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Kugadzwa kwemhando dzemhuka dzekuchinja kwemodic (MC) hwaro hwakakosha pakudzidza nezveMC. Tsuro chena makumi mashanu nemana dzeNew Zealand dzakakamurwa kuva boka resham-operation, boka remhasuru (ME group) uye boka renucleus pulposus implantation (NPE group). Muboka reNPE, intervertebral disc yakafumurwa nenzira yekuvhiya yeanterolateral lumbar uye tsono yakashandiswa kuboora muviri weL5 vertebral pedyo neplate yekupedzisira. NP yakabviswa kubva kuL1/2 intervertebral disc nesirinji ndokubayiwa mairi. Kuboora gomba mubhonzo re subchondral. Maitiro ekuvhiya uye nzira dzekuboora muboka remhasuru neboka resham-operation zvaive zvakafanana nezviri muboka reNP implantation. Muboka reME, chidimbu chemhasuru chakaiswa mugomba, nepo muboka resham-operation, hapana chakaiswa mugomba. Mushure mekuvhiyiwa, MRI scanning uye molecular biological testing zvakaitwa. Chiratidzo muboka reNPE chakachinja, asi hapana shanduko yakajeka yechiratidzo muboka resham-operation neboka reME. Kuongorora kwehistological kwakaratidza kuti kuwanda kwenyama kusina kujairika kwakaonekwa panzvimbo yakaisirwa, uye kuratidzwa kweIL-4, IL-17 uye IFN-γ kwakawedzera muboka reNPE. Kuiswa kweNP mubhonzo re subchondral kunogona kuumba muenzaniso wemhuka weMC.
Kuchinja kweModic (MC) maronda emapfupa emusana uye mabhonzo ari pedyo anoonekwa pamagnetic resonance imaging (MRI). Anowanzoitika kune vanhu vane zviratidzo zvine chekuita nazvo1. Zvidzidzo zvakawanda zvakasimbisa kukosha kweMC nekuda kwekubatana kwayo nekurwadziwa kwemusana (LBP)2,3. de Roos et al.4 naModic et al.5 vakazvimiririra vakatanga vatsanangura mhando nhatu dzakasiyana dzezviratidzo zve subchondral mumapfupa emusana. Kuchinja kweModic type I kunonyanya kuoneka paT1-weighted (T1W) sequences uye hyperintense paT2-weighted (T2W). Kukuvara uku kunoratidza mafissure endplates uye ma vascular granulation tissue ari pedyo mubhonzo. Kuchinja kweModic type II kunoratidza chiratidzo chakakwirira paT1W neT2W sequences. Murudzi urwu rwekukuvadzwa, kuparadzwa kwe endplate kunogona kuwanikwa, pamwe nekutsiviwa kwemafuta emusana ari pedyo. Kuchinja kweModic type III kunoratidza chiratidzo chakaderera muT1W neT2W sequences. Sclerotic lesions inoenderana nemabhonzo akaonekwa6. MC inoonekwa sechirwere chemusana uye inobatanidzwa zvakanyanya nezvirwere zvakawanda zvinokanganisa musana7,8,9.
Tichifunga nezveruzivo rwuripo, zvidzidzo zvakawanda zvakapa ruzivo rwakadzama nezvechikonzero uye nzira dzezvirwere zveMC. Albert nevamwe vake vakati MC inogona kukonzerwa nekuchekwa kwediski8. Hu nevamwe vake vakati MC inokonzerwa nekuora kwakanyanya kwediski10. Kroc vakataura nezvepfungwa yekuti "kuputika kwediski yemukati," iyo inotaura kuti kukuvara kwediski kunodzokororwa kunogona kutungamira kukubvaruka kwemicroscope muplate. Mushure mekuumbwa kwecleft, kuparadzwa kweplate nenucleus pulposus (NP) kunogona kukonzera mhinduro ye autoimmune, izvo zvinotungamira kukukura kweMC11. Ma nevamwe vake vakagovana maonero akafanana uye vakataura kuti NP-induced autoimmune ine basa guru mu pathogenesis yeMC12.
