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ʻO ka hoʻokumu ʻana i nā hiʻohiʻona holoholona o ka hoʻololi hoʻololi (MC) he kumu koʻikoʻi no ke aʻo ʻana i ka MC. Ua māhele ʻia he kanalimakūmāhā mau rabbits keʻokeʻo o New Zealand i hui hoʻopunipuni, pūʻulu hoʻokomo ʻiʻo (ME pūʻulu) a me ka pūʻulu hoʻokomo ʻana o ka nucleus pulposus (hui NPE). I loko o ka hui NPE, ua hōʻike ʻia ka disc intervertebral e ka anterolateral lumbar surgical approach a ua hoʻohana ʻia kahi nila no ke kī ʻana i ke kino vertebral L5 kokoke i ka pā hope. Lawe ʻia ʻo NP mai ka L1 / 2 intervertebral disc e kahi syringe a hoʻokomo ʻia i loko. ʻO ka ʻeli ʻana i lua ma ka iwi subchondral. ʻO nā kaʻina hana a me nā hana wili i loko o ka hui implantation muscle a me ka hui sham-operation ua like ia me nā mea i loko o ka hui NP implantation. I loko o ka pūʻulu ME, ua hoʻokomoʻia kahi'āpanaʻiʻo i loko o ka lua,ʻoiai ma ka hui hana hoʻopunipuni,ʻaʻohe mea i hoʻokomoʻia i loko o ka lua. Ma hope o ka hana ʻana, ua hana ʻia ka nānā ʻana i ka MRI a me ka hoʻāʻo ʻana i ka biological molecular. Ua hoʻololi ka hōʻailona ma ka hui NPE, akā ʻaʻohe hoʻololi hōʻailona maopopo i ka hui sham-operation a me ka hui ME. Ua hōʻike ʻia ka nānā ʻana o ka mōʻaukala i ka hoʻonui ʻia ʻana o ka ʻiʻo ma ka wahi implantation, a ua hoʻonui ʻia ka ʻōlelo o IL-4, IL-17 a me IFN-γ i ka hui NPE. ʻO ka hoʻokomo ʻana o NP i loko o ka iwi subchondral hiki ke hana i kahi ʻano holoholona o MC.
ʻO nā hoʻololi hoʻololi (MC) he mau liona o nā ʻaoʻao hope vertebral a me ka momona o ka iwi e pili ana i ʻike ʻia ma ka magnetic resonance imaging (MRI). He mea maʻamau ia i nā poʻe me nā hōʻailona pili1. Ua hoʻoikaika nui nā haʻawina i ke koʻikoʻi o MC ma muli o kona hui pū ʻana me ka ʻeha hope haʻahaʻa (LBP)2,3. de Roos et al.4 a me Modic et al.5 i wehewehe mua i ʻekolu mau ʻano like ʻole o ka subchondral hōʻailona abnormalities i ka iwi iwi vertebral. ʻO nā hoʻololi ʻano ʻano I he hypointense ma nā kaʻina T1-kaumaha (T1W) a me ka hyperintense ma nā kaʻina T2-kaumaha (T2W). Hōʻike kēia ʻeha i nā ʻāpana hope a me ka ʻiʻo granulation vascular pili i ka iwi iwi. Hōʻike nā hoʻololi modic type II i ka hōʻailona kiʻekiʻe ma nā kaʻina T1W a me T2W. I loko o kēia ʻano lesion, hiki ke ʻike ʻia ka luku ʻana i ka endplate, a me ka hoʻololi ʻana i ka momona histological o ka iwi iwi pili. Hōʻike nā hoʻololi modic type III i ka hōʻailona haʻahaʻa ma nā kaʻina T1W a me T2W. Ua ʻike ʻia nā ʻeha sclerotic e pili ana i nā papa hope. Manaʻo ʻia ʻo MC he maʻi pathological o ka spine a pili pū me nā maʻi degenerative he nui o ka spine7,8,9.
Ke noʻonoʻo nei i ka ʻikepili i loaʻa, ua hāʻawi kekahi mau noiʻi i nā ʻike kikoʻī i ka etiology a me nā hana pathological o MC. ʻO Albert et al. Ua manaʻo ʻia ʻo MC paha ke kumu o ka disc herniation8. Hu et al. ua hoʻopili ʻia ʻo MC i ka degeneration disc koʻikoʻi10. Hoʻopuka ʻo Kroc i ka manaʻo o ka "internal disc rupture," e ʻōlelo ana e hiki ke alakaʻi i ka trauma disc repetitive i nā microtears ma ka endplate. Ma hope o ka hoʻokumu ʻia ʻana o ka cleft, hiki i ka luku ʻana i ka endplate e ka nucleus pulposus (NP) ke hoʻoulu i kahi pane autoimmune, kahi e alakaʻi hou ai i ka hoʻomohala ʻana o MC11. Ma et al. kaʻana like i ka manaʻo like a hōʻike ʻo NP-induced autoimmunity i ke kuleana nui i ka pathogenesis o MC12.
