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Kukhazikitsidwa kwa mitundu ya nyama ya kusintha kwa modic (MC) ndi maziko ofunikira pophunzira za MC. Akalulu 54 oyera a ku New Zealand adagawidwa m'magulu a sham-operation, gulu la kuyika minofu (ME group) ndi gulu la nucleus pulposus implantation (NPE group). Mu gulu la NPE, disc ya intervertebral disc idawonekera pogwiritsa ntchito njira ya opaleshoni ya anterolateral lumbar ndipo singano idagwiritsidwa ntchito kuboola thupi la vertebral la L5 pafupi ndi mbale yomaliza. NP idachotsedwa mu disc ya intervertebral ya L1/2 ndi syringe ndikuyilowetsamo. Kuboola dzenje mu fupa la subchondral. Njira zochitira opaleshoni ndi njira zoboola mu gulu la kuyika minofu ndi gulu la kuyika minofu zinali zofanana ndi zomwe zili mu gulu la kuyika minofu la NP. Mu gulu la ME, chidutswa cha minofu chidayikidwa mu dzenje, pomwe mu gulu la kuyika minofu, palibe chomwe chidayikidwa mu dzenje. Pambuyo pa opaleshoni, MRI scanning ndi mayeso a biological a molecular adachitika. Chizindikiro mu gulu la NPE chidasintha, koma panalibe kusintha koonekeratu kwa chizindikiro mu gulu la kuyika minofu ndi gulu la ME. Kuwona kwa histological kunawonetsa kuti kuchulukana kwa minofu kosazolowereka kunawonedwa pamalo oikira, ndipo kuwonekera kwa IL-4, IL-17 ndi IFN-γ kunawonjezeka mu gulu la NPE. Kuika NP mu fupa la subchondral kumatha kupanga chitsanzo cha nyama cha MC.
Kusintha kwa Modic (MC) ndi zilonda za m'mphepete mwa vertebral endplates ndi fupa lapafupi lomwe limawonekera pa magnetic resonance imaging (MRI). Ndizofala kwambiri mwa anthu omwe ali ndi zizindikiro zogwirizana1. Kafukufuku wambiri wagogomezera kufunika kwa MC chifukwa chogwirizana ndi ululu wa msana (LBP)2,3. de Roos et al.4 ndi Modic et al.5 poyamba adafotokoza mitundu itatu yosiyanasiyana ya zolakwika za chizindikiro cha subchondral mu fupa la vertebral. Kusintha kwa Modic type I kumakhala kochepa kwambiri pa T1-weighted (T1W) sequences ndi hyperintense pa T2-weighted (T2W). Kutupa kumeneku kumawonetsa ma fissure endplates ndi minofu ya vascular granulation yoyandikana nayo mu fupa la fupa. Kusintha kwa Modic type II kumawonetsa chizindikiro chachikulu pa T1W ndi T2W sequences zonse. Mu mtundu uwu wa zilonda, kuwonongeka kwa endplate kungapezeke, komanso kusintha kwa mafuta a histological a fupa lapafupi. Kusintha kwa Modic type III kumawonetsa chizindikiro chochepa mu T1W ndi T2W sequences. Zilonda za sclerotic zofanana ndi ma endplates zawonedwa6. MC imaonedwa kuti ndi matenda a msana ndipo imagwirizana kwambiri ndi matenda ambiri osachiritsika a msana7,8,9.
Poganizira zomwe zilipo, maphunziro angapo apereka chidziwitso chatsatanetsatane pa chifukwa ndi njira zochizira matenda a MC. Albert et al. adanenanso kuti MC ikhoza kuyambitsidwa ndi herniation ya disc8. Hu et al. adati MC imayambitsidwa ndi kuwonongeka kwakukulu kwa disc10. Kroc adapereka lingaliro la "kuphulika kwa disc yamkati," komwe kumanena kuti kuvulala mobwerezabwereza kwa disc kungayambitse misozi yaying'ono mu endplate. Pambuyo pa kupangika kwa cleft, kuwonongeka kwa endplate ndi nucleus pulposus (NP) kungayambitse yankho la autoimmune, zomwe zimapangitsa kuti MC11 ikule. Ma et al. adagawana malingaliro ofanana ndipo adanenanso kuti NP-induced autoimmune imagwira ntchito yofunika kwambiri pakukula kwa MC12.
