人羊水祖细胞的分离和基因修饰

Isolation and gene modification of amniotic fluid derived progenitor cells

摘要:

探讨从孕中期羊水中分离出人羊水祖细胞的有效方法和FIX基因修饰后的效果，为血友病B的产前治疗提供可行的基础。从镜下分离出呈致密克隆生长的梭形细胞集落，经培养传代后，通过第3代慢病毒载体将hFIX导入该细胞，经酶联免疫反应(ELISA)等方法检测hFIX的表达并检测凝血活性。用这种方法得到的羊水祖细胞呈成纤维细胞样，倍增时间为39.05 h，该细胞在不仅在蛋白水平表达干细胞表面分子SSEA4，TRA1-60，在基因水平还可检测到NANOG，OCT4，SOX2mRNA的表达。羊水祖细胞导入hFIX cDNA后，能合成并分泌hFIX蛋白，传代后48 h在上清液中的浓度为20.37%±2.77%，凝血活性16.42%±1.78%。上清液中的浓度在第4天达到平台期，为50.35%±5.42%，凝血活性可达45.34%±4.67%。ELISA检测显示转染后的羊水细胞表达的hFIX蛋白的水平呈现基本稳定趋势，波动幅度较小；同时检测FIX凝血活性也与蛋白浓度呈正相关。从羊水中可以分离得到具有多能性祖细胞，转染了hFIX的羊水祖细胞在体外能持续合成有凝血功能的hFIX蛋白，为血友病B产前治疗的新方法提供了实验依据。

Abstract:

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% ±2.77% two days after passage, with clotting activity of 16.42% ±1.78%. The amount of hFIX:Ag reached a plateau of 50.35% ±5.42%, with clotting activity 45.34% ±4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.