CAJ | 학술논문

组织构建%软骨组织构建%兔%髓核细胞%体外培养%聚集蛋白聚糖%Ⅱ型胶原%椎间盘退变%细胞培养%省级基金%组织构建图片文章組織構建%軟骨組織構建%兔%髓覈細胞%體外培養%聚集蛋白聚糖%Ⅱ型膠原%椎間盤退變%細胞培養%省級基金%組織構建圖片文章
조직구건%연골조직구건%토%수핵세포%체외배양%취집단백취당%Ⅱ형효원%추간반퇴변%세포배양%성급기금%조직구건도편문장
tissue construction%cartilage tissue construction%rabbits%nucleus pulposus cells%in vitro culture%aggrecan%type Ⅱ col agen%intervertebral disc degeneration%cel culture%provincial grants-supported paper%tissue construction photographs-containing paper

背景:椎间盘退变是个慢性、复杂的过程,然而椎间盘退变其发生机制尚未完全阐明,很难自行修复.近年来研究细胞移植治疗椎间盘退行性变尚处在实验室阶段.研究髓核细胞的生物学性状可为研究椎间盘退变机制、组织工程构建椎间盘、基因治疗等提供理论依据.目的:研究兔不同代次髓核细胞的生物学特性,旨在找出合适的种子细胞去治疗椎间盘退变性疾病.方法:从新西兰大耳白兔椎间盘髓核组织中,分离并培养髓核细胞同时进行培养传代,对原代及第3,4代髓核细胞进行苏木精-伊红染色观察细胞形态学变化;甲苯胺蓝染色和免疫细胞化学法检测髓核细胞内聚集蛋白聚糖和Ⅱ型胶原的表达;反转录 PCR 法测定Ⅱ型胶原和聚合蛋白聚糖 mRNA 的表达水平,观察各代髓核细胞生物学特性的变化.结果与结论:兔椎间盘髓核细胞可以在体外培养并进行传代,原代髓核细胞一般需7 d 左右贴壁,形状呈类圆形或多角形,原代和第3代髓核细胞都呈圆形或多角形,活力较强,苏木精-伊红染色后细胞核被染成均一蓝黑色,胞浆呈现淡粉色;髓核细胞经过甲苯胺蓝染色后,胞浆内呈现天蓝色,通过Ⅱ型胶原免疫组织化学染色后,胞浆内表现为黄褐色沉淀.到第4代细胞出现退变,Ⅱ型胶原和聚合蛋白聚糖mRNA 的表达水平较前几代细胞显著下降.前3代的髓核细胞代谢旺盛,表型一致,聚集蛋白聚糖和Ⅱ型胶原表达正常,传第4代后髓核细胞开始出现衰老、退变.
배경:추간반퇴변시개만성、복잡적과정,연이추간반퇴변기발생궤제상미완전천명,흔난자행수복.근년래연구세포이식치료추간반퇴행성변상처재실험실계단.연구수핵세포적생물학성상가위연구추간반퇴변궤제、조직공정구건추간반、기인치료등제공이론의거.목적:연구토불동대차수핵세포적생물학특성,지재조출합괄적충자세포거치료추간반퇴변성질병.방법:종신서란대이백토추간반수핵조직중,분리병배양수핵세포동시진행배양전대,대원대급제3,4대수핵세포진행소목정-이홍염색관찰세포형태학변화;갑분알람염색화면역세포화학법검측수핵세포내취집단백취당화Ⅱ형효원적표체;반전록 PCR 법측정Ⅱ형효원화취합단백취당 mRNA 적표체수평,관찰각대수핵세포생물학특성적변화.결과여결론:토추간반수핵세포가이재체외배양병진행전대,원대수핵세포일반수7 d 좌우첩벽,형상정류원형혹다각형,원대화제3대수핵세포도정원형혹다각형,활력교강,소목정-이홍염색후세포핵피염성균일람흑색,포장정현담분색;수핵세포경과갑분알람염색후,포장내정현천람색,통과Ⅱ형효원면역조직화학염색후,포장내표현위황갈색침정.도제4대세포출현퇴변,Ⅱ형효원화취합단백취당mRNA 적표체수평교전궤대세포현저하강.전3대적수핵세포대사왕성,표형일치,취집단백취당화Ⅱ형효원표체정상,전제4대후수핵세포개시출현쇠로、퇴변.
BACKGROUND: Intervertebral disc degeneration is a chronic and complicate procedure, but its mechanism is stil unclear and difficult for self-repairs. Recent studies addressing stem cel transplantation in the treatment of disc degeneration disease are stil limited to the laboratory study. Research on biological properties of nucleus pulposus cells can provide theoretical basis for studying the mechanism underlying intervertebral disc degeneration, construction of the intervertebral disc by tissue engineering, and gene therapy. OBJECTIVE: To investigate the characteristics of different generations of rabbit nucleus pulposus cells, searching for the best suitable seed cells to treat degenerative disc diseases. METHODS: Nucleus pulposus cells from New Zealand white rabbits were separated, cultured and then passaged. The morphological changes of primary, passages 3 and 4 nucleus pulposus cells were observed by hematoxylin-eosin staining under an inverted microscope. The biological properties of rabbit nucleus pulposus cells were observed. Aggrecan and type Ⅱ colagen expressions w ere detected by toluidine blue and immunocytochemistry staining, respectively. Type nucleus pulposus cells were detected by reverse transcription-PCR. RESULTS AND CONCLUSION: Rabbit nucleus pulposus cells were successful y cultured and passaged in vitro. Primary nucleus pulposus cells were round or polygonal, and the average adherence time was 7 days. The first and third generations of nucleus pulposus cells were round or polygonal, and have strong vitality. Hematoxylin-eosin staining showed that nuclei were in a uniform blue-black, and cytoplasm showed light pink. The cytoplasm of nucleus pulposus cells was sky blue stained for toluidine blue staining, and type II col agen immunohistochemical staining showed the cytoplasm of nucleus pulposus cells displayed yel owish-brown. Passage 4 nucleus pulposus cells appeared with degeneration, and type mRNA expression was significantly decreased compared with previous generations. The first three generations of nucleus pulposus cells were exuberant in metabolism and showed consistent phenotypes and normal expression of aggrecan and type Ⅱ col agen. Passage 4 nucleus pulposus cells began to age and degenerate.