Masero emuviri anodzivirira zvirwere, kunyanya maCD4+ T helper lymphocytes, anoita basa rakakosha mukukura kwe autoimmune13. Chikamu cheTh17 chakawanikwa munguva pfupi yapfuura chinoburitsa proinflammatory cytokine IL-17, chinokurudzira chemokine expression, uye chinokurudzira T cells munhengo dzakakuvara kuti dzigadzire IFN-γ14. Th2 cells dzinoitawo basa rakasiyana mukukura kwe immune responses. Kubuda kweIL-4 semumiriri weTh2 cell kunogona kutungamira kumigumisiro yakaipa ye immunopathological15.
Kunyangwe zvidzidzo zvakawanda zvekiriniki zvakaitwa paMC16,17,18,19,20,21,22,23,24, kuchine kushomeka kwemamodeli ekuyedza emhuka akakodzera anogona kutevedzera maitiro eMC anowanzoitika muvanhu uye anogona kushandiswa kuferefeta chikonzero kana marapirwo matsva akadai sekurapa kwakanangana. Kusvika pari zvino, mhando shoma dzemhuka dzeMC dzakashumwa kuti dzinodzidza mashandiro echirwere.
Zvichibva padzidziso ye autoimmune yakataurwa naAlbert naMa, chidzidzo ichi chakagadzira modhi yeMC yetsuro iri nyore uye inogona kudzokororwa kuburikidza nekuisa NP yega pedyo neplate yemusana yakaboorwa. Zvimwe zvinangwa ndezvekutarisa hunhu hwe histological hwemhando dzemhuka uye kuongorora mashandiro chaiwo eNP mukukura kweMC. Kuti tiite izvi, tinoshandisa matekiniki akadai se molecular biology, MRI, uye histological studies kudzidza kufambira mberi kweMC.
Tsuro mbiri dzakafa nekubuda ropa panguva yekuvhiyiwa, uye tsuro ina dzakafa panguva yekuvhiyiwa kweMRI. Tsuro dzakasara makumi mana nemasere dzakararama uye hadzina kuratidza zviratidzo zvehunhu kana zvetsinga mushure mekuvhiyiwa.
MRI inoratidza kuti simba rechiratidzo chenyama yakaputirwa mumaburi akasiyana rakasiyana. Simba rechiratidzo chemuviri weL5 vertebral muboka reNPE rakachinja zvishoma nezvishoma pamavhiki gumi nemaviri, gumi nematanhatu uye makumi maviri mushure mekuiswa (T1W sequence yakaratidza chiratidzo chidiki, uye T2W sequence yakaratidza chiratidzo chakasanganiswa pamwe nechiratidzo chidiki) (Mufananidzo 1C), nepo kuoneka kweMRI kwemamwe mapoka maviri ezvikamu zvakaputirwa kwakaramba kwakadzikama munguva imwe chete (Mufananidzo 1A, B).
(A) Kuongororwa kweMRIs dzinotevedzana dzemusana werutsanga panguva nhatu. Hapana zviratidzo zvisina kunaka zvakawanikwa mumifananidzo yeboka rekushanda kwesham. (B) Hunhu hwezviratidzo zvemuviri wemusana muboka reME hwakafanana nehweboka rekushanda kwesham, uye hapana shanduko huru yezviratidzo inoonekwa panzvimbo yekuisa sham nekufamba kwenguva. (C) Muboka reNPE, chiratidzo chidiki chinoonekwa zvakajeka muT1W sequence, uye chiratidzo chakasanganiswa nechiratidzo chidiki zvinoonekwa zvakajeka muT2W sequence. Kubva panguva yemavhiki gumi nemaviri kusvika panguva yemavhiki makumi maviri, zviratidzo zvepamusoro zvishoma nezvishoma zvakakomberedza zviratidzo zvishoma muT2W sequence zvinoderera.
