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Ukusekwa kweemodeli zezilwanyana zotshintsho lwe-modic (MC) sisiseko esibalulekileyo sokufunda i-MC. Iimvundla ezimhlophe ezingamashumi amahlanu anesine zaseNew Zealand zahlulwe zaba liqela le-sham-operation, iqela le-muscle implantation (ME group) kunye neqela le-nucleus pulposus implantation (NPE group). Kwiqela le-NPE, i-intervertebral disc yavezwa ngendlela yotyando lwe-anterolateral lumbar kwaye kwasetyenziswa inaliti ukubhoboza umzimba we-vertebral we-L5 kufutshane nepleyiti yokugqibela. I-NP yakhutshwa kwi-disc ye-intervertebral ye-L1/2 ngesirinji yaza yafakwa kuyo. Ukubhoboza umngxuma kwithambo le-subchondral. Iinkqubo zotyando kunye neendlela zokubhoboza kwiqela le-muscle implantation kunye neqela le-sham-operation zazifana nezo zikwiqela le-NP implantation. Kwiqela le-ME, isiqwenga semisipha safakwa emngxunyeni, ngelixa kwiqela le-sham-operation, akukho nto yafakwa emngxunyeni. Emva kotyando, kwenziwa i-MRI scanning kunye novavanyo lwe-molecular biological. Isignali kwiqela le-NPE yatshintsha, kodwa akukho tshintsho lucacileyo lwesignali kwiqela le-sham-operation kunye neqela le-ME. Uphononongo lwe-histological lubonise ukuba ukwanda kwezicubu okungaqhelekanga kubonwe kwindawo yokufakelwa, kwaye ukubonakaliswa kwe-IL-4, IL-17 kunye ne-IFN-γ kwandisiwe kwiqela le-NPE. Ukufakelwa kwe-NP kwithambo le-subchondral kunokwenza imodeli yesilwanyana ye-MC.
Utshintsho lweModic (MC) zizilonda zeepleyiti ze-vertebral endplates kunye ne-bone marrow ekufutshane ebonakala kwi-magnetic resonance imaging (MRI). Ziqhelekile kakhulu kubantu abaneempawu ezinxulumene nazo1. Izifundo ezininzi zigxininise ukubaluleka kwe-MC ngenxa yokunxibelelana kwayo nentlungu esezantsi yomqolo (LBP)2,3. u-de Roos et al.4 kunye no-Modic et al.5 ngokuzimeleyo baqale bachaza iintlobo ezintathu ezahlukeneyo ze-subchondral signal abnormalities kwi-vertebral bone marrow. Utshintsho lwe-Modic type I luyi-hypointense kwi-T1-weighted (T1W) sequences kunye ne-hyperintense kwi-T2-weighted (T2W) sequences. Olu tshintsho lubonisa ii-fissure endplates kunye ne-vascular granulation tissue ekufutshane kwi-bone marrow. Utshintsho lwe-Modic type II lubonisa uphawu oluphezulu kuzo zombini ii-T1W kunye ne-T2W sequences. Kolu hlobo lwesilonda, ukutshatyalaliswa kwe-endplate kunokufumaneka, kunye nokutshintshwa kwamafutha e-histological kwi-bone marrow ekufutshane. Utshintsho lwe-Modic type III lubonisa uphawu oluphantsi kwi-T1W kunye ne-T2W sequences. Izilonda ze-Sclerotic ezihambelana nee-endplates zibonwe6. I-MC ithathwa njengesifo esibangela izifo zomqolo kwaye inxulunyaniswa ngokusondeleyo nezifo ezininzi eziwohlokayo zomqolo7,8,9.
Ukuqwalasela idatha ekhoyo, izifundo ezininzi zibonelele ngolwazi oluneenkcukacha malunga ne-etiology kunye neendlela ze-pathological ze-MC. U-Albert et al. bacebisa ukuba i-MC inokubangelwa yi-disc herniation8. U-Hu et al. bathi i-MC ibangelwa kukonakala okukhulu kwediski10. UKroc ucebise ingcamango "yokuqhekeka kwediski yangaphakathi," ethi ukwenzakala okuphindaphindiweyo kwediski kunokukhokelela kwiincindi kwi-endplate. Emva kokwakheka kwe-cleft, ukutshatyalaliswa kwe-endplate yi-nucleus pulposus (NP) kunokubangela impendulo ye-autoimmune, ekhokelela ekuphuhlisweni kwe-MC11. U-Ma et al. babenembono efanayo kwaye baxela ukuba i-NP-induced autoimmune idlala indima ebalulekileyo kwi-pathogenesis ye-MC12.
