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Ukusungulwa kwamamodeli wezilwane we-Modic Change (MC) yisisekelo esibalulekile sokutadisha i-MC. Onogwaja abamhlophe baseNew New Zen asezingeni eliphakeme bahlukaniswe ngeqembu le-Sham-Operation Group, iqembu lokufakwa kwemisipha (iqembu leqembu) neqembu le-npeus pulpposantation Group (NPE Group). Eqenjini le-NPE, i-degnerteral disc yavezwa yi-anterolateral lumbar ukusondela kuhlinzwa i-antenterle I-NP ikhishwe kwi-L1 / 2 yokungenelela ye-InveryerteBral disc ngesirinji futhi ifakwe kuyo. Ukumba umgodi ethanjeni elingaphansi. Izinqubo zokuhlinza kanye nezindlela zokumba eqenjini lokufakwa kwemisipha kanye neqembu le-Sham-Operation belifana nalawo aseqenjini le-NP Lokufakwa. Eqenjini lemina, kwafakwa ucezu lwesicubu emgodini, ngenkathi eseqenjini le-Sham-Operation, akukho lutho olubekwe emgodini. Ngemuva kokuhlinzwa, ukuskena kwe-MRI nokuhlolwa kwemvelo kwemvelo kwenziwa. Isiginali eqenjini le-NPE lashintsha, kodwa kwakungekho shintsho lwesibonakaliso esisobala eqenjini le-Sham-Operation neqembu lemi. Ukubhekwa kwe-Hism kukhombisa ukuthi ukwanda kwezicubu okungajwayelekile kwaqashelwa endaweni yokufakelwa, kanye nenkulumo ye-IL-4, iL-17 ne-Ifn-γ yandiswa eqenjini le-NPE. Ukufakwa kwe-NP kuthambo le-subchondral angakha imodeli yezilwane ye-MC.
Izinguquko ze-Modic (MC) yizilonda ze-vertebral explates kanye nomongo wamathambo aseduze abonakala ku-Magnetic Resonance imaging (MRI). Zivame kakhulu kubantu abanezimpawu ezihlangene1. Izifundo eziningi zigcizelele ukubaluleka kwe-MC ngenxa yokuhlangana kwayo ngezinhlungu eziphansi emuva (LBP) 2,3. De Roos et al.4 noModic et al.5 ngokuzimela kuqala uchaze izinhlobo ezintathu ezahlukene zezinhlobo ezintathu zokuphazamiseka kwesiginali esisezingeni eliphansi eVertebral Bone Markow. Uhlobo lwe-Modic engilishintshayo i-hypypense ku ukulandelana kwe-T1-Isisindo (T1W) kanye ne-hyperintense ku ukulandelana kwe-T2-Isisindo (T2W). Le i-lesion iveza ama-fissure ama-relplates kanye nezicubu zomzimba we-vascular eziseduzane emmosheni wamathambo. Uhlobo lwe-Modic Type II lubonisa isinali ephezulu kuzo zombili ukulandelana kwe-T1W kanye ne-T2W. Kulolu hlobo lwesilonda, ukubhujiswa okuphelele kungatholakala, kanye nokubuyiselwa okunamafutha emthonjeni we-The Woone Markow. Uhlobo lohlobo lwe-Modic III lukhombisa isinali ephansi ku-T1W kanye nokulandelana kwe-T2W. Izilonda ze-Sclerotic ezihambelana nezimali eziphelele zibonwe6. I-MC ibhekwa njengesifo se-pathological somgogodla futhi sihlotshaniswa kakhulu nezifo eziningi eziwohlokayo ze-SPINE7,8,9.