Maselo a chitetezo chamthupi, makamaka ma lymphocyte othandizira a CD4+ T, amachita gawo lofunika kwambiri pakukula kwa chitetezo chamthupi13. Gulu la Th17 lomwe lapezeka posachedwa limapanga cytokine IL-17 yotupa, limalimbikitsa kutulutsa kwa chemokine, komanso limalimbikitsa maselo a T m'ziwalo zowonongeka kuti apange IFN-γ14. Maselo a Th2 amachitanso gawo lapadera pakukula kwa mayankho a chitetezo chamthupi. Kuwonekera kwa IL-4 ngati selo loyimira Th2 kungayambitse zotsatira zoyipa za chitetezo chamthupi15.
Ngakhale kuti maphunziro ambiri azachipatala achitika pa MC16,17,18,19,20,21,22,23,24, pakadalibe kusowa kwa zitsanzo zoyenera zoyesera nyama zomwe zingatsanzire njira ya MC yomwe imachitika kawirikawiri mwa anthu ndipo ingagwiritsidwe ntchito kufufuza zomwe zimayambitsa kapena mankhwala atsopano monga chithandizo cholunjika. Mpaka pano, zitsanzo zochepa chabe za nyama za MC zanenedwa kuti zimaphunzira njira zomwe zimayambitsa matenda.
Kutengera ndi chiphunzitso cha autoimmune chomwe Albert ndi Ma adapereka, kafukufukuyu adakhazikitsa chitsanzo chosavuta komanso chobwerezabwereza cha MC cha kalulu mwa kuyika NP yokha pafupi ndi mbale yobowoledwa ya vertebral end. Zolinga zina ndikuwona mawonekedwe a histological a zitsanzo za nyama ndikuwunika njira zenizeni za NP pakukula kwa MC. Pachifukwa ichi, timagwiritsa ntchito njira monga biology ya mamolekyulu, MRI, ndi maphunziro a histological kuti tiphunzire kupita patsogolo kwa MC.
Akalulu awiri anamwalira chifukwa cha kutuluka magazi panthawi ya opaleshoni, ndipo akalulu anayi anamwalira panthawi ya opaleshoni ya MRI. Akalulu 48 otsalawo adapulumuka ndipo sanawonetse zizindikiro za khalidwe kapena za mitsempha atachitidwa opaleshoni.
MRI ikuwonetsa kuti mphamvu ya chizindikiro cha minofu yolumikizidwa m'mabowo osiyanasiyana ndi yosiyana. Mphamvu ya chizindikiro cha thupi la msana la L5 mu gulu la NPE inasintha pang'onopang'ono patatha milungu 12, 16 ndi 20 mutalowa (T1W sequence inawonetsa chizindikiro chochepa, ndipo T2W sequence inawonetsa chizindikiro chosakanikirana kuphatikiza chizindikiro chochepa) (Chithunzi 1C), pomwe mawonekedwe a MRI a magulu ena awiri a ziwalo zolumikizidwa anakhalabe okhazikika panthawi yomweyi (Chithunzi 1A, B).
(A) Ma MRI otsatizana a msana wa kalulu pa nthawi zitatu. Palibe zolakwika za chizindikiro zomwe zapezeka pazithunzi za gulu la opaleshoni ya sham. (B) Makhalidwe a chizindikiro cha thupi la vertebral mu gulu la ME ndi ofanana ndi omwe ali mu gulu la opaleshoni ya sham, ndipo palibe kusintha kwakukulu kwa chizindikiro komwe kumawonedwa pamalo opachikika pakapita nthawi. (C) Mu gulu la NPE, chizindikiro chotsika chimawonekera bwino mu sequence ya T1W, ndipo chizindikiro chosakanikirana ndi chizindikiro chotsika chimawonekera bwino mu sequence ya T2W. Kuyambira nthawi ya masabata 12 mpaka nthawi ya masabata 20, zizindikiro zapamwamba zosakhazikika zozungulira zizindikiro zotsika mu sequence ya T2W zimachepa.
