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Kafa samfuran dabbobi na canjin yanayi (MC) muhimmin tushe ne na nazarin MC. An raba zomaye fararen fata hamsin da huɗu na New Zealand zuwa rukunin tiyatar sham-operation, rukunin dasa tsoka (ƙungiyar ME) da ƙungiyar dasa nucleus pulposus (ƙungiyar NPE). A cikin ƙungiyar NPE, an fallasa faifan intervertebral ta hanyar tiyatar lumbar anterolateral kuma an yi amfani da allura don huda jikin ƙashin baya na L5 kusa da farantin ƙarshe. An cire NP daga faifan intervertebral na L1/2 ta hanyar sirinji sannan aka allura a ciki. Ana haƙa rami a cikin ƙashin subchondral. Hanyoyin tiyata da hanyoyin haƙa a cikin ƙungiyar dasa tsoka da ƙungiyar tiyatar sham-operation sun yi daidai da waɗanda ke cikin ƙungiyar dasa tsoka ta NP. A cikin ƙungiyar ME, an sanya wani yanki na tsoka a cikin ramin, yayin da a cikin ƙungiyar tiyatar sham-operation, babu abin da aka sanya a cikin ramin. Bayan tiyatar, an yi gwajin MRI da gwajin kwayoyin halitta. Siginar da ke cikin ƙungiyar NPE ta canza, amma babu wani canji a bayyane a cikin ƙungiyar tiyatar sham-operation da ƙungiyar ME. Binciken da aka yi a kan ƙwayoyin halitta ya nuna cewa an lura da yawan ƙwayoyin halitta marasa kyau a wurin da aka dasa su, kuma an ƙara yawan bayyanar IL-4, IL-17 da IFN-γ a cikin ƙungiyar NPE. Dasa NP a cikin ƙashin subchondral na iya samar da samfurin dabba na MC.
Canje-canjen Modic (MC) raunuka ne na ƙarshen vertebral da kuma ɓargon ƙashi da ke kusa da su waɗanda ake iya gani a hoton maganadisu (MRI). Suna da yawa a cikin mutanen da ke da alamun alaƙa1. Nazari da yawa sun jaddada mahimmancin MC saboda alaƙarsa da ƙarancin ciwon baya (LBP)2,3. de Roos et al.4 da Modic et al.5 sun fara bayyana nau'ikan rashin daidaituwar siginar subchondral guda uku a cikin ɓargon ƙashi na vertebral. Canje-canjen Modic nau'in I sune hypointense akan jerin T1-weighted (T1W) da hyperintense akan jerin T2-weighted (T2W). Wannan raunin yana bayyana ƙarshen endplates na fissure da kyallen granulation na jijiyoyin jini da ke kusa a cikin ɓargon ƙashi. Canje-canjen Modic nau'in II suna nuna babban sigina akan jerin T1W da T2W. A cikin wannan nau'in rauni, ana iya samun lalata farantin ƙarshe, da kuma maye gurbin kitse na histological na ɓargon ƙashi da ke kusa. Canje-canjen Modic nau'in III suna nuna ƙarancin sigina a cikin jerin T1W da T2W. An lura da raunukan Sclerotic da suka dace da farantin ƙarshe6. Ana ɗaukar MC a matsayin wata cuta ta ƙashin baya kuma tana da alaƙa da cututtuka da yawa na ƙashin baya7,8,9.
Idan aka yi la'akari da bayanan da ake da su, bincike da dama sun bayar da cikakken bayani game da asalin cutar da kuma hanyoyin da MC ke bi wajen magance cutar. Albert da abokan aikinsa sun ba da shawarar cewa MC na iya faruwa ne sakamakon lalacewar diski8. Hu da abokan aikinsa sun danganta MC da mummunan lalacewar diski10. Kroc ya gabatar da manufar "fashewar diski na ciki," wanda ya bayyana cewa maimaita raunin diski na iya haifar da ƙananan hawaye a cikin farantin ƙarshe. Bayan samuwar tsagewa, lalata farantin ƙarshe ta hanyar nucleus pulposus (NP) na iya haifar da amsawar autoimmune, wanda hakan ke ƙara haifar da ci gaban MC11. Ma da abokan aikinsa sun raba irin wannan ra'ayi kuma sun ba da rahoton cewa garkuwar jiki da NP ke haifarwa tana taka muhimmiyar rawa a cikin cutar MC12.