ʻO nā pūnaewele ʻōnaehana pale, ʻo ia hoʻi nā lymphocytes kōkua CD4 + T, he kuleana koʻikoʻi i ka pathogenesis o ka autoimmunity13. ʻO ka subset Th17 i ʻike hou ʻia e hoʻopuka i ka cytokine proinflammatory IL-17, hoʻoikaika i ka ʻōlelo chemokine, a hoʻoulu i nā cell T i loko o nā kino poino e hana i ka IFN-γ14. ʻO nā pūnaewele Th2 kekahi hana kūikawā i ka pathogenesis o nā pane immune. ʻO ka hōʻike ʻana o IL-4 ma ke ʻano he ʻelele Th2 hiki ke alakaʻi i nā hopena immunopathological koʻikoʻi15.
ʻOiai ua nui nā haʻawina hoʻokolohua i mālama ʻia ma MC16,17,18,19,20,21,22,23,24, aia nō ka nele o nā hiʻohiʻona hoʻokolohua holoholona kūpono e hiki ke hoʻohālike i ke kaʻina hana MC i hana pinepine ʻia i loko o ke kanaka a hiki ke hoʻohana ʻia e noiʻi i ka etiology a i ʻole nā lāʻau lapaʻau hou e like me ka therapy targeted. I kēia lā, ua hōʻike ʻia he liʻiliʻi wale nō nā hiʻohiʻona holoholona o MC e aʻo i nā hana pathological lalo.
Ma muli o ke kumumanaʻo autoimmune i manaʻo ʻia e Albert lāua ʻo Ma, ua hoʻokumu kēia haʻawina i kahi hiʻohiʻona rabbit MC maʻalahi a hiki ke hana hou ʻia e ka autotransplanting NP kokoke i ka pā hope vertebral drilled. ʻO nā pahuhopu ʻē aʻe e nānā i nā hiʻohiʻona histological o nā hiʻohiʻona holoholona a loiloi i nā mīkini kikoʻī o NP i ka hoʻomohala ʻana o MC. I kēia hopena, hoʻohana mākou i nā ʻenehana e like me ka molecular biology, MRI, a me nā haʻawina histological e aʻo i ka holomua o MC.
Ua make ʻelua rapeti i ke kahe koko i ka wā o ka ʻoki ʻana, a ʻehā mau lākeke i make i ka wā maʻi maʻi ma MRI. Ua ola nā lākeke 48 i koe a ʻaʻole hōʻike i nā hōʻailona hana a i ʻole neurological ma hope o ke ʻoki ʻana.
Hōʻike ka MRI i ka ʻokoʻa o ka hōʻailona hōʻailona o ka ʻiʻo i hoʻokomo ʻia i nā lua like ʻole. ʻO ka ikaika o ka hōʻailona o ka L5 vertebral kino i loko o ka hui NPE ua hoʻololi mālie i ka 12, 16 a me 20 mau wiki ma hope o ka hoʻokomoʻana (T1W sequence i hōʻike i ka hōʻailona haʻahaʻa, aʻo ka T2W sequence i hōʻike i ka hōʻailona hui pū me ka hōʻailona haʻahaʻa) (Fig. 1C), aʻo nā hiʻohiʻona MRI ʻO nā pūʻulu ʻelua o nā ʻāpana i hoʻopili ʻia i noho paʻa i ka manawa like (Fig. 1A, B).
(A) Lunamaka'āinana sequential MRIs o ka rabbit lumbar spine ma 3 manawa manawa. ʻAʻohe hōʻailona hōʻailona i loaʻa i nā kiʻi o ka hui hana hoʻopunipuni. (B) ʻO nā hiʻohiʻona hōʻailona o ke kino vertebral i ka hui ME e like me nā mea i loko o ka hui sham-operation, a ʻaʻole ʻike ʻia kahi hoʻololi hōʻailona koʻikoʻi ma ka pūnaewele hoʻokomo i ka manawa. (C) Ma ka pūʻulu NPE, ʻike maopopo ʻia ka hōʻailona haʻahaʻa ma ke kaʻina T1W, a ʻike maopopo ʻia ka hōʻailona hui ʻia a me ka hōʻailona haʻahaʻa i ke kaʻina T2W. Mai ka 12-week period a hiki i ka 20-week period, nā hōʻailona kiʻekiʻe sporadic e hoʻopuni ana i nā hōʻailona haʻahaʻa i ke kaʻina T2W.
