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Nhazi nke ụdị anụmanụ nke mgbanwe modic (MC) bụ ihe ndabere dị mkpa maka ịmụ MC. Iri ise na anọ New Zealand White rabbits kewara n'ime otu sham-operation group, muscle implantation group (ME group) na nucleus pulposus implantation group (NPE group). N'ime otu NPE, a na-ekpughe diski intervertebral site na usoro ịwa ahụ nke anterolateral lumbar na a na-eji agịga mee ka L5 vertebral ahụ dị nso na efere njedebe. A na-ewepụta NP na diski intervertebral L1/2 site na sirinji wee tinye ya n'ime ya. Na-egwupụta oghere n'ime ọkpụkpụ subchondral. Usoro ịwa ahụ na ụzọ mkpọpu n'ime ìgwè ndị na-emepụta ahụ ike na ndị na-arụ ọrụ sham bụ otu ihe ahụ dị na NP. N'ime ìgwè ME, a na-etinye otu anụ ahụ n'ime oghere, ebe n'ime otu ọrụ sham, ọ dịghị ihe ọ bụla etinyere n'ime oghere ahụ. Mgbe arụchara ọrụ ahụ, a na-eme nyocha MRI na nyocha nke mkpụrụ ndụ ihe nketa. Ihe mgbaàmà dị na otu NPE gbanwere, ma ọ dịghị mgbanwe mgbaàmà doro anya na ndị ọrụ sham-operation na otu ME. Nnyocha akụkọ ihe mere eme gosiri na a na-ahụ mmụba anụ ahụ na-adịghị mma na ebe a na-etinye ya, na nkwupụta nke IL-4, IL-17 na IFN-γ na-abawanye na otu NPE. Ịkụnye NP n'ime ọkpụkpụ subchondral nwere ike ịmepụta ụdị anụmanụ nke MC.
Mgbanwe Modic (MC) bụ ọnya nke vertebral endplates na ụmị ọkpụkpụ dị n'akụkụ a na-ahụ anya na imaging resonance magnet (MRI). Ha na-adịkarị n'ime ndị mmadụ nwere ihe mgbaàmà metụtara1. Ọtụtụ ọmụmụ ekwupụtala mkpa MC dị n'ihi njikọ ya na obere mgbu azụ (LBP) 2,3. de Roos et al.4 na Modic et al.5 n'adabereghị na mbụ kọwara ụdị atọ dị iche iche nke mgbaàmà subchondral dị na ụmị ọkpụkpụ vertebral. Ụdị Modic M na-agbanwe bụ hypointense na usoro T1-weighted (T1W) yana hyperintense na usoro T2-weighted (T2W). Ọnya a na-ekpughe ihe njedebe fissure na anụ ahụ granulation vaskụla dị n'akụkụ ụmị ọkpụkpụ. Mgbanwe ụdị Modic II na-egosi mgbama dị elu na ma usoro T1W na T2W. N'ime ụdị ọnya a, enwere ike ịchọta mbibi endplate, yana nnọchi abụba nke akụkọ ihe mere eme nke ụmị ọkpụkpụ dị n'akụkụ. Mgbanwe Modic ụdị III na-egosi mgbaama dị ala na usoro T1W na T2W. Achọpụtala ọnya Sclerotic kwekọrọ na njedebe njedebe6. A na-ewere MC dị ka ọrịa na-adịghị ala ala nke ọkpụkpụ azụ na-ejikọta ya na ọtụtụ ọrịa mmebi nke ọkpụkpụ azụ7,8,9.
N'ịtụle data dịnụ, ọtụtụ ọmụmụ enyela nkọwa zuru ezu banyere etiology na usoro pathological nke MC. Albert et al. tụrụ aro na enwere ike bute MC site na diski herniation8. Hu et al. ekwuputara MC na mmebi diski siri ike10. Kroc tụpụtara echiche nke "mgbawa diski dị n'ime," nke na-ekwu na trauma diski ugboro ugboro nwere ike iduga microtears na njedebe. Mgbe nchara nchara, mbibi endplate site na nucleus pulposus (NP) nwere ike ịkpalite nzaghachi autoimmune, nke na-eduga n'ihu na mmepe nke MC11. Ma et al. na-ekerịta echiche yiri nke ahụ wee kọọ na autoimmunity nke NP kpatara na-arụ ọrụ dị mkpa na pathogenesis nke MC12.
