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Kukhazikitsidwa kwa mitundu ya zinyama za modic change (MC) ndi maziko ofunikira pophunzirira MC. Akalulu makumi asanu ndi anayi a New Zealand White adagawidwa mu gulu la sham-operation, gulu loyika minofu (ME gulu) ndi nucleus pulposus implantation group (gulu la NPE). Mu gulu la NPE, disc intervertebral disc idavumbulutsidwa ndi njira ya opaleshoni ya anterolateral lumbar ndipo singano idagwiritsidwa ntchito poboola thupi la vertebral L5 pafupi ndi mbale yomaliza. NP inachotsedwa ku L1 / 2 intervertebral disc ndi syringe ndi jekeseni mmenemo. Kubowola dzenje mu fupa la subchondral. Njira zopangira opaleshoni ndi kubowola m'magulu opangira minofu ndi gulu la sham-operation zinali zofanana ndi zomwe zili mu gulu la NP implantation. Mu gulu la ME, chidutswa cha minofu chinayikidwa mu dzenje, pamene mu gulu la sham-operation, palibe chomwe chinayikidwa mu dzenje. Opaleshoniyo itachitika, kuyezetsa kwa MRI ndi kuyezetsa kwachilengedwe kwa maselo kudachitika. Chizindikiro mu gulu la NPE chinasintha, koma panalibe kusintha kwachizindikiro chodziwikiratu mu gulu la sham-operation ndi gulu la ME. Kuwona kwa histological kunawonetsa kuti kufalikira kwa minofu yachilendo kunawonedwa pamalo oyikidwa, ndipo mawu a IL-4, IL-17 ndi IFN-γ adawonjezeka mu gulu la NPE. Kuyika kwa NP mu fupa la subchondral kumatha kupanga mtundu wa nyama wa MC.
Kusintha kwa Modic (MC) ndi zotupa za vertebral endplates ndi mafupa oyandikana nawo omwe amawonekera pazithunzi za magnetic resonance (MRI). Amapezeka mwa anthu omwe ali ndi zizindikiro zofananira1. Maphunziro ambiri adatsindika kufunika kwa MC chifukwa cha kuyanjana kwake ndi ululu wopweteka kwambiri (LBP) 2,3. de Roos et al.4 ndi Modic et al.5 mwayekha poyamba adalongosola mitundu itatu yosiyana ya zizindikiro za subchondral zosaoneka bwino m'mafupa a vertebral. Kusintha kwa mtundu wa Modic I ndi hypointense pamatsatidwe a T1-weighted (T1W) ndi hyperintense pamatsatidwe a T2-weighted (T2W). Chotupa ichi chikuwonetsa minyewa yamkati ndi mitsempha yoyandikana nayo m'mafupa. Kusintha kwa mtundu wa Modic II kumawonetsa chizindikiro chachikulu pamatsatidwe onse a T1W ndi T2W. Mu mtundu uwu wa zilonda, chiwonongeko cha endplate chikhoza kupezeka, komanso histological mafuta m'malo mwa m'mafupa oyandikana nawo. Kusintha kwa mtundu wa Modic III kumawonetsa chizindikiro chochepa mumayendedwe a T1W ndi T2W. Zotupa za sclerotic zomwe zimayenderana ndi ma endplates zawonedwa6. MC imatengedwa kuti ndi matenda a msana ndipo imagwirizana kwambiri ndi matenda ambiri osokonezeka a msana7,8,9.
Poganizira zomwe zilipo, maphunziro angapo apereka zidziwitso zatsatanetsatane za etiology ndi njira zamatenda a MC. Albert ndi al. adanenanso kuti MC ikhoza kuyambitsidwa ndi disc herniation8. Hu et al. akuti MC ndi kuwonongeka kwakukulu kwa disc10. Kroc adapereka lingaliro la "kuphulika kwa disc yamkati," yomwe imanena kuti kubwerezabwereza kwa disc kungayambitse ma microtears kumapeto. Pambuyo popanga mng'alu, chiwonongeko cha endplate ndi nucleus pulposus (NP) chingayambitse kuyankha kwa autoimmune, zomwe zimatsogolera ku chitukuko cha MC11. Ma et al. adagawana malingaliro ofananawo ndipo adanenanso kuti NP-induced autoimmunity imathandizira kwambiri pathogenesis ya MC12.
