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Nhazi nke ụdị anụmanụ nke mgbanwe modic (MC) bụ ihe dị mkpa maka ịmụ MC. E kewara oke bekee ọcha iri ise na anọ nke New Zealand n'ime otu sham-operation, otu muscle implantation (ME group) na otu nucleus pulposus implantation (NPE group). N'ime otu NPE, a hụrụ diski intervertebral site na ụzọ ịwa ahụ lumbar anterolateral ma jiri agịga kụpụ ahụ vertebral L5 nso efere njedebe. E si na diski intervertebral L1/2 wepụta NP ma gbanye ya n'ime ya. Ịkụpụta oghere n'ọkpụkpụ subchondral. Usoro ịwa ahụ na ụzọ ịgbapụ n'ime otu itinye akwara na otu sham-operation bụ otu ihe ahụ dị na otu itinye akwara NP. N'ime otu ME, e tinyere otu akwara n'ime oghere ahụ, ebe n'ime otu ọrụ sham-operation, etinyeghị ihe ọ bụla n'ime oghere ahụ. Mgbe a wachara ahụ, e mere nyocha MRI na nnwale bayọlọjik molekul. Mgbaàmà dị na otu NPE gbanwere, mana enweghị mgbanwe mgbaàmà doro anya na otu ọrụ sham-operation na otu ME. Nnyocha histological gosiri na a hụrụ mmụba anụ ahụ na-adịghị mma n'ebe a kụnyere ya, nakwa na ngosipụta nke IL-4, IL-17 na IFN-γ mụbara n'ime 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 nke a na-ahụ na magnet resonance imaging (MRI). Ha na-adịkarị n'ime ndị nwere mgbaàmà ndị metụtara ya1. Ọtụtụ ọmụmụ ihe emeela ka mkpa nke MC pụta ìhè n'ihi njikọ ya na obere mgbu azụ (LBP)2,3. de Roos et al.4 na Modic et al.5 buru ụzọ kọwaa ụdị atọ dị iche iche nke nsogbu mgbaàmà subchondral na ụmị ọkpụkpụ vertebral. Mgbanwe ụdị Modic I bụ hypointense na usoro T1-weighted (T1W) na hyperintense na usoro T2-weighted (T2W). Ọrịa a na-ekpughe endplates fissure na anụ ahụ granulation vascular dị n'akụkụ na ụmị ọkpụkpụ. Mgbanwe ụdị Modic II na-egosi signal dị elu na usoro T1W na T2W. N'ụdị mmerụ ahụ a, enwere ike ịchọta mbibi endplates, yana mgbanwe histological fatty nke ụmị ọkpụkpụ dị n'akụkụ. Mgbanwe ụdị Modic III na-egosi signal dị ala na usoro T1W na T2W. A hụla ọnya Sclerotic nke kwekọrọ na endplates6. A na-ewere MC dị ka ọrịa na-efe efe nke ọkpụkpụ azụ ma nwee njikọ chiri anya na ọtụtụ ọrịa na-emebi emebi nke ọkpụkpụ azụ7,8,9.
N'ịtụle data dịnụ, ọtụtụ nnyocha enyela nghọta zuru ezu banyere isi ihe kpatara ya na usoro ọrịa nke MC. Albert na ndị otu ya tụrụ aro na MC nwere ike ịbụ site na mmụba diski8. Hu na ndị otu ya kwuru na MC kpatara nnukwu mmebi diski10. Kroc tụrụ aro echiche nke "mgbawa diski dị n'ime," nke na-ekwu na mmerụ ahụ diski ugboro ugboro nwere ike ibute obere anya mmiri na mpempe akwụkwọ njedebe. Mgbe e guzobere mgbawa, mbibi nke nucleus pulposus (NP) nwere ike ịkpalite mmeghachi omume autoimmune, nke na-edugakwa na mmepe nke MC11. Ma na ndị otu ya kerịta echiche yiri nke ahụ ma kọọ na autoimmunity nke NP kpatara na-arụ ọrụ dị mkpa na pathogenesis nke MC12.