Uma kucatshangelwa imininingwane etholakalayo, izifundo eziningana zinikeze imininingwane eningiliziwe kwizindlela ze-Etiology kanye nezindlela ze-MC. U-Albert et al. Kuphakanyiswe ukuthi i-MC ingahle ibangelwa yi-Disc Herniation8. Hu et al. I-MC ebekelwe i-DICK DEGEINETER10. UKroc uhlongoza umqondo we- "Disc Disc Langaphakathi," esho ukuthi i-reptitive disc trauma ingahle iholele kuma-micronotes esiqeshini. Ngemuva kokwakheka kwe-Cleft, kukhuthaze ukubhujiswa yi-nucleus pulporus (np) kungadala impendulo ye-autoimmune, okuholela phambili ekuthuthukisweni kwe-MC11. Ma et al. Uhlanganyele umbono ofanayo futhi wabika ukuthi i-autoimmunity ye-NP-eyenziwe nge-NP idlala indima ebalulekile kwi-pathogenesis ye-MC12.
Amaseli amasosha omzimba, ikakhulukazi ama-CD4 + T Helper lymphocytes, adlale indima ebalulekile kwi-pathogenesion ye-autoogenity13. I-th17 esanda kutholwa ikhiqiza i-proinflammatotory IL-17, ikhuthaza ukuvezwa kwama-chemokine, futhi ivuse amaseli e-T ngezitho ezilimele ukukhiqiza i-IFN-γ14. Amaseli we-TH2 abuye adlale indima eyingqayizivele kwi-pathogenesis yezimpendulo zokuzivikela komzimba. Ukuvezwa kwe-IL-4 njengeseli elimele i-TH2 cell kungaholela emiphumeleni emibi ye-immunopathological15.
Yize kuqhutshelwe izifundo eziningi zemitholampilo ku-MC16,17,18,19,20,22,29,20,23,19,20,24,24, kusekhona ukuntuleka kwamamodeli afanelekayo wezilwane angenza i-MC evame ukwenzeka kubantu futhi ingaba isetshenziselwe ukuphenya i-etiology noma izindlela zokwelapha ezintsha njengokulashwa okuhlosiwe. Kuze kube manje, amamodeli ambalwa wezilwane we-MC okubikwa ukuthi afundze afundza tingcosana.
Ngokusekelwe embonweni ye-autoimmune ephakanyiswe ngu-Albert noMa, lolu cwaningo lusungula imodeli elula ye-MC yoMvundla ye-AutoTransplant Ezinye izinhloso ukubheka izici zomlando zamamodeli wezilwane futhi kuhlolwe izindlela ezithile ze-NP ekuthuthukisweni kwe-MC. Kuze kube manje, sisebenzisa amasu anjenge-molecular biology, i-MRI, kanye nezifundo ze-Histoloroid ukutadisha ukuthuthuka kwe-MC.
Onogwaja ababili babulawa ukopha ngesikhathi sokuhlinzwa, kanti onogwaja abane bafa ngesikhathi se-anesthesia ngesikhathi se-MRI. Onogwaja abangama-48 abasele basinda futhi abakhombisanga izibonakaliso zokuziphatha noma zokuziphatha kwemizwa ngemuva kokuhlinzwa.
I-MRI ikhombisa ukuthi ukuqina kwesiginali zezicubu ezishumekiwe ezigodini ezahlukahlukene zehlukile. Ukuqina kwesiginali komzimba we-L5 Vertebral eqenjini le-NPE kushintshe kancane kancane ngamaviki ayi-12, angu-16 nangama-20 kuboniswe isibonakaliso esiphansi, futhi ukulandelana kwe-T2W kuboniswe isibonakaliso esixubile kanye nesiginali ephansi) (Umdwebo 1c), ngenkathi ubukeka be-MRI Kwamanye amaqembu amabili ezingxenye ezishumekiwe ahlala ezinzile ngesikhathi esifanayo (Fig. 1a, b).
. Akunazimpawu zokukhishwa kwezimpawu ezitholwe ezithombeni zeqembu le-Sham-Operation. . . Kusukela esikhathini samasonto ayi-12 kuya esikhathini samasonto angama-20, amasignali aphezulu aphezulu azungeze amasiginali aphansi ngokuncipha kokulandelana kwe-T2W.