Hiki ke ʻike ʻia ka hyperplasia iwi ʻike ma ka wahi implantation o ke kino vertebral i ka hui NPE, a ʻoi aku ka wikiwiki o ka hyperplasia iwi mai 12 a hiki i nā wiki 20 (Fig. 2C) i hoʻohālikelike ʻia me ka hui NPE, ʻaʻohe loli nui i ʻike ʻia i ka vertebral modeled. kino; Hui Sham a me ME hui (Fig. 2C) 2A,B).
(A) ʻO ka ʻili o ke kino vertebral ma ka ʻāpana i hoʻokomo ʻia he maʻalahi loa, hoʻōla maikaʻi ka lua, ʻaʻohe hyperplasia i ke kino vertebral. (B) ʻO ke ʻano o ka pūnaewele i hoʻokomo ʻia i loko o ka pūʻulu ME ua like ia me ka pūʻulu hana sham, a ʻaʻohe hoʻololi ʻike ʻia i ke ʻano o ka pūnaewele i hoʻokomo ʻia i ka manawa. (C) Ua loaʻa ka hyperplasia iwi ma kahi i hoʻokomo ʻia ma ka hui NPE. Ua piʻi wikiwiki ka hyperplasia iwi a ua hoʻonui ʻia ma waena o ka disc intervertebral i ke kino vertebral contralateral.
Hāʻawi ka ʻikepili moʻolelo i ka ʻike kikoʻī e pili ana i ka hana iwi. Hōʻike ke kiʻi 3 i nā kiʻi o nā ʻāpana postoperative i hoʻopaʻa ʻia me ka H&E. I loko o ka hui sham-operation, ua hoʻonohonoho maikaʻiʻia nā chondrocytes aʻaʻole iʻikeʻia ka hoʻonuiʻana o ke kelepona (Fig. 3A). Ua like ke kūlana o ka hui ME me ka hui hoʻopunipuni (Fig 3B). Eia naʻe, i loko o ka pūʻulu NPE, uaʻikeʻia ka nui o nā chondrocytes a me ka hoʻonuiʻana o nā pūnaewele NP-like ma kahi implantation (Fig 3C);
(A) Hiki ke ʻike ʻia ʻo Trabeculae ma kahi kokoke i ka pā hope, ua hoʻonohonoho pono ʻia nā chondrocytes me ka nui a me ke ʻano like ʻole a ʻaʻohe hoʻonui (40 mau manawa). (B) ʻO ke kūlana o ka pūnaewele implantation i loko o ka hui ME e like me ka hui sham. Hiki ke ʻike ʻia nā trabeculae a me nā chondrocytes, akā ʻaʻohe ʻike ʻia o ka hoʻonui ʻia ʻana ma ka wahi implantation (40 mau manawa). (B) Hiki ke ʻike ʻia ka ulu nui ʻana o nā chondrocytes a me NP-like cell, a ʻaʻole like ke ʻano a me ka nui o nā chondrocytes (40 mau manawa).
Uaʻikeʻia ka'ōlelo o interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, a me interferon γ (IFN-γ) mRNA ma nā hui NPE a me ME. I ka hoʻohālikelike ʻia ʻana o nā pae hōʻike o nā genes target, ua hoʻonui nui ʻia nā hōʻike gene o IL-4, IL-17, a me IFN-γ i ka hui NPE i hoʻohālikelike ʻia me nā mea o ka hui ME a me ka hui hana sham (Fig. 4) (P <0.05). Ke hoʻohālikelike ʻia me ka pūʻulu hana sham, ua piʻi iki nā pae hōʻike o IL-4, IL-17, a me IFN-γ i loko o ka hui ME a ʻaʻole i hiki i ka loli helu (P> 0.05).
ʻO ka hōʻike mRNA o IL-4, IL-17 a me IFN-γ i loko o ka hui NPE i hōʻike i kahi kiʻekiʻe kiʻekiʻe ma mua o ka poʻe i loko o ka hui hana sham a me ka hui ME (P <0.05).
I ka hoʻohālikelike ʻana, ʻaʻole i hōʻike ʻia nā pae hōʻike i ka hui ME ʻaʻohe ʻokoʻa nui (P>0.05).