Kwayoyin garkuwar jiki, musamman ƙwayoyin lymphocytes na CD4+ T, suna taka muhimmiyar rawa wajen haifar da garkuwar jiki13. An gano ɓangaren Th17 kwanan nan yana samar da cytokine mai kumburi IL-17, yana haɓaka bayyanar chemokine, kuma yana ƙarfafa ƙwayoyin T a cikin gabobin da suka lalace don samar da IFN-γ14. Kwayoyin Th2 kuma suna taka muhimmiyar rawa a cikin yanayin amsawar garkuwar jiki. Bayyanar IL-4 a matsayin wakili na ƙwayoyin Th2 na iya haifar da mummunan sakamako na rigakafi15.
Duk da cewa an gudanar da bincike da yawa na asibiti kan MC16,17,18,19,20,21,22,23,24, har yanzu akwai rashin samfuran gwaji na dabbobi masu dacewa waɗanda za su iya kwaikwayon tsarin MC wanda ke faruwa akai-akai a cikin mutane kuma ana iya amfani da su don bincika asalin cutar ko sabbin magunguna kamar maganin da aka yi niyya. Zuwa yanzu, ƙananan samfuran dabbobi na MC ne kawai aka ruwaito don yin nazarin hanyoyin cututtukan da ke haifar da cutar.
Bisa ga ka'idar garkuwar jiki da Albert da Ma suka gabatar, wannan binciken ya kafa wani samfurin zomo mai sauƙi kuma mai sake haifuwa ta hanyar dasa NP kusa da farantin ƙarshen ƙashin baya da aka haƙa. Sauran manufofin sune lura da halayen histological na samfuran dabbobi da kuma kimanta takamaiman hanyoyin NP a cikin ci gaban MC. Don wannan, muna amfani da dabaru kamar ilmin halitta na kwayoyin halitta, MRI, da nazarin histological don nazarin ci gaban MC.
Zomaye biyu sun mutu sakamakon zubar jini a lokacin tiyata, kuma zomaye huɗu sun mutu a lokacin maganin sa barci a lokacin MRI. Sauran zomaye 48 sun tsira kuma ba su nuna wata alama ta ɗabi'a ko ta jijiyoyi ba bayan tiyatar.
MRI ya nuna cewa ƙarfin siginar kyallen da aka saka a cikin ramuka daban-daban ya bambanta. Ƙarfin siginar jikin ƙashin baya na L5 a cikin ƙungiyar NPE ya canza a hankali a makonni 12, 16 da 20 bayan sakawa (jerin T1W ya nuna ƙarancin sigina, kuma jerin T2W ya nuna haɗin sigina da ƙarancin sigina) (Hoto na 1C), yayin da bayyanar MRI na sauran ƙungiyoyi biyu na sassan da aka saka ya kasance mai daidaito a cikin wannan lokacin (Hoto na 1A, B).
(A) Wakiltar MRIs na kashin baya na zomo a lokutan lokaci 3. Ba a sami wata matsala ta sigina a hotunan ƙungiyar tiyatar sham ba. (B) Halayen siginar jikin ƙashi a cikin ƙungiyar ME sun yi kama da na ƙungiyar tiyatar sham, kuma ba a lura da wani muhimmin canji na sigina a wurin da aka saka a tsawon lokaci ba. (C) A cikin ƙungiyar NPE, ana iya ganin ƙaramin sigina a cikin jerin T1W, kuma siginar gauraye da ƙaramin sigina suna bayyane a cikin jerin T2W. Daga lokacin makonni 12 zuwa lokacin makonni 20, manyan sigina da ke kewaye da ƙananan sigina a cikin jerin T2W suna raguwa.