Ma cell a chitetezo chamthupi, makamaka ma CD4 + T othandizira ma lymphocyte, amagwira ntchito yofunika kwambiri pakuyambitsa matenda a autoimmunity13. Gawo la Th17 lomwe lapezeka posachedwa limapanga proinflammatory cytokine IL-17, imalimbikitsa kufotokoza kwa chemokine, ndipo imapangitsa maselo a T mu ziwalo zowonongeka kuti apange IFN-γ14. Maselo a Th2 amakhalanso ndi gawo lapadera pa matenda a chitetezo cha mthupi. Kufotokozera kwa IL-4 ngati woyimilira Th2 cell kungayambitse zotsatira zoopsa za immunopathological15.
Ngakhale maphunziro azachipatala ambiri achitika pa MC16,17,18,19,20,21,22,23,24, pakalibe kusowa kwa zitsanzo zoyenera zoyeserera za nyama zomwe zimatha kutsanzira njira ya MC yomwe imachitika kawirikawiri mwa anthu ndipo imatha amagwiritsidwa ntchito pofufuza etiology kapena mankhwala atsopano monga mankhwala omwe akuwunikira. Mpaka pano, ndi nyama zochepa chabe za MC zomwe zanenedwa kuti zimaphunzira njira zomwe zimayambitsa matenda.
Kutengera chiphunzitso cha autoimmune chomwe Albert ndi Ma adapereka, kafukufukuyu adakhazikitsa njira yosavuta komanso yobwereketsa ya kalulu MC mwa autotransplanting NP pafupi ndi mbale yobowoleza ya vertebral end plate. Zolinga zina ndikuwona mawonekedwe a histological a zinyama ndikuwunika njira zenizeni za NP pakukula kwa MC. Kuti izi zitheke, timagwiritsa ntchito njira monga molekyulu ya biology, MRI, ndi maphunziro a histological kuti tiphunzire momwe MC ikuyendera.
Akalulu awiri anafa ndi magazi pa nthawi ya opaleshoni, ndipo akalulu anayi anafa panthawi ya opaleshoni pa MRI. Akalulu otsala a 48 adapulumuka ndipo sanawonetse zizindikiro za khalidwe kapena minyewa pambuyo pa opaleshoni.
MRI imasonyeza kuti mphamvu ya chizindikiro cha minofu yomwe ili m'mabowo osiyanasiyana ndi yosiyana. Chizindikiro champhamvu cha L5 vertebral body mu gulu la NPE chinasintha pang'onopang'ono pa 12, 16 ndi masabata a 20 pambuyo pa kuyika (T1W yotsatizana inasonyeza chizindikiro chochepa, ndipo mndandanda wa T2W umasonyeza chizindikiro chosakanikirana kuphatikizapo chizindikiro chochepa) (Mkuyu 1C), pamene maonekedwe a MRI mwa magulu ena awiri a magawo ophatikizidwa anakhalabe okhazikika panthawi yomweyi (mkuyu 1A, B).
(A) Oyimira sequential MRIs a kalulu lumbar msana pa 3 nthawi. Palibe zosokoneza zazizindikiro zomwe zidapezeka muzithunzi za gulu la sham-operation. (B) Makhalidwe a chizindikiro cha thupi la vertebral mu gulu la ME ndi ofanana ndi omwe ali mu gulu la sham-operation, ndipo palibe kusintha kwakukulu kwa chizindikiro komwe kumawoneka pa malo oyikapo pakapita nthawi. (C) Mu gulu la NPE, chizindikiro chochepa chikuwoneka bwino mu mndandanda wa T1W, ndipo chizindikiro chosakanikirana ndi chizindikiro chochepa chikuwonekera bwino mu mndandanda wa T2W. Kuyambira nthawi ya masabata a 12 mpaka nthawi ya masabata a 20, zizindikiro zapamwamba zowonongeka zozungulira zizindikiro zochepa muzotsatira za T2W zimachepa.