Kuwanda kwemapfupa kuri pachena kunogona kuonekwa panzvimbo yekuisa muviri wemusana muboka reNPE, uye kuwedzera kwemapfupa kunoitika nekukurumidza kubva pamavhiki gumi nemaviri kusvika makumi maviri (Mufananidzo 2C) zvichienzaniswa neboka reNPE, hapana shanduko yakakosha inoonekwa mumiviri yemusana yakafananidzwa; boka reSham neboka reME (Mufananidzo 2C) 2A,B).
(A) Pamusoro pemuviri wemusana uri pachikamu chakaiswa wakatsetseka, buri rinopora zvakanaka, uye hapana hyperplasia mumuviri wemusana. (B) Chimiro chenzvimbo yakaisirwa muboka reME chakafanana necheboka rekushanda kwenhema, uye hapana shanduko iri pachena pakuonekwa kwenzvimbo yakaisirwa nekufamba kwenguva. (C) Hyperplasia yemapfupa yakaitika panzvimbo yakaisirwa muboka reNPE. Hyperplasia yemapfupa yakawedzera nekukurumidza uye yakatopfuura nepakati pe intervertebral disc kuenda kumuviri wemusana uri padivi.
Kuongorora histological kunopa ruzivo rwakadzama nezvekuumbwa kwemapfupa. Mufananidzo 3 unoratidza mifananidzo yezvikamu zvepashure pekuvhiyiwa zvakasvibiswa neH&E. Muboka resham-operation, ma chondrocytes akanga akarongeka zvakanaka uye hapana kuwanda kwemasero kwakaonekwa (Mufananidzo 3A). Mamiriro ezvinhu muboka reME akafanana neaya ari muboka resham-operation (Mufananidzo 3B). Zvisinei, muboka reNPE, huwandu hwakawanda hwema chondrocytes uye kuwanda kwemasero akafanana neNP zvakaonekwa panzvimbo yekuisa mapfupa (Mufananidzo 3C);
(A) Trabeculae inogona kuonekwa pedyo neplate yekupedzisira, ma chondrocyte akarongwa zvakanaka nehukuru nechimiro chemasero zvakafanana uye hapana kuwanda (ka40). (B) Mamiriro enzvimbo yekuisirwa muboka reME akafanana neaya eboka remanyepo. Trabeculae nema chondrocyte anogona kuonekwa, asi hapana kuwanda kuri pachena panzvimbo yekuisirwa (ka40). (B) Zvinogona kuonekwa kuti ma chondrocyte nema NP-like cells anowedzera zvakanyanya, uye chimiro nehukuru hwema chondrocyte hazvina kuenzana (ka40).
Kuonekwa kwe interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, uye interferon γ (IFN-γ) mRNA kwakaonekwa mumapoka ese eNPE neME. Pakaenzaniswa mwero wekutaura kwemajini akananga, kuratidzwa kwemajini eIL-4, IL-17, uye IFN-γ kwakawedzera zvakanyanya muboka reNPE zvichienzaniswa neaya eboka reME neboka rekushanda kwesham (Mufananidzo 4) (P < 0.05). Zvichienzaniswa neboka rekushanda kwesham, mwero wekutaura kweIL-4, IL-17, uye IFN-γ muboka reME wakawedzera zvishoma chete uye hauna kusvika pakuchinja kwenhamba (P > 0.05).
Kuratidzwa kwemRNA kweIL-4, IL-17 uye IFN-γ muboka reNPE kwakaratidza mafambiro akakwira zvakanyanya kupfuura ari muboka rekushanda kwenhema neboka reME (P < 0.05).
Kusiyana neizvi, mwero wekutaura muboka reME hauna kuratidza mutsauko wakakosha (P>0.05).
