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目的探讨改良体外光化学治疗(ECP)方法提高淋巴细胞凋亡诱导率的效果。方法以无菌离体移植肝供者脾脏作为实验材料,分别以改良和传统ECP方法制备供者脾脏淋巴细胞悬液(SP),所得SP经8-甲氧基补骨脂素(8-MOP)联合A波段长波紫外线(UVA)照射(PUVA)、8-MOP、UVA处理,并分别设对照组;不同处理的SP于37℃、50 mL/L CO2孵箱中培养过夜(6~8 h),通过观察细胞形态学改变并用流式细胞术检测凋亡率,比较各组间淋巴细胞凋亡率的差异。结果改良ECP方法制备的SP,经PUVA、8-MOP、UVA处理早期凋亡率和总凋亡率分别为(95.33±3.03)%和(97.10±2.12)%,(23.39±4.55)%和(36.32±6.63)%,(66.98±3.60)%和(68.65±4.35)%,与对照组(12.82±1.86%和13.4±2.65%)相比,差异显著,具有统计学意义(P<0.01);传统ECP法各实验组早期及总凋亡率分别为(79.73±4.21)%和(82.70±4.13)%,(61.42±2.28)%和(68.91±2.18)%,(19.30±1.78)%和(28.06±1.88)%,(10.84±0.98)%和(12.77±1.22)%,组间比较差异显著,具有统计学意义(P<0.01);改良法与传统ECP法相比,早期和总凋亡率有显著性差异(P<0.01),而晚期凋亡率无显著性差异(P>0.05)。结论改良型ECP方法可简便、安全、高效提高离体脾脏淋巴细胞早期凋亡率,为进一步研究ECP诱导的树突细胞免疫调节作用奠定基础。
Objective To explore the effect of improving the in vitro photochemical therapy (ECP) method to increase the rate of lymphocyte apoptosis induction. Methods The donor spleen was transplanted into the spleen of the donor liver in vitro. The spleen lymphocyte suspension (SP) was prepared by modified and traditional ECP methods. The obtained SP was treated with 8-methoxy psoralen (8-MOP (PUVA), 8-MOP and UVA, respectively. The SPs were incubated in a 50 mL / L CO2 incubator at 37 ° C overnight (6-8 h ). The morphological changes of the cells were observed and the apoptotic rate was detected by flow cytometry. The differences of lymphocyte apoptosis rates among the groups were compared. Results The results showed that the early apoptotic rate and the total apoptotic rate of SP prepared by modified ECP method were (95.33 ± 3.03)% and (97.10 ± 2.12)%, (23.39 ± 4.55)% and ( (36.32 ± 6.63)%, (66.98 ± 3.60)% and (68.65 ± 4.35)% respectively, which were significantly different from those in the control group (12.82 ± 1.86% and 13.4 ± 2.65%, P <0.01). The early and total apoptotic rates in the experimental group were (79.73 ± 4.21)% and (82.70 ± 4.13)%, (61.42 ± 2.28)% and (68.91 ± 2.18)%, (19.30 ± 1.78)% and 28.06 ± 1.88)%, (10.84 ± 0.98)% and (12.77 ± 1.22)%, respectively. There was significant difference between the two groups (P <0.01). Compared with the traditional ECP method, There was significant difference (P <0.01), while the late apoptosis rate was no significant difference (P> 0.05). Conclusion The modified ECP method can increase the early apoptotic rate of spleen lymphocytes in vitro easily, safely and efficiently, which lays the foundation for further study on the immune regulation of dendritic cells induced by ECP.