Mkpụrụ ndụ sistemu na-alụso ọrịa ọgụ, ọkachasị CD4+ T lymphocytes na-enyere aka, na-arụ ọrụ dị egwu na pathogenesis nke autoimmunity13. Ihe ntinye Th17 achọpụtara n'oge na-adịbeghị anya na-emepụta cytokine proinflammatory IL-17, na-akwalite okwu chemokine, ma na-akpali mkpụrụ ndụ T na akụkụ ahụ mebiri emebi iji mepụta IFN-γ14. Mkpụrụ ndụ Th2 na-arụkwa ọrụ pụrụ iche na pathogenesis nke nzaghachi mgbochi. Nkwupụta nke IL-4 dị ka onye nnọchi anya Th2 cell nwere ike ibute nsonaazụ immunopathological siri ike15.
Ọ bụ ezie na ọtụtụ ọmụmụ ụlọ ọgwụ emeela na MC16,17,18,19,20,21,22,23,24, a ka nwere ụkọ ụdị nnwale anụmanụ kwesịrị ekwesị nke nwere ike iṅomi usoro MC nke na-emekarị n'ime mmadụ ma nwee ike ịbụ. eji enyocha etiology ma ọ bụ ọgwụgwọ ọhụrụ dịka ọgwụgwọ ezubere iche. Ruo ugbu a, ọ bụ naanị ụdị anụmanụ ole na ole nke MC ka akọọrọ ka ha mụọ usoro pathological dị n'okpuru.
Dabere n'echiche autoimmune nke Albert na Ma tụpụtara, ọmụmụ ihe a hibere ụdị oke bekee MC dị mfe na nke a na-emegharịgharị site na ịgbanye onwe ya NP n'akụkụ efere ngwụcha vertebral gbawara agbawa. Ebumnobi ndị ọzọ bụ ileba anya na njirimara akụkọ ihe mere eme nke ụdị anụmanụ ma nyochaa kpọmkwem usoro NP na mmepe nke MC. Iji mezuo nke a, anyị na-eji usoro dị ka usoro ihe ọmụmụ ihe omimi, MRI, na ihe ọmụmụ akụkọ ihe mere eme iji mụọ ọganihu nke MC.
Oke bekee abụọ nwụrụ n'ihi ọbara ọgbụgba n'oge ịwa ahụ, na oke bekee anọ nwụrụ n'oge anesthesia n'oge MRI. Oke bekee 48 fọdụrụnụ dị ndụ ma ọ nweghị akara omume ma ọ bụ akwara ozi mgbe a wachara ya ahụ.
MRI na-egosi na ike mgbaàmà nke anụ ahụ agbakwunyere na oghere dị iche iche dị iche. Mmetụta mgbaàmà nke L5 vertebral ahụ na NPE otu ji nwayọọ nwayọọ gbanwee na 12, 16 na 20 izu mgbe ntinye (usoro T1W gosipụtara akara ngosi dị ala, na usoro T2W gosipụtara akara mgbagwoju anya tinyere akara ngosi dị ala) (Fig 1C), ebe MRI na-apụta. nke abụọ ọzọ dị iche iche nke agbakwunyere akụkụ nọgidere dịtụ kwụsiri ike n'otu oge ahụ (Fig. 1A, B).
(A) MRI na-anọchite anya usoro nke oke bekee lumbar na oge 3. Ọnweghị ihe mgbaama ahụ ahụrụ n'onyinyo nke otu ndị na-arụ ọrụ sham. (B) Njirimara mgbaàmà nke vertebral ahụ na ME otu yiri ndị na-arụ ọrụ sham, na ọ dịghị ịrịba mgbanwe mgbanwe mgbanwe a na-ahụ na embedring na oge. (C) N'ime otu NPE, akara ngosi dị ala na-apụta nke ọma na usoro T1W, na akara ngosi agwakọtara na akara ngosi dị ala na-ahụ anya na usoro T2W. Site na oge izu iri na abụọ ruo oge izu 20, akara ngosi dị elu na-agbakarị gburugburu akara ngosi dị ala na usoro T2W na-ebelata.