Kuongorora kweWestern blot kwakaitwa uchishandisa ma antibodies anowanikwa mumusika kurwisa IL-4 neIL-17 kusimbisa maitiro ekutaura kwemRNA akachinjwa. Sezvakaratidzwa muMifananidzo 5A,B, zvichienzaniswa neboka reME neboka rekushanda kwesham, huwandu hwemapuroteni eIL-4 neIL-17 muboka reNPE hwakawedzera zvakanyanya (P < 0.05). Zvichienzaniswa neboka rekushanda kwesham, huwandu hwemapuroteni eIL-4 neIL-17 muboka reME hwakakundikanawo kusvika pakuchinja kwakakosha (P> 0.05).
(A) Mapuroteni eIL-4 neIL-17 muboka reNPE aive akakwira zvakanyanya kupfuura ari muboka reME neboka replacebo (P < 0.05). (B) Western blot histogram.
Nekuda kwehuwandu hushoma hwemasampuli evanhu akawanikwa panguva yekuvhiyiwa, zvidzidzo zvakajeka uye zvakadzama nezve pathogenesis yeMC zvakaoma. Takaedza kugadzira modhi yemhuka yeMC kuti tidzidze mashandiro ayo ezvirwere. Panguva imwe chete, kuongororwa kweX-ray, kuongororwa kweHistological uye kuongororwa kweMolecular Biological zvakashandiswa kutevedzera nzira yeMC inokonzerwa neNP autograft. Nekuda kweizvozvo, modhi yeNP implantation yakaguma nekuchinja zvishoma nezvishoma musimba rechiratidzo kubva pamavhiki gumi nemaviri kusvika pamavhiki makumi maviri (mixed low signal in T1W sequences uye low signal in T2W sequences), zvichiratidza shanduko dzematishu, uye kuongororwa kweHistological neMolecular Biological kwakasimbisa mhedzisiro yekudzidza kweX-ray.
Mhedzisiro yekuedza uku inoratidza kuti shanduko dzekuona nedzehistological dzakaitika panzvimbo yekukanganiswa kwemuviri wemusana muboka reNPE. Panguva imwe chete, kuratidzwa kwemajini eIL-4, IL-17 neIFN-γ, pamwe neIL-4, IL-17 neIFN-γ zvakaonekwa, zvichiratidza kuti kukanganiswa kwetishu ye nucleus pulposus mumuviri wemusana kunogona kukonzera shanduko dzakawanda dzechiratidzo nechimiro. Zviri nyore kuona kuti hunhu hwezviratidzo zvemiviri yemusana yemuenzaniso wemhuka (chiratidzo chidiki muT1W sequence, chiratidzo chakasanganiswa uye chiratidzo chidiki muT2W sequence) zvakafanana zvikuru nemasero emusana wemunhu, uye hunhu hweMRI hunosimbisawo kucherechedzwa kwehistology uye gross anatomy, kureva kuti, shanduko mumasero emuviri wemusana dzinofambira mberi. Kunyangwe mhinduro yekuzvimba inokonzerwa nekukuvara kwakanyanya inogona kuonekwa nguva pfupi mushure mekuboorwa, mhedzisiro yeMRI yakaratidza kuti kuchinja kwezviratidzo kuri kuwedzera zvishoma nezvishoma kwakaonekwa mavhiki gumi nemaviri mushure mekuboorwa uye kwakaramba kuripo kusvika mavhiki makumi maviri pasina zviratidzo zvekupora kana kudzoserwa kweshanduko dzeMRI. Zvakawanikwa izvi zvinoratidza kuti autologous vertebral NP inzira yakavimbika yekusimbisa progressive MV mutsuro.