I-HyPreplasia esobala yethambo ingabonakala endaweni yokufakelwa komzimba we-vertebral eqenjini le-NPE, futhi i-hyperplasia yethambo yenzeka ngokushesha kusuka kumaviki ayi-12 kuye kwangama-20, alukho ushintsho olubalulekile olubonwa ku-vertebral ebekiwe imizimba; I-Sham Group ne-Me Group (Fig. 2c) 2a, b).
(A) Ingaphezulu lomzimba we-vertebral engxenyeni efakwe kahle ibushelelezi kakhulu, umgodi upholisa kahle, futhi akukho hyperplasia emzimbeni we-vertebral. . (C) Kwenzeke i-hyperplasia yethambo engxenyeni efakiwe eqenjini le-NPE. I-hyperplasia yethambo inyuke ngokushesha futhi yandiswa nge-disc ye-intervertebral emzimbeni we-vertebral.
Ukuhlaziywa komlando kuhlinzeka ngemininingwane eminingi enemininingwane mayelana nokwakheka kwamathambo. Umdwebo 3 ukhombisa izithombe zezigaba ze-postoperative ezinamatshe nge-H & E. Eqenjini le-Sham-Operation, amaClondrocyte ahlelwe kahle futhi akukho ukwanda kwamaseli atholakele (Fig. 3a). Isimo eqenjini laseMi sasifana nalokho eqenjini le-Sham-Operation (Fig. 3b). Kodwa-ke, eqenjini le-NPE, inani elikhulu lama-chondocyte kanye nesakhiwo samaseli we-NP-like abonwa endaweni yokufakelwa (Fig. 3c);
. . I-trabeculae kanye nama-chondocytes kungabonakala, kepha akukho ukwanda okusobala endaweni yokufakelwa (amahlandla angama-40). .
Inkulumo ye-Impeluukin 4 (IL-4) MRNA, I-Impelekin 17 (IL-17) Mrna, kanye ne-Interferon γ (IFN-γ Lapho amazinga ezvel of incepge aqhathaniswa, izinkulumo zofuzo ze-IL-4, IL-17, ne-Ifn-γ - zikhuphuke kakhulu eqenjini le-NPE uma liqhathaniswa neqembu le-MAMAM (Fig. 4) (P <0.05). Uma kuqhathaniswa neqembu le-Sham Operation, amazinga egama elithi IL-4, iL-17, kanye ne-Ifn-γ eqenjini lami lenyuke kancane kuphela futhi abafinyelelanga ushintsho lwezibalo (P> 0.05).
Inkulumo ye-MRNA ye-IL-4, i-IL-17 ne-Ifn-γ eqenjini le-NPE akhombisa ukuthambekela okuphakeme kakhulu kunalabo abaseqenjini le-Sham Operation kanye neqembu le-MEMI (P <0.05).
Ngokuphambene nalokho, amazinga enkulumo eqenjini lemi awakhombisanga mehluko omkhulu (p> 0.05).
Ukuhlaziywa kwe-Western Blot kwenziwa kusetshenziswa ama-antibodies atholakala kwezentengiselwano ngokumelene ne-IL-4 ne-IL-17 ukuze aqinisekise iphethini elishintshiwe lenkulumba. Njengoba kukhonjisiwe kwizibalo 5A, B, uma kuqhathaniswa neqembu le-me kanye ne-SHAM Operation Group, amaprotheni ama-IL-4 ne-IL-17 eqenjini le-NPE lakhuphuka kakhulu (p <0.05). Uma kuqhathaniswa neqembu le-Sham Operation, amaprotheni ama-IL-4 ne-IL-17 eqenjini lemine futhi ahlulekile ukufinyelela izinguquko ezibalulekile ngokwezibalo (P> 0.05).
. (B) I-Western Blot Histogram.