Ua hana ʻia ka loiloi Western blot me ka hoʻohana ʻana i nā antibodies i kūʻai ʻia e kūʻē iā IL-4 a me IL-17 e hōʻoia i ke ʻano hōʻike mRNA i hoʻololi ʻia. E like me ka mea i hōʻike ʻia ma nā Kiʻi 5A,B, i hoʻohālikelike ʻia me ka hui ME a me ka hui hana sham, ua hoʻonui nui ʻia nā pae protein o IL-4 a me IL-17 i ka hui NPE (P <0.05). Ke hoʻohālikelike ʻia me ka pūʻulu hana sham, ʻaʻole hiki i nā pae protein o IL-4 a me IL-17 i ka hui ME ke hiki i nā loli koʻikoʻi (P> 0.05).
(A) Ua ʻoi aku ka kiʻekiʻe o nā pae protein o IL-4 a me IL-17 i ka hui NPE ma mua o nā mea i loko o ka hui ME a me ka hui placebo (P <0.05). (B) Histogram blot komohana.
Ma muli o ka liʻiliʻi o nā laʻana kanaka i loaʻa i ka wā o ke kaʻa ʻana, ʻano paʻakikī nā haʻawina a me nā kikoʻī e pili ana i ka pathogenesis o MC. Ua hoʻāʻo mākou e hoʻokumu i kahi hoʻohālike holoholona o MC e aʻo i kāna mau hana pathological. Ma ka manawa like, ua hoʻohana ʻia ka loiloi radiological, ka loiloi histological a me ka loiloi biological molecular e hahai i ke ʻano o ka MC i hoʻokomo ʻia e NP autograft. ʻO ka hopena, ua loaʻa i ke kumu hoʻohālike NP implantation ka hoʻololi mālie i ka ikaika o ka hōʻailona mai 12-week a 20-week time point (hui pū ʻia ka hōʻailona haʻahaʻa i nā kaʻina T1W a me ka hōʻailona haʻahaʻa i nā kaʻina T2W), e hōʻike ana i nā loli kiko, a me ka histological a me ka molecular. ua hōʻoia nā loiloi olaola i nā hopena o ka noiʻi radiological.
Hōʻike nā hopena o kēia hoʻokolohua i nā hoʻololi hiʻohiʻona a me ka histological i loaʻa ma ke kahua o ka hewa kino vertebral i ka hui NPE. I ka manawa like, ua ʻike ʻia ka ʻōlelo o IL-4, IL-17 a me IFN-γ genes, a me IL-4, IL-17 a me IFN-γ, e hōʻike ana i ka hewa o ka autologous nucleus pulposus tissue i ka vertebral. hiki i ke kino ke hoʻololi i nā hōʻailona a me nā loli. He mea maʻalahi kaʻikeʻana i nāʻano hōʻailona o nā kino vertebral o keʻano holoholona (hōʻailona haʻahaʻa ma ke kaʻina T1W, ka hōʻailona hui a me ka hōʻailona haʻahaʻa ma ke kaʻina T2W) heʻano like loa ia me nā pūnaewele vertebral kanaka, a me nāʻano MRI pū kekahi e hōʻoia i nāʻike o ka histology a me ka anatomy gross,ʻo ia hoʻi, ke holomua nei nā hoʻololi o nā pūnaewele kino vertebral. ʻOiai hiki ke ʻike koke ʻia ka pane ʻeha i hoʻokumu ʻia e ka trauma nui ma hope o ka puncture, ua hōʻike nā hopena MRI i ka piʻi nui ʻana o nā hoʻololi hōʻailona i 12 mau pule ma hope o ka puncture a hoʻomau ʻia a hiki i nā wiki he 20 me ka ʻole o nā hōʻailona o ka hoʻihoʻi ʻana a i ʻole ka hoʻololi ʻana o nā loli MRI. Hōʻike kēia mau hopena i ka autologous vertebral NP he ala hilinaʻi no ka hoʻokumu ʻana i ka MV holomua i nā rapeti.