Kuchuluka kwa mafupa koonekeratu kumatha kuwoneka pamalo oikira thupi la msana mu gulu la NPE, ndipo kuchuluka kwa mafupa kumachitika mwachangu kuyambira masabata 12 mpaka 20 (Chithunzi 2C) poyerekeza ndi gulu la NPE, palibe kusintha kwakukulu komwe kumawoneka m'matupi a msana omwe apangidwa; Gulu la Sham ndi gulu la ME (Chithunzi 2C) 2A,B).
(A) Pamwamba pa thupi la msana pa gawo loikidwa ndi losalala kwambiri, dzenje limachira bwino, ndipo palibe hyperplasia m'thupi la msana. (B) Mawonekedwe a malo oikidwa mu gulu la ME ndi ofanana ndi a gulu la opaleshoni yachinyengo, ndipo palibe kusintha koonekeratu pa mawonekedwe a malo oikidwa pakapita nthawi. (C) Hyperplasia ya mafupa inachitika pamalo oikidwa mu gulu la NPE. Hyperplasia ya mafupa inakula mofulumira ndipo inafalikira kudzera mu intervertebral disc kupita ku thupi la msana la contralateral.
Kusanthula kwa histological kumapereka chidziwitso chatsatanetsatane chokhudza kupangika kwa mafupa. Chithunzi 3 chikuwonetsa zithunzi za magawo a pambuyo pa opaleshoni okhala ndi H&E. Mu gulu la sham-operation, ma chondrocyte anali okonzedwa bwino ndipo palibe kuchuluka kwa maselo komwe kunapezeka (Chithunzi 3A). Mkhalidwe mu gulu la ME unali wofanana ndi wa gulu la sham-operation (Chithunzi 3B). Komabe, mu gulu la NPE, kuchuluka kwa ma chondrocyte ndi kuchuluka kwa maselo ofanana ndi NP kunawonedwa pamalo opatulira (Chithunzi 3C);
(A) Ma trabeculae amatha kuwoneka pafupi ndi mbale yomaliza, ma chondrocyte amakonzedwa bwino ndi kukula ndi mawonekedwe ofanana a maselo ndipo palibe kuchulukana (nthawi 40). (B) Mkhalidwe wa malo oikiramo maselo mu gulu la ME ndi wofanana ndi wa gulu lachinyengo. Ma trabeculae ndi ma chondrocyte amatha kuwoneka, koma palibe kuchulukana koonekeratu pamalo oikiramo maselo (nthawi 40). (B) Zitha kuwoneka kuti ma chondrocyte ndi maselo ofanana ndi NP amachulukana kwambiri, ndipo mawonekedwe ndi kukula kwa ma chondrocyte sizofanana (nthawi 40).
Kuwonetsedwa kwa interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, ndi interferon γ (IFN-γ) mRNA kunawonedwa m'magulu onse a NPE ndi ME. Pamene kuchuluka kwa majini omwe amafunidwa kunayerekezeredwa, kuchuluka kwa majini a IL-4, IL-17, ndi IFN-γ kunawonjezeka kwambiri mu gulu la NPE poyerekeza ndi gulu la ME ndi gulu la opareshoni ya sham (Chithunzi 4) (P < 0.05). Poyerekeza ndi gulu la opareshoni ya sham, kuchuluka kwa IL-4, IL-17, ndi IFN-γ mu gulu la ME kunawonjezeka pang'ono chabe ndipo sikunafike pakusintha kwa ziwerengero (P > 0.05).
Kufotokozera kwa mRNA kwa IL-4, IL-17 ndi IFN-γ mu gulu la NPE kunawonetsa chizolowezi chachikulu kwambiri kuposa cha gulu la chinyengo ndi gulu la ME (P < 0.05).
Mosiyana ndi zimenezi, kuchuluka kwa mawu m'gulu la ME sikunasonyeze kusiyana kwakukulu (P>0.05).