Ana iya ganin hyperplasia na ƙashi a wurin dasawa na jikin ƙashi a cikin ƙungiyar NPE, kuma hyperplasia na ƙashi yana faruwa da sauri daga makonni 12 zuwa 20 (Hoto na 2C) idan aka kwatanta da ƙungiyar NPE, babu wani muhimmin canji da aka gani a jikin ƙashi da aka yi wa ƙira; ƙungiyar Sham da ƙungiyar ME (Hoto na 2C) 2A,B).
(A) Fuskar jikin ƙashin baya a ɓangaren da aka dasa tana da santsi sosai, ramin yana warkewa sosai, kuma babu hyperplasia a jikin ƙashin baya. (B) Siffar wurin da aka dasa a cikin ƙungiyar ME yayi kama da na ƙungiyar tiyatar sham, kuma babu wani canji a bayyane a bayyanar wurin da aka dasa akan lokaci. (C) Hyperplasia na ƙashi ya faru a wurin da aka dasa a cikin ƙungiyar NPE. Hyperplasia na ƙashin ya ƙaru da sauri har ma ya faɗaɗa ta cikin diski na intervertebral zuwa jikin ƙashin baya na gefe.
Binciken Histological ya ba da ƙarin bayani game da samuwar ƙashi. Hoto na 3 ya nuna hotunan sassan bayan tiyata da aka yi wa fenti da H&E. A cikin ƙungiyar tiyatar sham, ƙwayoyin chondrocytes sun shirya sosai kuma ba a gano haɓakar ƙwayoyin ba (Hoto na 3A). Yanayin da ke cikin ƙungiyar ME yayi kama da na ƙungiyar tiyatar sham (Hoto na 3B). Duk da haka, a cikin ƙungiyar NPE, an lura da adadi mai yawa na ƙwayoyin chondrocytes da yaduwar ƙwayoyin NP a wurin da aka dasa (Hoto na 3C);
(A) Ana iya ganin Trabeculae kusa da farantin ƙarshe, an shirya ƙwayoyin chondrocytes cikin tsari mai kyau tare da girman ƙwayoyin halitta iri ɗaya da siffa iri ɗaya kuma babu yaduwa (sau 40). (B) Yanayin wurin dasawa a cikin ƙungiyar ME yayi kama da na ƙungiyar sham. Ana iya ganin ƙwayoyin Trabeculae da chondrocytes, amma babu wani bayyanar yaduwa a wurin dasawa (sau 40). (B) Ana iya ganin cewa ƙwayoyin chondrocytes da ƙwayoyin NP suna ƙaruwa sosai, kuma siffa da girman ƙwayoyin chondrocytes ba su daidaita ba (sau 40).
An lura da bayyanar mRNA na interleukin 4 (IL-4), mRNA na interleukin 17 (IL-17), da kuma mRNA na interferon γ (IFN-γ) a cikin ƙungiyoyin NPE da ME. Lokacin da aka kwatanta matakan bayyanar kwayoyin halittar da aka yi niyya, an ƙara yawan bayyanar kwayoyin halittar IL-4, IL-17, da IFN-γ sosai a cikin ƙungiyar NPE idan aka kwatanta da na ƙungiyar ME da ƙungiyar aikin sham (Hoto na 4) (P < 0.05). Idan aka kwatanta da ƙungiyar aikin sham, matakan bayyanar IL-4, IL-17, da IFN-γ a cikin ƙungiyar ME sun ƙaru kaɗan kaɗan kuma ba su kai ga canjin ƙididdiga ba (P > 0.05).
Bayyanar mRNA na IL-4, IL-17 da IFN-γ a cikin ƙungiyar NPE ta nuna yanayin da ya fi girma fiye da na ƙungiyar aikin sham da ƙungiyar ME (P < 0.05).