Chodziwika bwino cha fupa cha hyperplasia chikhoza kuwonedwa pa malo opangira thupi la vertebral mu gulu la NPE, ndipo fupa la hyperplasia limapezeka mofulumira kuchokera ku 12 mpaka masabata a 20 (mkuyu 2C) poyerekeza ndi gulu la NPE, palibe kusintha kwakukulu komwe kumawoneka mumtundu wa vertebral. matupi; Gulu la Sham ndi gulu la ME (mkuyu 2C) 2A, B).
(A) Pamwamba pa thupi la vertebral pa gawo loyikidwa ndi losalala kwambiri, dzenje limachiritsa bwino, ndipo palibe hyperplasia mu thupi la vertebral. (B) Maonekedwe a malo oikidwa mu gulu la ME ndi ofanana ndi omwe ali mu gulu la opareshoni ya sham, ndipo palibe kusintha koonekeratu kwa maonekedwe a malo oikidwa pakapita nthawi. (C) Bone hyperplasia inachitika pa malo oikidwa mu gulu la NPE. Mphuno ya hyperplasia inakula mofulumira ndipo imapitirira kupyolera mu intervertebral disc kupita ku thupi la vertebral contralateral.
Kusanthula kwa histological kumapereka chidziwitso chatsatanetsatane chokhudza mapangidwe a mafupa. Chithunzi 3 chikuwonetsa zithunzi za zigawo zapambuyo pa opaleshoni zodetsedwa ndi H&E. Mu gulu la sham-operation, ma chondrocyte adakonzedwa bwino ndipo palibe kufalikira kwa maselo komwe kunapezeka (Mkuyu 3A). Zomwe zili mu gulu la ME zinali zofanana ndi zomwe zili m'gulu la anthu ochita zachiwerewere (mkuyu 3B). Komabe, mu gulu la NPE, chiwerengero chachikulu cha chondrocytes ndi kuchuluka kwa maselo a NP-ngati anawonedwa pa malo opangira (mkuyu 3C);
(A) Trabeculae imatha kuwonedwa pafupi ndi mbale yomaliza, ma chondrocyte amakonzedwa bwino ndi kukula kwa selo limodzi ndi mawonekedwe ndipo palibe kufalikira (nthawi 40). (B) Mkhalidwe wa malo oyikidwa mu gulu la ME ndi ofanana ndi gulu la sham. Ma trabeculae ndi chondrocytes amatha kuwoneka, koma palibe kufalikira koonekeratu pamalo oyikapo (nthawi 40). (B) Zitha kuwoneka kuti ma chondrocytes ndi maselo a NP amachuluka kwambiri, ndipo mawonekedwe ndi kukula kwa chondrocytes ndizosiyana (nthawi 40).
Mawu a interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, ndi interferon γ (IFN-γ) mRNA adawonedwa m'magulu onse a NPE ndi ME. Pamene mafotokozedwe amtundu wa jini amafananizidwa, ma jini a IL-4, IL-17, ndi IFN-γ adawonjezeka kwambiri mu gulu la NPE poyerekeza ndi gulu la ME ndi gulu la opaleshoni ya sham (Mkuyu 4). (P <0.05). Poyerekeza ndi gulu la opareshoni ya sham, mafotokozedwe a IL-4, IL-17, ndi IFN-γ mu gulu la ME adakwera pang'ono ndipo sanafikire kusintha kwa ziwerengero (P> 0.05).
Mafotokozedwe a mRNA a IL-4, IL-17 ndi IFN-γ mu gulu la NPE adawonetsa machitidwe apamwamba kwambiri kuposa omwe ali mu gulu la opareshoni ya sham ndi gulu la ME (P <0.05).
Mosiyana ndi izi, milingo ya mawu mu gulu la ME sinawonetse kusiyana kwakukulu (P> 0.05).