Mkpụrụ ndụ ndị na-alụso ọrịa ọgụ, ọkachasị mkpụrụ ndụ ndị na-enyere aka na CD4+ T, na-arụ ọrụ dị oke mkpa na mmepe nke ọrịa autoimmunity13. Nkewa Th17 achọpụtara n'oge na-adịbeghị anya na-emepụta cytokine proinflammatory IL-17, na-akwalite ngosipụta chemokine, ma na-akpali mkpụrụ ndụ T n'ime akụkụ ahụ mebiri emebi iji mepụta IFN-γ14. Mkpụrụ ndụ Th2 na-arụkwa ọrụ pụrụ iche na mmepe nke nzaghachi ahụ ji alụso ọrịa ọgụ. Ngosipụta nke IL-4 dị ka mkpụrụ ndụ Th2 nnọchite anya nwere ike ibute nnukwu nsonaazụ immunopathological15.
Ọ bụ ezie na e meela ọtụtụ nnyocha ahụike na MC16,17,18,19,20,21,22,23,24, mana enwebeghị ụdị nnwale anụmanụ kwesịrị ekwesị nke nwere ike iṅomi usoro MC nke na-emekarị n'ime mmadụ ma enwere ike iji ya nyochaa isi ihe kpatara ya ma ọ bụ ọgwụgwọ ọhụrụ dịka ọgwụgwọ ezubere iche. Ruo ugbu a, ọ bụ naanị ụdị anụmanụ ole na ole nke MC ka akọọrọ iji mụọ usoro ọrịa dị n'okpuru.
Dabere na ozizi autoimmune nke Albert na Ma tụrụ aro, ọmụmụ ihe a guzobere ụdị MC oke bekee dị mfe ma nwee ike ịmụgharị site na ịgbanye NP n'akụkụ efere njedebe vertebral a gwuru egwu. Ebumnuche ndị ọzọ bụ ileba anya na njirimara histological nke ụdị anụmanụ ma nyochaa usoro kpọmkwem nke NP na mmepe nke MC. Iji mezuo nke a, anyị na-eji usoro dị ka molekul bayọlọji, MRI, na ọmụmụ histological iji mụọ ọganihu nke MC.
Oke bekee abụọ nwụrụ n'ihi ọbara ọgbụgba n'oge a na-awa ahụ, oke bekee anọ nwụrụ n'oge a na-akụnwụ ahụ n'oge MRI. Oke bekee iri anọ na asatọ fọdụrụ dị ndụ ma ha egosighi ihe mgbaàmà ọ bụla gbasara omume ma ọ bụ akwara ozi mgbe a wachara ha ahụ.
MRI na-egosi na ike mgbaàmà nke anụ ahụ e tinyere n'ime oghere dị iche iche dị iche. Ike mgbaàmà nke ahụ L5 vertebral na otu NPE gbanwere nwayọ nwayọ na izu 12, 16 na 20 mgbe etinyere ya (usoro T1W gosiri obere mgbaàmà, usoro T2W gosikwara akara agwakọtara yana obere mgbaàmà) (Foto 1C), ebe ọdịdị MRI nke otu abụọ ndị ọzọ nke akụkụ ndị a gbanyere mkpọrọgwụ nọgidere na-adịgide adịgide n'otu oge ahụ (Foto 1A, B).
(A) MRI nke ọkpụkpụ azụ oke bekee na-anọchite anya n'usoro ihe ngosi ugboro atọ. Ahụghị ihe mgbaàmà ọ bụla na onyonyo nke otu ọrụ ịwa ahụ. (B) Àgwà mgbaàmà nke ahụ vertebral na otu ME yiri nke dị na otu ọrụ ịwa ahụ, a hụghịkwa mgbanwe mgbaàmà dị mkpa na ebe a na-etinye ya ka oge na-aga. (C) Na otu NPE, a na-ahụ mgbaàmà dị ala nke ọma na usoro T1W, a na-ahụkwa mgbaàmà agwakọtara na mgbaàmà dị ala nke ọma na usoro T2W. Site na oge izu iri na abụọ ruo oge izu iri abụọ, mgbaàmà dị elu na-agbagharị agbagharị gbara akara dị ala na usoro T2W na-ebelata.