Ngenxa yenani elilinganiselwe lamasampula abantu atholwe ngesikhathi sokuhlinzwa, izifundo ezicacile nezinemininingwane kwi-pathogenesis ye-MC zinzima ngandlela thile. Sizame ukusungula imodeli yezilwane ye-MC ukutadisha izindlela zayo ezingaba khona ze-pathological. Ngasikhathi sinye, ukuhlolwa kwe-radiological, ukuhlolwa kwe-histolorological kanye nokuhlolwa kwama-molecular biological kwasetshenziselwa ukulandela inkambo ye-MC edonswa yi-NP AutoGrod. Ngenxa yalokhu, imodeli yokufakwa kwe-NP iholele ekushintsheni kancane kancane ekujuleni kwamanani esignali kusuka kumaphoyinti wesikhathi samasonto ayi-12 kuya ku-20 Ukuhlaziywa kwemvelo kuqinisekise imiphumela yocwaningo lwe-radiological.
Imiphumela yalokhu kuhlolwa kukhombisa ukuthi izinguquko ezibukwayo nezingokomlando zenzeke endaweni yokwephulwa komzimba we-vertebral eqenjini le-NPE. Ngasikhathi sinye, inkulumo ye-IL-4, i-17 ne-17 ne-IFN-γ γ γ γ γ - kanye ne-IL-4, i-IL-17 ne-Ifn-γ nayo i-Ifn-γ. umzimba ungadala uchungechunge lwezinguquko zesiginali kanye ne-morphological. Kulula ukuthola ukuthi izici zesiginali zemizimba ye-vertebral yemodeli yezilwane (isinali ephansi ngokulandelana kwe-T1W, isiginali exubile kanye nesiginali ephansi ngokulandelana kwabantu, kanye nezimpawu ze-MRI nazo Qinisekisa lokho okubukwayo kwe-Histology kanye ne-anatomy ephelele, okungukuthi, izinguquko ezisezingeni eliphakeme lama-vertebral zithuthuka. Yize impendulo yokuvuvukala okubangelwa usizi olukhulu kungahle kuvele kungekudala ngemuva kokubhoboza izinguquko zokubhoboza kwavela kaningi amasonto ayi-12 ngemuva kokushintshwa kwamasonto angama-20 ngaphandle kwezimpawu zoshintsho lwe-MRI. Le miphumela iphakamisa ukuthi i-vertebral ye-vertebral ye-autolovous yindlela ethembekile yokusungula i-MV ethuthukayo enonogwaja.
Le modeli yokubhoboza idinga ikhono elanele, isikhathi, kanye nemizamo yokuhlinzwa. Ekuhlolweni kokuqala, ukuhlukaniswa noma ukukhuthaza ngokweqile kwezakhiwo ze-paravertebral ligaments kungahle kube nomphumela wokwakheka kwama-vertebral ama-osteophytes. Ukunakekelwa kufanele kuthathwe ukungalimazi noma ukucasula ama-disc aseduze. Njengoba ukujula kokungena kokungena kufanele kulawulwe ukuthola imiphumela engaguquki futhi yenziwa kabusha, senza ipulaki ngesandla ngokusika umgodi wenaliti elingu-3 mm. Usebenzisa le plugi kuqinisekisa ukujula kokushayela okufanayo emzimbeni we-vertebral. Ekuhlolweni kokuqala, odokotela abahlinzayo abathathu ababambe iqhaza ekusebenzeni bathola izinaliti ezi-16 ze-Cauge kalula ukusebenza nazo izinaliti ezi-18-geuge noma ezinye izindlela. Ukugwema ukopha ngokweqile ngesikhathi sokumba, ukubamba inaliti kusesikhashana kuzohlinzeka ngomgodi ofanelekile wokufaka, kuphakamisa ukuthi isilinganiso esithile se-MC singalawulwa ngale ndlela.