Modhi iyi yekuboora inoda hunyanzvi hwakakwana, nguva, uye kushanda nesimba pakuvhiya. Mukuedza kwekutanga, kupatsanurwa kana kukurudzirwa zvakanyanya kwezvikamu zveparavertebral ligamentous zvinogona kukonzera kuumbwa kwemapfupa emapfupa emapfupa. Kungwarira kunofanira kuitwa kuti kusakuvadza kana kutsamwisa madiski ari pedyo. Sezvo kudzika kwekupinda kuchifanira kudzorwa kuti tiwane mhedzisiro yakafanana uye inodzokororwa, takagadzira chivharo nemaoko nekucheka sheath yetsono yakareba 3 mm. Kushandisa chivharo ichi kunovimbisa kudzika kwakafanana kwekuboora mumuviri wemapfupa emapfupa. Mukuedza kwekutanga, vanachiremba vatatu vemapfupa vaibatanidzwa mukuvhiya vakawana tsono dze16-gauge dziri nyore kushanda nadzo kupfuura tsono dze18-gauge kana dzimwe nzira. Kudzivirira kubuda ropa zvakanyanya panguva yekuboora, kubata tsono kwenguva yakati kuchapa gomba rakanyatsokodzera, zvichiratidza kuti mwero wakati weMC unogona kudzorwa nenzira iyi.
Kunyangwe zvidzidzo zvakawanda zvakatarisa MC, zvishoma zvinozivikanwa nezve etiology uye pathogenesis yeMC25,26,27. Zvichibva pane zvidzidzo zvedu zvekare, takaona kuti autoimmune ine basa guru mukuvapo uye kukura kweMC12. Chidzidzo ichi chakaongorora huwandu hweIL-4, IL-17, uye IFN-γ, idzo dziri nzira huru dzekusiyanisa masero eCD4+ mushure mekukurudzirwa kweantigen. Mukudzidza kwedu, zvichienzaniswa neboka risina kunaka, boka reNPE raive nehuwandu hwakawanda hweIL-4, IL-17, uye IFN-γ, uye huwandu hweprotein hweIL-4 neIL-17 hwaive hwakakwirawo.
Panyaya yekiriniki, kuratidzwa kweIL-17 mRNA kwakawedzera mumasero eNP kubva kuvarwere vane disc herniation28. Kuwedzera kwemazinga ekutaura kweIL-4 neIFN-γ kwakawanikwawo mumuenzaniso we acute non-compressive disc herniation zvichienzaniswa neanodzora hutano29. IL-17 ine basa guru mukuzvimba, kukuvara kwenyama muzvirwere zve autoimmune30 uye inowedzera mhinduro yemuviri kuIFN-γ31. Kukuvara kwenyama kwakawedzerwa neIL-17 kwakaratidzwa muMRL/lpr mice32 uye mbeva dzinobatwa ne autoimmune33. IL-4 inogona kudzivirira kuratidzwa kwe proinflammatory cytokines (senge IL-1β neTNFα) uye macrophage activation34. Zvakataurwa kuti mRNA expression yeIL-4 yakanga yakasiyana muboka reNPE zvichienzaniswa neIL-17 neIFN-γ panguva imwe chete; Kuratidzwa kwemRNA kweIFN-γ muboka reNPE kwaive kwakakwira zvakanyanya kupfuura mune mamwe mapoka. Nokudaro, kugadzirwa kweIFN-γ kunogona kuva murevereri wemhinduro yekuzvimba inokonzerwa neNP intercalation. Zvidzidzo zvakaratidza kuti IFN-γ inogadzirwa nemarudzi akasiyana-siyana emasero, anosanganisira masero emhando yekutanga ekubatsira T, masero ekuuraya echisikigo, uye macrophages35,36, uye i cytokine inokosha inodzivirira kuzvimba inokurudzira mhinduro dzemuviri dzekudzivirira chirwere.