Iiseli zenkqubo yomzimba yokuzikhusela, ngakumbi ii-CD4+ T helper lymphocytes, zidlala indima ebalulekileyo kwi-pathogenesis ye-autoimmune13. I-Th17 subset efunyenwe kutshanje ivelisa i-cytokine IL-17 ebangela ukuvuvukala, ikhuthaza ukubonakaliswa kwe-chemokine, kwaye ivuselela iiseli ze-T kwizitho ezonakeleyo ukuba zivelise i-IFN-γ14. Iiseli ze-Th2 zikwadlala indima ekhethekileyo kwi-pathogenesis yeempendulo zomzimba. Ukubonakaliswa kwe-IL-4 njengeseli ye-Th2 emeleyo kunokukhokelela kwimiphumo emibi ye-immunopathological15.
Nangona izifundo ezininzi zeklinikhi zenziwe kwi-MC16,17,18,19,20,21,22,23,24, kusekho ukunqongophala kweemodeli ezifanelekileyo zovavanyo lwezilwanyana ezinokulinganisa inkqubo ye-MC eyenzeka rhoqo ebantwini kwaye zinokusetyenziselwa ukuphanda imvelaphi okanye unyango olutsha olufana nonyango olujoliswe kulo. Ukuza kuthi ga ngoku, zimbalwa kuphela iimodeli zezilwanyana ze-MC eziye zaxelwa ukuba zifunde iindlela ezisisiseko zesifo.
Ngokusekelwe kwithiyori yokuzikhusela komzimba ecetyiswe nguAlbert noMa, olu phononongo luseke imodeli elula nephinda ivele ye-MC yomvundla ngokuyitshintsha ngokuzenzekelayo i-NP kufutshane nepleyiti yokugqibela yomqolo eyombiweyo. Ezinye iinjongo kukujonga iimpawu ze-histological zeemodeli zezilwanyana kunye nokuvavanya iindlela ezithile ze-NP ekuphuhlisweni kwe-MC. Ngenxa yesi sizathu, sisebenzisa iindlela ezifana ne-molecular biology, i-MRI, kunye nezifundo ze-histological ukufunda inkqubela phambili ye-MC.
Iimvundla ezimbini zafa ngenxa yokopha ngexesha lotyando, kwaye ezine zafa ngexesha lotyando lwe-MRI. Ezinye iimvundla ezingama-48 zasinda kwaye azizange zibonise zimpawu zokuziphatha okanye ze-neurological emva kotyando.
I-MRI ibonisa ukuba amandla esignali yezicubu ezifakwe kwimingxunya eyahlukeneyo ahlukile. Amandla esignali yomzimba we-L5 vertebral kwiqela le-NPE atshintsha kancinci kwiiveki ezili-12, ezili-16 nezingama-20 emva kokufakwa (ulandelelwano lwe-T1W lubonise uphawu oluphantsi, kwaye ulandelelwano lwe-T2W lubonise uphawu oluxutyiweyo kunye nophawu oluphantsi) (Umzobo 1C), ngelixa ukubonakala kwe-MRI kwamanye amaqela amabini eendawo ezifakwe kwi-intanethi kwahlala kuzinzile ngexesha elifanayo (Umzobo 1A, B).
(A) Ii-MRI ezilandelelanayo ezimele umqolo we-lumbar spine kwiindawo ezi-3. Akukho zimpawu ezingaqhelekanga ezifunyenweyo kwimifanekiso yeqela le-sham-operation. (B) Iimpawu zesignali yomzimba we-vertebral kwiqela le-ME ziyafana nezo zikwiqela le-sham-operation, kwaye akukho tshintsho lubalulekileyo lwesignali olubonwa kwindawo yokufaka ngokuhamba kwexesha. (C) Kwiqela le-NPE, isignali ephantsi ibonakala ngokucacileyo kulandelelwano lwe-T1W, kwaye isignali exutyiweyo kunye nesignali ephantsi ibonakala ngokucacileyo kulandelelwano lwe-T2W. Ukususela kwixesha leeveki ezili-12 ukuya kwixesha leeveki ezingama-20, imiqondiso ephezulu ejikelezayo yesignali ephantsi kulandelelwano lwe-T2W iyancipha.
