人工髋关节无菌性松动相关蛋白因子的筛选和慢病毒介导拮抗蛋白的干细胞表达研究

发布时间：2018-05-23 15:03

本文选题：关节置换术 + 假体无菌性松动　；参考：《上海交通大学》2014年博士论文

【摘要】：【目的】对于与无菌性松动相关的生物标记物的高质量研究进行系统评价,为无菌性松动诊断研究中的生物标志物的合理选择提供科学依据。对接受了金属对金属的表面髋关节置换、金属对聚乙烯和陶瓷对陶瓷的全髋置换术患者的临床结果进行回顾性随访比较,明确不同磨擦界面与骨溶解的关系,也为无菌性松动的早期诊断研究的合理分组提供依据。明确多种骨溶解或骨重建的生物标记物与人工髋关节假体早期无菌性松动的关系,及其诊断价值。明确慢病毒介导的白介素1受体拮抗蛋白(Interleukin 1 receptor antagonist,IL-1Ra)基因是否能够在小鼠骨髓间充质干细胞体外稳定的表达。【方法】经过系统检索纳入了32篇纵向研究,采用牛津循证医学中心证据水平和Newcastle-Ottawa质量评价量表评价研究质量,通过证据综合分析评价每种标记物对于无菌性松动的诊断效能和推荐级别。选择154例(171髋)施行全髋关节置换术的患者,包括27髋的金属对金属表面置换(第1)组,41例的陶瓷对陶瓷全髋置换(第2)组和103髋的金属对聚乙烯全髋置换(第3)组。记录年骨溶解率和术后并发症等指标。选择105例研究对象,包括31例拟行关节翻修术的患者(晚期松动组);15例具有临床及影像学松动表现但不需要翻修手术的患者(早期松动组);19例无松动表现的患者(稳定假体组);40例健康成人。用酶免法检测血浆骨保护素(Osteoprotegerin,OPG),细胞核因子κB受体活化因子配基(Receptor activator of the nuclear factorκB ligand,RANKL),Ⅰ型胶原N末端肽(Cross-linked N-telopeptide of type I collagen,NTX),I型前胶原羧基端肽(Carboxy terminal propeptide of type I procollagen,PICP),肿瘤坏死因子(Tumor necrosis factor,TNF)α和白介素(Interleukin-1,IL)1β的含量。各标记物的血浆水平进行组间单独(SPSS软件)和综合(Reviewmanager软件)的统计分析。介导IL-1Ra/cop GFP基因的慢病毒载体构建后转染小鼠骨髓间充质干细胞,采用RT-q PCR和Western bolt检测IL-1Ra的体外表达。用CCK-8试剂盒检测细胞活力,并用台盘蓝染色和MTT分析检测干细胞生长能力。【结果】48种生物标记物或细胞因子中,只有NTX有中等证据,推荐级别为B;IL-1β,TNF-α,IL-8,抗酒石酸酸性磷酸酶,前列环素E2和骨钙素有有限证据,推荐级别为C。经过平均5年的随访,校正后第1组的年骨溶解率显著低于第2和3组(p值均0.0001)。第1组的术后并发症最少,并且没有金属过敏反应。晚期松动组与对照组相比只有IL-1β和OPG的差异有显著性(均为p0.05),血浆IL-1β诊断效能最强。6项标记物血浆水平的联合分析不但在晚期松动组和稳定组,而且在稳定组和健康组之间差异也有显著性(p值均为0.02),并且各组内6项标记物之间的异质性极低(I2=0)。介导IL-1Ra/cop GFP基因的第三代慢病毒载体构建良好,转染小鼠骨髓间充质干细胞后能稳定表达IL-1Ra,细胞增殖周期短,但是早期生长能力好,而且相比未转染的细胞活力更好。【结论】与人工关节假体无菌性松动相关的生物标记物中IL-1β,TNF-a,IL-8,抗酒石酸酸性磷酸酶,前列环素E2和骨钙素,尤其是NTX有中等证据,可以推荐作为可靠的检测标记物。三种全髋置换的界面材料中金属对金属表面置换的骨溶解进展最少,而且没有金属颗粒副反应。6项生物标记物中IL-1β诊断效能最强。在传统的体检和影像学检查认为假体稳定的全髋术后患者中多项标记物的联合分析有显著升高。慢病毒转染的小鼠骨髓间充质干细胞能稳定表达外源性IL-1Ra,细胞活力和增殖能力好。
[Abstract]:[Objective] to systematically evaluate the high quality study of biomarkers associated with aseptic loosening to provide a scientific basis for the rational selection of biomarkers in the diagnosis of aseptic loosening. The clinical results were followed up and compared to clarify the relationship between the different friction interfaces and osteolysis, and to provide the basis for the rational grouping of early diagnostic studies for aseptic loosening. Whether the interleukin 1 receptor antagonist (Interleukin 1 receptor antagonist, IL-1Ra) gene is able to express in vitro stable expression in mouse bone marrow mesenchymal stem cells in vitro. [Methods] 32 longitudinal studies were included through systematic retrieval, and the evaluation of evidence water level and Newcastle-Ottawa quality assessment scale in Oxford evidence-based medicine center was used. Quality, the diagnostic efficacy and recommended level of each marker for aseptic loosening were evaluated through a comprehensive analysis of evidence. 