强直性脊柱炎髋关节韧带组织培养模型的建立及腺病毒转染的实验研究  

Establishment of culture models for hip joint tendons of ankylosing spondylitis and the effect of adenovirus-mediated gene transfer

作　　者：李虎[1] 李儒军[1] 曹宸喜 王白成 林剑浩[1] 陶可[1,3] LI Hu;LI Rujun;CAO Chenxi;WANG Baicheng;LIN Jianhao;TAO Ke(Institute of Arthritis, Peking University People's Hospital, Beijing 100044;School of 8-year Clinical Medicine, Peking University Health Science Center, Beijing 100191, China;Center of Experimental Orthopedics, Saarland University Medical Center, Homburg/Saar D-66424, Germany)

机构地区：[1]北京大学人民医院骨关节科关节炎诊疗中心,北京100044 [2]北京大学医学部,北京100191 [3]德国洪堡医学院实验骨科研究所,萨尔d66424

出　　处：《中华骨与关节外科杂志》2018年第12期932-939,共8页Chinese Journal of Bone and Joint Surgery

基　　金：国家自然科学基金资助项目(81641175;81672183);国家重点研发计划项目(2016YFC1101800;2017YFC0108003);国家卫计委研究资助项目;北京市自然科学基金资助项目(7182172);北京市科委重大项目(D171100003217002;2171100002717094);北京大学人民医院院内发展基金资助项目(RDY2018-04;RDY2016-15);2015年国际骨关节炎研究协会(OARSI)访问学者奖学金;2016年国际软骨修复协会(ICRS)奖学金;2016年北京厚爱关节健康公益基金会青年学者奖学金

摘　　要：背景:强直性脊柱炎(AS)是一种与炎症紧密相关但病因尚未完全阐明的慢性进行性结缔组织疾病。目的:本研究拟通过构建和比较AS韧带组织体外原位(in situ)、2D细胞贴壁体外(in vitro)及3D水凝胶体外培养模型,旨在探索更适合模拟AS韧带体外的培养模型。方法:AS髋关节韧带组织取自我科4例重度AS拟行髋关节置换的患者,平均年龄(44.0±4.1)岁。髋关节组织块常规消化获得单细胞,并行倒置相差显微镜观察和抗vimentin免疫荧光染色(IFC)鉴定检查。对上述髋关节韧带组织与成纤维细胞分别行组织块原位、体外2D贴壁和3D海藻酸盐水凝胶法培养,重组腺病毒(rAd)5-lacZ转染上述标本14 d后,分别采用大体观察、HE染色、Hoechst 33258实验、X-gal染色、抗β-gal免疫组织化学染色和酶联免疫吸附实验(ELISA)等对细胞形态与增殖能力、病毒转染效率和致炎性细胞因子表达等进行检测。结果:第2代AS髋关节韧带细胞抗vimentin IFC检测均出现明显的红色荧光,结果阳性,表明分离培养的细胞为成纤维细胞。rAd5-lacZ转染上述3种模型14 d后,倒置相差显微镜下观察HE染色后的细胞形态并计数单位面积下的细胞数、Hoechst 33258检测细胞DNA含量和ELISA法检测细胞分泌功能提示,3种模型中以2D平面的细胞增殖效率和致炎性细胞因子分泌能力最强(F=12.21、14.91、282.67、350.18,P<0.05或0.01)、但细胞"去分化"现象亦最明显,而AS韧带组织原位培养和成纤维细胞3D海藻酸盐水凝胶培养条件下的细胞增殖效率和致炎性细胞因子表达能力相似(t=1.37、0.71、1.44、1.83,P>0.05),细胞形态前后无明显变化。抗β-gal免疫组织化学染色并计数阳性细胞数/光镜下的总细胞数得出rAd-lacZ转染3种模型效率相似,分别为65.84%、72.26%和75.39%(F=0.873,P>0.05)。结论:韧带组织原位培养和成纤维细胞3D水凝胶培养能更好地模拟AS韧带成纤维细胞体内生存环Background: Ankylosing spondylitis (AS) is a chronic progressive connective tissue disease closely related to in-flammation, yet the entire etiology has not been completely elucidated. Objective: To construct and compare the in situ ex-plant, 2D and 3D hydrogel in vitro cultured models for hip joint tendons of AS and investigate which one is more suitable for AS tendons in vitro culture. Methods: Tendon specimens of AS hip joint were extracted from 4 AS patients with severe hip de-formities aging of (44.0±4.1) years old. Specimens were exposed to type II collagenase and obtained single cell suspensions, and phase contrast microscopy and anti-vimentin immunofluorescence staining (IFC) were applied to evaluate the cells. The specimens and fibroblasts were divided and cultured as in situ explants, adherent two-dimensional (2D) cell monolayers and three-dimensional (3D) alginate hydrogel in vitro respectively, and the recombinant adenovirus (rAd)-lacZ (E. coli beta-galac-tosidase gene) was used to transduce these above-mentioned models for 14 days. Cell morphology and proliferation (cellulari-ty), viral transfection efficiency and secretion of pro-inflammatory cytokines were estimated by Haemotoxylin and Eosin (HE) staining, Hoechst 33258 test, enzyme-linked immunosorbent assay (ELISA), respectively. Results: The second passage of AS tendon cells was positive against anti-vimentin primary antibody, indicating that the cells were fibroblasts. After transduction with rAd5-lacZ for 14 days, HE staining, Hoechst 33258 and ELISA assays indicated that rAd5-lacZ gene transfer could pro-mote cell proliferation and enhance secretion of pro-inflammatory cytokines, with 2D model exhibiting the strongest cell prolif-eration and highest secretion of pro-inflammatory cytokines among these three models (F=12.21, 14.91, 282.67, 350.18, P< 0.05 or 0.01),while similar results of cell proliferation and secretion of pro-inflammatory cytokines and few changes in pheno-type were found in explant and 3D alginate hydrogel models (t=1.37, 0

关 键 词：强直性脊柱炎 培养模型 韧带组织 成纤维细胞 去分化 腺病毒 转染 

分 类 号：R593.23[医药卫生—内科学] R684[医药卫生—临床医学]