Yize izifundo eziningi zibhekiswe ku-MC, kuncane ezaziwa nge-Etiology nePathogeneis ye-MC25,26,27. Ngokusekelwe ezifundweni zethu zangaphambili, sathola ukuthi ama-autoimmunity adlala indima ebalulekile ekwenzeni nasekuthuthukisweni kwe-MC12. Lolu cwaningo luhlolisise inkulumo enkulu ye-IL-4, iL-17, kanye ne-Ifn-γ-γ, okuyindlela ehlukene yokuhlukanisa imigwaqo yamaseli we-CD4 + ngemuva kokuvuselelwa kwe-Antigen. Esifundweni sethu, uma kuqhathaniswa neqembu elingelona iqiniso, iqembu le-NPE lalinenkulumo ephezulu ye-IL-4, iL-17, ne-Ifn-, kanye namaphrotheni we-IL-4 ne-IL-17 ayephakeme.
Ngokwenyama, i-IL-17 nkulunkulu ye-MRNA yandiswa kumaseli we-NP avela ezigulini ezine-Disc Herniation28. Amazinga akhuphuke ama-IL-4 ne-IFN-γ atholakala emodeni ye-Disc Herniation engenangqondo enobunzima uma iqhathaniswa nezilawuli ezinempilo29. I-IL-17 idlala indima ebalulekile ekuvuvukeni, ukulimala kwezicubu ezifweni ze-autoimmune. Ukulimala kwezicubu ezithuthukisiwe ze-IL-17 I-IL-4 ingavimba inkulumo yama-cytokines proinfatory (njenge-IL-1 ne-TNFCE) kanye ne-macrophage activation34. Kubikwa ukuthi ukuvezwa kwe-MRNA kwe-IL-4 kwakuhlukile eqenjini le-NPE kuqhathaniswa ne-IL-17 ne-Ifn-γ ngasikhathi sinye; Inkulumo ye-MRNA ye-Ifn-γ eqenjini le-NPE laliphakeme kakhulu kunalokho kwamanye amaqembu. Ngakho-ke, ukukhiqizwa kwe-IFN-γ kungaba ngumlamuli wempendulo yokuvuvukala odonswe yi-NP yokungenisa. Ucwaningo luye lwabonisa ukuthi i-IFN-γ ikhiqizwa yizinhlobo eziningi zeseli, kufaka phakathi amaseli we-Type 1 Hulumende we-T, amaseli wemvelo wokubulala, kanye nama-macrophages35,36, futhi kuyindlela esemqoka ukuvuvukala kwe-cytotas ekhuthaza izimpendulo zokuzivikela komzimba37.
Lolu cwaningo lubonisa ukuthi impendulo ye-autoimmune ingabandakanyeka ekwenzeni nasekuthuthukisweni kwe-MC. Lumama et al. Kutholakale ukuthi izici zesiginali ze-MC kanye ne-NP evelele ziyafana ku-MRI, futhi zombili zibonisa isinali ephezulu ku-T2W Sequence38. Amanye ama-cytokines aqinisekisiwe ukuthi ahlotshaniswe kakhulu nokuvela kwe-MC, njenge-IL-139. Ma et al. Kuphakanyiswe ukuthi ukukhiqizwa okuphezulu noma okuphansi kwe-NP kungahle kube nethonya elikhulu ekwenzeni kanye nokuthuthukiswa kwe-MC12. IBobechko40 neHerzbein et al.41 babike ukuthi i-NP iyisicubu esingenakugwenywa esingakwazi ukungena ekusakazweni kwemithambo kusuka ekuzalweni. I-NP Protorions yethula imizimba yakwamanye amazwe ekunikezelweni kwegazi, ngaleyo ndlela iqondiswe ukuphendulwa kwe-autoimmune yasendaweni42. Ukusabela kwe-autoimmune kungakunxenxa inani elikhulu lezinto zomzimba, futhi lapho lezi zinto ziqhubeka ngokuqhubekayo izicubu, zingadala izinguquko ekusayiniwe43. Kulolu cwaningo, ukucindezelwa kwe-Overexpression ye-IL-4, iL-17 ne-Ifn-γ-γ ziyizinto ezijwayelekile zomzimba, zifakazela ubuhlobo obuseduze phakathi kwe-NP ne-MCS44. Le modeli yezilwane ilingisa kahle i-NP Preaking kanye nokungena epuletini lokuphela. Le nqubo iphinde yaveza umthelela we-autoimmunity ku-MC.