Pono kēia kŘkohu puncture i ke akamai, ka manawa, a me ka ho'oikaika kino. Ma nā ho'āʻo mua, hiki i ka weheʻana a iʻole ka hoʻoulu nuiʻana o nā hale ligamentous paravertebral i ka hanaʻana i nā osteophytes vertebral. Pono e mālama ʻia ʻaʻole e hōʻino a hoʻonāukiuki paha i nā disc pili. No ka mea, pono e hoomaluia ka hohonu o ke komo ana no ka loaa ana o na hopena kupaa a hiki ke hana hou ia, ua hana lima makou i ka pulupulu ma ka oki ana i ka hili o ka nila 3 mm ka loa. ʻO ka hoʻohana ʻana i kēia plug e hōʻoia i ka hohonu o ka wili ʻana i loko o ke kino vertebral. Ma nā hoʻokolohua mua, ʻekolu mau kauka lapaʻau orthopedic i komo i ka hana i ʻike i nā nila he 16-gauge maʻalahi ke hana me nā nila 18-gauge a i ʻole nā ʻano hana ʻē aʻe. No ka pale ʻana i ke kahe nui ʻana o ke koko i ka wā e wili ai, ʻo ka hoʻopaʻa ʻana i ka nila no kekahi manawa e hāʻawi i kahi lua hoʻokomo kūpono, me ka manaʻo e hiki ke hoʻomalu ʻia kahi degere o MC ma kēia ala.
ʻOiai he nui nā haʻawina i manaʻo i ka MC, ʻike liʻiliʻi e pili ana i ka etiology a me ka pathogenesis o MC25,26,27. Ma muli o kā mākou mau haʻawina mua, ua ʻike mākou he kuleana nui ka autoimmunity i ka hanana a me ka hoʻomohala ʻana o MC12. Ua nānā kēia haʻawina i ka hōʻike quantitative o IL-4, IL-17, a me IFN-γ, ʻo ia nā ala ʻokoʻa nui o nā cell CD4+ ma hope o ka hoʻoulu ʻana i ka antigen. I kā mākou noiʻi ʻana, i hoʻohālikelike ʻia me ka hui maikaʻi ʻole, ʻoi aku ka kiʻekiʻe o ka hui NPE o IL-4, IL-17, a me IFN-γ, a ʻoi aku ka kiʻekiʻe o nā pae protein o IL-4 a me IL-17.
ʻO ka maʻi maʻi, hoʻonui ʻia ka hōʻike IL-17 mRNA i nā cell NP mai nā maʻi me ka disc herniation28. Hoʻonui ʻia nā pae hōʻike IL-4 a me IFN-γ i kahi ʻano herniation disc non-compressive i hoʻohālikelike ʻia me nā controls29 olakino. He kuleana koʻikoʻi ka IL-17 i ka mumū, ka ʻeha o ka ʻiʻo i nā maʻi autoimmune30 a hoʻonui i ka pane kūlohelohe iā IFN-γ31. Ua hōʻike ʻia ka hōʻeha ʻana o ka ʻiʻo IL-17 i hoʻonui ʻia ma MRL/lpr mice32 a me nā mice33 hiki ke hoʻomaʻamaʻa autoimmunity. Hiki i ka IL-4 ke kāohi i ka hōʻike o nā cytokine proinflammatory (e like me IL-1β a me TNFα) a me ka macrophage activation34. Ua hōʻikeʻia heʻokoʻa ka'ōlelo mRNA o IL-4 i ka hui NPE i hoʻohālikelikeʻia me IL-17 a me IFN-γ i ka manawa like; ʻO ka'ōlelo mRNA o IFN-γ i loko o ka hui NPE iʻoi aku ka kiʻekiʻe ma mua o nā hui'ē aʻe. No laila, hiki i ka hana IFN-γ ke lilo i mea hoʻolaha o ka pane inflammatory i hoʻokomoʻia e ka NP intercalation. Ua hōʻike nā haʻawina e hana ʻia ʻo IFN-γ e nā ʻano cell he nui, e komo pū ana me ke ʻano 1 helper T cell, nā mea pepehi maoli, a me macrophages35,36, a he kī pro-inflammatory cytokine e hāpai ana i nā pane kūlohelohe37.