Kusanthula kwa Western blot kunachitika pogwiritsa ntchito ma antibodies omwe amapezeka m'masitolo motsutsana ndi IL-4 ndi IL-17 kuti atsimikizire kusintha kwa mawonekedwe a mRNA. Monga momwe zasonyezedwera mu Zithunzi 5A, B, poyerekeza ndi gulu la ME ndi gulu la opaleshoni ya sham, kuchuluka kwa mapuloteni a IL-4 ndi IL-17 mu gulu la NPE kunawonjezeka kwambiri (P < 0.05). Poyerekeza ndi gulu la opaleshoni ya sham, kuchuluka kwa mapuloteni a IL-4 ndi IL-17 mu gulu la ME kunalepheranso kufika pakusintha kwakukulu (P> 0.05).
(A) Kuchuluka kwa mapuloteni a IL-4 ndi IL-17 m'gulu la NPE kunali kwakukulu kwambiri kuposa omwe ali m'gulu la ME ndi gulu la placebo (P < 0.05). (B) Western blot histogram.
Chifukwa cha kuchuluka kochepa kwa zitsanzo za anthu zomwe zapezeka panthawi ya opaleshoni, maphunziro omveka bwino komanso atsatanetsatane okhudza matenda a MC ndi ovuta pang'ono. Tinayesa kukhazikitsa chitsanzo cha nyama cha MC kuti tiphunzire njira zake zomwe zingatheke zochizira matenda. Nthawi yomweyo, kuwunika kwa radiological, kuwunika kwa histological ndi kuwunika kwa mamolekyulu a zamoyo zinagwiritsidwa ntchito kutsatira njira ya MC yoyambitsidwa ndi NP autograft. Zotsatira zake, chitsanzo cha NP implantation chinapangitsa kusintha pang'onopang'ono kwa mphamvu ya chizindikiro kuchokera pa masabata 12 mpaka masabata 20 (kusakaniza chizindikiro chochepa mu T1W sequences ndi chizindikiro chochepa mu T2W sequences), kusonyeza kusintha kwa minofu, ndipo kuwunika kwa histological ndi mamolekyulu a zamoyo kunatsimikizira zotsatira za kafukufuku wa radiological.
Zotsatira za kuyesera kumeneku zikusonyeza kuti kusintha kwa maso ndi za histological kunachitika pamalo pomwe thupi la vertebral linaphwanya mu gulu la NPE. Nthawi yomweyo, kuwonetsedwa kwa majini a IL-4, IL-17 ndi IFN-γ, komanso majini a IL-4, IL-17 ndi IFN-γ kunawonedwa, zomwe zikusonyeza kuti kuphwanya kwa minofu ya autologous nucleus pulposus m'thupi la vertebral kungayambitse kusintha kwa zizindikiro ndi mawonekedwe. N'zosavuta kupeza kuti makhalidwe a zizindikiro za matupi a vertebral a mtundu wa nyama (chizindikiro chochepa mu T1W sequence, chizindikiro chosakanikirana ndi chizindikiro chochepa mu T2W sequence) ndi ofanana kwambiri ndi a maselo a vertebral a anthu, ndipo makhalidwe a MRI amatsimikiziranso kuwona kwa histology ndi gross anatomy, ndiko kuti, kusintha kwa maselo a vertebral body kumapita patsogolo. Ngakhale kuti kutupa komwe kumachitika chifukwa cha kuvulala kwakukulu kungawonekere atangobowoka, zotsatira za MRI zasonyeza kuti kusintha kwa zizindikiro komwe kumawonjezeka pang'onopang'ono kunawonekera milungu 12 pambuyo pobowoka ndipo kunapitirira mpaka milungu 20 popanda zizindikiro za kuchira kapena kusinthika kwa kusintha kwa MRI. Zotsatirazi zikusonyeza kuti autologous vertebral NP ndi njira yodalirika yokhazikitsira MV yopita patsogolo mwa akalulu.
