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目的:研究离体热处理和顺铂联合热处理对术中回收血红细胞功能的影响及其中混杂的肝肿瘤细胞株(HepG2)的杀灭作用。创新点:(1)采用多种评价指标研究了不同时间离体热处理对术中回收血中混杂的HepG2的杀灭作用及对红细胞的影响,并确定了对红细胞安全且能有效杀灭HepG2的离体热处理时间。(2)从多个角度评价了离体顺铂联合热处理对术中回收血红细胞的影响及对其中混杂的HepG2的杀灭作用,确定了该方案中对红细胞安全且能有效去除HepG2的顺铂浓度。方法:采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)、5-乙炔基-2’脱氧尿嘧啶核苷(EdU)和平板克隆形成评估HepG2的细胞存活率、DNA复制率和克隆形成能力(图1和3);从红细胞渗透脆性、携氧能力(2,3-二磷酸甘油酸(2,3-DPG)、半饱和氧分压(P50))、能量代谢(Na+-K+-ATPase、pH)、膜完整性(游离血红蛋白(Hb)、血清K+和Na+浓度、细胞膜磷脂酰丝氨酸外翻比例)等角度评估红细胞功能(图2和4;表3)。结论:肝肿瘤术中回收血经离体顺铂联合热处理(42°C,50μg/ml)60min后,能有效清除其中混杂的HepG2,但对红细胞无显著影响,值得体内进一步研究顺铂热处理有效应用于肿瘤手术自体血液回输的安全方案。
OBJECTIVE: To study the effect of ex vivo heat treatment and cisplatin combined with heat treatment on the recovery of intraoperative red blood cell function and the killing effect of HepG2. Innovative points: (1) Using a variety of evaluation indicators to study the in vitro heat treatment of intraoperative recovery of blood mixed HepG2 killing effect and the impact on the red blood cells, and to determine the red blood cells safe and can effectively kill HepG2 Exothermal treatment time. (2) To evaluate the effect of cisplatin combined with heat treatment on intraoperative recovery of red blood cells and the killing effect of HepG2 mixed with cisplatin from multiple perspectives, and to determine the effect of cisplatin on HepG2 cells that is safe and can effectively remove HepG2 concentration. Methods: MTT, 5-ethynyl-2’-deoxyuridine (EdU) and 3-chlorothiazole Plate cloning formation assessed HepG2 cell viability, DNA replication and clonogenicity (Figures 1 and 3); from fragile erythrocyte infiltration, oxygen carrying capacity (2,3-diphosphoglycerate (2,3-DPG) (P50), energy metabolism (Na + -K + -ATPase, pH), membrane integrity (free hemoglobin (Hb), serum K + and Na + concentration and phosphatidylserine eversion ratio) (Figures 2 and 4; Table 3). CONCLUSION: HepG2 can effectively remove HepG2 mixed with Cisplatin combined with heat treatment (42 ° C, 50μg / ml) in liver tumor during operation, but has no significant effect on erythrocytes, which is worthy of further study on cisplatin heat treatment in vivo Safety solution for autologous blood transfusion in tumor surgery.