A pụrụ ịhụ hyperplasia ọkpụkpụ doro anya n'ebe a na-etinye ahụ vertebral n'ime otu NPE, hyperplasia ọkpụkpụ na-apụtakwa ngwa ngwa site na izu iri na abụọ ruo iri abụọ (Fig. 2C) ma e jiri ya tụnyere otu NPE, a hụghị mgbanwe dị ukwuu n'ahụ vertebral ndị a tụrụ atụ; otu Sham na otu ME (Fig. 2C) 2A,B).
(A) Elu ahụ vertebral dị n'akụkụ e tinyere ya dị nnọọ nro, oghere ahụ na-agwọ nke ọma, ọ dịghịkwa hyperplasia n'ahụ vertebral. (B) Ọdịdị nke ebe e tinyere ya n'ime otu ME yiri nke dị n'òtù ọrụ aghụghọ, ọ dịghịkwa mgbanwe doro anya n'ọdịdị nke ebe e tinyere ya ka oge na-aga. (C) Hyperplasia ọkpụkpụ mere n'ebe e tinyere ya n'ime otu NPE. Hyperplasia ọkpụkpụ mụbara ngwa ngwa ma gbasaa site na diski intervertebral ruo n'ahụ vertebral dị n'akụkụ.
Nnyocha Histological na-enye ozi zuru ezu gbasara nhazi ọkpụkpụ. Foto nke 3 na-egosi foto nke akụkụ ndị e tinyere H&E n'ime ahụ mgbe a wachara ahụ. N'ime otu ịwa ahụ sham-operation, chondrocytes ahụ dị mma nke ọma, achọpụtaghịkwa mmụba nke mkpụrụ ndụ (Fig. 3A). Ọnọdụ dị na otu ME yiri nke dị na otu ịwa ahụ sham-operation (Fig. 3B). Agbanyeghị, n'ime otu NPE, a hụrụ ọtụtụ chondrocytes na mmụba nke mkpụrụ ndụ dị ka NP na ebe a kụnyere ha (Fig. 3C);
(A) A na-ahụ Trabeculae n'akụkụ efere njedebe, a na-ahazi chondrocytes nke ọma na nha na ọdịdị nke mkpụrụ ndụ ahụ, ọ dịghịkwa amụba (ugboro 40). (B) Ọnọdụ ebe a na-etinye ihe n'ime otu ME yiri nke otu sham. A na-ahụ Trabeculae na chondrocytes, mana ọ dịghị mmụba doro anya na ebe a na-etinye ihe (ugboro 40). (B) A na-ahụ na chondrocytes na mkpụrụ ndụ dị ka NP na-amụba nke ukwuu, ọdịdị na nha nke chondrocytes adịghị nhata (ugboro 40).
A hụrụ mRNA interleukin 4 (IL-4) mRNA, mRNA interleukin 17 (IL-17), na mRNA interferon γ (IFN-γ) n'ime otu NPE na ME. Mgbe a tụnyere ọkwa ngosipụta nke mkpụrụ ndụ ihe nketa ebumnuche, ngosipụta mkpụrụ ndụ ihe nketa nke IL-4, IL-17, na IFN-γ mụbara nke ukwuu na otu NPE ma e jiri ya tụnyere nke otu ME na otu ọrụ sham (Foto 4) (P < 0.05). Ma e jiri ya tụnyere otu ọrụ sham, ọkwa ngosipụta nke IL-4, IL-17, na IFN-γ n'ime otu ME mụbara naanị ntakịrị ma eruteghị mgbanwe ọnụọgụgụ (P > 0.05).
Ngosipụta mRNA nke IL-4, IL-17 na IFN-γ n'ime otu NPE gosiri usoro dị elu karịa nke ndị nọ na otu ọrụ sham na otu ME (P < 0.05).
N'ụzọ dị iche, ọkwa ngosipụta n'ime otu ME egosighi nnukwu ọdịiche (P>0.05).