I-bone hyperplasia ecacileyo inokubonwa kwindawo yokufakelwa komzimba we-vertebral kwiqela le-NPE, kwaye i-bone hyperplasia yenzeka ngokukhawuleza ukusuka kwiiveki ezili-12 ukuya kwezingama-20 (Umzobo 2C) xa kuthelekiswa neqela le-NPE, akukho tshintsho lubalulekileyo lubonwa kwimizimba ye-vertebral emodeliweyo; Iqela le-Sham kunye neqela le-ME (Umzobo 2C) 2A,B).
(A) Umphezulu womzimba wethambo lomqolo kwindawo efakelweyo uthambile kakhulu, umngxuma uphola kakuhle, kwaye akukho hyperplasia emzimbeni wethambo lomqolo. (B) Imilo yendawo efakelweyo kwiqela le-ME ifana naleyo ikwiqela lokusebenza kwe-sham, kwaye akukho tshintsho lucacileyo kwimbonakalo yendawo efakelweyo ngokuhamba kwexesha. (C) I-Bone hyperplasia yenzeka kwindawo efakelweyo kwiqela le-NPE. I-bone hyperplasia yanda ngokukhawuleza yaze yanwenwela kwi-intervertebral disc ukuya kumzimba wethambo lomqolo ochaseneyo.
Uhlalutyo lwe-Histological lubonelela ngolwazi oluthe kratya malunga nokwakheka kwamathambo. Umfanekiso 3 ubonisa iifoto zamacandelo emva kotyando anombala we-H&E. Kwiqela le-sham-operation, ii-chondrocytes zazicwangciswe kakuhle kwaye akukho ukwanda kweeseli okufunyenweyo (Umzobo 3A). Imeko kwiqela le-ME yayifana naleyo kwiqela le-sham-operation (Umzobo 3B). Nangona kunjalo, kwiqela le-NPE, inani elikhulu lee-chondrocytes kunye nokwanda kweeseli ezifana ne-NP kwabonwa kwindawo yokufakelwa (Umzobo 3C);
(A) IiTrabeculae zinokubonwa kufutshane nepleyiti yokugqibela, ii-chondrocytes zicwangciswe kakuhle ngobukhulu kunye nemilo yeeseli ezifanayo kwaye akukho ukwanda (amaxesha angama-40). (B) Imeko yendawo yokufaka kwi-ME group ifana neyeqela le-fake. IiTrabeculae kunye nee-chondrocytes zinokubonwa, kodwa akukho ukwanda okucacileyo kwindawo yokufaka kwi-implantation (amaxesha angama-40). (B) Kuyabonakala ukuba ii-chondrocytes kunye neeseli ezifana ne-NP ziyanda kakhulu, kwaye imo kunye nobukhulu bee-chondrocytes azilingani (amaxesha angama-40).
Ukubonakaliswa kwe-interleukin 4 (IL-4) mRNA, i-interleukin 17 (IL-17) mRNA, kunye ne-interferon γ (IFN-γ) mRNA kubonwe kumaqela e-NPE kunye ne-ME. Xa amanqanaba okubonakaliswa kwezakhi zofuzo ekujoliswe kuzo ethelekiswa, ukubonakaliswa kwezakhi zofuzo ze-IL-4, IL-17, kunye ne-IFN-γ kwandiswe kakhulu kwiqela le-NPE xa kuthelekiswa nalawo eqela le-ME kunye neqela lokusebenza kwe-sham (Umzobo 4) (P < 0.05). Xa kuthelekiswa neqela lokusebenza kwe-sham, amanqanaba okubonakaliswa kwe-IL-4, IL-17, kunye ne-IFN-γ kwiqela le-ME anyuke kancinci kwaye awazange afikelele kutshintsho lwezibalo (P > 0.05).
Ukubonakaliswa kwe-mRNA kwe-IL-4, IL-17 kunye ne-IFN-γ kwiqela le-NPE kubonise umkhwa ophezulu kakhulu kunabo bakwiqela lokusebenza ngobuxoki kunye neqela le-ME (P < 0.05).
Ngokwahlukileyo koko, amanqanaba okubonakaliswa kwiqela le-ME awabonisanga mahluko abalulekileyo (P>0.05).