Njengoba kulindeleke, le modeli yezilwane isinikeza ipulatifomu engaba khona ukuze ifunde i-MC. Kodwa-ke, le modeli isenokulinganiselwa okuthile: Okokuqala, ngesikhathi sesigaba sokubheka izilwane, abanye onogwaja abasezingeni eliphakathi kudingeka bahlolisiswe ukuhlolwa kwe-biology kanye ne-moleculate biology, ngakho-ke ezinye izilwane "ziphuma ngokusetshenziswa" ngokuhamba kwesikhathi. Okwesibili, yize amaphuzu amathathu ahlelwe kulolu cwaningo, ngeshwa, sasifanekisela uhlobo olulodwa lwe-MC (uhlobo lwe-Modic engilishintshayo), ngakho-ke akwanele ukumela inqubo yokuthuthukiswa kwezifo komuntu, namaphoyinti wesikhathi esengeziwe adinga ukusethwa kuwo Bheka kangcono zonke izinguquko zesiginali. Okwesithathu, izinguquko ezisesimweni sezicubu zingakhonjiswa ngokucacile ngokubala umlando, kepha ezinye izindlela ezikhethekile zingaveza kangcono ushintsho ezingozini ezingahambisanayo kule modeli. Isibonelo, i-microscopy ekhanyayo ekhanyayo yasetshenziselwa ukuhlaziya ukwakheka kwe-fibrocartilage e-rabitntertebral discs45. Imiphumela yesikhathi eside ye-NP ku-MC ne-ExpPlate idinga ukutadisha okwengeziwe.
Onogwaja abamhlophe baseNew Zealand abanama-50 abeseNew Zealand (Isisindo esingama-2,5-3 kg, aneminyaka engu-3-3,5 ehlukaniswe ngehlukaniswe nge-SHAM Operation Group, iqembu lokufakwa kwemisipha (iqembu le-Irve Root Implantation (NPE). Zonke izinqubo zokuhlola zavunywa yiKomidi Lezimilo zesibhedlela saseTianjin, futhi izindlela zokuhlola zenziwa ngokuqinile ngemihlahlandlela evunyelwe.
Enye intuthuko yenziwe kwinqubo yokuhlinzwa ye-S. Sobamima 46. Unogwaja ngamunye wafakwa endaweni elandelanayo ye-recumbency kanye nendawo engaphandle yama-lumbar inveryertebral discs (Ivds) ivezwe kusetshenziswa indlela yokubuyisa kabusha ye-postereolateral. Unogwaja ngamunye wanikezwa i-General Anesthesia (20% urethane, 5 ml / kg nge-endlebe vein). I-incinasion yesikhumba ende yenziwe evela emaphethelweni aphansi amambambo e-pelvic brim, 2 cm ventral emisipha ye-paravertebral. Umgogodla ofanele we-antelateral ovela ku-L1 kuye ku-L6 evezwa ngokuhlukile okubukhali nokufiphala kwezicubu ezingaphansi ngokweqile, izicubu ze-retropperitoneal, kanye nemisipha (Fig. 6a). Izinga le-disc linqunywe kusetshenziswa i-pelvic brim njengophawu lomhlaba we-anatomical weleveli ye-L5-L6 Disc. Sebenzisa inaliti yokubhoboza engu-16-gege ukuze ushaye umgodi eduze kwepuleti lokuphela le-L5 Vertebra ekujuleni kwe-3 mm (Fig. 6b). Sebenzisa isirinji sesirinji esingu-5 Susa i-nucleus pulpopos noma izicubu ngokuya ngezidingo zeqembu ngalinye. Ngemuva kokuthi umgodi wokudonsa ujule, kufakwa ama-sutures ansundu ku-fascia esijulile, i-fascia evulekile kanye nesikhumba, unakekele ukuthi ungalimazi izicubu zomzimba we-vertebral ngesikhathi sokuhlinzwa.