Enwere ike ịhụ hyperplasia ọkpụkpụ doro anya na ntinye nke vertebral ahụ na òtù NPE, na hyperplasia ọkpụkpụ na-apụta ngwa ngwa site na 12 ruo 20 izu (Fig 2C) ma e jiri ya tụnyere ìgwè NPE, ọ dịghị mgbanwe dị ịrịba ama na vertebral e mere ka ọ dị. ahu; Otu Sham na ME otu (Fig 2C) 2A,B).
(A) Elu nke vertebral ahụ na akụkụ etinyere ya dị ezigbo mma, oghere ahụ na-agwọ nke ọma, ọ dịghịkwa hyperplasia na vertebral ahụ. (B) Ọdịdị nke saịtị etinyere na ME otu yiri nke ahụ na-arụ ọrụ sham, ọ dịghịkwa mgbanwe doro anya na ọdịdị nke saịtị a na-etinye n'ime oge. (C) Ọkpụkpụ hyperplasia nke ọkpụkpụ mere na saịtị etinyere na otu NPE. Ọkpụkpụ hyperplasia nke ọkpụkpụ mụbara ngwa ngwa na ọbụna gbatịa site na intervertebral diski ruo n'ahụ vertebral contralateral.
Nyocha akụkọ ihe mere eme na-enye nkọwa zuru ezu banyere ọkpụkpụ ọkpụkpụ. Onyonyo 3 na-egosi foto nke akụkụ ndị a rụchara arụ ọrụ nke nwere ntụpọ H&E. N'ime otu ọrụ sham-operation, a na-ahazi chondrocytes nke ọma na ọ dịghị mmụba cell ahụrụ (Fig 3A). Ọnọdụ dị na ME otu yiri nke ahụ na-arụ ọrụ sham (Fig 3B). Otú ọ dị, na otu NPE, a na-ahụ ọnụ ọgụgụ buru ibu nke chondrocytes na mmụba nke mkpụrụ ndụ NP dị ka ebe a na-etinye ya (Fig 3C);
(A) Enwere ike ịhụ trabeculae n'akụkụ efere ngwụcha, a na-ahazi chondrocytes nke ọma na nha sel edo edo na ọdịdị na enweghị mmụba (ugboro 40). (B) Ọnọdụ ebe a na-etinye ihe ọkụkụ na ME otu yiri nke otu sham. Enwere ike ịhụ trabeculae na chondrocytes, ma ọ dịghị mmụba doro anya na ebe a na-etinye ya (ugboro 40). (B) Enwere ike ịhụ na chondrocytes na sel ndị dị ka NP na-amụba nke ọma, na ọdịdị na nha nke chondrocytes enweghị isi (ugboro 40).
A hụrụ okwu nke interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, na interferon γ (IFN-γ) mRNA na ma otu NPE na ME. Mgbe atụnyere usoro okwu nke mkpụrụ ndụ ihe nketa, okwu mkpụrụ ndụ nke IL-4, IL-17, na IFN-γ na-abawanye nke ọma na otu NPE ma e jiri ya tụnyere ndị ME na otu ọrụ sham (Fig 4). (P <0.05). E jiri ya tụnyere otu ọrụ sham, ọkwa ọkwa nke IL-4, IL-17, na IFN-γ na ME otu mụbara ntakịrị ma ghara iru mgbanwe ọnụ ọgụgụ (P> 0.05).
Okwu mRNA nke IL-4, IL-17 na IFN-γ na otu NPE gosipụtara omume dị elu nke ukwuu karịa ndị nọ na otu ọrụ sham na otu ME (P <0.05).
N'ụzọ dị iche, ọkwa ọkwa dị na òtù ME egosighi ọdịiche dị ịrịba ama (P> 0.05).