Chitsanzo choboola ichi chimafuna luso lokwanira, nthawi, komanso kuyesetsa kwa opaleshoni. Poyesa koyambirira, kuduladula kapena kusonkhezera kwambiri mapangidwe a paravertebral ligamentous kungayambitse mapangidwe a vertebral osteophytes. Samalani kuti musawononge kapena kukwiyitsa ma disc omwe ali pafupi. Popeza kuya kwa kulowa kuyenera kulamulidwa kuti mupeze zotsatira zofanana komanso zobwerezabwereza, tinapanga pulagi pamanja podula chivundikiro cha singano yayitali ya 3 mm. Kugwiritsa ntchito pulagi iyi kumatsimikizira kuya kofanana kwa kuboola m'thupi la vertebral. Poyesa koyambirira, madokotala atatu a mafupa omwe adachita opaleshoniyi adapeza kuti singano za gauge 16 ndizosavuta kugwira ntchito kuposa singano za gauge 18 kapena njira zina. Kuti tipewe kutuluka magazi kwambiri panthawi yoboola, kugwira singano chete kwakanthawi kumapereka dzenje loyenera kwambiri lolowera, zomwe zikusonyeza kuti mlingo winawake wa MC ukhoza kulamulidwa mwanjira iyi.
Ngakhale kuti maphunziro ambiri akuyang'ana kwambiri MC, zochepa zomwe zimadziwika za chifukwa chake MC25,26,27 imayambitsa komanso momwe imafalikira. Kutengera ndi maphunziro athu am'mbuyomu, tapeza kuti chitetezo chamthupi chimagwira ntchito yofunika kwambiri pakukula ndi kufalikira kwa MC12. Kafukufukuyu adafufuza kuchuluka kwa IL-4, IL-17, ndi IFN-γ, zomwe ndi njira zazikulu zosiyanitsira maselo a CD4+ pambuyo poyambitsa antigen. Mu kafukufuku wathu, poyerekeza ndi gulu loipa, gulu la NPE linali ndi kuchuluka kwa IL-4, IL-17, ndi IFN-γ, ndipo kuchuluka kwa mapuloteni a IL-4 ndi IL-17 kunalinso kwakukulu.
Mwachipatala, kuchuluka kwa IL-17 mRNA kumawonjezeka m'maselo a NP kuchokera kwa odwala omwe ali ndi disc herniation28. Kuwonjezeka kwa IL-4 ndi IFN-γ expression milingo kunapezekanso mu acute non-compressive disc herniation model poyerekeza ndi athanzi controls29. IL-17 imagwira ntchito yofunika kwambiri pa kutupa, kuvulala kwa minofu m'matenda a autoimmune30 ndipo imawonjezera chitetezo chamthupi ku IFN-γ31. Kuvulala kwa minofu komwe kumachitika chifukwa cha IL-17 kwanenedwa mu MRL/lpr mice32 ndi mbewa zomwe zimakhudzidwa ndi autoimmune33. IL-4 ikhoza kuletsa kuwonetsedwa kwa ma cytokines oyambitsa kutupa (monga IL-1β ndi TNFα) ndi macrophage activation34. Zinanenedwa kuti kuwonetsedwa kwa mRNA kwa IL-4 kunali kosiyana mu gulu la NPE poyerekeza ndi IL-17 ndi IFN-γ panthawi yomweyo; Kuwonetsedwa kwa mRNA kwa IFN-γ mu gulu la NPE kunali kwakukulu kwambiri kuposa m'magulu ena. Chifukwa chake, kupanga kwa IFN-γ kungakhale mkhalapakati wa yankho lotupa lomwe limayambitsidwa ndi NP intercalation. Kafukufuku wasonyeza kuti IFN-γ imapangidwa ndi mitundu yosiyanasiyana ya maselo, kuphatikizapo maselo othandizira amtundu woyamba a T, maselo opha tizilombo toyambitsa matenda, ndi macrophages35,36, ndipo ndi cytokine yofunika kwambiri yoteteza kutupa yomwe imalimbikitsa chitetezo cha mthupi37.