Kusanthula kwa blot kumadzulo kunachitika pogwiritsa ntchito ma antibodies omwe amapezeka pamalonda motsutsana ndi IL-4 ndi IL-17 kuti atsimikizire mawonekedwe osinthika a mRNA. Monga momwe tawonetsera mu Zithunzi 5A, B, poyerekeza ndi gulu la ME ndi gulu la opareshoni ya sham, mapuloteni a IL-4 ndi IL-17 mu gulu la NPE adawonjezeka kwambiri (P <0.05). Poyerekeza ndi gulu la opareshoni yabodza, ma protein a IL-4 ndi IL-17 mu gulu la ME nawonso adalephera kufikira kusintha kwakukulu (P> 0.05).
(A) Mapuloteni a IL-4 ndi IL-17 mu gulu la NPE anali apamwamba kwambiri kuposa omwe ali mu gulu la ME ndi gulu la placebo (P <0.05). (B) West blot histogram.
Chifukwa cha chiwerengero chochepa cha zitsanzo za anthu zomwe zimapezedwa panthawi ya opaleshoni, maphunziro omveka bwino komanso atsatanetsatane pa matenda a MC ndi ovuta. Tidayesa kukhazikitsa mtundu wa nyama wa MC kuti tiphunzire momwe angapangire ma pathological. Panthawi imodzimodziyo, kuyesa kwa radiological, histological evaluation ndi molecular biological evaluation idagwiritsidwa ntchito potsatira njira ya MC yoyambitsidwa ndi NP autograft. Zotsatira zake, NP implantation model inachititsa kusintha kwapang'onopang'ono kwa mphamvu yazizindikiro kuchokera ku 12-sabata mpaka 20-masabata a nthawi (chizindikiro chochepa chosakanikirana mumayendedwe a T1W ndi chizindikiro chochepa mumayendedwe a T2W), kusonyeza kusintha kwa minofu, ndi histological and molecular. kuwunika kwachilengedwe kunatsimikizira zotsatira za kafukufuku wama radiology.
Zotsatira za kuyesaku zikuwonetsa kuti kusintha kwazithunzi ndi histological kunachitika pamalo ophwanya thupi la vertebral mu gulu la NPE. Panthawi imodzimodziyo, mawu a IL-4, IL-17 ndi IFN-γ majini, komanso IL-4, IL-17 ndi IFN-γ adawonedwa, kusonyeza kuti kuphwanya kwa autologous nucleus pulposus tissue mu vertebral. thupi lingayambitse mndandanda wa zizindikiro ndi kusintha kwa morphological. N'zosavuta kupeza kuti zizindikiro za matupi amtundu wamtundu wa nyama (chizindikiro chochepa mu mndandanda wa T1W, chizindikiro chosakanikirana ndi chizindikiro chochepa mu mndandanda wa T2W) ndi ofanana kwambiri ndi maselo amtundu waumunthu, komanso makhalidwe a MRI. kutsimikizira kuziona histology ndi aakulu anatomy, ndiye kuti, kusintha kwa vertebral thupi maselo ndi pang'onopang'ono. Ngakhale kuyankha kotupa komwe kumabwera chifukwa cha kuvulala koopsa kumatha kuwoneka atangotha kupunthwa, zotsatira za MRI zikuwonetsa kuti kusintha kwazizindikiro komwe kumawonjezeka pang'onopang'ono kunawonekera masabata a 12 pambuyo pa kuphulika ndikupitilira mpaka masabata a 20 popanda zizindikiro za kuchira kapena kusintha kwa kusintha kwa MRI. Zotsatirazi zikusonyeza kuti autologous vertebral NP ndi njira yodalirika yokhazikitsira MV yopita patsogolo mu akalulu.