Hōʻike kēia haʻawina e pili ana ka pane autoimmune i ka hanana a me ka hoʻomohala ʻana o MC. Luoma et al. ʻike ʻia nā ʻano hōʻailona o MC a me ka NP koʻikoʻi e like me ka MRI, a hōʻike ʻelua i ka hōʻailona kiʻekiʻe ma T2W sequence38. Ua hōʻoia ʻia kekahi mau cytokines e pili kokoke ana i ka hanana ʻana o MC, e like me IL-139. Ma et al. Ua manaʻo ʻia ʻo ka piʻi ʻana i luna a i lalo paha o ka NP ke mana nui i ka hanana a me ka hoʻomohala ʻana o MC12. Ua hōʻike ʻo Bobechko40 a me Herzbein et al.41 ʻo ka NP he ʻiʻo immunotolerant ʻaʻole hiki ke komo i ke kahe koko mai ka hānau ʻana. Hoʻokomo ka NP protrusions i nā kino ʻē i loko o ka hāʻawi koko, a laila e hoʻopili i nā hopena autoimmune kūloko42. Hiki i nā hopena autoimmune ke hoʻoulu i ka nui o nā mea palekana, a ke hōʻike mau ʻia kēia mau mea i nā ʻiʻo, hiki iā lākou ke hoʻololi i ka hōʻailona43. Ma kēia noiʻi ʻana, ʻo ka overexpression o IL-4, IL-17 a me IFN-γ nā mea maʻamau maʻamau, e hōʻoia hou ana i ka pilina pili ma waena o NP a me MCs44. Hoʻohālike maikaʻi kēia ʻano holoholona i ka holomua NP a komo i ka pā hope. Ua hōʻike hou kēia kaʻina hana i ka hopena o ka autoimmunity ma MC.
E like me ka mea i manaʻo ʻia, hāʻawi kēia ʻano holoholona iā mākou i kahi kahua hiki ke aʻo i ka MC. Eia nō naʻe, he mau palena ko kēia kŘkohu: ʻo ka mea mua, i ka wā o ka nānā ʻana i nā holoholona, pono e hoʻokaʻawale ʻia kekahi mau lākeke waena no ka hoʻāʻo ʻana i ka mōʻaukala a me ka molecular biology, no laila e "haʻule ʻole" kekahi mau holoholona i ka manawa. ʻO ka lua, ʻoiai ʻekolu mau manawa i hoʻonohonoho ʻia i loko o kēia haʻawina, akā naʻe, ua hoʻohālike wale mākou i hoʻokahi ʻano MC (Modic type I change), no laila ʻaʻole lawa ka hōʻike ʻana i ke kaʻina hana hoʻomohala ʻana i ka maʻi kanaka, a ʻoi aku ka nui o nā manawa e pono e hoʻonohonoho i. e nānā pono i nā hoʻololi hōʻailona a pau. ʻO ke kolu, hiki ke hōʻike maopopo ʻia nā hoʻololi ʻana o ka ʻiʻo e ka histological staining, akā hiki i kekahi mau loea kūikawā ke hōʻike maikaʻi i nā loli microstructural o kēia kükohu. No ka laʻana, ua hoʻohana ʻia ka microscopy māmā polarized e kālailai i ka hoʻokumu ʻana o ka fibrocartilage i loko o nā disc intervertebral rabbit45. ʻO nā hopena lōʻihi o ka NP ma ka MC a me ka endplate e pono ai ke aʻo hou.
He kanalimakūmāhā kāne ʻo New Zealand nā lapiti keʻokeʻo (ke kaumaha ma kahi o 2.5-3 kg, makahiki 3-3.5 mahina) i hoʻokaʻawale ʻia i ka hui hana sham, pūʻulu implantation muscle (hui ME) a me ka pūʻulu implantation root root (NPE group). Ua'āponoʻia nā kaʻina hana hoʻokolohua a pau e ke Kōmike Ethics o ka Halemai Tianjin, a ua hanaʻia nāʻano hoʻokolohua e like me nā kuhikuhi i'āponoʻia.
Ua hana ʻia kekahi mau hoʻomaikaʻi ʻana i ke ʻano hana ʻokiʻoki a S. Sobajima 46. Hoʻonoho ʻia kēlā me kēia rabbit i kahi kūlana recumbency lateral a ua hōʻike ʻia ka ʻili o mua o ʻelima lumbar intervertebral discs (IVDs) me ka hoʻohana ʻana i kahi ala retroperitoneal posterolateral. Hāʻawi ʻia kēlā me kēia rabbit i ka maʻi maʻi maʻamau (20% urethane, 5 ml/kg ma o ka vein pepeiao). Ua hana ʻia kahi ʻoki ʻili lōʻihi mai ka ʻaoʻao haʻahaʻa o nā iwi ʻaoʻao a hiki i ka pelvic brim, 2 cm ventral i nā ʻiʻo paravertebral. Ua hōʻike ʻia ka spine anterolateral ʻākau mai L1 a i L6 e ka ʻokiʻoki ʻana a me ka blunt o ka ʻiʻo subcutaneous ma luna, ka ʻiʻo retroperitoneal, a me nā ʻiʻo (Fig. 6A). Ua hoʻoholo ʻia ka pae diski me ka hoʻohana ʻana i ka pelvic brim ma ke ʻano he hōʻailona anatomical no ka pae diski L5-L6. E ho ohana i ka nila 16 ana e wili ai i puka ma kahi kokoke i ka papa hope o ka vertebra L5 a hiki i ka hohonu o 3 mm (Fig. 6B). E hoʻohana i kahi syringe 5-ml e hoʻoulu i ka autologous nucleus pulposus ma ka L1-L2 intervertebral disc (Fig. 6C). Wehe i ka nucleus pulposus a i ʻole ka ʻiʻo e like me nā koi o kēlā me kēia hui. Ma hope o ka hohonu ʻana o ka lua drill, kau ʻia nā sutures absorbable ma ka fascia hohonu, fascia superficial a me ka ʻili, me ka mālama pono ʻole e hōʻino i ka ʻiʻo periosteal o ke kino vertebral i ka wā o ke kaʻina.