Kafukufukuyu akusonyeza kuti yankho la autoimmune lingakhale lothandiza pakuchitika ndi kukula kwa MC. Luoma ndi anzake adapeza kuti zizindikiro za MC ndi NP yodziwika bwino ndizofanana pa MRI, ndipo zonse zimasonyeza chizindikiro chapamwamba mu T2W sequence38. Ma cytokines ena atsimikiziridwa kuti amagwirizana kwambiri ndi kuchitika kwa MC, monga IL-139. Ma ndi anzake adanenanso kuti kutuluka kwa NP mmwamba kapena pansi kungakhale ndi mphamvu yaikulu pa kuchitika ndi kukula kwa MC12. Bobechko40 ndi Herzbein ndi anzake adanenanso kuti NP ndi minofu yosalekerera chitetezo chamthupi yomwe singalowe m'mitsempha yamagazi kuyambira kubadwa. Kutuluka kwa NP kumabweretsa matupi akunja m'magazi, motero kumayambitsa zochitika za autoimmune zakomweko42. Zochita za autoimmune zimatha kuyambitsa zinthu zambiri zoteteza chitetezo chamthupi, ndipo pamene zinthuzi zimawonekera nthawi zonse ku minofu, zimatha kuyambitsa kusintha kwa zizindikiro43. Mu kafukufukuyu, kuwonetsa kwambiri IL-4, IL-17 ndi IFN-γ ndi zinthu zoteteza chitetezo chamthupi, zomwe zimatsimikiziranso ubale wapakati pa NP ndi MCs44. Chitsanzo cha nyama ichi chimatsanzira bwino kupita patsogolo kwa NP ndi kulowa mu mbale yomaliza. Njirayi inavumbulutsanso momwe chitetezo chamthupi cha munthu chimakhudzira MC.
Monga momwe tikuyembekezerera, chitsanzo cha nyama ichi chimatipatsa malo oti tiphunzire za MC. Komabe, chitsanzochi chili ndi zofooka zina: choyamba, panthawi yowonera nyama, akalulu ena apakatikati amafunika kuphedwa kuti akayesedwe ka histological ndi molecular biology, kotero nyama zina "zimasiya kugwiritsa ntchito" pakapita nthawi. Kachiwiri, ngakhale kuti nthawi zitatu zakhazikitsidwa mu kafukufukuyu, mwatsoka, tinangopanga chitsanzo cha mtundu umodzi wa MC (Modic type I change), kotero sizokwanira kuyimira njira yopangira matenda a anthu, ndipo nthawi zambiri ziyenera kukhazikitsidwa kuti tiwone bwino kusintha konse kwa chizindikiro. Kachitatu, kusintha kwa kapangidwe ka minofu kumatha kuwonetsedwa bwino ndi histological staining, koma njira zina zapadera zitha kuwulula bwino kusintha kwa microstructural mu chitsanzo ichi. Mwachitsanzo, polarized light microscopy idagwiritsidwa ntchito pofufuza kapangidwe ka fibrocartilage mu rabbit intervertebral discs45. Zotsatira za NP za nthawi yayitali pa MC ndi endplate zimafuna kuphunzira kwina.
Akalulu oyera amphongo 54 aku New Zealand (olemera pafupifupi makilogalamu 2.5-3, azaka zapakati pa miyezi 3-3.5) adagawidwa mwachisawawa m'magulu awiri: gulu la opaleshoni ya chinyengo, gulu la kuyika minofu (gulu la ME) ndi gulu la kuyika mizu ya mitsempha (gulu la NPE). Njira zonse zoyesera zidavomerezedwa ndi Komiti ya Makhalidwe Abwino ya Chipatala cha Tianjin, ndipo njira zoyesera zidachitika motsatira malangizo ovomerezeka.
Kusintha kwina kwachitika pa njira yopangira opaleshoni ya S. Sobajima 46. Kalulu aliyense anaikidwa pamalo ogona kumbuyo ndipo pamwamba pa ma disc asanu otsatizana a lumbar intervertebral (IVDs) anaonekera pogwiritsa ntchito njira ya posterolateral retroperitoneal. Kalulu aliyense anapatsidwa mankhwala oletsa ululu (20% urethane, 5 ml/kg kudzera m'mitsempha ya khutu). Kucheka khungu kwa nthawi yayitali kunapangidwa kuchokera m'mphepete mwa nthiti mpaka m'mphepete mwa chiuno, 2 cm ventral kupita ku minofu ya paravertebral. Msana wakumanja wa anterolateral kuchokera ku L1 mpaka L6 unaonekera mwa kudula minofu ya subcutaneous yomwe inali pamwamba, minofu ya retroperitoneal, ndi minofu (Chithunzi 6A). Mlingo wa disc unadziwika pogwiritsa ntchito m'mphepete mwa chiuno ngati chizindikiro cha anatomical cha mulingo wa disc wa L5-L6. Gwiritsani ntchito singano ya 16-gauge puncture kuti muboole dzenje pafupi ndi mbale yomaliza ya vertebra ya L5 mpaka kuya kwa 3 mm (Chithunzi 6B). Gwiritsani ntchito sirinji ya 5-ml kuti mutulutse nucleus pulposus ya autologous mu L1-L2 intervertebral disc (Chithunzi 6C). Chotsani nucleus pulposus kapena minofu malinga ndi zofunikira za gulu lililonse. Pambuyo poti dzenje lobowola lazama, ma suture otha kuyamwa amayikidwa pa deep fascia, superficial fascia ndi khungu, ndikusamala kuti asawononge minofu ya periosteal ya thupi la vertebral panthawi ya opaleshoni.