Chidzidzo ichi chinoratidza kuti mhinduro ye autoimmune inogona kunge iri chikamu chekuitika uye kukura kwe MC. Luoma nevamwe vakaona kuti hunhu hwechiratidzo che MC ne NP yakakurumbira hwakafanana paMRI, uye ese ari maviri anoratidza chiratidzo chakakwira muT2W sequence38. Mamwe ma cytokines akasimbiswa kuti ane hukama hwakasimba nekuitika kwe MC, senge IL-139. Ma nevamwe vakataura kuti kubuda kwe NP kumusoro kana pasi kunogona kuve nepesvedzero huru pakuitika uye kukura kwe MC12. Bobechko40 naHerzbein nevamwe.41 vakataura kuti NP itembu isingakwanise kuzvidzivirira mumuviri isingakwanise kupinda muropa kubva pakuzvarwa. Kubuda kwe NP kunounza miviri yekunze muropa, nokudaro zvichikonzera maitiro e autoimmune emunharaunda42. Kubuda kwe autoimmune kunogona kukonzera huwandu hwakawanda hwezvinhu zve immune, uye kana zvinhu izvi zvikaramba zvichiratidzwa kune tishu, zvinogona kukonzera shanduko mu signaling43. Muchidzidzo ichi, kuwanda kwe IL-4, IL-17 uye IFN-γ zvinhu zve immune, zvichiratidza hukama hwepedyo pakati pe NP ne MCs44. Iyi mhando yemhuka inotevedzera zvakanaka kubuda kweNP uye kupinda muplate yekupedzisira. Maitiro aya akaburitsa pachena kukanganiswa kwe autoimmune immune system pa MC.
Sezvaitarisirwa, muenzaniso uyu wemhuka unotipa mukana wekudzidza nezveMC. Zvisinei, muenzaniso uyu uchiri nemiganhu: chekutanga, panguva yekutarisa mhuka, dzimwe tsuro dzepakati nepakati dzinoda kuurayiwa kuti dziongororwe histological uye molecular biology, saka dzimwe mhuka "dzinorega kushandiswa" nekufamba kwenguva. Chechipiri, kunyangwe nguva nhatu dzakaiswa muchidzidzo ichi, zvinosuwisa kuti takangotevedzera mhando imwe chete yeMC (Modic type I change), saka hazvina kukwana kumiririra maitiro ekukura kwechirwere chemunhu, uye nguva dzakawanda dzinofanira kuiswa kuti tione zviri nani shanduko dzese dzechiratidzo. Chechitatu, shanduko muchimiro chetishu dzinogona kuratidzwa zvakajeka ne histological staining, asi mamwe matekiniki akasarudzika anogona kuratidza zviri nani shanduko dze microstructural mumuenzaniso uyu. Semuenzaniso, polarized light microscopy yakashandiswa kuongorora kuumbwa kwefibrocartilage mu rabbit intervertebral discs45. Mhedzisiro yenguva refu yeNP paMC ne endplate inoda kudzidza kwakawanda.
Tsuro chena dzechirume makumi mashanu neina dzekuNew Zealand (dzinorema anenge 2.5-3 kg, dzine makore 3-3.5) dzakakamurwa zvisina tsarukano muboka rekushanda kwenhema, boka rekuisa tsandanyama (ME group) uye boka rekuisa nemidzi yetsinga (NPE group). Maitiro ese ekuyedza akatenderwa neKomiti yeMaitiro eChipatara cheTianjin, uye nzira dzekuyedza dzakaitwa zvichienderana nemirairo yakatenderwa.
Kuvandudzwa kwakaitwa panzira yekuvhiya yaS. Sobajima 46. Tsuro yega yega yakaiswa panzvimbo yekurara kwemuviri uye pamusoro pema disc mashanu akatevedzana e lumbar intervertebral discs (IVDs) akafumurwa uchishandisa nzira ye posterolateral retroperitoneal. Tsuro yega yega yakapihwa anesthesia yakajairika (20% urethane, 5 ml/kg kuburikidza netsinga yenzeve). Kuchekwa kweganda kwenguva refu kwakaitwa kubva kumucheto wezasi wembabvu kusvika kumucheto wepelvic, 2 cm ventral kusvika kumhasuru dzeparavertebral. Muzongoza wekurudyi kubva kuL1 kusvika kuL6 wakafumurwa nekupatsanurwa kwakapinza uye kwakapfava kwe subcutaneous tissue, retroperitoneal tissue, uye tsandanyama (Mufananidzo 6A). Chikamu che disc chakaongororwa uchishandisa pelvic brim sechiratidzo che anatomical che level ye disc yeL5-L6. Shandisa tsono ye 16-gauge puncture kuboora buri pedyo neplate yekupedzisira ye L5 vertebra kusvika pakadzika kwe 3 mm (Mufananidzo 6B). Shandisa sirinji ye5-ml kuburitsa nucleus pulposus muL1-L2 intervertebral disc (Mufananidzo 6C). Bvisa nucleus pulposus kana tsandanyama zvichienderana nezvinodiwa neboka rega rega. Mushure mekunge gomba rekuboorera radzika, sutures dzinonyudzwa dzinoiswa pa deep fascia, surface fascia neganda, uchingwarira kuti usakuvadze periosteal tissue yemuviri wemuzongoza panguva yekuvhiyiwa.