Uhlalutyo lwe-Western blot lwenziwe kusetyenziswa ii-antibodies ezithengiswayo nxamnye ne-IL-4 kunye ne-IL-17 ukuqinisekisa ipatheni yokubonakaliswa kwe-mRNA etshintshileyo. Njengoko kubonisiwe kwiMifanekiso 5A,B, xa kuthelekiswa neqela le-ME kunye neqela lokusebenza kwe-sham, amanqanaba eeproteni ze-IL-4 kunye ne-IL-17 kwiqela le-NPE anyuswe kakhulu (P < 0.05). Xa kuthelekiswa neqela lokusebenza kwe-sham, amanqanaba eeproteni ze-IL-4 kunye ne-IL-17 kwiqela le-ME nawo asilele ukufikelela kutshintsho olubalulekileyo ngokwezibalo (P> 0.05).
(A) Amanqanaba eproteni ye-IL-4 kunye ne-IL-17 kwiqela le-NPE ayephezulu kakhulu kunalawo akwiqela le-ME kunye neqela le-placebo (P < 0.05). (B) I-Western blot histogram.
Ngenxa yenani elincinci leesampuli zabantu ezifunyenwe ngexesha lotyando, izifundo ezicacileyo nezineenkcukacha malunga ne-pathogenesis ye-MC zinzima kancinci. Sizame ukuseka imodeli yesilwanyana ye-MC ukuze sifunde iindlela zayo ezinokubakho ze-pathological. Kwangaxeshanye, uvavanyo lwe-radiological, uvavanyo lwe-histological kunye novavanyo lwe-molecular biological zisetyenzisiwe ukulandela ikhondo le-MC elibangelwa yi-NP autograft. Ngenxa yoko, imodeli yokufakelwa kwe-NP ibangele utshintsho oluncinci kubunzulu besignali ukusuka kwiiveki ezili-12 ukuya kwiiveki ezingama-20 (isignali ephantsi exutyiweyo kwi-T1W sequences kunye nesignali ephantsi kwi-T2W sequences), ebonisa utshintsho kwizicubu, kwaye uvavanyo lwe-histological kunye ne-molecular biological luqinisekisile iziphumo zophando lwe-radiological.
Iziphumo zolu vavanyo zibonisa ukuba utshintsho olubonakalayo nolwe-histological lwenzeka kwindawo apho umzimba we-vertebral uphazamiseke khona kwiqela le-NPE. Kwangaxeshanye, ukubonakaliswa kwe-IL-4, IL-17 kunye ne-IFN-γ genes, kunye ne-IL-4, IL-17 kunye ne-IFN-γ kwabonwa, okubonisa ukuba ukwaphulwa kwe-autologous nucleus pulposus tissue emzimbeni we-vertebral kunokubangela uthotho lweempawu kunye notshintsho lwe-morphological. Kulula ukufumanisa ukuba iimpawu zezibonakaliso zemizimba ye-vertebral yemodeli yesilwanyana (uphawu oluphantsi kulandelelwano lwe-T1W, uphawu oluxutyiweyo kunye nophawu oluphantsi kulandelelwano lwe-T2W) zifana kakhulu nezo zeeseli ze-vertebral zabantu, kwaye iimpawu ze-MRI nazo ziqinisekisa ukubonwa kwe-histology kunye ne-gross anatomy, oko kukuthi, utshintsho kwiiseli zomzimba we-vertebral luyaqhubeka. Nangona impendulo yokuvuvukala ebangelwa kukwenzakala okukhulu inokuvela kungekudala emva kokubhoboza, iziphumo ze-MRI zibonise ukuba utshintsho lwezibonakaliso olukhula ngokuqhubekayo luvele kwiiveki ezili-12 emva kokubhoboza kwaye lwaqhubeka ukuya kwiiveki ezingama-20 ngaphandle kweempawu zokubuyela kwimeko yesiqhelo okanye ukuguquka kotshintsho lwe-MRI. Ezi ziphumo zibonisa ukuba i-autologous vertebral NP yindlela ethembekileyo yokuseka i-MV eqhubekayo kwimivundla.