(A) Disiki ya L5–L6 imaonekera kudzera mu njira ya posterolateral retroperitoneal. (B) Gwiritsani ntchito singano ya 16-gauge kuboola dzenje pafupi ndi endplate ya L5. (C) Ma Autologous MF amakololedwa.
Mankhwala oletsa ululu anaperekedwa ndi 20% urethane (5 ml/kg) kudzera m'mitsempha ya khutu, ndipo ma X-ray a msana wa lumbar anabwerezedwanso pa masabata 12, 16, ndi 20 pambuyo pa opaleshoni.
Akalulu anaperekedwa nsembe mwa kubayidwa ketamine (25.0 mg/kg) ndi sodium pentobarbital (1.2 g/kg) m'mitsempha patatha milungu 12, 16, ndi 20 kuchokera pamene opaleshoni inachitika. Msana wonse unachotsedwa kuti aunikenso histological ndipo kusanthula kwenikweni kunachitika. Quantitative reverse transcription (RT-qPCR) ndi Western blotting zinagwiritsidwa ntchito kuzindikira kusintha kwa zinthu zoteteza thupi.
Kuyezetsa kwa MRI kunachitika mwa akalulu pogwiritsa ntchito maginito a 3.0 T clinical magnet (GE Medical Systems, Florence, SC) okhala ndi cholandirira cha orthogonal limb coil. Akalulu anapatsidwa urethane wa 20% (5 mL/kg) kudzera m'mitsempha ya khutu kenako n’kuikidwa chagadama mkati mwa maginitowo ndi dera la lumbar loyang’ana pa coil yozungulira ya mainchesi 5 (GE Medical Systems). Zithunzi za Coronal T2-weighted localizer (TR, 1445 ms; TE, 37 ms) zinapezedwa kuti zidziwitse komwe kuli lumbar disc kuyambira L3–L4 mpaka L5–L6. Zidutswa za Sagittal plane T2-weighted zinapezedwa ndi izi: fast spin-echo sequence ndi repetition time (TR) ya 2200 ms ndi echo time (TE) ya 70 ms, matrix; visual field ya 260 ndi 8 stimuli; Cutting thickness was 2 mm, gap was 0.2 mm.
Pambuyo poti chithunzi chomaliza chajambulidwa ndipo kalulu womaliza waphedwa, ma disc a sham, minofu yolumikizidwa, ndi NP zinachotsedwa kuti ziwunikidwe ndi histological. Minofu inakhazikika mu formalin ya 10% yopanda calcium kwa sabata imodzi, yochotsedwa ndi ethylenediaminetetraacetic acid, ndipo parafini inadulidwa. Ma blocks a minofu anaikidwa mu parafini ndikudulidwa m'magawo a sagittal (5 μm makulidwe) pogwiritsa ntchito microtome. Magawo anapakidwa hematoxylin ndi eosin (H&E).
Pambuyo posonkhanitsa ma disc a intervertebral kuchokera ku akalulu m'gulu lililonse, RNA yonse idachotsedwa pogwiritsa ntchito UNIQ-10 column (Shanghai Sangon Biotechnology Co., Ltd., China) motsatira malangizo a wopanga ndi ImProm II reverse transcription system (Promega Inc., Madison, WI, USA). Reverse transcription idachitika.