E jiri ọgwụ mgbochi ndị dị n'ahịa mee nyocha Western blot iji kwado usoro ngosipụta mRNA agbanwere. Dịka egosiri na Foto 5A,B, ma e jiri ya tụnyere otu ME na otu ọrụ sham, ọkwa protein nke IL-4 na IL-17 na otu NPE mụbara nke ukwuu (P < 0.05). Ma e jiri ya tụnyere otu ọrụ sham, ọkwa protein nke IL-4 na IL-17 na otu ME erughịkwa mgbanwe dị mkpa na ọnụọgụgụ (P> 0.05).
(A) Ọkwa protein nke IL-4 na IL-17 n'ime otu NPE dị elu nke ukwuu karịa nke dị na otu ME na otu placebo (P < 0.05). (B) Western blot histogram.
N'ihi obere ọnụọgụgụ nke ihe nlele mmadụ e nwetara n'oge ịwa ahụ, ọmụmụ ihe doro anya na nke zuru ezu gbasara ọrịa MC siri ike. Anyị gbalịrị ịmepụta ihe nlereanya anụmanụ nke MC iji mụọ usoro ọrịa nwere ike ime. N'otu oge ahụ, e jiri nyocha redio, nyocha histological na nyocha bayọlọjik molekul soro usoro MC nke NP autograft kpaliri. N'ihi ya, ihe nlereanya ntinye NP butere mgbanwe nwayọ nwayọ na ike mgbaàmà site na izu iri na abụọ ruo isi oge izu iri abụọ (mgbaàmà dị ala agwakọtara na usoro T1W na obere akara na usoro T2W), na-egosi mgbanwe anụ ahụ, na nyocha bayọlọjik histological na molekul kwadoro nsonaazụ nke ọmụmụ redio.
Nsonaazụ nke nnwale a na-egosi na mgbanwe anya na histological mere n'ebe mmebi ahụ vertebral dị na otu NPE. N'otu oge ahụ, a hụrụ ngosipụta nke mkpụrụ ndụ ihe nketa IL-4, IL-17 na IFN-γ, yana IL-4, IL-17 na IFN-γ, na-egosi na mmebi nke anụ ahụ autologous nucleus pulposus na ahụ vertebral nwere ike ibute usoro mgbanwe mgbaàmà na morphological. Ọ dị mfe ịchọpụta na njirimara mgbaàmà nke ahụ vertebral nke ụdị anụmanụ (akara dị ala na usoro T1W, akara agwakọtara na akara dị ala na usoro T2W) yiri nke mkpụrụ ndụ vertebral mmadụ, njirimara MRI na-akwadokwa ihe a hụrụ na histology na anatomy buru ibu, ya bụ, mgbanwe na mkpụrụ ndụ vertebral na-aga n'ihu. Ọ bụ ezie na nzaghachi mkpali nke mmerụ ahụ dị oke njọ kpatara nwere ike ịpụta ozugbo a gbanyere ya, nsonaazụ MRI gosiri na mgbanwe mgbaàmà na-abawanye ụba na-apụta izu iri na abụọ mgbe a gbanyere ya ma nọgide ruo izu iri abụọ na-enweghị ihe ịrịba ama ọ bụla nke mgbake ma ọ bụ mgbanwe nke mgbanwe MRI. Nsonaazụ ndị a na-egosi na autologous vertebral NP bụ ụzọ a pụrụ ịtụkwasị obi maka ịmepụta MV na-aga n'ihu na oke bekee.
Ụdị mgbapu a chọrọ nkà zuru oke, oge, na mgbalị ịwa ahụ. N'ime nnwale mbụ, ịgbawa ma ọ bụ mkpali gabigara ókè nke akụkụ paravertebral ligamentous nwere ike ibute mmepe nke osteophytes vertebral. Ekwesịrị ịkpachara anya ka a ghara imebi ma ọ bụ kpasuo diski ndị dị nso. Ebe ọ bụ na a ga-achịkwa omimi nke ịbanye n'ime ahụ iji nweta nsonaazụ na-agbanwe agbanwe na nke a pụrụ imegharị, anyị ji aka mee plọg site n'ibipụ mkpuchi agịga ogologo 3 mm. Iji plọg a na-eme ka omimi ịgbapụ otu n'ahụ vertebral dị. Na nnwale mbụ, ndị dọkịta atọ na-awa ahụ orthopedic tinyere aka na ọrụ ahụ chọpụtara na agịga 16 dị mfe iji rụọ ọrụ karịa agịga 18 ma ọ bụ ụzọ ndị ọzọ. Iji zere ọbara ọgbụgba gabigara ókè n'oge ịgbapụ, ijide agịga ahụ ruo oge ụfọdụ ga-enye oghere ntinye kwesịrị ekwesị, na-egosi na enwere ike ijikwa ọkwa MC n'ụzọ dị otu a.