Sabanin haka, matakan bayyana a cikin ƙungiyar ME ba su nuna wani bambanci mai mahimmanci ba (P>0.05).
An gudanar da nazarin Western blot ta amfani da magungunan rigakafi da ake samu a kasuwa don magance IL-4 da IL-17 don tabbatar da yanayin bayyanar mRNA da aka canza. Kamar yadda aka nuna a cikin Figures 5A,B, idan aka kwatanta da ƙungiyar ME da ƙungiyar aikin sham, matakan furotin na IL-4 da IL-17 a cikin ƙungiyar NPE sun ƙaru sosai (P < 0.05). Idan aka kwatanta da ƙungiyar aikin sham, matakan furotin na IL-4 da IL-17 a cikin ƙungiyar ME suma sun kasa cimma manyan canje-canje a kididdiga (P> 0.05).
(A) Matakan furotin na IL-4 da IL-17 a cikin rukunin NPE sun fi na ƙungiyar ME da ƙungiyar placebo girma sosai (P < 0.05). (B) Tarihin Western blot.
Saboda ƙarancin adadin samfuran ɗan adam da aka samu yayin tiyata, bincike mai zurfi da cikakken bayani kan cutar MC yana da ɗan wahala. Mun yi ƙoƙarin kafa samfurin dabba na MC don nazarin hanyoyin cututtukan da ke iya tasowa. A lokaci guda, an yi amfani da kimantawar rediyo, kimantawar histological da kimantawar kwayoyin halitta don bin tsarin MC wanda NP autograft ya haifar. Sakamakon haka, samfurin dasa NP ya haifar da canji a hankali a cikin ƙarfin sigina daga makonni 12 zuwa makonni 20 na lokaci (gauraye ƙarancin sigina a cikin jerin T1W da ƙarancin sigina a cikin jerin T2W), yana nuna canje-canjen nama, kuma kimantawar histological da kwayoyin halitta sun tabbatar da sakamakon binciken rediyo.
Sakamakon wannan gwajin ya nuna cewa canje-canje na gani da na tarihi sun faru ne a wurin da aka samu keta haddin jikin ƙashi a cikin ƙungiyar NPE. A lokaci guda, an lura da bayyanar kwayoyin halittar IL-4, IL-17 da IFN-γ, da kuma kwayoyin halittar IL-4, IL-17 da IFN-γ, wanda ke nuna cewa keta haddin kwayar halittar pulposus ta autologous a cikin jikin ƙashi na iya haifar da jerin canje-canje na sigina da na siffa. Yana da sauƙi a gano cewa halayen siginar jikin ƙashi na samfurin dabba (ƙarancin sigina a cikin jerin T1W, siginar gauraye da ƙarancin sigina a cikin jerin T2W) suna kama da na ƙwayoyin ƙashi na ɗan adam, kuma halayen MRI kuma suna tabbatar da lura da histology da babban tsarin jiki, wato, canje-canje a cikin ƙwayoyin jikin ƙashi na iya ci gaba. Kodayake amsawar kumburi da rauni mai tsanani ya haifar na iya bayyana jim kaɗan bayan huda, sakamakon MRI ya nuna cewa canje-canjen sigina da ke ƙaruwa a hankali sun bayyana makonni 12 bayan huda kuma sun ci gaba har zuwa makonni 20 ba tare da wata alamar murmurewa ko juyawar canje-canjen MRI ba. Waɗannan sakamakon sun nuna cewa autologous vertebral NP hanya ce mai inganci don gano ci gaba MV a cikin zomaye.