Emere nyocha Western blot site na iji ọgwụ mgbochi dị na azụmaahịa megide IL-4 na IL-17 iji kwado ụkpụrụ okwu mRNA gbanwere. Dị ka e gosiri na ọnụ ọgụgụ 5A, B, ma e jiri ya tụnyere òtù ME na otu ọrụ sham, ọkwa protein nke IL-4 na IL-17 na ìgwè NPE mụbara nke ukwuu (P <0.05). E jiri ya tụnyere otu ọrụ sham, ọkwa protein nke IL-4 na IL-17 n'ime otu ME erubeghịkwa mgbanwe dị ịrịba ama (P> 0.05).
(A) Ogo protein nke IL-4 na IL-17 na otu NPE dị elu karịa ndị nọ na ME na placebo otu (P <0.05). (B) Histogram nke Western blot.
N'ihi ọnụ ọgụgụ dị nta nke ihe nlele mmadụ nwetara n'oge ịwa ahụ, nchọpụta doro anya na nke zuru ezu banyere pathogenesis nke MC siri ike. Anyị nwara iguzobe ụdị anụmanụ nke MC iji mụọ usoro ọrịa nwere ike ime. N'otu oge ahụ, a na-eji nyocha redio, nyocha akụkọ ihe mere eme na nyocha mkpụrụ ndụ ihe nketa na-agbaso usoro MC nke NP autograft kpatara. N'ihi ya, ụdị ntinye nke NP mere ka mgbanwe nke ntanye na ike mgbaàmà site na 12-izu ruo 20-izu oge (ngwakọta ala dị ala na usoro T1W na akara ngosi dị ala na usoro T2W), na-egosi mgbanwe anụ ahụ, na akụkọ ihe mere eme na molecular. Nleba anya nke ndu kwadoro nchoputa nke nyocha redio.
Nsonaazụ nke nnwale a na-egosi na mgbanwe anya na akụkọ ihe mere eme mere na saịtị nke mmebi ahụ vertebral na otu NPE. N'otu oge ahụ, a hụrụ okwu nke IL-4, IL-17 na IFN-γ genes, yana IL-4, IL-17 na IFN-γ, na-egosi na mmebi nke autologous nucleus pulposus tissue na vertebral. ahụ nwere ike ịkpata usoro mgbama na mgbanwe ọdịdị ọdịdị. Ọ dị mfe ịchọpụta na njirimara mgbaàmà nke akụkụ vertebral nke ụdị anụmanụ (mgbama dị ala na usoro T1W, akara agwakọtara na akara dị ala na usoro T2W) dị nnọọ ka nke mkpụrụ ndụ vertebral mmadụ, na njirimara MRI nwekwara. gosi na nlebanya nke histology na oke anatomy, ya bụ, mgbanwe nke mkpụrụ ndụ vertebral ahụ na-aga n'ihu. Ọ bụ ezie na nzaghachi mkpali na-akpata site na nnukwu trauma nwere ike ịpụta n'oge na-adịghị anya mgbe nkwụsịtụ gasịrị, nsonaazụ MRI gosipụtara na mgbanwe mgbaàmà na-arịwanye elu na-aga n'ihu na-apụta 12 izu mgbe nkwụsịtụ na-adịgide ruo izu 20 n'enweghị ihe ịrịba ama ọ bụla nke mgbake ma ọ bụ ntụgharị nke mgbanwe MRI. Nsonaazụ ndị a na-egosi na autologous vertebral NP bụ ụzọ a pụrụ ịdabere na ya maka iguzobe MV na-aga n'ihu na oke bekee.