Mtundu wokhomerera uwu umafuna luso lokwanira, nthawi, ndi kuyesetsa kuchita opaleshoni. M'mayesero oyambirira, dissection kapena kukondoweza kwambiri kwa paravertebral ligamentous structures kungapangitse mapangidwe a vertebral osteophytes. Chisamaliro chiyenera kutengedwa kuti chisawononge kapena kukwiyitsa ma disks oyandikana nawo. Popeza kuya kwa kulowa kuyenera kuwongoleredwa kuti tipeze zotsatira zofananira komanso zobwereketsa, tidapanga pulagi pamanja podula singano yayitali ya 3 mm. Kugwiritsa ntchito pulagiyi kumatsimikizira kuya kwa yunifolomu m'thupi la vertebral. Poyesa koyambirira, madokotala atatu a mafupa omwe adagwira nawo ntchitoyo adapeza singano za 16-gauge zosavuta kugwira ntchito ndi singano za 18-gauge kapena njira zina. Pofuna kupewa kutaya magazi kwambiri pobowola, kusunga singanoyo kwakanthawi kumapereka dzenje lolowera, kutanthauza kuti mlingo wina wa MC ukhoza kuwongoleredwa motere.
Ngakhale maphunziro ambiri alunjika ku MC, ndizochepa zomwe zimadziwika za etiology ndi pathogenesis ya MC25,26,27. Kutengera ndi maphunziro athu am'mbuyomu, tidapeza kuti autoimmunity imagwira ntchito yofunika kwambiri pakuchitika komanso kukula kwa MC12. Kafukufukuyu adafufuza kuchuluka kwa IL-4, IL-17, ndi IFN-γ, zomwe ndizo njira zazikulu zosiyanitsira ma CD4 + pambuyo pa kukondoweza kwa antigen. Mu phunziro lathu, poyerekeza ndi gulu loipa, gulu la NPE linali ndi mawu apamwamba a IL-4, IL-17, ndi IFN-γ, ndipo mapuloteni a IL-4 ndi IL-17 anali apamwamba.
Zachipatala, mawu a IL-17 mRNA akuwonjezeka m'maselo a NP kuchokera kwa odwala omwe ali ndi disc herniation28. Kuwonjezeka kwa mawu a IL-4 ndi IFN-γ kunapezekanso mumtundu wovuta wosakanizika wa disc herniation poyerekeza ndi maulamuliro athanzi29. IL-17 imagwira ntchito yofunika kwambiri pa kutupa, kuvulala kwa minofu m'matenda a autoimmune30 ndipo kumapangitsa kuti chitetezo cha mthupi chitetezeke ku IFN-γ31. Kuvulala kwa minofu ya IL-17-mediated kwanenedwa mu MRL/lpr mice32 ndi mbewa zodziteteza ku autoimmunity-susceptible mbewa33. IL-4 ikhoza kulepheretsa kufotokoza kwa proinflammatory cytokines (monga IL-1β ndi TNFα) ndi macrophage activation34. Zinanenedwa kuti mawu a mRNA a IL-4 anali osiyana mu gulu la NPE poyerekeza ndi IL-17 ndi IFN-γ pa nthawi yomweyo; Mawu a mRNA a IFN-γ mu gulu la NPE anali apamwamba kwambiri kuposa omwe ali m'magulu ena. Chifukwa chake, kupanga kwa IFN-γ kungakhale mkhalapakati wa kuyankha kotupa komwe kumayambitsidwa ndi kulumikizana kwa NP. Kafukufuku wasonyeza kuti IFN-γ imapangidwa ndi mitundu yambiri ya maselo, kuphatikizapo maselo a T othandizira amtundu wa 1, maselo akupha zachilengedwe, ndi macrophages35,36, ndipo ndi cytokine yofunika kwambiri yomwe imalimbikitsa chitetezo cha mthupi37.
