论文部分内容阅读
目的构建脐血巨核祖细胞/胃癌SGC7901细胞融合体产生功能性血小板。方法免疫磁珠分选人脐带血CD34+细胞,通过RPMI1640培养基体外静态培养,加入定向分化因子TPO、FL、IL-3、IL-6产生巨核祖细胞;复苏胃癌SGC7901细胞;采用聚乙二醇(PEG)化学融合法制备巨核祖细胞/胃癌SGC7901细胞融合体,HAT筛选系统分选融合细胞,继续培养;流式细胞仪检测培养孔上层液中类血小板颗粒表面CD41、CD62p表达,血小板聚集试验、粘附试验检测血小板功能,瑞氏染色观察颗粒形态。结果免疫磁珠分选的CD34+细胞培养7 d可增殖80倍,与胃癌SGC7901细胞株经PEG化学融合法融合成巨核祖细胞/胃癌细胞,融合率为(36.91±1.08)%;融合细胞培养10 d可增殖4倍,产生(6.1±1.21)×106/ml的类血小板颗粒;瑞氏染色后观察,其形态与正常血小板相似。类血小板颗粒表达血小板特异性标志CD41及血小板活化后标志CD62p,与正常血小板比较,CD62p在类血小板颗粒中表达略降低:正常血小板(74.11±1.71)%、类血小板颗粒(71.04±1.64)%;CD41表达明显降低:正常血小板(88.83±3.26)%、类血小板颗粒(72.51±2.79)%(t=6.59,P<0.05);聚集试验表明具有正常血小板相似的聚集功能;血小板粘附试验:正常血小板(40.43±1.63%)、类血小板颗粒(35.53±4.06%)。结论通过PEG细胞化学融合法构建的脐带血巨核祖细胞/胃癌SGC7901细胞融合体能产生功能性血小板。
Objective To construct functional platelets of human cord blood megakaryocyte progenitor cells / gastric cancer SGC7901 cells fusion. Methods Human umbilical cord blood CD34 + cells were sorted by magnetic beads. The cells were cultured in vitro by RPMI1640 medium, and the megakaryocyte progenitor cells were induced by adding targeting differentiation factor TPO, FL, IL-3 and IL-6. The gastric cancer SGC7901 cells were resuscitated. (PEG). The fusion cells of megakaryocyte progenitor cells / gastric cancer SGC7901 cells were prepared. The fusion cells were sorted by HAT screening system and cultured continuously. The expression of CD41 and CD62p on the surface of platelets in culture supernatant was detected by flow cytometry. , Adhesion test to detect platelet function, Wright’s staining observed particle morphology. Results CD34 + cells sorted by immunomagnetic beads for 7 days proliferated 80 times and fused with giant cell nuclear antigen (GSP) / gastric cancer cells by PEG chemical fusion method with SGC7901 gastric cancer cell line, the fusion rate was (36.91 ± 1.08)%. Fusion cell culture 10 d can proliferate 4 times, resulting in (6.1 ± 1.21) × 106 / ml of platelet-like particles; Wright’s staining after observation, its morphology and normal platelets similar. Platelet-derived platelet-specific marker CD41 and platelet-activating marker CD62p were slightly decreased in platelet-like platelets compared with normal platelets: normal platelets (74.11 ± 1.71)% and platelet-like platelets (71.04 ± 1.64)%; (88.83 ± 3.26)%, platelet-like granules (72.51 ± 2.79)% (t = 6.59, P <0.05). The aggregation test showed similar platelet aggregation function. The platelet adhesion test was normal Platelet (40.43 ± 1.63%), platelet-like particles (35.53 ± 4.06%). CONCLUSION: Fusion cord blood megakaryocyte progenitor cells / gastric cancer SGC7901 cells constructed by chemical fusion of PEG cells can produce functional platelets.