Nlereanya ntapu a chọrọ nka zuru oke, oge, na mbọ ịwa ahụ. N'ime nnwale ndị mbụ, nbipu ma ọ bụ oke mkpali nke akụkụ ahụ ligamentous paravertebral nwere ike ime ka e guzobe osteophytes vertebral. Ekwesịrị ịkpachapụ anya ka ị ghara imebi ma ọ bụ kpasuo diski ndị dị n'akụkụ. Ebe ọ bụ na a ghaghị ịchịkwa omimi nke ntinye iji nweta nsonaazụ na-agbanwe agbanwe na nke a na-emegharị, anyị na-eji aka mee plọg site na bepụ n'ọbọ nke agịga ogologo 3 mm. Iji plọg a na-eme ka o doo anya omimi mkpọpu otu n'ime ahụ vertebral. N'ime nyocha nke mbụ, ndị dọkịta na-awa ahụ atọ na-arụ ọrụ na-arụ ọrụ na-achọpụta na agịga nlele 16 dị mfe iji rụọ ọrụ karịa agịga 18 ma ọ bụ ụzọ ndị ọzọ. Iji zere ọbara ọgbụgba dị ukwuu n'oge a na-egwupụta ihe, ijide agịga ka ọ dị ntakịrị ga-enye oghere ntinye nke dabara adaba, na-atụ aro na a ga-achịkwa ụfọdụ ogo MC n'ụzọ dị otú a.
Ọ bụ ezie na ọtụtụ ọmụmụ ihe ezubere iche maka MC, amachaghị banyere etiology na pathogenesis nke MC25,26,27. Dabere na ọmụmụ anyị gara aga, anyị chọpụtara na autoimmunity na-arụ ọrụ dị mkpa na omume na mmepe nke MC12. Ọmụmụ ihe a nyochara okwu ọnụ ọgụgụ nke IL-4, IL-17, na IFN-γ, bụ ụzọ dị iche iche dị iche iche nke sel CD4 + mgbe mkpali antigen gasịrị. N'ime ọmụmụ ihe anyị, ma e jiri ya tụnyere ndị na-adịghị mma, otu NPE nwere nkwupụta dị elu nke IL-4, IL-17, na IFN-γ, na ọkwa protein nke IL-4 na IL-17 dịkwa elu.
Na ụlọ ọgwụ, IL-17 mRNA okwu na-abawanye na sel NP site na ndị ọrịa nwere diski herniation28. A hụkwara ọkwa nkwupụta IL-4 na IFN-γ na nnukwu ụdị diski diski na-adịghị mkpakọ ma e jiri ya tụnyere njikwa ahụike29. IL-17 na-arụ ọrụ dị mkpa na mbufụt, mmerụ anụ ahụ na ọrịa autoimmune30 ma na-eme ka nzaghachi mgbochi na IFN-γ31 dịkwuo elu. A kọwo mmerụ ahụ anụ ahụ na-agbasa IL-17 na MRL/lpr ụmụ oke32 na autoimmunity-susceptible mice33. IL-4 nwere ike igbochi okwu nke cytokines proinflammatory (dị ka IL-1β na TNFa) na macrophage activation34. A kọrọ na okwu mRNA nke IL-4 dị iche na otu NPE tụnyere IL-17 na IFN-γ n'otu oge ahụ; Okwu mRNA nke IFN-γ n'ime otu NPE dị elu karịa nke ahụ na otu ndị ọzọ. Ya mere, mmepụta IFN-γ nwere ike ịbụ onye ogbugbo nke nzaghachi mkpali nke NP na-ebute. Nnyocha egosiwo na IFN-γ na-emepụta site na ọtụtụ ụdị cell, gụnyere ụdị 1 na-enyere aka T sel, mkpụrụ ndụ egbu egbu anụ ahụ, na macrophages35,36, na bụ isi pro-inflammatory cytokine nke na-akwalite nzaghachi mgbochi37.