Ọ bụ ezie na ọtụtụ nnyocha ezubere maka MC, a maghị ihe gbasara isi ihe kpatara ya na ọrịa nke MC25,26,27. Dabere na ọmụmụ ihe anyị gara aga, anyị chọpụtara na ọrịa onwe onye na-arụ ọrụ dị mkpa na mmepe na mmepe nke MC12. Ọmụmụ ihe a lere anya na ngosipụta ọnụọgụ nke IL-4, IL-17, na IFN-γ, nke bụ ụzọ isi kewaa mkpụrụ ndụ CD4+ mgbe mkpali antigen gasịrị. N'ọmụmụ ihe anyị, ma e jiri ya tụnyere otu na-adịghị mma, otu NPE nwere ngosipụta dị elu nke IL-4, IL-17, na IFN-γ, ọkwa protein nke IL-4 na IL-17 dịkwa elu.
N'ihe gbasara ahụike, a na-amụba mmịpụta mRNA IL-17 na mkpụrụ ndụ NP site n'aka ndị ọrịa nwere mmịpụta diski28. A hụkwara mmụba nke ọkwa mmịpụta IL-4 na IFN-γ na ụdị mmịpụta diski dị oke njọ ma e jiri ya tụnyere njikwa ahụike29. IL-17 na-arụ ọrụ dị mkpa na mbufụt, mmerụ ahụ anụ ahụ na ọrịa autoimmune30 ma na-eme ka nzaghachi ahụ ji alụso ọrịa ọgụ dịkwuo mma na IFN-γ31. Akọpụtala mmerụ ahụ anụ ahụ IL-17 nke na-akwalite na ụmụ oke MRL/lpr32 na ụmụ oke nwere ike ịnwe mmịpụta autoimmune33. IL-4 nwere ike igbochi ngosipụta nke cytokines proinflammatory (dị ka IL-1β na TNFα) na macrophage activation34. A kọrọ na mRNA ngosipụta nke IL-4 dị iche na otu NPE ma e jiri ya tụnyere IL-17 na IFN-γ n'otu oge ahụ; MRNA ngosipụta nke IFN-γ na otu NPE dị elu karịa nke dị na otu ndị ọzọ. Ya mere, mmepụta IFN-γ nwere ike ịbụ onye na-eme ihe n'etiti nzaghachi mkpali nke NP intercalation kpatara. Nnyocha egosila na ọtụtụ ụdị mkpụrụ ndụ na-emepụta IFN-γ, gụnyere mkpụrụ ndụ T nke na-arụ ọrụ nke 1, mkpụrụ ndụ ihe nketa na-egbu egbu, na macrophages35,36, ọ bụkwa cytokine dị mkpa nke na-akwalite mmeghachi omume mgbochi ọrịa37.