Wannan samfurin huda yana buƙatar isasshen ƙwarewa, lokaci, da ƙoƙarin tiyata. A cikin gwaje-gwajen farko, yankewa ko motsa jiki mai yawa na tsarin ligamentous na paravertebral na iya haifar da samuwar osteophytes na vertebral. Ya kamata a yi taka tsantsan kada a lalata ko kuma a fusata faifan da ke kusa. Tunda zurfin shigar ciki dole ne a sarrafa shi don samun sakamako mai daidaito da sake haifuwa, mun yi toshe da hannu ta hanyar yanke murfin allura mai tsawon mm 3. Yin amfani da wannan toshe yana tabbatar da zurfin haƙa rami ɗaya a jikin ƙashin baya. A cikin gwaje-gwajen farko, likitocin ƙashi uku da suka shiga aikin sun gano allurar ma'auni 16 sun fi sauƙin aiki da su fiye da allurar ma'auni 18 ko wasu hanyoyi. Don guje wa zubar jini mai yawa yayin haƙa rami, riƙe allurar a tsaye na ɗan lokaci zai samar da ramin sakawa mafi dacewa, yana nuna cewa ana iya sarrafa wani matakin MC ta wannan hanyar.
Duk da cewa bincike da yawa sun yi niyya ga MC, ba a san komai game da asalin cutar da kuma tushen cutar ba. Dangane da binciken da muka yi a baya, mun gano cewa garkuwar jiki tana taka muhimmiyar rawa wajen faruwa da kuma ci gaban MC12. Wannan binciken ya binciki yadda kwayoyin halitta na IL-4, IL-17, da IFN-γ ke bambanta ƙwayoyin halitta na CD4+ bayan ƙarfafa antigen. A cikin bincikenmu, idan aka kwatanta da ƙungiyar da ba ta da kyau, ƙungiyar NPE tana da mafi girman bayyanar IL-4, IL-17, da IFN-γ, kuma matakan furotin na IL-4 da IL-17 suma sun fi yawa.
A asibiti, bayyanar mRNA ta IL-17 ta ƙaru a cikin ƙwayoyin NP daga marasa lafiya da ke da cutar diski28. An kuma sami ƙaruwar matakan bayyanar IL-4 da IFN-γ a cikin wani nau'in cutar diski mai tsanani wanda ba shi da matsewa idan aka kwatanta da kula da lafiya29. IL-17 tana taka muhimmiyar rawa a cikin kumburi, raunin nama a cikin cututtukan autoimmune30 kuma tana haɓaka amsawar garkuwar jiki ga IFN-γ31. An ba da rahoton ƙaruwar raunin nama da IL-17 ke haifarwa a cikin berayen MRL/lpr32 da beraye masu saurin kamuwa da cutar autoimmune33. IL-4 na iya hana bayyanar cytokines masu kumburi (kamar IL-1β da TNFα) da kunna macrophage34. An ruwaito cewa bayyanar mRNA na IL-4 ya bambanta a cikin ƙungiyar NPE idan aka kwatanta da IL-17 da IFN-γ a lokaci guda; Bayyanar mRNA na IFN-γ a cikin ƙungiyar NPE ya fi girma sosai fiye da na sauran ƙungiyoyi. Saboda haka, samar da IFN-γ na iya zama mai shiga tsakani na martanin kumburi da NP intercalation ya haifar. Bincike ya nuna cewa ana samar da IFN-γ ta nau'ikan ƙwayoyin halitta da yawa, gami da ƙwayoyin T masu taimakawa nau'in 1 da aka kunna, ƙwayoyin kashe ƙwayoyin halitta, da macrophages35,36, kuma babban cytokine ne mai hana kumburi wanda ke haɓaka martanin garkuwar jiki37.
