纳米级氧化石墨烯对人红细胞和凝血功能影响的体外研究

In vitro study of the impacts of nanoscale graphene oxide on human erythrocyte and coagulation function

目的探讨纳米级(<100 nm)氧化石墨烯(GO)对人红细胞和凝血功能的影响。方法分别利用原子力显微镜、激光粒度仪和傅里叶红外光谱仪表征或检测商品化GO的形貌、水力学直径和分子功能基团;真空静脉采集6名健康志愿者外周血作为测试对象,用0.9%NaCl溶液将GO配置成浓度为(0—1.0)mg/mL的GO实验样品,将不同浓度GO实验样品分别与红细胞、贫血小板血浆(PPP)和抗凝全血混合后37℃孵育;检测红细胞形态和相对红细胞溶血率以分析GO和红细胞相互作用,测定活化部分凝血酶时间(APTT)、凝血酶原时间(PT)及血栓弹力图(TEG)以分析GO对凝血功能的影响。结果研究用的GO为单原子层结构,直径12.2—75.8 nm,在生理盐水中良好分散;红细胞显微形态:GO呈浓度依赖性地改变红细胞的形态和聚集行为;相对红细胞溶血率随GO浓度增大而增大,GO终浓度0.5 mg/mL时相对红细胞溶血率(3.23±0.23)%;血浆凝血测试:GO终浓度(0.1—0.5)mg/mL时APTT(s)由27.6±1.64延长至39.8±3.92(P<0.01),而不同浓度GO对PT影响不明显(P>0.05);TEG检测:GO未改变全血的凝血功能。结论纳米级(<100 nm) GO具有良好的红细胞相容性,且不影响全血凝血功能,具备更广的生物医学应用价值。

Objective to study the impacts of nanoscale graphene oxide(GO)(<100 nm) on human erythrocyte and coagulation function. Methods The morphology, hydraulic diameter and function groups of GO were characterized by atomic force microscopy, laser particle analyzer and Fourier infrared spectrometer respectively. Peripheral blood sample was collected from six healthy volunteers. GO was prepared into GO experimental samples with a concentration ranged from 0—1.0 mg/mL using 0.9% NaCl saline. GO experimental samples were added into erythrocyte, poor platelet plasma(PPP) and whole blood respectively and incubated together at 37℃. The interactions between erythrocyte and GO were characterized by erythrocyte morphology and relative hemolytic rate. The effects of GO on coagulation function were evaluated through activated partial thrombin time(APTT), prothrobin time(PT) and thromboelastography(TEG). Results The studied GO possessed a single atomic layer, with a diameter range of 12.2—75.8 nm and showed good dispersion in physiological saline. The images of erythrocyte morphology showed that GO can affect the morphology and aggregation of erythrocyte in a concentration-dependent manner. The relative hemolytic rate rose with the concentration of GO. The relative hemolytic rate was(3.23±0.23)% when the final concentration of GO reached to 0.5 mg/mL. GO with final concentrations ranged from 0.1 mg/mL to 0.5 mg/mL prolonged the APTT(s) from(27.6±1.6)s to(39.8±3.92)s significantly(P<0.01), while there was no significant effect on the PT for GO with the studied concentrations(P>0.05). TEG tests showed that GO had no observed effects on coagulation capability of the whole blood. Conclusion Nanoscale GO(<100 nm) presents good erythrocyte compatibility and does not affect the whole blood coagulation function, suggesting its potential biomedical application values.