Nnyocha a na-egosi na mmeghachi omume autoimmune nwere ike itinye aka na mmepe na mmepe nke MC. Luoma na ndị otu ya chọpụtara na njirimara mgbaàmà nke MC na NP a ma ama yiri nke MRI, ha abụọ na-egosikwa akara dị elu na usoro T2W38. E gosila na ụfọdụ cytokines nwere njikọ chiri anya na ihe omume nke MC, dị ka IL-139. Ma na ndị otu ya tụrụ aro na mmụba elu ma ọ bụ ala nke NP nwere ike inwe nnukwu mmetụta na mmepe na mmepe nke MC12. Bobechko40 na Herzbein na ndị otu ya kọrọ na NP bụ anụ ahụ na-adịghị anabata immunosuppression nke na-enweghị ike ịbanye na mgbasa ọbara site na ọmụmụ. Mgbapụta NP na-ewebata ahụ mba ọzọ n'ime ọbara, si otú a na-eme ka mmeghachi omume autoimmune mpaghara dị na mpaghara dị iche iche dị. Mmeghachi omume autoimmune nwere ike ịkpalite ọtụtụ ihe mgbochi ọrịa, mgbe ihe ndị a na-apụta ìhè mgbe niile na anụ ahụ, ha nwere ike ibute mgbanwe na mgbaama43. N'ọmụmụ ihe a, oke mmụba nke IL-4, IL-17 na IFN-γ bụ ihe ndị na-egbochi ọrịa nkịtị, na-egosikwa mmekọrịta chiri anya dị n'etiti NP na MCs44. Ụdị anụmanụ a na-eṅomi ọganihu NP na ntinye ya n'ime efere njedebe. Usoro a kpughekwara mmetụta nke mgbochi ọrịa onwe onye na MC.
Dịka a tụrụ anya ya, ụdị anụmanụ a na-enye anyị ikpo okwu nwere ike ịmụ MC. Agbanyeghị, ụdị a ka nwere ụfọdụ oke: nke mbụ, n'oge usoro nlele anụmanụ, ụfọdụ oke bekee dị n'etiti kwesịrị ka e gbuo ha maka nnwale histological na molekul bayọlọji, yabụ ụfọdụ anụmanụ "anaghị eji ha eme ihe" ka oge na-aga. Nke abụọ, ọ bụ ezie na e debere isi ihe atọ n'ọmụmụ ihe a, ọ dị mwute ikwu na anyị mere naanị otu ụdị MC (ụdị Modic I gbanwere), yabụ na ọ ezughị iji nọchite anya usoro mmepe ọrịa mmadụ, a ga-edobekwa isi ihe ndị ọzọ iji hụ mgbanwe mgbaàmà niile nke ọma. Nke atọ, enwere ike igosi mgbanwe ndị dị na nhazi anụ ahụ nke ọma site na histological staining, mana ụfọdụ usoro pụrụ iche nwere ike ikpughe mgbanwe microstructural na ụdị a nke ọma. Dịka ọmụmaatụ, ejiri mikroskopi ọkụ polarized iji nyochaa nhazi nke fibrocartilage na diski intervertebral rabbit45. Mmetụta ogologo oge nke NP na MC na endplate chọrọ ọmụmụ ihe ọzọ.
E kewara oke bekee ọcha iri ise na anọ nke New Zealand (nke dị ihe dị ka kilogram 2.5-3, dị ọnwa 3-3.5) n'ụzọ na-enweghị usoro, otu ịwa ahụ aghụghọ, otu ịkụnye akwara (ìgwè ME) na otu ịkụnye mgbọrọgwụ akwara (òtù NPE). Kọmitii Omume nke Ụlọ Ọgwụ Tianjin kwadoro usoro nnwale niile, e mekwara usoro nnwale ahụ n'ụzọ doro anya dịka ntuziaka akwadoro si dị.
E meela ụfọdụ mgbanwe na usoro ịwa ahụ nke S. Sobajima 46. E tinyere oke bekee ọ bụla n'ọnọdụ mpụta n'akụkụ, e wee jiri usoro posterolateral retroperitoneal kpughee elu ihu nke diski intervertebral lumbar ise na-esochi onwe ha (IVDs) site na iji usoro posterolateral retroperitoneal. E nyere oke bekee ọ bụla ọgwụ mgbu izugbe (20% urethane, 5 ml/kg site na akwara ntị). E gbubiri akpụkpọ ahụ ogologo oge site na nsọtụ ala nke ọgịrịga ruo n'akụkụ pelvic, 2 cm ventral ruo akwara paravertebral. E kpughere ọkpụkpụ azụ anterolateral aka nri site na L1 ruo L6 site na nkewa dị nkọ na nke na-adịghị mma nke anụ ahụ dị n'okpuru ala, anụ ahụ retroperitoneal, na akwara (Fig. 6A). E jiri pelvic brim chọpụta ọkwa diski ahụ dị ka ihe nnọchianya anatomical maka ọkwa diski L5-L6. Jiri agịga puncture 16-gauge iji gwuo oghere dị nso na efere njedebe nke vertebra L5 ruo omimi nke 3 mm (Fig. 6B). Jiri sirinji 5-ml mee ka akwara autologous dị n'ime diski intervertebral L1-L2 (Foto 6C). Wepụ akwara nucleus ma ọ bụ akwara dịka otu ọ bụla chọrọ si dị. Mgbe e mechachara oghere ahụ miri emi, a na-etinye ihe ndị a na-amịkọrọ n'ime akwara miri emi, akwara elu na akpụkpọ ahụ, na-akpachara anya ka a ghara imebi anụ ahụ periosteal nke vertebral n'oge ịwa ahụ.