Kafukufukuyu akuwonetsa kuti kuyankha kwa autoimmune kumatha kukhudzidwa ndi zomwe zimachitika komanso kukula kwa MC. Luoma et al. anapeza kuti zizindikiro za MC ndi NP zodziwika ndizofanana pa MRI, ndipo zonsezi zimasonyeza chizindikiro chapamwamba mu mndandanda wa T2W38. Ma cytokines ena atsimikiziridwa kuti amagwirizana kwambiri ndi zochitika za MC, monga IL-139. Ma et al. adanenanso kuti kukwera kapena kutsika kwa NP kungakhale ndi chikoka chachikulu pazochitika ndi chitukuko cha MC12. Bobechko40 ndi Herzbein et al.41 adanena kuti NP ndi minofu ya immunotolerant yomwe singalowe mu mitsempha ya mitsempha kuyambira kubadwa. NP protrusions imayambitsa matupi akunja m'magazi, motero amayanjanitsa machitidwe a autoimmune am'deralo42. Zochita za autoimmune zimatha kuyambitsa kuchuluka kwa chitetezo chamthupi, ndipo zinthu izi zikawonetsedwa mosalekeza ku minofu, zimatha kuyambitsa kusintha kwa signing43. Mu kafukufukuyu, kufotokoza mopambanitsa kwa IL-4, IL-17 ndi IFN-γ ndizodziwikiratu zomwe zimayambitsa chitetezo chamthupi, kutsimikiziranso ubale wapamtima pakati pa NP ndi MCs44. Nyama yamtunduwu imatsanzira bwino NP ndikulowa mu mbale yomaliza. Izi zidawonetsanso zotsatira za autoimmunity pa MC.
Monga zikuyembekezeredwa, mtundu wa nyama uwu umatipatsa mwayi wophunzirira MC. Komabe, chitsanzochi chimakhalabe ndi zolepheretsa: choyamba, panthawi yoyang'ana nyama, akalulu ena apakati amafunika kuthandizidwa kuti ayese zamoyo ndi maselo, kotero nyama zina "zimasiya kugwiritsidwa ntchito" pakapita nthawi. Kachiwiri, ngakhale kuti nthawi zitatu zakhazikitsidwa mu phunziroli, mwatsoka, tinangopanga mtundu umodzi wa MC (Modic mtundu wa ine kusintha), kotero sikokwanira kuimira ndondomeko ya chitukuko cha matenda aumunthu, ndipo nthawi zambiri ziyenera kukhazikitsidwa. bwino kuyang'ana kusintha konse kwa chizindikiro. Chachitatu, kusintha kwa minofu kumatha kuwonetsedwa momveka bwino ndi madontho a histological, koma njira zina zapadera zimatha kuwulula kusintha kwapang'onopang'ono kwachitsanzo ichi. Mwachitsanzo, polarized light microscopy idagwiritsidwa ntchito kusanthula mapangidwe a fibrocartilage mu akalulu intervertebral discs45. Zotsatira za nthawi yayitali za NP pa MC ndi endplate zimafunikira kuphunzira kwina.
Akalulu oyera makumi asanu ndi anayi aamuna a New Zealand (olemera pafupifupi 2.5-3 kg, zaka 3-3.5 miyezi) adagawidwa mwachisawawa kukhala gulu la opaleshoni ya sham, gulu loyika minofu (ME gulu) ndi gulu la mitsempha ya mitsempha (gulu la NPE). Njira zonse zoyesera zidavomerezedwa ndi Komiti ya Ethics ya Chipatala cha Tianjin, ndipo njira zoyesera zidachitika motsatira malangizo ovomerezeka.
Kusintha kwina kwapangidwa ku njira ya opaleshoni ya S. Sobajima 46 . Kalulu aliyense anayikidwa mu lateral recumbency udindo ndi anterior pamwamba pa zisanu zotsatizana lumbar intervertebral discs (IVDs) anaonekera pogwiritsira ntchito posterolateral retroperitoneal njira. Kalulu aliyense amapatsidwa mankhwala oletsa ululu wamba (20% urethane, 5 ml/kg kudzera mtsempha wa khutu). Kupaka khungu kwautali kunapangidwa kuchokera m'munsi mwa nthiti mpaka m'mphepete mwa chiuno, 2 masentimita kupita kumtunda kupita ku minofu ya paravertebral. Kumanja kwa anterolateral msana kuchokera ku L1 kupita ku L6 kunawonetsedwa ndi kugawanika kwakuthwa komanso kosasunthika kwa minofu ya subcutaneous, minofu ya retroperitoneal, ndi minofu (mkuyu 6A). Mulingo wa disc udatsimikiziridwa pogwiritsa ntchito m'mphepete mwa pelvic ngati chizindikiro chamtundu wa L5-L6 disc. Gwiritsani ntchito singano ya 16-gauge kuti mubowole dzenje pafupi ndi mapeto a L5 vertebra mpaka kuya kwa 3 mm (mkuyu 6B). Gwiritsani ntchito syringe ya 5-ml kuti mukhumbe autologous nucleus pulposus mu L1-L2 intervertebral disc (Mkuyu 6C). Chotsani nyukiliya pulposus kapena minofu molingana ndi zofunikira za gulu lirilonse. Pambuyo pobowola zakuya, sutures absorbable amaikidwa pa kuya fascia, pamwamba fascia ndi khungu, kusamala kuti kuwononga periosteal minofu ya vertebral thupi pa opaleshoni.