(A) Hōʻike ʻia ka diski L5-L6 ma o kahi ala hope retroperitoneal. (B) E hoʻohana i ka nila 16-gauge e wili i ka lua ma kahi kokoke i ka papa hope L5. (C) Ua ʻohi ʻia nā MF Autologous.
Hoʻohana ʻia ka anesthesia maʻamau me 20% urethane (5 ml / kg) i lawelawe ʻia ma o ka vein pepeiao, a ua hana hou ʻia nā radiograph spine lumbar ma 12, 16, a me 20 mau pule ma hope o ka hana.
Ua mōhai ʻia nā rabbit e ka intramuscular injection o ketamine (25.0 mg / kg) a me ka sodium pentobarbital intravenous (1.2 g / kg) ma 12, 16 a me 20 mau pule ma hope o ka hana ʻana. Ua wehe ʻia ka spine holoʻokoʻa no ka nānā ʻana i ka histological a ua hana ʻia ka loiloi maoli. Ua hoʻohana ʻia ka quantitative reverse transcription (RT-qPCR) a me Western blotting e ʻike i nā loli i nā mea pale.
Ua hana ʻia nā hoʻokolohua MRI ma nā lapeti me ka hoʻohana ʻana i kahi 3.0 T clinical magnet (GE Medical Systems, Florence, SC) i hoʻolako ʻia me kahi mea hoʻokipa limb coil orthogonal. Ua hoʻomaʻamaʻa ʻia nā rapeti me ka 20% urethane (5 mL/kg) ma o ka vein pepeiao a laila waiho ʻia i lalo i loko o ka magnet me ka ʻāpana lumbar i hoʻopaʻa ʻia i ka 5-inihi anawaena circular surface coil (GE Medical Systems). Ua loaʻa iā Coronal T2-weighted localizer kiʻi (TR, 1445 ms; TE, 37 ms) e wehewehe i kahi o ka disc lumbar mai L3-L4 a i L5-L6. Ua loaʻa nā ʻāpana paona T2-kaumaha Sagittal plane me kēia mau hoʻonohonoho: ke kaʻina spin-echo wikiwiki me ka manawa hou (TR) o 2200 ms a me kahi manawa echo (TE) o 70 ms, matrix; kahua ʻike o 260 a me ʻewalu hoʻoulu; ʻO ka mānoanoa o ka ʻoki ʻana he 2 mm, ʻo ka ʻāpana he 0.2 mm.
Ma hope o ke kiʻi ʻia ʻana o ke kiʻi hope loa a pepehi ʻia ka lapeti hope loa, ua wehe ʻia ka sham, muscle-embedded, a me nā disc NP no ka nānā ʻana i ka histological. Hoʻopaʻa ʻia nā ʻupena i 10% neutral buffered formalin no 1 pule, decalcified me ka ethylenediaminetetraacetic acid, a me ka ʻāpana paraffin. Hoʻokomo ʻia nā poloka kiko i loko o ka paraffin a ʻokiʻoki i loko o nā ʻāpana sagittal (5 μm mānoanoa) me ka microtome. Ua hoʻopaʻa ʻia nā ʻāpana me ka hematoxylin a me eosin (H&E).
Ma hope o ka hōʻiliʻili ʻana i nā disc intervertebral mai nā rabbit i kēlā me kēia hui, ua unuhi ʻia ka huina RNA me ka hoʻohana ʻana i kahi kolamu UNIQ-10 (Shanghai Sangon Biotechnology Co., Ltd., Kina) e like me nā ʻōlelo a ka mea hana a me kahi ʻōnaehana transcription ImProm II (Promega Inc. , Madison, WI, USA). Ua hana ʻia ka palapala hoʻohuli.