Le modeli yokubhoboza ifuna ubuchule obaneleyo, ixesha, kunye nomgudu wotyando. Kwizilingo zokuqala, ukuqhaqha okanye ukuvuselela kakhulu izakhiwo ze-paravertebral ligamentous kunokubangela ukwakheka kwe-vertebral osteophytes. Kufuneka kuthathwe ingqalelo ukuba kungonakalisa okanye kuphazamise iidiski ezikufutshane. Ekubeni ubunzulu bokungena kufuneka bulawulwe ukuze kufunyanwe iziphumo ezihambelanayo neziphindaphindayo, senze iplagi ngesandla ngokusika isikhongozeli senaliti ende eyi-3 mm. Ukusebenzisa le plagi kuqinisekisa ubunzulu bokubhoboza obufanayo emzimbeni we-vertebral. Kwizilingo zokuqala, oogqirha abathathu bamathambo ababandakanyekayo kolu tyando bafumanise ukuba kulula ukusebenza ngeenaliti ze-16-gauge kuneenaliti ze-18-gauge okanye ezinye iindlela. Ukuze kuthintelwe ukopha kakhulu ngexesha lokubhoboza, ukubamba inaliti inganyakazi okwethutyana kuya kubonelela ngomngxuma ofanelekileyo wokufaka, nto leyo ebonisa ukuba inqanaba elithile le-MC linokulawulwa ngale ndlela.
Nangona izifundo ezininzi zijolise kwi-MC, kuncinci okwaziwayo malunga ne-etiology kunye ne-pathogenesis ye-MC25,26,27. Ngokusekelwe kwizifundo zethu zangaphambili, sifumanise ukuba ukuzikhusela komzimba kudlala indima ebalulekileyo ekudalweni nasekuphuhlisweni kwe-MC12. Olu phononongo luhlolisise ukubonakaliswa kobuninzi kwe-IL-4, IL-17, kunye ne-IFN-γ, ezizezona ndlela ziphambili zokwahlulahlula iiseli ze-CD4+ emva kokukhuthazwa kwe-antigen. Kuphononongo lwethu, xa kuthelekiswa neqela elingalunganga, iqela le-NPE lalinokubonakaliswa okuphezulu kwe-IL-4, IL-17, kunye ne-IFN-γ, kwaye amanqanaba eeprotheyini ze-IL-4 kunye ne-IL-17 nawo ayephezulu.
Ngokwezonyango, ukubonakaliswa kwe-mRNA ye-IL-17 kuyanda kwiiseli ze-NP ezivela kwizigulana ezine-disc herniation28. Ukwanda kwamanqanaba okubonakaliswa kwe-IL-4 kunye ne-IFN-γ kufunyenwe nakwimodeli ye-disc herniation engeyiyo ecinezelweyo xa kuthelekiswa nolawulo oluphilileyo29. I-IL-17 idlala indima ebalulekileyo ekuvuvukeni, ukwenzakala kwezicubu kwizifo zokuzikhusela komzimba30 kwaye iphucula impendulo yomzimba kwi-IFN-γ31. Ukwanda kokulimala kwezicubu okubangelwa yi-IL-17 kuye kwaxelwa kwi-MRL/lpr mice32 kunye neempuku ezichaphazeleka yi-autoimmune33. I-IL-4 inokuthintela ukubonakaliswa kwee-cytokines ezibangela ukuvuvukala (ezifana ne-IL-1β kunye ne-TNFα) kunye nokusebenza kwe-macrophage34. Kuxelwe ukuba ukubonakaliswa kwe-mRNA kwe-IL-4 kwahlukile kwiqela le-NPE xa kuthelekiswa ne-IL-17 kunye ne-IFN-γ ngexesha elinye; Ukubonakaliswa kwe-mRNA kwe-IFN-γ kwiqela le-NPE kwakuphezulu kakhulu kunakwamanye amaqela. Ke ngoko, ukuveliswa kwe-IFN-γ kunokuba ngumlamli wempendulo yokuvuvukala ebangelwa yi-NP intercalation. Izifundo zibonise ukuba i-IFN-γ iveliswa ziintlobo ezininzi zeeseli, kuquka ii-activated type 1 helper T cells, natural killer cells, kunye nee-macrophages35,36, kwaye yi-cytokine ephambili ekhuthaza ukuvuvukala ekhuthaza iimpendulo zomzimba37.