(A) I-L5-L6 disc ivezwa ngendlela ye-posterelateral retrouperitoneal. (B) Sebenzisa inaliti engu-16-gege yokumba umgodi eduze kwe-L5 endPlate. (C) Ama-MFs azenzakalelayo avunwa.
I-General Anesthesia yanikezwa ngo-20% urethane (5 ml / kg) olawulwa ngethambo lendlebe, futhi ama-radine we-lumbar spine aphindwa ku-12, 16, namaviki angama-20 eposi.
Onogwaja bahlatshwa umhlatshelo we-intramuscular ye-ketamine (25.0 mg / kg) kanye ne-intravenous sodium pentobarbital (1,2 g / kg) kumaviki ayi-12, 16 kanye nama-20 ngemuva kokuhlinzwa. Umgogodla wonke wasuswa ekuhlaziyweni komlando nokuhlaziywa kwangempela. Ukubhalwa Okubukeko Okuningi (i-RT-QPCR) kanye nokugcwala kwentshonalanga kwakusetshenziselwa ukuthola izinguquko ezintweni zokuzivikela komzimba.
Ukuhlolwa kwe-MRI kwenziwa enogwaja kusetshenziswa inqwaba ye-3.0 t Clinical (izinhlelo zezokwelapha, iFlorence, SC) ifakwe isithonjana se-Orthogonal Limb Coil. Onogwaja babuhlungu abane-20% i-urethane (5 ml / kg) nge-endlebe vein bese bebekwa amandla phakathi kwezilwane ezibucayi abanezindawo eziyizindilinga ze-lumbar cendile (GE Medical Systems). Izithombe zeCoronal T2-Siedied Localizer (tr, 1445 ms; te, 37 ms) zithathwe ukuchaza indawo ye-lumbar disc kusuka ku-L3-L4 kuya ku-L5-L6. I-Sagittal Plane T2-Sied Slices etholwe ngezilungiselelo ezilandelayo: Ukulandelana okusheshayo kwe-spin-echo nge-Repetition Isikhathi (TR) ka-2200 ms kanye nesikhathi esingu-70 MS, Matrix; Insimu ebonakalayo engama-260 nesishiyagalombili ashukumisayo; Ubukhulu bokusika kwakungu-2 mm, igebe lalingu-0.2 mm.
Ngemuva kokuthi kuthathwe isithombe sokugcina kanti unogwaja wokugcina wabulawa, i-sham, ama-misches ashunyezwe izicubu, kanye ne-NP ama-disc asuswe ekuhlolweni komlando. Izicubu zazilungisiwe ku-10% ezingathathi hlangothi Amabhulokhi wezicubu afakwe kuphalafini futhi asikwe ezingxenyeni ze-sagittal (5 μm ubukhulu) usebenzisa i-microtome. Izingxenye zazinqunyelwe nge-hematoxylin ne-eosin (H & E).
Ngemuva kokuqoqa ama-discs wokungenelela kusuka onogwaja ngamunye eqenjini ngalinye, i-RNA ephelele yayikhishwe kusetshenziswa ikholomu ye-UniQ-10 (iShanghai Sango biotechnology Co, Ltd. , Madison, Wi, USA). Ukubuyela emuva kwenziwa.
