组织工程与重建外科杂志
組織工程與重建外科雜誌
조직공정여중건외과잡지
JOURNAL OF TISSUE ENGINEERING AND RECONSTRUCTIVE SURGERY
2015年
1期
6-9
,共4页
牛血清白蛋白%酶联免疫吸附测定%组织工程骨%残余量
牛血清白蛋白%酶聯免疫吸附測定%組織工程骨%殘餘量
우혈청백단백%매련면역흡부측정%조직공정골%잔여량
Bovine serum albumin%Enzyme-linked immenosorbent assay%Tissue engineered bone%Residual
目的：检测体外构建的组织工程骨中的牛血清白蛋白（Bovine serum albumin，BSA）残余量，并探讨减少残余量的方法。方法体外分离培养hBMSCs，将第2代细胞消化、离心、洗涤3次，获取细胞洗涤液样品。取第2代hBMSCs接种于β-TCP支架材料，体外成骨诱导2周，构建组织工程骨。生理盐水浸洗3次，获取洗涤液样品。然后加入PBS，37℃振荡浸提24 h，获取浸提液样品。同法获取未接种细胞的单纯支架材料的浸提液样品作为对照。采用酶联免疫法检测洗涤液与组织工程骨样品浸提液中BSA的残余量，观察洗涤次数与洗涤液中BSA含量的变化关系。结果随洗涤次数增多，洗涤液与浸提液中的BSA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浸提液中BSA残余量分别为（19.54±6.70） ng和（15.67±5.49） ng，单位重量的残余量分别为（0.656±0.213） ng/mg和（0.796±0.205） ng/mg，两组无显著差异（P>0.05）。结论酶联免疫法适用于组织工程骨中BSA残余量的检测。因为支架材料较细胞更易吸附BSA，现有条件下构建的组织工程骨BSA残余量仍然较高，需要进一步探索降低残余量的方法。
目的：檢測體外構建的組織工程骨中的牛血清白蛋白（Bovine serum albumin，BSA）殘餘量，併探討減少殘餘量的方法。方法體外分離培養hBMSCs，將第2代細胞消化、離心、洗滌3次，穫取細胞洗滌液樣品。取第2代hBMSCs接種于β-TCP支架材料，體外成骨誘導2週，構建組織工程骨。生理鹽水浸洗3次，穫取洗滌液樣品。然後加入PBS，37℃振盪浸提24 h，穫取浸提液樣品。同法穫取未接種細胞的單純支架材料的浸提液樣品作為對照。採用酶聯免疫法檢測洗滌液與組織工程骨樣品浸提液中BSA的殘餘量，觀察洗滌次數與洗滌液中BSA含量的變化關繫。結果隨洗滌次數增多，洗滌液與浸提液中的BSA含量明顯降低。酶聯免疫法測定的組織工程骨與單純支架材料浸提液中BSA殘餘量分彆為（19.54±6.70） ng和（15.67±5.49） ng，單位重量的殘餘量分彆為（0.656±0.213） ng/mg和（0.796±0.205） ng/mg，兩組無顯著差異（P>0.05）。結論酶聯免疫法適用于組織工程骨中BSA殘餘量的檢測。因為支架材料較細胞更易吸附BSA，現有條件下構建的組織工程骨BSA殘餘量仍然較高，需要進一步探索降低殘餘量的方法。
목적：검측체외구건적조직공정골중적우혈청백단백（Bovine serum albumin，BSA）잔여량，병탐토감소잔여량적방법。방법체외분리배양hBMSCs，장제2대세포소화、리심、세조3차，획취세포세조액양품。취제2대hBMSCs접충우β-TCP지가재료，체외성골유도2주，구건조직공정골。생리염수침세3차，획취세조액양품。연후가입PBS，37℃진탕침제24 h，획취침제액양품。동법획취미접충세포적단순지가재료적침제액양품작위대조。채용매련면역법검측세조액여조직공정골양품침제액중BSA적잔여량，관찰세조차수여세조액중BSA함량적변화관계。결과수세조차수증다，세조액여침제액중적BSA함량명현강저。매련면역법측정적조직공정골여단순지가재료침제액중BSA잔여량분별위（19.54±6.70） ng화（15.67±5.49） ng，단위중량적잔여량분별위（0.656±0.213） ng/mg화（0.796±0.205） ng/mg，량조무현저차이（P>0.05）。결론매련면역법괄용우조직공정골중BSA잔여량적검측。인위지가재료교세포경역흡부BSA，현유조건하구건적조직공정골BSA잔여량잉연교고，수요진일보탐색강저잔여량적방법。
Objective To detect the residual bovine serum albumin (BSA) in tissue engineered bone constructed conventionally in vitro byβ-TCP and to investigate new methods to reduce it. Methods Human bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured in vitro. BMSCs of passage 2 were digested, after centrifugation and washing, the washing liquid of the cells was obtained. And BMSCs of passage 2 were inoculated in β-TCP scaffolds and induced osteogenesis in vitro for 2 weeks to construct tissue engineered bone. The tissue engineered bone was rinsed in 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 without cells inoculated simultaneously was served as the control. Residual BSA content in the extracted liquor and the washing liquid samples were detected by ELISA, and the change relationship of BSA content with times of washing was observed. Results BSA content in the dilution liquid decreased as washing times increased. The average residual BSA in tissue engineered bone and in scaffold material detected by ELISA was (19.54±6.70) ng and (15.67±5.49) ng respectively, which was normalized as (0.656±0.213) ng/mg and (0.796±0.205) ng/mg, showing no significant difference between two groups (P>0.05, n=10). Conclusion 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, further studies of reducing BSA residual are still needed.