(A) Dhisiki reL5–L6 rinoburitswa pachena kuburikidza nenzira ye posterolateral retroperitoneal. (B) Shandisa tsono ine gauge 16 kuboora buri pedyo neL5 endplate. (C) Autologous MFs dzinotorwa.
Anesthesia yakaitwa ne20% urethane (5 ml/kg) kuburikidza netsinga dzenzeve, uye radiographs dzemusana welumbar dzakadzokororwa pamavhiki gumi nemaviri, gumi nematanhatu, uye makumi maviri mushure mekuvhiyiwa.
Tsuro dzakabayiwa nekubaiwa ketamine (25.0 mg/kg) ne sodium pentobarbital (1.2 g/kg) mutsinga mushure memavhiki gumi nemaviri, gumi nematanhatu, uye makumi maviri mushure mekuvhiyiwa. Muzongoza wese wakabviswa kuti uongororwe histological uye ongororo chaiyo yakaitwa. Quantitative reverse transcription (RT-qPCR) uye Western blotting zvakashandiswa kuona shanduko mu immune factors.
Kuongororwa kweMRI kwakaitwa mutsuro uchishandisa 3.0 T clinical magnet (GE Medical Systems, Florence, SC) ine orthogonal limb coil receiver. Tsuro dzakaiswa anesthesia ne20% urethane (5 mL/kg) kuburikidza netsinga yenzeve ndokuzoiswa mudumbu mukati memagnet ne lumbar region yakatarisana ne 5-inch dhayamita circular surface coil (GE Medical Systems). Coronal T2-weighted localizer images (TR, 1445 ms; TE, 37 ms) dzakatorwa kuti dzitsanangure nzvimbo ye lumbar disc kubva kuL3–L4 kusvika kuL5–L6. Sagittal plane T2-weighted slices dzakatorwa nezvirongwa zvinotevera: fast spin-echo sequence ine repetition time (TR) ye 2200 ms uye an echo time (TE) ye 70 ms, matrix; visual field ye 260 uye eight stimuli; Cutting field yaive 2 mm, gap yaive 0.2 mm.
Mushure mekunge mufananidzo wekupedzisira watorwa uye tsuro yekupedzisira yaurayiwa, sham, tsandanyama dzakaiswa mutsandanyama, uye ma NP discs zvakabviswa kuti zviongororwe histological. Matumba akaiswa mu 10% neutral buffered formalin kwevhiki imwe, akabviswa calcium ne ethylenediaminetetraacetic acid, uye akapatsanurwa paraffin. Matumba ematimba akaiswa mu paraffin ndokuchekwa kuita zvikamu zve sagittal (5 μm gobvu) uchishandisa microtome. Zvikamu zvakaiswa mavara ne hematoxylin ne eosin (H&E).
Mushure mekuunganidza ma disc e intervertebral disc kubva mutsuro dziri muboka rega rega, huwandu hweRNA hwakatorwa uchishandisa UNIQ-10 column (Shanghai Sangon Biotechnology Co., Ltd., China) zvichienderana nemirairo yemugadziri uye sisitimu yeImProm II reverse transcription (Promega Inc., Madison, WI, USA). Kunyora reverse transcription kwakaitwa.