Wannan binciken ya nuna cewa amsawar autoimmune na iya shiga cikin faruwar da ci gaban MC. Luoma da abokan aikinsa sun gano cewa halayen siginar MC da fitaccen NP suna kama da juna akan MRI, kuma duka suna nuna babban sigina a cikin jerin T2W38. An tabbatar da cewa wasu cytokines suna da alaƙa da faruwar MC, kamar IL-139. Ma da abokan aikinsa sun ba da shawarar cewa fitowar NP sama ko ƙasa na iya yin babban tasiri akan faruwar da ci gaban MC12. Bobechko40 da Herzbein da abokan aikinsa sun ba da rahoton cewa NP nama ne mai jure garkuwar jiki wanda ba zai iya shiga zagayawar jini ba tun daga haihuwa. Fitowar NP yana shigar da jikin waje cikin wadatar jini, ta haka ne ke daidaita halayen autoimmune na gida42. Halayen autoimmune na iya haifar da adadi mai yawa na abubuwan garkuwar jiki, kuma lokacin da waɗannan abubuwan ke ci gaba da fallasa ga kyallen takarda, suna iya haifar da canje-canje a cikin siginar43. A cikin wannan binciken, yawan bayyanar IL-4, IL-17 da IFN-γ sune abubuwan da suka fi dacewa da garkuwar jiki, wanda hakan ke ƙara tabbatar da alaƙar da ke tsakanin NP da MCs44. Wannan samfurin dabba ya yi koyi da nasarar NP da kuma shiga cikin farantin ƙarshe. Wannan tsari ya ƙara bayyana tasirin garkuwar jiki ga MC.
Kamar yadda aka zata, wannan samfurin dabba yana ba mu damar yin nazarin MC. Duk da haka, wannan samfurin har yanzu yana da wasu ƙuntatawa: na farko, a lokacin lokacin lura da dabbobi, wasu zomaye masu matsakaicin mataki suna buƙatar a kashe su don gwajin ilimin halittar jiki da kwayoyin halitta, don haka wasu dabbobi "suka daina amfani" akan lokaci. Na biyu, kodayake an saita maki uku a cikin wannan binciken, abin takaici, mun yi samfurin nau'in MC ɗaya kawai (Modic type I change), don haka bai isa ya wakilci tsarin ci gaban cututtukan ɗan adam ba, kuma ana buƙatar saita ƙarin maki lokaci don lura da duk canje-canjen sigina mafi kyau. Na uku, canje-canje a cikin tsarin nama za a iya nuna su a sarari ta hanyar tabo na histological, amma wasu dabaru na musamman na iya bayyana canje-canjen microstructural a cikin wannan samfurin mafi kyau. Misali, an yi amfani da na'urar hangen nesa mai haske don nazarin samuwar fibrocartilage a cikin faifan intervertebral na zomo45. Tasirin NP na dogon lokaci akan MC da farantin ƙarshe yana buƙatar ƙarin bincike.
An raba zomaye fari maza hamsin da huɗu na New Zealand (nauyinsu ya kai kimanin kilogiram 2.5-3, masu watanni 3-3.5) bazuwar zuwa rukunin tiyatar bogi, rukunin dashen tsoka (ƙungiyar ME) da ƙungiyar dashen tushen jijiyoyi (ƙungiyar NPE). Kwamitin Ɗa'a na Asibitin Tianjin ya amince da dukkan hanyoyin gwaji, kuma an gudanar da hanyoyin gwaji bisa ƙa'idodi da aka amince da su.
An yi wasu gyare-gyare ga dabarar tiyata ta S. Sobajima 46. An sanya kowane zomo a matsayin wurin hutawa a gefe kuma an fallasa saman gaban faifan intervertebral guda biyar a jere a lumbar (IVDs) ta amfani da hanyar posterolateral retroperitoneal. An yi wa kowane zomo maganin sa barci na gaba ɗaya (20% urethane, 5 ml/kg ta hanyar jijiyar kunne). An yi yanke fata mai tsayi daga ƙasan haƙarƙari zuwa gefen ƙugu, 2 cm ventral zuwa tsokoki na paravertebral. An fallasa kashin baya na dama daga L1 zuwa L6 ta hanyar kaifi da ƙyalli na nama na subcutaneous da ke saman, nama na retroperitoneal, da tsokoki (Hoto na 6A). An ƙayyade matakin diski ta amfani da gefen ƙugu a matsayin alamar jiki don matakin diski na L5-L6. Yi amfani da allurar huda mai girman 16 don haƙa rami kusa da farantin ƙarshen vertebrae na L5 zuwa zurfin 3 mm (Hoto na 6B). Yi amfani da sirinji mai nauyin 5-ml don fitar da sinadarin pulposus na tsakiya mai aiki a cikin faifan intervertebral na L1-L2 (Hoto na 6C). Cire ƙwayar pulposus ko tsoka ta tsakiya bisa ga buƙatun kowane rukuni. Bayan an zurfafa ramin haƙa ramin, ana sanya ɗinki masu shaye-shaye a kan zurfin fascia, fascia na sama da fata, ana kula da kada a lalata kyallen jikin ƙashin baya yayin tiyata.