(A) Diski ya L5-L6 imawululidwa kudzera mu njira ya posterolateral retroperitoneal. (B) Gwiritsani ntchito singano ya 16-gauge kuboola dzenje pafupi ndi L5 endplate. (C) Ma Autologous MF amakololedwa.
Mankhwala osokoneza bongo amaperekedwa ndi 20% urethane (5 ml / kg) yoyendetsedwa kudzera m'mitsempha ya khutu, ndipo ma radiographs a lumbar spine anabwerezedwa pa 12, 16, ndi masabata a 20 pambuyo pa opaleshoni.
Akalulu anaperekedwa nsembe ndi jekeseni wa intramuscular wa ketamine (25.0 mg / kg) ndi intravenous sodium pentobarbital (1.2 g / kg) pa 12, 16 ndi masabata a 20 pambuyo pa opaleshoni. Msana wonse unachotsedwa chifukwa cha histological analysis ndipo kusanthula kwenikweni kunachitika. Quantitative reverse transcription (RT-qPCR) ndi Western blotting anagwiritsidwa ntchito kuti azindikire kusintha kwa chitetezo cha mthupi.
Kuyeza kwa MRI kunkachitidwa mu akalulu pogwiritsa ntchito maginito a 3.0 T (GE Medical Systems, Florence, SC) okhala ndi cholandira cha orthogonal coil. Akalulu amagonekedwa ndi 20% urethane (5 mL/kg) kudzera pa mtsempha wa khutu ndiyeno anayikidwa pamwamba pa maginito ndi dera la lumbar lokhazikika pa 5-inch diameter circular surface coil (GE Medical Systems). Zithunzi za Coronal T2-zolemera za localizer (TR, 1445 ms; TE, 37 ms) zinapezedwa kuti zifotokoze malo a lumbar disc kuchokera ku L3-L4 mpaka L5-L6. Magawo olemera a ndege a Sagittal T2 adapezedwa ndi makonzedwe otsatirawa: kutsatizana kofulumira kwa spin-echo ndi nthawi yobwereza (TR) ya 2200 ms ndi nthawi ya echo (TE) ya 70 ms, matrix; malo owonera a 260 ndi zolimbikitsa zisanu ndi zitatu; Makulidwe odula anali 2 mm, kusiyana kwake kunali 0.2 mm.
Pambuyo pa chithunzi chomaliza chinatengedwa ndipo kalulu wotsiriza anaphedwa, sham, ophatikizidwa minofu, ndi NP discs anachotsedwa kufufuza histological. Minofu inakhazikitsidwa mu 10% yopanda ndale ya formalin kwa sabata la 1, decalcified ndi ethylenediaminetetraacetic acid, ndi parafini yogawidwa. Zotchingira minofu zidayikidwa mu parafini ndikudula magawo a sagittal (5 μm wandiweyani) pogwiritsa ntchito microtome. Magawo anali odetsedwa ndi hematoxylin ndi eosin (H&E).
Pambuyo posonkhanitsa ma intervertebral discs kuchokera ku akalulu mu gulu lirilonse, RNA yonse idachotsedwa pogwiritsa ntchito chigawo cha UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., China) molingana ndi malangizo a wopanga ndi ImProm II reverse transcript system (Promega Inc. , Madison, WI, USA). Kutembenuza kwachitika.