Ọmụmụ ihe a na-egosi na nzaghachi autoimmune nwere ike itinye aka na ihe omume na mmepe nke MC. Luoma et al. chọpụtara na njirimara mgbaàmà nke MC na NP a ma ama yiri na MRI, ma ha abụọ na-egosi akara ngosi dị elu na usoro T2W38. Ụfọdụ cytokines ekwenyela na ha nwere njikọ chiri anya na ihe omume MC, dịka IL-139. Ma et al. tụrụ aro na mgbago elu ma ọ bụ ala nke NP nwere ike inwe mmetụta dị ukwuu na omume na mmepe nke MC12. Bobechko40 na Herzbein et al.41 kọrọ na NP bụ anụ ahụ na-edozi ahụ nke na-enweghị ike ịbanye na mgbasa ozi vaskụla site na ọmụmụ. NP protrusions na-ewebata ahụ ndị mba ọzọ n'ime ọbara, si otú a na-edozi mmeghachi omume autoimmune mpaghara42. Mmeghachi omume nke autoimmune nwere ike ime ka ọnụ ọgụgụ buru ibu nke ihe na-alụso ọrịa ọgụ, na mgbe ihe ndị a na-aga n'ihu na anụ ahụ, ha nwere ike ime mgbanwe na mgbaàmà43. N'ime ọmụmụ ihe a, nkwubi okwu nke IL-4, IL-17 na IFN-γ bụ ihe ndị na-adịghị ahụkebe, na-egosipụtakwa mmekọrịta chiri anya n'etiti NP na MCs44. Ụdị anụmanụ a na-eṅomi nke ọma NP ọganihu na ịbanye n'ime efere njedebe. Usoro a gosikwara mmetụta nke autoimmunity na MC.
Dị ka a tụrụ anya, ụdị anụmanụ a na-enye anyị ohere iji mụọ MC. Otú ọ dị, ihe nlereanya a ka nwere ụfọdụ njedebe: nke mbụ, n'oge oge nleba anya anụmanụ, ọ dị mkpa ka euthanized ụfọdụ oke bekee dị n'etiti etiti maka nyocha ihe omimi nke akụkọ ihe mere eme na molecular, ya mere, ụfọdụ anụmanụ "na-ada ada" ka oge na-aga. Nke abuo, ọ bụ ezie na e debere isi oge atọ n'ime ọmụmụ ihe a, ọ dị mwute ikwu na anyị mere otu ụdị MC (ụdị Modic m mgbanwe), n'ihi ya, ezughị ezu iji gosipụta usoro mmepe nke ọrịa mmadụ, ma ọ dị mkpa ka etinyekwuo oge. ka mma hụ mgbanwe mgbama niile. Nke atọ, mgbanwe n'ụdị anụ ahụ nwere ike igosi n'ụzọ doro anya site na ntụpọ akụkọ ihe mere eme, ma ụfọdụ usoro pụrụ iche nwere ike ikpughe mgbanwe microstructural na ụdị a. Dịka ọmụmaatụ, a na-eji microscopy ìhè polarized iji nyochaa nhazi nke fibrocartilage na diski intervertebral rabbit45. Mmetụta ogologo oge nke NP na MC na endplate chọrọ ọmụmụ ihe ọzọ.
Iri ise na anọ nwoke New Zealand ọcha rabbits (ibu ihe dị ka 2.5-3 n'arọ, afọ 3-3.5 ọnwa) na-enweghị usoro kewara n'ime sham ọrụ otu, muscle implantation otu (ME otu) na akwara mgbọrọgwụ implantation otu (NPE otu). Kọmitii ụkpụrụ omume nke ụlọ ọgwụ Tianjin kwadoro usoro nnwale niile, a na-emekwa usoro nnwale ahụ n'ụzọ siri ike dabere na ntuziaka akwadoro.
Emeela ụfọdụ ndozi na usoro ịwa ahụ nke S. Sobajima 46 . A na-etinye oke bekee ọ bụla n'ọnọdụ nkwụsịtụ n'akụkụ na ihu ihu nke diski intervertebral lumbar ise (IVDs) na-esote na-ekpughere site na iji usoro retroperitoneal posterolateral. A na-enye oke bekee ọ bụla mgbakasị izugbe (20% urethane, 5 ml/kg site na akwara ntị). A na-eme mbepụ akpụkpọ anụ ogologo oge site na nsọtụ ala nke ọgịrịga ruo n'ọnụ pelvic, 2 cm ventral na akwara paravertebral. Ekpughere azụ azụ azụ nke ọma site na L1 ruo L6 site na nhụsianya dị nkọ na nke na-adịghị mma nke anụ ahụ dị n'okpuru anụ ahụ, anụ ahụ retroperitoneal, na mọzụlụ (Fig. 6A). Ekpebiri ọkwa diski ahụ site na iji pelvic brim dị ka akara anatomical maka ọkwa diski L5-L6. Jiri agịga mkpị 16-nlele iji kpoo oghere dị nso na njedebe njedebe nke vertebra L5 ruo omimi nke 3 mm (Fig 6B). Jiri sirinji 5-ml iji mee ka ọkpụkpụ autologous pulposus dị na L1-L2 intervertebral diski (Fig 6C). Wepu pulposus nucleus ma ọ bụ akwara dị ka ihe ndị otu ọ bụla chọrọ si dị. Mgbe oghere a na-egwupụta ihe na-emikpu, a na-etinye sutures na-adọrọ adọrọ na fascia miri emi, fascia elu na akpụkpọ anụ, na-elezi anya ka ọ ghara imebi anụ ahụ periosteal nke vertebral ahụ n'oge ịwa ahụ.