Ua hana ʻia ʻo RT-qPCR me ka Prism 7300 (Applied Biosystems Inc., USA) a me SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) e like me nā ʻōlelo a ka mea hana. ʻO ka nui o ka hopena PCR he 20 μl a loaʻa iā 1.5 μl o ka cDNA diluted a me 0.2 μM o kēlā me kēia primer. Ua hoʻolālā ʻia nā Primers e OligoPerfect Designer (Invitrogen, Valencia, CA) a hana ʻia e Nanjing Golden Stewart Biotechnology Co., Ltd. (Kina) (Table 1). Ua hoʻohana ʻia nā kūlana kaʻa kaʻa wela: ka hana hoʻāla polymerase mua ma 94 ° C no 2 min, a laila 40 mau pōʻai o 15 s i kēlā me kēia ma 94 ° C no ka denaturation template, annealing no 1 min ma 60 ° C, extension, a me fluorescence. Ua hana ʻia nā ana no 1 min ma 72 ° C. Ua hoʻonui ʻia nā laʻana āpau i ʻekolu manawa a ua hoʻohana ʻia ka waiwai awelika no ka nānā ʻana RT-qPCR. Ua kālailai ʻia ka ʻikepili hoʻonui me ka FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). ʻO IL-4, IL-17, a me IFN-γ hōʻike i hoʻohālikelike ʻia i ka mana endogenous (ACTB). Ua helu ʻia nā pae hōʻike pili o ka mRNA i hoʻohana ʻia me ke ʻano 2-ΔΔCT.
Ua unuhi ʻia ka nui o ka protein mai nā ʻiʻo me ka hoʻohana ʻana i kahi homogenizer kiko ma RIPA lysis buffer (loaʻa ka protease a me ka phosphatase inhibitor cocktail) a laila centrifuged ma 13,000 rpm no 20 min ma 4 ° C e wehe i nā ʻōpala kiko. Hoʻouka ʻia he kanalima micrograms o ka protein i kēlā me kēia ala, hoʻokaʻawale ʻia e 10% SDS-PAGE, a laila hoʻoneʻe ʻia i kahi membrane PVDF. Hana ʻia ka pale ʻana i ka waiū maloʻo ʻole 5% ma Tris-buffered saline (TBS) i loaʻa ka 0.1% Ma waena o 20 no 1 h ma ka lumi wela. Hoʻokomo ʻia ka membrane me ka rabbit anti-decorin primary antibody (diluted 1:200; Boster, Wuhan, Kina) (diluted 1:200; Bioss, Beijing, Kina) i ka pō ma 4 ° C a hana i nā lā ʻelua; me ka lua antibody (kao anti-rabbit immunoglobulin G ma 1:40,000 dilution) hui me horseradish peroxidase (Boster, Wuhan, Kina) no 1 hola ma ka lumi wela. Ua ʻike ʻia nā hōʻailona Western blot e ka hoʻonui ʻana i ka chemiluminescence ma ka membrane chemiluminescent ma hope o ka hoʻomālamalama ʻana o X-ray. No ka nānā ʻana i ka densitometric, ua nānā ʻia nā blots a helu ʻia me ka hoʻohana ʻana i ka polokalamu BandScan a ua hōʻike ʻia nā hopena e like me ka ratio o ka immunoreactivity gene target i ka immunoreactivity tubulin.
Ua hana ʻia nā helu helu me ka hoʻohana ʻana i ka polokalamu polokalamu SPSS16.0 (SPSS, USA). Ua hōʻike ʻia nā ʻikepili i hōʻiliʻili ʻia i ka wā o ke aʻo ʻana ma ke ʻano he mean ± standard deviation (mean ± SD) a ua kālailai ʻia me ka hoʻohana ʻana i nā ana hoʻohālikelike hou ʻana o ke ʻano hoʻohālikelike (ANOVA) e hoʻoholo i nā ʻokoʻa ma waena o nā hui ʻelua. ʻO ka P <0.05 i manaʻo ʻia he koʻikoʻi.
No laila, ʻo ka hoʻokumu ʻana i kahi hiʻohiʻona holoholona o MC ma o ka hoʻokomo ʻana i nā NP autologous i loko o ke kino vertebral a me ka nānā ʻana i ka macroanatomical, ka nānā ʻana MRI, ka loiloi histological a me ka molecular biological analysis hiki ke lilo i mea hana nui no ka loiloi a hoʻomaopopo ʻana i nā hana o ke kanaka MC a me ka hoʻomohala ʻana i ka therapeutic hou. mau hana.
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