氨基乙酰丙酸光动力疗法清除的混合淋巴细胞杀伤作用研究

目的对氨基乙酰丙酸(5-aminolevulinicacid,ALA)光动力疗法(ALA-PDT)清除激活淋巴细胞的机制以及ALA-PDT处理后的混合淋巴细胞对同种异体淋巴细胞及白血病细胞的杀伤作用进行研究。方法将同种异体外周血单个核细胞按单向混合淋巴细胞培养方法制备单向混合淋巴细胞(MLC细胞)。对MLC细胞进行ALA-PDT处理后,将其与制备MLC细胞时使用的刺激细胞、第三方正常人PBMCs以及K562细胞、HL60细胞1∶1混合培养48h。MTT比色法检测细胞活性,并计算ALA-PDT处理后的MLC细胞对不同细胞的杀伤率。用流式细胞仪检测ALA-PDT处理对MLC细胞的杀伤情况。结果ALA-PDT处理后的MLC细胞对刺激细胞的杀伤率明显低于第三方正常人PB-MCs、K562细胞和HL60细胞的杀伤率,分别为32·4%±1·16%比78·1%±0·952%、75·5%±2·40%和77·0%±1·13%(P<0·001),ALA-PDT处理后的MLC细胞对HL60细胞、第三方正常人PBMCs、K562细胞之间也存在差异。ALA-PDT清除激活淋巴细胞以凋亡为主。结论ALA-PDT可能通过诱导细胞凋亡选择性清除同种异体激活的淋巴细胞,使针对同种异体、淋巴细胞介导的排斥反应减轻,同时也保留了对第三方正常人PBMCs以及肿瘤细胞的杀伤特性。

Study on cytotoxicity of ALA-PDT treated mixed lymphocytes

Objective To determine the mechanism of 5-aminolevulinic acid(ALA)photodynamic therapy(PDT)and to test the cytotoxicities by ALA-PDT treated one way mixed lymphocytes culture(MLC)cells on peripheral blood mononuclear cells(PBMCs), K562 cells and HL60 cells.Methods Allogenic PBMCs were treated on one-way mixed lymphocyte culture, which were then treated with ALA-PDT. The ALA-PDT treated MLC cells were harvested and cultured with stimulators, other PBMCS, K562 and HL60 at a ratio of 1:1 for 48 hours. MTT assays were used to detect the cytotoxicities of MLC cells on stimulators, other PBMCs, K562 and HL60. Cellular apoptosis of ALA-PDT treated MLC cells were determined by annexin V-FITC and propidium iodide staining flow cytometry. Results The kill rate of ALA-PDT treated MLC cells on stimulators was apparently lower than those on other PBMCs, K562 and HL60(32.4%±1.6% versus 78.1%±0.952%,75.5%±2.40% and 77.0%±1.13% respectively)(P<0.001).The kill rates on other PBMCs, K562 and HL60 were different from each other. The cellular apoptosis of MLC cells was the main results induced by ALA-PDT. Conclusions ALA-PDT may selectively purse MLC cells through cellular apoptosis, which relieves the rejection induced by lymphocytes and retains the cytotoxicities of these lymphocytes on other PBMCs and tumor cells.