) ngokwemiyalelo yomenzi kanye nohlelo lwe-ImProm II reverse transcription (Promega Inc., eMadison, eWI, e-USA). Kwenziwe i-reverse transcription.
I-RT-qPCR yenziwe kusetshenziswa i-Prism 7300 (Applied Biosystems Inc., USA) kanye ne-SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) ngokwemiyalelo yomenzi. Umthamo wokusabela kwe-PCR wawungu-20 μl futhi wawuqukethe i-1.5 μl ye-cDNA exutshwe kanye ne-0.2 μM ye-primer ngayinye. Ama-primer aklanywa yi-OligoPerfect Designer (Invitrogen, Valencia, CA) futhi akhiqizwa yi-Nanjing Golden Stewart Biotechnology Co., Ltd. (China) (Ithebula 1). Kusetshenziswe izimo ezilandelayo zokujikeleza kokushisa: isinyathelo sokuqala sokwenza i-polymerase isebenze ku-94°C imizuzu emi-2, bese kuba imijikelezo engama-40 yamasekhondi ayi-15 ngalinye ku-94°C ukuze kuguqulwe i-template, kufakwe i-annealing imizuzu eyi-1 ku-60°C, kunwetshwe, futhi kukhanye. ukulinganisa kwenziwa imizuzu eyi-1 ku-72°C. Zonke amasampula akhuliswe kathathu futhi inani elimaphakathi lasetshenziswa ekuhlaziyweni kwe-RT-qPCR. Idatha yokukhulisa ihlaziywe kusetshenziswa i-FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). Ukubonakaliswa kwezakhi zofuzo ze-IL-4, IL-17, kanye ne-IFN-γ kwalungiswa kwaba yi-endogenous control (ACTB). Amazinga okubonakaliswa okuhlobene kwe-target mRNA abalwe kusetshenziswa indlela ye-2-ΔΔCT.
Iphrotheni ephelele yakhishwa ezicutshini kusetshenziswa i-homogenizer yezicubu ku-RIPA lysis buffer (equkethe i-protease kanye ne-phosphatase inhibitor cocktail) yabe isifakwa ku-centrifuge ku-13,000 rpm imizuzu engama-20 ku-4°C ukuze kususwe udoti wezicubu. Ama-microgram angamashumi amahlanu ephrotheni alayishwa ngomzila ngamunye, ahlukaniswe yi-10% SDS-PAGE, bese edluliselwa ku-membrane ye-PVDF. Ukuvimba kwenziwa kubisi olomile olungenamafutha angu-5% ku-Tris-buffered saline (TBS) equkethe i-0.1% Tween 20 ihora eli-1 ekushiseni kwegumbi. I-membrane yafakwa ku-antibody yokuqala ye-rabbit anti-decorin (exutshwe ngu-1:200; Boster, Wuhan, China) (exutshwe ngu-1:200; Bioss, Beijing, China) ubusuku bonke ku-4°C futhi yasabela ezinsukwini zesibili; nge-antibody yesibili (i-goat anti-rabbit immunoglobulin G ku-1:40,000 dilution) kuhlanganiswe ne-horseradish peroxidase (Boster, Wuhan, China) ihora eli-1 ekushiseni kwegumbi. Izimpawu ze-Western blot zitholwe ngokwanda kwe-chemiluminescence ku-membrane ye-chemiluminescent ngemuva kokushiswa kwe-X-ray. Ekuhlaziyweni kwe-densitometric, ama-blots askeniwe futhi alinganiswa kusetshenziswa isofthiwe ye-BandScan futhi imiphumela ivezwe njengesilinganiso se-target gene immunoreactivity ku-tubulin immunoreactivity.
Izibalo zezibalo zenziwe kusetshenziswa iphakheji yesofthiwe ye-SPSS16.0 (SPSS, USA). Idatha eqoqwe ngesikhathi socwaningo ivezwe njenge-mean ± standard deviation (mean ± SD) futhi yahlaziywa kusetshenziswa ukuhlaziywa kwe-variance yezindlela eziphindaphindwayo eyodwa (ANOVA) ukuze kutholakale umehluko phakathi kwamaqembu amabili. I-P < 0.05 ibhekwe njengebalulekile ngokwezibalo.
Ngakho-ke, ukusungulwa kwemodeli yesilwane ye-MC ngokufaka ama-NP e-autologous emzimbeni womgogodla nokwenza ukubonwa kwe-macroanatomical, ukuhlaziywa kwe-MRI, ukuhlolwa kwe-histological kanye nokuhlaziywa kwe-molecular biological kungaba ithuluzi elibalulekile lokuhlola nokuqonda izindlela ze-MC yomuntu kanye nokuthuthukisa izindlela ezintsha zokwelapha.
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