(A) A na-ekpughe diski L5-L6 site na ụzọ retroperitoneal posterolateral. (B) Jiri agịga 16 ga-egwupụta oghere dị nso na njedebe L5. (C) A na-egbute ndị MF nke akpaaka.
A na-eji ọgwụ 20% urethane (5 ml / kg) na-elekọta ya site na vein ntị, a na-emegharị redio nke lumbar na 12, 16, na 20 izu mgbe ọ gasịrị.
A na-achụ oke bekee site na intramuscular injection nke ketamine (25.0 mg / kg) na sodium pentobarbital intravenous (1.2 g / kg) na 12, 16 na 20 izu mgbe ịwachara ahụ. E wepụrụ ọkpụkpụ azụ dum maka nyocha akụkọ ihe mere eme na nyocha nke ọma. A na-eji ntụgharị ntụgharị ọnụọgụ ọnụọgụ (RT-qPCR) na nchapụta ọdịda anyanwụ chọpụta mgbanwe na ihe ndị na-alụso ọrịa ọgụ.
A na-eme nyocha MRI na oke bekee site na iji 3.0 T clinical magnet (GE Medical Systems, Florence, SC) nke nwere ihe nkwụnye eriri ụkwụ nke orthogonal. Ejiri 20% urethane (5 mL / kg) were 20% urethane mechie oke bekee site na akwara ntị wee tinye ya n'ime magnet na mpaghara lumbar dabere na eriri okirikiri okirikiri 5-inch (GE Medical Systems). A na-enweta ihe oyiyi mpaghara mpaghara Coronal T2 (TR, 1445 ms; TE, 37 ms) iji kọwaa ebe diski lumbar si na L3-L4 ruo L5-L6. E nwetara Mpekere T2 dị arọ nke Sagittal na ntọala ndị a: usoro ntụgharị ngwa ngwa na oge nkwughachi (TR) nke 2200 ms na oge echo (TE) nke 70 ms, matrix; ebe a na-ahụ anya nke 260 na mkpali asatọ; Ọkpụkpụ ịkpụ bụ 2 mm, ọdịiche ahụ bụ 0.2 mm.
Mgbe e mechara foto ikpeazụ ma gbuo oke bekee ikpeazụ, e wepụrụ ihe nhụsianya ahụ, ihe nkedo akwara na NP diski maka nyocha akụkọ ihe mere eme. Edobere anụ ahụ n'ime 10% na-anọpụ iche formalin buffered maka izu 1, mebie ya na ethylenediaminetetraacetic acid, na akụkụ paraffin. A na-etinye ihe mgbochi anụ ahụ na paraffin wee bee n'ime akụkụ sagittal (oke 5 μm) site na iji microtome. Ejiri hematoxylin na eosin (H&E) metọọ akụkụ nke akụkụ ahụ.
Mgbe anakọtara diski intervertebral site na oke bekee n'ime otu ọ bụla, a na-ewepụta RNA mkpokọta site na iji kọlụm UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., China) dịka ntuziaka onye nrụpụta na sistemụ ntụgharị nke ImProm II (Promega Inc. , Madison, WI, USA). Emere ntụgharị ntụgharị.