Olu phononongo lubonisa ukuba impendulo ye-autoimmune inokuba negalelo ekwenzekeni nasekuphuhlisweni kwe-MC. ULuoma nabanye bafumanise ukuba iimpawu zesignali ye-MC kunye ne-NP ebalaseleyo ziyafana kwi-MRI, kwaye zombini zibonisa uphawu oluphezulu kulandelelwano lwe-T2W38. Ezinye ii-cytokines ziqinisekisiwe ukuba zinxulumene ngokusondeleyo nokwenzeka kwe-MC, njenge-IL-139. UMa nabanye bacebisa ukuba ukunyuka okanye ukwehla kwe-NP kunokuba nefuthe elikhulu ekwenzekeni nasekuphuhlisweni kwe-MC12. UBobechko40 kunye noHerzbein nabanye41 baxele ukuba i-NP sisicwili esinganyamezeliyo kwi-immunosimplicity esingenakungena kwi-vascular circulation ukususela ekuzalweni. Ii-NP protrusions zingenisa imizimba yangaphandle kwi-blood supply, ngaloo ndlela zilawula iimpendulo ze-autoimmune zasekuhlaleni42. Iimpendulo ze-Autoimmune zinokubangela inani elikhulu lezinto zokuzikhusela, kwaye xa ezi zinto ziqhubeka zibonakaliswa kwizicubu, zinokubangela utshintsho kwi-signaling43. Kolu phononongo, ukuvezwa kakhulu kwe-IL-4, IL-17 kunye ne-IFN-γ zizinto eziqhelekileyo zokuzikhusela, nto leyo ebonisa ngakumbi ubudlelwane obusondeleyo phakathi kwe-NP kunye ne-MC44. Le modeli yesilwanyana ixelisa kakuhle ukuphuphuma kwe-NP kunye nokungena kwipleyiti yokugqibela. Le nkqubo ikwatyhile impembelelo yokuzikhusela komzimba kwi-MC.
Njengoko bekulindelwe, le modeli yesilwanyana isinika iqonga elinokwenzeka lokufunda i-MC. Nangona kunjalo, le modeli isenemida ethile: okokuqala, ngexesha lesigaba sokujonga izilwanyana, ezinye iimbila ezikwinqanaba eliphakathi kufuneka zibulawe ukuze kuvavanywe i-histological kunye ne-molecular biology, ngoko ke ezinye izilwanyana "ziyayeka ukusetyenziswa" ngokuhamba kwexesha. Okwesibini, nangona amanqaku amathathu exesha ebekwe kolu phononongo, ngelishwa, senze imodeli yohlobo olunye lwe-MC (utshintsho lohlobo lwe-Modic I), ngoko ke akwanelanga ukumela inkqubo yophuhliso lwesifo somntu, kwaye amanqaku amaninzi exesha kufuneka asetwe ukuze kubonwe ngcono zonke utshintsho lwesignali. Okwesithathu, utshintsho kwisakhiwo sezicubu lunokubonakaliswa ngokucacileyo yi-histological staining, kodwa ezinye iindlela ezikhethekileyo zinokutyhila ngcono utshintsho lwe-microstructural kule modeli. Umzekelo, i-polarized light microscopy yasetyenziswa ukuhlalutya ukwakheka kwe-fibrocartilage kwi-rabbit intervertebral discs45. Iziphumo zexesha elide ze-NP kwi-MC nakwi-endplate zifuna uphando olongezelelweyo.
Iinkunzi ezingamashumi amahlanu anesine zemivundla emhlophe yaseNew Zealand (ezinobunzima obumalunga ne-2.5-3 kg, ezineminyaka eyi-3-3.5 ubudala) zahlulwe ngokungacwangciswanga zaba liqela lotyando lwenkohliso, iqela lokufakelwa kwemisipha (iqela le-ME) kunye neqela lokufakelwa kweengcambu zemithambo-luvo (iqela le-NPE). Zonke iinkqubo zovavanyo zamkelwe yiKomiti yoBulungisa yeSibhedlele saseTianjin, kwaye iindlela zovavanyo zenziwa ngokuhambelana nezikhokelo ezivunyiweyo.
Kuye kwenziwa uphuculo oluthile kwindlela yotyando lwe-S. Sobajima 46. Umvundla ngamnye wabekwa kwindawo yokulala ecaleni kwaye umphezulu wangaphambili weediski ezintlanu ezilandelelanayo ze-lumbar intervertebral discs (IVDs) wavezwa kusetyenziswa indlela ye-posterolateral retroperitoneal. Umvundla ngamnye wanikwa i-anesthesia jikelele (20% urethane, 5 ml/kg nge-vein yendlebe). Kwenziwe ukusika ulusu olude ukusuka kumphetho ongezantsi weembambo ukuya kumphetho we-pelvic, i-2 cm ventral ukuya kwimisipha ye-paravertebral. Umqolo wasekunene we-anterolateral ukusuka kwi-L1 ukuya kwi-L6 wavezwa ngokuqhekeka okubukhali nokungacacanga kwezicubu ezingaphantsi komhlaba eziphezu, izicubu ze-retroperitoneal, kunye nemisipha (Umzobo 6A). Inqanaba le-disc lamiselwa kusetyenziswa umphetho we-pelvic njengophawu lwe-anatomical kwinqanaba le-disc le-L5-L6. Sebenzisa inaliti yokubhoboza eyi-16-gauge ukubhoboza umngxuma kufutshane nepleyiti yokugqibela ye-vertebra ye-L5 ukuya kubunzulu be-3 mm (Umzobo 6B). Sebenzisa isirinji ye-5-ml ukufutha i-autologous nucleus pulposus kwi-L1-L2 intervertebral disc (Umzobo 6C). Susa i-nucleus pulposus okanye umsipha ngokweemfuno zeqela ngalinye. Emva kokuba umngxuma wokubhoboza unzulu, imithungo efunxwayo ibekwa kwi-deep fascia, i-superficial fascia kunye nolusu, uqaphela ukuba ungonakalisi izicubu ze-periosteal zomzimba womqolo ngexesha lotyando.