RT-qPCR idachitidwa 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. Kuchuluka kwa PCR reaction kunali 20 μl ndipo kunali 1.5 μl ya cDNA yochepetsedwa ndi 0.2 μM ya primer iliyonse. Ma primer adapangidwa ndi OligoPerfect Designer (Invitrogen, Valencia, CA) ndipo adapangidwa ndi Nanjing Golden Stewart Biotechnology Co., Ltd. (China) (Table 1). Zinthu zotsatirazi zokhudzana ndi kutentha zidagwiritsidwa ntchito: sitepe yoyamba yoyambitsa polymerase pa 94°C kwa mphindi ziwiri, kenako ma cycle 40 a 15 s iliyonse pa 94°C kuti template isinthe, annealing kwa mphindi imodzi pa 60°C, extension, ndi fluorescence. Miyeso idachitika kwa mphindi imodzi pa 72°C. Zitsanzo zonse zinakulitsidwa katatu ndipo mtengo wapakati unagwiritsidwa ntchito pofufuza za RT-qPCR. Deta yokulitsa inasanthulidwa pogwiritsa ntchito FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). IL-4, IL-17, ndi IFN-γ gene expression zinasinthidwa kukhala endogenous control (ACTB). Ma levels of relative expression a target mRNA anawerengedwa pogwiritsa ntchito njira ya 2-ΔΔCT.
Mapuloteni onse adachotsedwa m'maselo pogwiritsa ntchito homogenizer ya minofu mu RIPA lysis buffer (yomwe ili ndi protease ndi phosphatase inhibitor cocktail) kenako adayikidwa pa centrifuge pa 13,000 rpm kwa mphindi 20 pa 4°C kuti achotse zinyalala za minofu. Ma microgram makumi asanu a mapuloteni adayikidwa pa msewu uliwonse, kulekanitsidwa ndi 10% SDS-PAGE, kenako adasamutsidwa ku nembanemba ya PVDF. Kutseka kunachitika mu mkaka wouma wopanda mafuta wa 5% mu Tris-buffered saline (TBS) wokhala ndi 0.1% Tween 20 kwa ola limodzi kutentha kwa chipinda. Nembanembayo idayikidwa ndi anti-decorin primary antibody ya kalulu (yomwe idachepetsedwa 1:200; Boster, Wuhan, China) (yomwe idachepetsedwa 1:200; Bioss, Beijing, China) usiku wonse pa 4°C ndipo idachitapo kanthu pa masiku achiwiri; ndi antibody yachiwiri (mbuzi yotsutsana ndi kalulu G pa 1:40,000 dilution) pamodzi ndi horseradish peroxidase (Boster, Wuhan, China) kwa ola limodzi kutentha kwa chipinda. Zizindikiro za Western blot zinapezeka ndi kuwonjezeka kwa chemiluminescence pa nembanemba ya chemiluminescent pambuyo pa X-ray radiation. Pa kusanthula kwa densitometry, ma blots adasanthulidwa ndikuyesedwa pogwiritsa ntchito pulogalamu ya BandScan ndipo zotsatira zake zidafotokozedwa ngati chiŵerengero cha target gene immunoreactivity ndi tubulin immunoreactivity.
Kuwerengera ziwerengero kunachitika pogwiritsa ntchito pulogalamu ya SPSS16.0 (SPSS, USA). Deta yomwe inasonkhanitsidwa panthawi ya kafukufukuyu inafotokozedwa ngati mean ± standard deviation (mean ± SD) ndipo inasanthulidwa pogwiritsa ntchito njira imodzi yobwerezabwereza kusanthula kusiyana kwa kusiyana (ANOVA) kuti adziwe kusiyana pakati pa magulu awiriwa. P < 0.05 inaonedwa kuti ndi yofunika kwambiri pa ziwerengero.
Motero, kukhazikitsidwa kwa chitsanzo cha nyama cha MC mwa kuika ma NP a autologous m'thupi la msana ndikuchita kafukufuku wa macroanatomical, kusanthula kwa MRI, kuwunika kwa histological ndi kusanthula kwa mamolekyulu a zamoyo kungakhale chida chofunikira poyesa ndikumvetsetsa njira za MC ya anthu ndikupanga njira zatsopano zochiritsira.
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Nthawi yotumizira: Disembala-13-2024