(A) A na-ekpughe diski L5–L6 site na ụzọ posterolateral retroperitoneal. (B) Jiri agịga 16-gauge gwuo oghere dị nso na njedebe L5. (C) A na-egbutu MF ndị na-adịghị ike.
E nyere ọgwụ mgbu izugbe site na iji 20% urethane (5 ml/kg) nke e nyere site na akwara ntị, e mekwara rediograf ọkpụkpụ azụ lumbar ugboro ugboro n'izu iri na abụọ, iri na isii, na iri abụọ mgbe a wachara ahụ.
A chụpụrụ oke bekee site n'ịgba ketamine n'ime ahụ (25.0 mg/kg) na sodium pentobarbital n'ime ọbara (1.2 g/kg) n'ime izu 12, 16 na 20 ka a wachara ha ahụ. E wepụrụ ọkpụkpụ azụ ahụ dum maka nyocha histological ma mee ezigbo nyocha. E jiri Quantitative reverse transcription (RT-qPCR) na Western blotting chọpụta mgbanwe na ihe ndị na-alụso ọrịa ọgụ.
E mere nyocha MRI n'ime oke bekee site na iji magnet 3.0 T clinical (GE Medical Systems, Florence, SC) nke nwere ihe nnata akwara ụkwụ orthogonal. E jiri urethane 20% (5 mL/kg) mee ka oke bekee nwee ahụ mgbu site na akwara ntị wee tinye ya n'elu magnet ahụ na mpaghara lumbar nke etiti ya na okirikiri elu okirikiri nke sentimita 5 (GE Medical Systems). E nwetara onyonyo localizer coronal T2-weighted (TR, 1445 ms; TE, 37 ms) iji kọwaa ebe diski lumbar dị site na L3-L4 ruo L5-L6. E nwetara iberibe T2-weighted plane sagittal site na ntọala ndị a: usoro spin-echo ngwa ngwa yana oge ugboro ugboro (TR) nke 2200 ms na oge echo (TE) nke 70 ms, matrix; ubi anya nke 260 na mkpali asatọ; Okpukpo ịkpụ bụ 2 mm, oghere ahụ bụ 0.2 mm.
Mgbe e sere foto ikpeazụ ma gbuo oke bekee ikpeazụ, ewepụrụ diski sham, nke akwara dị n'ime ya, na NP maka nyocha histological. E tinyere anụ ahụ na formalin nke nwere 10% neutral buffered ruo otu izu, jiri ethylenediaminetetraacetic acid decalcium, ma kewaa paraffin. E tinyere blọk anụ ahụ na paraffin ma bepụ ya n'ime akụkụ sagittal (5 μm ọkpụrụkpụ) site na iji microtome. E tinyere hematoxylin na eosin (H&E) n'ime akụkụ ndị ahụ.
Mgbe a chịkọtachara diski intervertebral site n'aka oke bekee n'ime otu ọ bụla, e jiri kọlụm UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., China) wepụta RNA zuru oke dịka ntuziaka nke onye nrụpụta si dị na sistemụ ntụgharị azụ ImProm II (Promega Inc., Madison, WI, USA). E mere ntụgharị azụ.
