应用酶联免疫法检测组织工程骨牛血清白蛋白残余量的实验研究

摘要

目的 检测常规体外构建的组织工程骨中牛血清白蛋白残余量,并探讨减少其残留量的方法.方法 体外分离培养人骨髓基质干细胞,取第2代细胞(P2)接种于完全脱钙骨支架材料并成骨诱导培养2周,体外构建组织工程骨.成骨诱导液为含10％胎牛血清的条件培养液,并于检测前1天更换为无血清的条件培养液.待测组织工程骨先经1∶100 (v∶v)生理盐水浸洗3次,然后加入磷酸盐缓冲液(PBS),37℃振荡浸提24 h,获取浸提液.同法获取未接种细胞的单纯支架材料的浸提液作为对照组.采用酶联免疫法检测样品浸提液中牛血清白蛋白的残余量,比较两者牛血清白蛋白残余量的差异.结果 经生理盐水洗涤3次后,洗涤液中的牛血清白蛋白含量明显降低.酶联免疫法测定的组织工程骨与单纯支架材料的牛血清白蛋白残余量分别为(15.57±5.82)ng和(14.85±5.06)ng,单位重量的牛血清白蛋白残余量分别为(0.254±0.088)ng/mg和(0.306±0.079)ng/mg,两组相比均无显著性差异(P＞ 0.05,n=10).结论 酶联免疫法适用于组织工程骨中牛血清白蛋白残余量的检测.因为支架材料较细胞更易吸附牛血清白蛋白,现有条件下构建的组织工程骨牛血清白蛋白残余量仍然较高,需要继续探索降低其残余含量的新方法.%Objective To detect the residual bovine serum albumin (BSA) in tissue engineered bone constructed conventionally in vitro by enzyme-linked immunosorbent assay (ELISA) and investigate methods to reduce it.Methods Human bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured in vitro,and BMSCs of passage 2 were inoculated in demineralized bone matrix scaffolds and induced osteogenesis in vitro for two weeks to construct tissue engineered bone.Osteogenesis inducing medium was conditioned medium containing 10％ fetal bovine serum,and was replaced with serum-free conditioned medium 1 day before detection.Then the tissue engineered bone was rinsed in 1∶100 (v∶v) saline for three times,put in PBS buffer and extracted for 24 hours at 37 ℃ to obtain the extracted liquor.The extracted liquor of scaffold alone without cells inoculated simultaneously served as the control.Residual BSA content in the extracted liquor were detected by ELISA and compared between the two groups.Results BSA content in the dilution liquid decreased after washing three times.The average residual BSA in tissue engineered bone and in scaffold material detected by ELISA was (15.57 ± 5.82) ng and (14.85 ± 5.06) ng,respectively,which was normalized as (0.254 ± 0.088) ng/mg and (0.306 ± 0.079) ng/mg,respectively,showing no significant difference between two groups (P ＞ 0.05,n =10).Conclusions ELISA method is suitable for the detection of the residual BSA in tissue engineered bone.As the scaffold materials absorb BSA more easily than the cells,the residual BSA in tissue engineered bone is still relatively high.Therefore,new methods of reducing BSA residual need to be explored.