RT-qPCR inachitidwa pogwiritsa ntchito Prism 7300 (Applied Biosystems Inc., USA) ndi SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) malinga ndi malangizo a wopanga. Voliyumu ya PCR inali 20 μl ndipo inali ndi 1.5 μl ya cDNA yosungunuka ndi 0.2 μM ya choyambirira chilichonse. Zoyambira zidapangidwa ndi OligoPerfect Designer (Invitrogen, Valencia, CA) ndikupangidwa ndi Nanjing Golden Stewart Biotechnology Co., Ltd. (China) (Table 1). Matenthedwe otsatirawa apanjinga apanjinga adagwiritsidwa ntchito: sitepe yoyamba ya polymerase activation pa 94 ° C kwa 2 min, kenako 40 mizunguliro ya 15 s iliyonse pa 94 ° C ya template denaturation, annealing kwa 1 min pa 60 ° C, kukulitsa, ndi fluorescence. kuyeza kunachitika kwa mphindi imodzi pa 72 ° C. Zitsanzo zonse zidakulitsidwa katatu ndipo mtengo wapakati unagwiritsidwa ntchito pakuwunika kwa RT-qPCR. Deta yokulitsa idawunikidwa pogwiritsa ntchito FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). IL-4, IL-17, ndi IFN-γ mawu a jini adasinthidwa kukhala endogenous control (ACTB). Miyezo yofananira ya chandamale ya mRNA idawerengedwa pogwiritsa ntchito njira ya 2-ΔΔCT.
Mapuloteni okwana anali otengedwa ku minofu pogwiritsa ntchito homogenizer ya minofu mu RIPA lysis buffer (yokhala ndi protease ndi phosphatase inhibitor cocktail) ndiyeno centrifuged pa 13,000 rpm kwa mphindi 20 pa 4 ° C kuchotsa zinyalala za minofu. Ma micrograms makumi asanu a mapuloteni adanyamulidwa pamseu, wolekanitsidwa ndi 10% SDS-PAGE, kenako amasamutsidwa ku nembanemba ya PVDF. Kutsekera kunkachitika mu 5% mkaka wouma wopanda mafuta mu Tris-buffered saline (TBS) wokhala ndi 0.1% Pakati pa 20 kwa 1 h kutentha kwapakati. Nembanembayo idakulungidwa ndi anti-decorin primary antibody (yochepetsedwa 1:200; Boster, Wuhan, China) (yochepetsedwa 1:200; Bioss, Beijing, China) usiku wonse pa 4 ° C ndikuchitapo masiku achiwiri; yokhala ndi chitetezo chamthupi (anti-rabbit immunoglobulin G pa 1:40,000 dilution) yophatikizidwa ndi horseradish peroxidase (Boster, Wuhan, China) kwa ola limodzi kutentha kwachipinda. Zizindikiro zaku Western blot zidadziwika ndi kuchuluka kwa chemiluminescence pa nembanemba ya chemiluminescent pambuyo pakuwunikira kwa X-ray. Pakuwunika kwa densitometric, ma blots adawunikidwa ndikuyesedwa pogwiritsa ntchito pulogalamu ya BandScan ndipo zotsatira zake zidawonetsedwa ngati chiŵerengero cha chandamale cha gene immunoreactivity to tubulin immunoreactivity.
Mawerengedwe owerengera adachitidwa pogwiritsa ntchito pulogalamu ya SPSS16.0 (SPSS, USA). Deta yomwe inasonkhanitsidwa panthawi yophunzirayi inafotokozedwa ngati ± kusiyana koyenera (kutanthauza ± SD) ndikuwunikidwa pogwiritsa ntchito njira imodzi yobwerezabwereza kusanthula kusiyana (ANOVA) kuti mudziwe kusiyana pakati pa magulu awiriwa. P <0.05 idawonedwa ngati yofunika kwambiri.
Choncho, kukhazikitsidwa kwa chitsanzo cha nyama cha MC mwa kuika NPs autologous mu thupi la vertebral ndikuchita macroanatomical observation, MRI analysis, histological evaluation ndi molecular biological analysis ingakhale chida chofunikira pofufuza ndi kumvetsetsa njira za MC yaumunthu ndikupanga chithandizo chatsopano. kulowererapo.
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