E jiri Prism 7300 (Applied Biosystems Inc., USA) na SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) mee RT-qPCR dịka ntuziaka onye nrụpụta si dị. Olu mmeghachi omume PCR bụ 20 μl ma nwee 1.5 μl nke cDNA a gwakọtara agwakọta na 0.2 μM nke primer ọ bụla. OligoPerfect Designer (Invitrogen, Valencia, CA) mepụtara primers ma Nanjing Golden Stewart Biotechnology Co., Ltd. (China) mepụtara ya (Tebụl 1). Ejiri ọnọdụ ịgbagharị okpomọkụ ndị a: nzọụkwụ mmalite nke polymerase na 94°C maka nkeji 2, wee jiri usoro 40 nke s 15 nke ọ bụla na 94°C maka denaturation template, annealing maka nkeji 1 na 60°C, ndọtị, na fluorescence. E mere nha maka nkeji 1 na 72°C. E mere ka ihe nlele niile dịkwuo elu ugboro atọ, e jirikwa uru nkezi mee ihe maka nyocha RT-qPCR. E jiri FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA) nyochaa data mmụba. E mere ka ngosipụta mkpụrụ ndụ ihe nketa IL-4, IL-17, na IFN-γ dị ka njikwa endogenous (ACTB). E jiri usoro 2-ΔΔCT gbakọọ ọkwa ngosipụta nke mRNA ebumnuche.
E si n'anụ ahụ wepụta protein zuru oke site na anụ ahụ site na iji ihe na-eme ka anụ ahụ dị n'ime ihe nchekwa RIPA lysis (nke nwere ihe na-egbochi protease na phosphatase) wee kpụọ ya na 13,000 rpm maka nkeji iri abụọ na 4°C iji wepụ ihe ndị mebiri emebi anụ ahụ. E tinyere microgram iri ise nke protein n'otu ụzọ, kewaa ya site na 10% SDS-PAGE, wee bufee ya na akpụkpọ ahụ PVDF. E tinyere mgbochi na mmiri ara ehi kpọrọ nkụ nke 5% na-abụghị abụba na saline Tris-buffered (TBS) nke nwere 0.1% Tween 20 maka awa 1 na okpomọkụ ụlọ. E tinyere ihe mgbochi antibody anti-decorin primary (nke a gwakọtara agwakọta 1:200; Boster, Wuhan, China) (nke a gwakọtara agwakọta 1:200; Bioss, Beijing, China) n'abalị na 4°C ma meghachi omume na ụbọchị nke abụọ; ya na ọgwụ mgbochi nke abụọ (mgbochi mgbochi rabbit immunoglobulin G na 1:40,000 dilution) jikọtara ya na horseradish peroxidase (Boster, Wuhan, China) ruo otu awa n'ọnọdụ okpomọkụ ụlọ. A chọpụtara akara Western blot site na mmụba nke chemiluminescence na akpụkpọ ahụ chemiluminescent mgbe e mechara radieshon X-ray. Maka nyocha densitometric, ejiri ngwanrọ BandScan nyochaa ma tụọ blots ahụ, e wee gosipụta nsonaazụ ya dị ka oke nke immunoreactivity mkpụrụ ndụ ihe nketa lekwasịrị anya na tubulin immunoreactivity.
E ji ngwugwu ngwanrọ SPSS16.0 (SPSS, USA) mee ngụkọ ọnụọgụgụ. E gosipụtara data anakọtara n'oge ọmụmụ ihe ahụ dị ka nkezi ± ọkọlọtọ divideration (nkezi ± SD) ma nyochaa ya site na iji otu ụzọ ugboro ugboro nyocha nke mgbanwe (ANOVA) iji chọpụta ọdịiche dị n'etiti otu abụọ ahụ. A na-ewere P < 0.05 dị ka ihe dị mkpa na ọnụọgụgụ.
Ya mere, ntọala nke ụdị anụmanụ nke MC site na itinye NPs onwe onye n'ime ahụ vertebral na ime nyocha macroanatomical, nyocha MRI, nyocha histological na nyocha biological molecular nwere ike ịghọ ngwaọrụ dị mkpa maka inyocha na ịghọta usoro nke MC mmadụ na ịmepụta ọgwụgwọ ọhụrụ.
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