154 patients (171 hips) underwent total hip replacement, including 27 hip metal surface replacement (first), 41 ceramic to ceramic total hip replacement (second) and 103 hip metal to polyethylene total hip replacement. Third) group. Record annual osteolysis and postoperative complications. Select 105 subjects, including 31 patients with prosthetic revision (late loosening group); 15 patients with clinical and imaging looseness but no refurbishment (early loosening group); 19 patients with no loosening (stable prosthesis); 40 healthy adults. Osteoprotegerin (OPG), nuclear factor kappa B receptor activating factor ligand (Receptor activator of the nuclear factor kappa B ligand, RANKL), and collagen carboxyl terminated peptide of type I collagen, were detected by enzyme immunoassay. Ocollagen, PICP), the content of tumor necrosis factor (Tumor necrosis factor, TNF) alpha and interleukin (Interleukin-1, IL) 1 beta. Statistical analysis of the plasma levels of each marker (SPSS software) and synthesis (Reviewmanager software). The mouse bone marrow mesenchymal stem cells were transfected after the lentivirus vector of IL-1Ra/cop GFP gene was mediated. RT-q PCR and Western bolt were used to detect the expression of IL-1Ra in vitro. Cell viability was detected by CCK-8 kit, and the growth ability of stem cells was detected with table blue staining and MTT analysis. [results] only NTX has medium evidence, and the recommended grade is B; IL-1 beta, TNF- alpha, IL-8, tartaric acid acid phosphatase, before 48 kinds of biomarkers or cytokines. There was limited evidence of cyclin E2 and osteocalcin. The recommended grade was C. after an average of 5 years of follow-up. The annual osteolysis rate in the first groups was significantly lower than that of second and third groups (P 0.0001). The first group had the least postoperative complications and no metal anaphylaxis. The difference between the late loosening group and the control group was significantly higher than the control group (P0 and OPG). .05), the combined analysis of plasma levels of the strongest.6 markers in plasma IL-1 beta diagnosis was not only in the late loosening group and the stable group, but also between the stable group and the healthy group (the p value was 0.02), and the heterogeneity between the 6 markers in each group was very low (I2=0). The third generation of the lentivirus vector of the IL-1Ra/cop GFP gene was mediated. Well constructed, transfected mouse bone marrow mesenchymal stem cells can express IL-1Ra steadily, the cell proliferation cycle is short, but early growth ability is good, and compared with untransfected cells, the cell viability is better. [Conclusion] IL-1 beta, TNF-a, IL-8, tartaric acid acid phosphatase, prostacyclin in the biomarkers associated with aseptic loosening of artificial joint prosthesis E2 and osteocalcin, especially NTX, have medium evidence, which can be recommended as a reliable detection marker. Metal surface replacement osteolysis is the least in three total hip replacement interface materials, and no metal particle side reaction in.6 biomarkers has the strongest diagnostic efficacy of IL-1 beta. In traditional physical examination and imaging examination The combined analysis of multiple markers in patients with stable hip prosthesis has been significantly increased. The mouse bone marrow mesenchymal stem cells transfected by lentivirus can stabilize the expression of exogenous IL-1Ra, and the cell viability and proliferation ability are good.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.17;R687.4

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