(A) Idiski ye-L5–L6 ivezwa ngendlela ye-posterolateral retroperitoneal. (B) Sebenzisa inaliti ye-16-gauge ukubhoboza umngxuma kufutshane ne-endplate ye-L5. (C) Ii-Autologous MFs ziyavunwa.
I-anesthesia eqhelekileyo yenziwe nge-20% ye-urethane (5 ml/kg) enikwe ngemithambo yendlebe, kwaye ii-radiographs zomqolo we-lumbar zaphindwa kwiiveki ezili-12, ezili-16, nezingama-20 emva kotyando.
Iimbila zanikelwa ngenaliti ye-ketamine (25.0 mg/kg) kunye ne-sodium pentobarbital (1.2 g/kg) kwi-intravenous weeks ezili-12, ezili-16 kunye nezingama-20 emva kotyando. Umqolo wonke wasuswa ukuze kuhlalutywe i-histological kwaye kwenziwa uhlalutyo lokwenyani. I-Quantitative reverse transcription (RT-qPCR) kunye ne-Western blotting zasetyenziswa ukubona utshintsho kwi-immune factors.
Uvavanyo lwe-MRI lwenziwe kwiimvundla kusetyenziswa i-3.0 T clinical magnet (GE Medical Systems, Florence, SC) exhotyiswe nge-orthogonal limb coil receiver. Iimvundla zafakwa i-anesthesia nge-20% urethane (5 mL/kg) nge-ear vein zaza zabekwa phantsi ngaphakathi kwi-magnet apho ummandla we-lumbar ugxile kwi-5-intshi ububanzi be-circular surface coil (GE Medical Systems). Imifanekiso ye-Coronal T2-weighted localizer (TR, 1445 ms; TE, 37 ms) yafunyanwa ukuchaza indawo ye-lumbar disc ukusuka kwi-L3–L4 ukuya kwi-L5–L6. Izilayi ze-Sagittal plane T2-weighted zafunyanwa ngezi zicwangciso zilandelayo: ulandelelwano olukhawulezayo lwe-spin-echo kunye nexesha lokuphindaphinda (TR) le-2200 ms kunye nexesha le-echo (TE) le-70 ms, i-matrix; intsimi ebonakalayo ye-260 kunye ne-stimuli ezisibhozo; Ubukhulu bokusika babuyi-2 mm, isithuba sasiyi-0.2 mm.
Emva kokuba kuthathwe ifoto yokugqibela kwaye kubulawe umvundla wokugqibela, iidiski zesham, ezifakwe kwimisipha, kunye ne-NP zasuswa ukuze kuhlolwe i-histological. Iitissues zafakwa kwi-formalin engathathi hlangothi eyi-10% kangangeveki e-1, zasuswa i-calcification nge-ethylenediaminetetraacetic acid, zaza zahlulwahlulwa ngeparafini. Iibhloko zetissue zafakwa kwiparafini zaza zanqunyulwa zaba ngamacandelo e-sagittal (5 μm ubukhulu) kusetyenziswa i-microtome. Iinxalenye zafakwa i-hematoxylin kunye ne-eosin (H&E).
Emva kokuqokelela iidiski ze-intervertebral kwiimbila kwiqela ngalinye, i-RNA iyonke yakhutshwa kusetyenziswa ikholamu ye-UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., eTshayina) ngokwemiyalelo yomenzi kunye nenkqubo ye-ImProm II reverse transcription (Promega Inc., eMadison, eWI, e-USA). Kwenziwe i-reverse transcription.