(A) Ana fallasa faifan L5-L6 ta hanyar amfani da hanyar posterolateral retroperitoneal. (B) Yi amfani da allura mai ma'auni 16 don haƙa rami kusa da farantin ƙarshe na L5. (C) Ana girbe MFs masu aiki da kansu.
An yi wa jijiyoyi allurar rigakafi ta gaba ɗaya da kashi 20% na urethane (5 ml/kg) da aka ba ta hanyar jijiya ta kunne, kuma an maimaita yin amfani da na'urar daukar hoton kashin baya ta lumbar a makonni 12, 16, da 20 bayan tiyata.
An yanka zomaye ta hanyar allurar ketamine (25.0 mg/kg) da kuma allurar sodium pentobarbital ta cikin jijiya (1.2 g/kg) a makonni 12, 16 da 20 bayan tiyata. An cire dukkan kashin baya don nazarin histological kuma an yi cikakken bincike. An yi amfani da ƙididdigar juzu'i (RT-qPCR) da Western blotting don gano canje-canje a cikin abubuwan da ke haifar da garkuwar jiki.
An yi gwajin MRI a cikin zomaye ta amfani da magnet na asibiti mai girman 3.0 T (GE Medical Systems, Florence, SC) wanda aka sanye da na'urar karɓar na'urar haɗin gwiwa ta orthogonal. An yi wa zomaye maganin sa barci da kashi 20% na urethane (5 mL/kg) ta hanyar jijiyar kunne sannan aka sanya su a kwance a cikin maganadisu tare da yankin lumbar da ke tsakiya a kan na'urar zagaye mai girman inci 5 (GE Medical Systems). An samo hotunan localizer na Coronal T2 mai nauyin nauyi (TR, 1445 ms; TE, 37 ms) don ayyana wurin da diski na lumbar yake daga L3-L4 zuwa L5-L6. An samo yanka masu nauyin T2 na jirgin sagittal tare da saitunan masu zuwa: jerin juyawa mai sauri tare da lokacin maimaitawa (TR) na 2200 ms da lokacin echo (TE) na 70 ms, matrix; filin gani na 260 da abubuwan ƙarfafawa guda takwas; Kauri na yanke shine 2 mm, rata shine 0.2 mm.
Bayan an ɗauki hoton ƙarshe kuma aka kashe zomo na ƙarshe, an cire faifan NP, waɗanda aka haɗa da tsoka, da kuma faifan NP don gwajin histological. An gyara nama a cikin formalin mai kauri 10% na tsawon mako 1, an cire sinadarin ethylenediaminetetraacetic acid, sannan aka raba paraffin. An saka tubalan nama a cikin paraffin kuma aka yanke su zuwa sassan sagittal (kauri 5 μm) ta amfani da microtome. An yi wa sassan fenti da hematoxylin da eosin (H&E).
Bayan tattara faifan intervertebral daga zomaye a kowace rukuni, an cire jimillar RNA ta amfani da ginshiƙin UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., China) bisa ga umarnin masana'anta da kuma tsarin kwafi na ImProm II (Promega Inc., Madison, WI, Amurka). An yi kwafi na baya.