Emere RT-qPCR site na iji Prism 7300 (Applied Biosystems Inc., USA) na SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) dịka ntuziaka onye nrụpụta siri dị. Olu mmeghachi omume PCR bụ 20 μl ma nwee 1.5 μl cDNA agbazere na 0.2 μM nke primer ọ bụla. Emebere primers site na OligoPerfect Designer (Invitrogen, Valencia, CA) nke Nanjing Golden Stewart Biotechnology Co., Ltd. (China) rụpụtara (Table 1). A na-eji ọnọdụ ịgba ígwè na-ekpo ọkụ na-esonụ: nzọụkwụ mbido polymerase mbụ na 94 ° C maka 2 min, mgbe ahụ 40 cycles nke 15 s ọ bụla na 94 ° C maka denaturation template, annealing maka 1 min na 60 ° C, ndọtị, na fluorescence. A na-eme nha maka nkeji 1 na 72 Celsius. A kwalitere nlele niile ugboro atọ ma jiri nkezi uru maka nyocha RT-qPCR. A na-enyocha data mmụba site na iji FlexStation 3 (Ngwaọrụ Molecular, Sunnyvale, CA, USA). IL-4, IL-17, na IFN-γ okwu mkpụrụ ndụ ka emebere ka njikwa endogenous (ACTB). A na-agbakọ ọkwa ngosipụta nke mRNA lekwasịrị anya site na iji usoro 2-ΔΔCT.
A na-ewepụta protein zuru ezu site na anụ ahụ site na iji anụ ahụ homogenizer na RIPA lysis buffer (nwere protease na phosphatase inhibitor mmanya) wee kechie ya na 13,000 rpm maka 20 min na 4 ° C iji wepụ anụ ahụ. Ebujuru micrograms nke protein iri ise n'otu ụzọ, kewapụrụ site na 10% SDS-PAGE, wee bufee ya na akpụkpọ anụ PVDF. Emere mgbochi na 5% mmiri ara ehi na-adịghị abụba na saline Tris-buffered (TBS) nwere 0.1% Tween 20 maka 1 h n'ime ụlọ okpomọkụ. A na-etinye akpụkpọ ahụ ahụ na bekee anti-decorin primary antibody (diluted 1:200; Boster, Wuhan, China) (gbasara 1:200; Bioss, Beijing, China) n'abali na 4 Celsius ma meghachi omume n'ụbọchị nke abụọ; nwere mgbochi nke abụọ (ewu mgbochi rabbit immunoglobulin G na 1:40,000 dilution) jikọtara ya na horseradish peroxidase (Boster, Wuhan, China) maka elekere 1 n'ime ụlọ. Achọpụtara akara ntụpọ ọdịda anyanwụ site na mmụba kemiluminescence na akpụkpọ ahụ chemiluminescent ka ọkụ ọkụ X-ray gasịrị. Maka nyocha densitometric, a na-enyocha ma gbakọọ ntụpọ site na iji ngwanrọ BandScan yana egosipụtara nsonaazụ ya dị ka oke nke mkpụrụ ndụ immunoreactivity ebumnuche na tubulin immunoreactivity.
Ejiri ngwugwu ngwanrọ SPSS16.0 (SPSS, USA) mee ngụkọta ọnụ ọgụgụ. E gosipụtara data anakọtara n'oge ọmụmụ ihe dị ka ± ọkọlọtọ ọkọlọtọ (pụtara ± SD) wee nyochaa site na iji otu ụzọ nyocha nke mgbanwe dị iche iche (ANOVA) iji chọpụta ọdịiche dị n'etiti otu abụọ ahụ. P <0.05 ka e weere dị ka ihe dị mkpa.
Ya mere, nguzobe nke ụdị anụmanụ nke MC site n'itinye autologous NPs n'ime vertebral ahụ ma na-eme nchọpụta macroanatomical, MRI analysis, histological evaluation and molecular biological analysis nwere ike ịghọ ngwá ọrụ dị mkpa maka nyochaa na ịghọta usoro nke MC mmadụ na ịmepụta ọgwụgwọ ọhụrụ. ntinye aka.
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