RT-qPCR yakaitwa uchishandisa Prism 7300 (Applied Biosystems Inc., USA) uye SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) zvichienderana nemirairo yemugadziri. Vhoriyamu yePCR reaction yaive 20 μl uye yaive ne1.5 μl ye diluted cDNA uye 0.2 μM ye primer yega yega. Ma primer akagadzirwa neOligoPerfect Designer (Invitrogen, Valencia, CA) uye akagadzirwa neNanjing Golden Stewart Biotechnology Co., Ltd. (China) (Tafura 1). Mamiriro anotevera ekupisa akashandiswa: danho rekutanga re polymerase activation pa94°C kwemaminitsi maviri, wozotevera ma cycles makumi mana e15 s imwe neimwe pa94°C kuti template denaturation iite, annealing kweminiti imwe pa60°C, extension, uye fluorescence. kuyerwa kwakaitwa kweminiti imwe pa72°C. Samples dzese dzakawedzerwa katatu uye avhareji yehuwandu yakashandiswa pakuongorora RT-qPCR. Data rekuwedzera rakaongororwa uchishandisa FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). IL-4, IL-17, uye IFN-γ gene expression zvakagadziriswa kune endogenous control (ACTB). Relative expression levels ye target mRNA yakaverengerwa uchishandisa nzira ye2-ΔΔCT.
Mapuroteni ese akatorwa kubva munyama achishandisa mushonga wekubvisa matishu muRIPA lysis buffer (ine protease ne phosphatase inhibitor cocktail) uye akazoiswa centrifuge pa13,000 rpm kwemaminitsi makumi maviri pa4°C kubvisa tsvina yematishu. Makirogiramu makumi mashanu emapuroteni akaiswa panzira imwe neimwe, akaparadzaniswa ne10% SDS-PAGE, ndokuzoendeswa kuPVDF membrane. Kuvhara kwakaitwa mumukaka wakaoma we5% nonfat muTris-buffered saline (TBS) ine 0.1% Tween 20 kweawa imwe chete patembiricha yemumba. Kamudzira kacho kakaiswa murabbit anti-decorin primary antibody (yakasanganiswa 1:200; Boster, Wuhan, China) (yakasanganiswa 1:200; Bioss, Beijing, China) husiku hwese pa4°C uye kakapindurwa pamazuva echipiri; ne secondary antibody (goat anti-rabbit immunoglobulin G pa 1:40,000 dilution) yakasanganiswa ne horseradish peroxidase (Boster, Wuhan, China) kweawa imwe chete patembiricha yemumba. Zviratidzo zveWestern blot zvakaonekwa nekuwedzera kwe chemiluminescence pa chemiluminescent membrane mushure mekuvhenekwa neX-ray. Pakuongorora kwe densitometric, mablots akaongororwa uye akayerwa uchishandisa BandScan software uye mhedzisiro yacho yakatsanangurwa sechiyero che target gene immunoreactivity kune tubulin immunoreactivity.
Kuverenga nhamba kwakaitwa uchishandisa software package yeSPSS16.0 (SPSS, USA). Data rakaunganidzwa panguva yekudzidza rakataurwa se mean ± standard deviation (mean ± SD) uye rakaongororwa uchishandisa one-way repeated measures analysis of variance (ANOVA) kuti vaone mutsauko uripo pakati pemapoka maviri aya. P < 0.05 yakaonekwa seyakakosha muhuwandu hwemashoko.
Saka, kuumbwa kwemuenzaniso wemhuka weMC nekuisa maNPs e autologous mumuviri wemuzongoza uye kuita observation ye macroanatomical, MRI analysis, histological evaluation uye molecular biological analysis zvinogona kuva chishandiso chakakosha chekuongorora nekunzwisisa mashandiro e MC yemunhu nekugadzira nzira itsva dzekurapa.
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Nguva yekutumira: Zvita-13-2024