An yi RT-qPCR ta amfani da Prism 7300 (Applied Biosystems Inc., Amurka) da SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, Amurka) bisa ga umarnin masana'anta. Girman amsawar PCR shine 20 μl kuma ya ƙunshi 1.5 μl na cDNA mai narkewa da 0.2 μM na kowane primer. An tsara primers ta OligoPerfect Designer (Invitrogen, Valencia, CA) kuma Nanjing Golden Stewart Biotechnology Co., Ltd. (China) ta ƙera (Tebur 1). An yi amfani da waɗannan yanayin zagayowar zafi: matakin kunnawa na farko na polymerase a 94°C na minti 2, sannan zagaye 40 na s 15 kowannensu a 94°C don cire samfurin, annealing na minti 1 a 60°C, faɗaɗawa, da haske. An yi ma'aunin na minti 1 a 72°C. An ƙara girman dukkan samfuran sau uku kuma an yi amfani da matsakaicin ƙimar don nazarin RT-qPCR. An yi nazarin bayanan ƙara girma ta amfani da FlexStation 3 (Na'urorin Molecular, Sunnyvale, CA, Amurka). An daidaita bayyanar kwayar halittar IL-4, IL-17, da IFN-γ zuwa ga ikon sarrafa endogenous (ACTB). An ƙididdige matakan bayyanar mRNA da aka yi niyya ta amfani da hanyar 2-ΔΔCT.
An cire jimlar furotin daga kyallen takarda ta amfani da nama homogenizer a cikin RIPA lysis buffer (wanda ke ɗauke da protease da phosphatase inhibitor cocktail) sannan a sanya shi a centrifuge a 13,000 rpm na tsawon minti 20 a 4°C don cire tarkacen nama. An ɗora micrograms hamsin na furotin a kowace layi, an raba su da 10% SDS-PAGE, sannan aka canja su zuwa membrane na PVDF. An yi toshewa a cikin madara busasshiyar madara mara kitse 5% a cikin Tris-buffered saline (TBS) wanda ke ɗauke da 0.1% Tween 20 na tsawon awa 1 a zafin ɗaki. An saka membrane ɗin da maganin rigakafi na zomo na farko (wanda aka narkar da 1:200; Boster, Wuhan, China) (wanda aka narkar da 1:200; Bioss, Beijing, China) cikin dare a 4°C kuma an mayar da martani a ranakun biyu; tare da maganin rigakafi na biyu (akuya anti-zomo immunoglobulin G a dilution 1:40,000) tare da horseradish peroxidase (Boster, Wuhan, China) na tsawon awa 1 a zafin ɗaki. An gano siginar Western blot ta hanyar ƙaruwar chemiluminescence akan membrane na chemiluminescent bayan hasken X-ray. Don nazarin densitometric, an duba blots kuma an auna su ta amfani da software na BandScan kuma an bayyana sakamakon a matsayin rabo na aikin rigakafi na kwayar halitta da aka yi niyya ga aikin rigakafi na tubulin.
An yi lissafin ƙididdiga ta amfani da fakitin software na SPSS16.0 (SPSS, Amurka). Bayanan da aka tattara a lokacin binciken an bayyana su a matsayin matsakaicin ± karkacewar daidaito (matsakaicin ± SD) kuma an yi nazari ta amfani da nazarin ma'auni na maimaitawa na hanya ɗaya (ANOVA) don tantance bambance-bambance tsakanin ƙungiyoyin biyu. An ɗauki P < 0.05 a matsayin mai mahimmanci a kididdiga.
Saboda haka, kafa samfurin dabba na MC ta hanyar dasa NPs masu kama da juna a cikin jikin ƙashi da kuma yin lura da macroanatomical, nazarin MRI, kimantawa ta tarihi da kuma nazarin kwayoyin halitta na iya zama muhimmin kayan aiki don tantancewa da fahimtar hanyoyin MC na ɗan adam da haɓaka sabbin hanyoyin magancewa.
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