I-RT-QPCR yenziwa kusetshenziswa i-prism 7300 (i-appled biosystems Inc., e-USA) ne-SYBR GREEN JUMP Start Taq Readlix (Sigma-Aldrich, St. I-USA) ngokwemiyalo yomenzi. Ivolumu yokuphendula ye-PCR yayiyi-20 μL futhi iqukethe 1.5 μl ye-CDNA ehlanganisiwe kanye ne-0.2 μm ye-Primer ngayinye. Ama-primers aklanywe ngu-Oligoperfect Designer (i-Invitrogen, Valencia, CA) futhi akhiqizwa yi-Nanjing Golden Stewart Biotechnology Co, Ltd. (China) (China Izimo ezilandelayo zokuhamba ngebhayisikile zisetshenzisiwe: Isinyathelo sokuqala se-polymerase activation isinyathelo ngo-94 ° C nge-2 min, bese kuthi imijikelezo engu-40 yangama-20 s nge-104 ° C ye-template denaturation, ukunxusa amaminithi ayi-60 ° C, ukunwetshwa, kanye ne-fluorescence. Izilinganiso zenziwa nge-1 min ku-72 ° C. Onke amasampula akhuliswa kathathu futhi inani elijwayelekile lalisetshenziselwa ukuhlaziya i-RT-QPCR. Idatha ye-Amplifashini yahlaziywa kusetshenziswa i-FlexStation 3 (amadivaysi ama-molecular, i-Sunnyvale, CA, USA). I-IL-4, i-IL-17, kanye ne-Ifn-γ γ γ γ γ γ γ γ γ γ γ γ γ uhlolo lwazo lalihlelwa ngokulawulwa okungapheli (i-ACTB). Amazinga wokubonisa okuhlobene kwelitshe mmna abalwa kusetshenziswa indlela engu-2-Δctct.
Amaprotheni aphelele akhishwe ezicubu ezisebenzisa i-homogenizer yezicubu eRipA lyrsis buffer (equkethe i-protease kanye ne-photoshifuse inhibitor i-cocktail) bese ekhipha i-ngrifug Ama-microgram angama-50 amaprotheni alayishwa ngomzila ngamunye, ahlukaniswe nge-10% ntS-ikhasi, bese adluliselwa kulwelwesi lwe-PVDF. Ukuvinjwa kwenziwa ngobisi olungu-5% olomile ubisi e-Tris-buffered saline (TBS) equkethe u-0.1% phakathi kwama-20 nge-1 h ekushiseni kwegumbi. Ulwelwelwe ulwelwesi lwaluhlaselwa ngonogwaja anti-decerin anti-primary anti-(discon; Nge-antibogy ye-Immunoglobulin yaseSekondari (I-Goat anti-Rabbit Immunoglobulin g ngo-1: 40,000 dilution) ihlanganiswe ne-horseradish peroxidase (groxidase (Goster, iWuhan, China) Ihora eli-1. Amasiginali we-Western Blot atholwe ngokunwetshwa kwe-chemiluminescence ku-membrane ye-chemilominescent ngemuva kwe-x-ray irradiation. Ngokuhlaziywa kwe-densitometric, ama-blots askenwa futhi ahlelwe kusetshenziswa isoftware ye-bandscan kanti imiphumela yavezwa njengesilinganiso sethagethi yohlobo lwethagethi ye-Immunororiaction.
Izibalo zezibalo zenziwa kusetshenziswa iphakethe lesoftware ye-SPSS16.0 (SPSS, USA). Idatha eqoqwe ngesikhathi socwaningo ivezwe njenge-HOST ± ukuphambuka okujwayelekile (kusho ± SD) bese kuhlaziywa kusetshenziswa izindlela eziphindaphindwayo eziphindaphindwayo (ANOVA) ukuthola umehluko phakathi kwamaqembu amabili. P <0.05 kubhekwa njengento ebalulekile ngokwezibalo.
Ngakho-ke, ukusungulwa kwemodeli yezilwane ye-MC ngokufaka i-autololous emzimbeni we-vertebral futhi wenze ukubonwa kwe-macroanatatomical, ukuhlaziya kwe-MRI, ukuhlolwa kwamangqamuzana, ukuhlaziya kwamangqamuzana kungaba yithuluzi elibalulekile lokuhlola abantu ukungenelela.
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