I-RT-qPCR yenziwe kusetyenziswa iPrism 7300 (Applied Biosystems Inc., USA) kunye neSYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) ngokwemiyalelo yomenzi. Umthamo wempendulo ye-PCR yayiyi-20 μl kwaye yayine-1.5 μl ye-cDNA exutyiweyo kunye ne-0.2 μM yeprimer nganye. Iiprimer zenziwe yi-OligoPerfect Designer (Invitrogen, Valencia, CA) kwaye zenziwe yiNanjing Golden Stewart Biotechnology Co., Ltd. (China) (Itheyibhile 1). Ezi meko zilandelayo zokujikeleza kobushushu zisetyenzisiwe: inyathelo lokuqala lokuqalisa i-polymerase kwi-94°C imizuzu emi-2, emva koko imijikelo engama-40 yemizuzwana eli-15 nganye kwi-94°C yokutshintsha itemplate, ukongeza umzuzu o-1 kwi-60°C, ulwandiso, kunye ne-fluorescence. ukulinganisa kwenziwe umzuzu o-1 kwi-72°C. Zonke iisampulu zandiswa kathathu kwaye ixabiso eliphakathi lasetyenziswa kuhlalutyo lwe-RT-qPCR. Idatha yokwandiswa yahlalutywa kusetyenziswa iFlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). I-IL-4, IL-17, kunye ne-IFN-γ gene expression zalungiswa kwi-endogenous control (ACTB). Amanqanaba okubonakaliswa okunxulumeneyo kwe-target mRNA abalwe kusetyenziswa indlela ye-2-ΔΔCT.
Iprotheyini iyonke ikhutshwe kwizicubu kusetyenziswa i-tissue homogenizer kwi-RIPA lysis buffer (equlethe i-protease kunye ne-phosphatase inhibitor cocktail) yaze yafakwa kwi-centrifuge kwi-13,000 rpm imizuzu engama-20 kwi-4°C ukususa ukungcola kwezicubu. Ii-micrograms ezingamashumi amahlanu zeprotheyini zafakwa kumzila ngamnye, zahlulwe yi-10% SDS-PAGE, zaze zadluliselwa kwi-membrane ye-PVDF. Ukuvimba kwenziwe kwi-5% yobisi olomileyo olungenamafutha kwi-Tris-buffered saline (TBS) equlethe i-0.1% Tween 20 iyure e-1 kubushushu begumbi. I-membrane yafakwa kwi-antibody ye-rabbit anti-decorin primary (exutywe yi-1:200; Boster, Wuhan, China) (exutywe yi-1:200; Bioss, Beijing, China) ubusuku bonke kwi-4°C kwaye yasabela kwiintsuku zesibini; kunye ne-antibody yesibini (i-goat anti-rabbit immunoglobulin G kwi-1:40,000 dilution) idityaniswe ne-horseradish peroxidase (eBoster, eWuhan, eTshayina) iyure e-1 kubushushu begumbi. Izibonakaliso ze-Western blot zifunyenwe ngokwanda kwe-chemiluminescence kwi-membrane ye-chemiluminescent emva kokukhanyiswa kwe-X-ray. Kuhlalutyo lwe-densitometric, ii-blots ziskeniwe kwaye zalinganiswa kusetyenziswa isoftware ye-BandScan kwaye iziphumo zichazwe njengomlinganiselo we-target gene immunoreactivity kwi-tubulin immunoreactivity.
Ubalo lwezibalo lwenziwe kusetyenziswa iphakheji yesoftware ye-SPSS16.0 (SPSS, e-USA). Idatha eqokelelwe ngexesha lophando ibonakaliswe njenge-mean ± standard deviation (mean ± SD) kwaye yahlalutywa kusetyenziswa uhlalutyo lwe-variance oluphindaphindwayo lwendlela enye (ANOVA) ukufumanisa umahluko phakathi kwamaqela amabini. I-P < 0.05 ithathwe njengebalulekileyo ngokwezibalo.
Ngoko ke, ukusekwa komzekelo wesilwanyana se-MC ngokufaka ii-NPs ze-autologous emzimbeni we-vertebral nokwenza uqwalaselo lwe-macroanatomical, uhlalutyo lwe-MRI, uvavanyo lwe-histological kunye nohlalutyo lwe-molecular biological kunokuba sisixhobo esibalulekileyo sokuvavanya nokuqonda iindlela ze-MC yomntu kunye nokuphuhlisa iindlela ezintsha zonyango.
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