纳秒脉冲治疗裸鼠恶性黑色素瘤的疗效观察

发布时间：2018-09-04 06:24

【摘要】：目的：评估纳秒级电脉冲治疗绿色荧光蛋白标记的恶性黑色素瘤细胞(A375-GFP)裸鼠皮下移植瘤的疗效。方法：通过细胞转染技术将带有GFP基因的慢病毒颗粒转染人恶性黑色素瘤A375细胞。荧光显微镜下筛选转染率达100%且荧光信号稳定的复感染指数（multiplicity of infection，MOI），建立稳定A375-GFP细胞株，设为转染组细胞A375-GFP。同时设立未转染组亲代细胞A375。通过四甲基偶氮唑蓝（MTT）法及倍增时间检测两组细胞增殖能力的改变，流式细胞学检测细胞周期分布的差异。建立裸鼠A375-GFP细胞皮下移植瘤模型，随机分为治疗组（n=6）和对照组（n=6）。纳秒脉冲电场(脉宽200ns、场强20kV/cm、频率5Hz，2000个脉冲,4个方位，每隔2d治疗1次，，共2次)治疗裸鼠皮下瘤；采用活体荧光成像技术检测裸鼠皮下瘤治疗前、治疗后48h、10d皮下瘤荧光变化；组织HE染色观察评估治疗后90d局部效果；数码相机观察随访治疗后局部疤痕变化。结果： 1.成功构建了稳定表达GFP的A375-GFP细胞。 2.通过MTT法检测细胞生长72h时OD值发现A375（1.066±0.514）及A375-GFP组细胞（1.023±0.810）比较差异没有统计学意义(P＞0.05)。A375-GFP细胞的倍增时间（28.9±1.51）h与A375细胞（29.04±1.04）h差异无统计学意义(P＞0.05)。流式细胞学检测示两组细胞周期分布差异无统计学意义(P＞0.05)。 3.稳定建立了可视化裸鼠皮下A375-GFP细胞移植瘤模型。 4.纳秒脉冲治疗裸鼠皮下移植瘤后，活体荧光成像仪检测示治疗组皮下移植瘤荧光强度及面积于治疗后48h大部分减少，至治疗后10d完全消失；对照组则逐渐增加，治疗组与对照组比较，差异具有统计学意义（F=1055.11，P0.0001）。nsPEF治疗后90d组织HE染色结果证实治疗组所有裸鼠皮下移植瘤完全消失。皮下移植瘤经nsPEF治疗后3-4d,治疗局部区域出现结痂，于2周内脱落，随时间推移，局部疤痕逐渐变浅，且肉眼未见原位新生瘤体。结论： 1.通过慢病毒转染可以获得稳定，高效且长期表达GFP的人黑色素瘤株A375细胞。 2.慢病毒转染的GFP对A375细胞的增殖能力未见明显影响，可反映亲代细胞的增殖能力，为后续实验研究奠定基础。 3.成功构建了可视化裸鼠皮下A375-GFP细胞移植瘤模型，通过电极板4个方向夹取肿瘤，并采用脉冲参数为200ns，5Hz，20kv/cm，2000个纳秒级电脉冲治疗皮下移植瘤2次后可以有效消融A375-GFP裸鼠皮下移植瘤，且疤痕少，操作简单，它可能成为浅表肿瘤临床微创治疗的一种新的途径。
[Abstract]:Aim: to evaluate the efficacy of nanosecond electric pulse in the treatment of malignant melanoma cells (A375-GFP) labeled with green fluorescent protein (GFP) in nude mice. Methods: lentivirus particles with GFP gene were transfected into human malignant melanoma A375 cells by cell transfection technique. The co-infection index (multiplicity of infection,MOI) with 100% transfection rate and stable fluorescence signal was screened under fluorescence microscope. The stable A375-GFP cell line was established as A375-GFP cell line. At the same time, the parent cells A375in the untransfected group were established. The proliferation ability of the two groups was detected by tetramethyl azolium blue (MTT) assay and doubling time, and the difference of cell cycle distribution was detected by flow cytometry. The model of subcutaneous transplantation of A375-GFP cells in nude mice was established and randomly divided into treatment group (nong6) and control group (nong6). Nanosecond pulsed electric field (pulse width 200ns, field intensity 20kV / cm, frequency 5Hz, 2000 pulses, 4 directions, 2 times every 2 days) was used to treat subcutaneous tumor in nude mice. Tissue HE staining was used to evaluate the local effect 90 days after treatment, and digital camera was used to observe the changes of local scar after treatment. Results: 1. A375-GFP cells stably expressing GFP were successfully constructed. The OD value of A375 cells (1.066 卤0.514) and A375-GFP group (1.023 卤0.810) was determined by MTT assay. There was no significant difference in doubling time between A375-GFP cells (28.9 卤1.51) h and A375 cells (29.04 卤1.04) h (P > 0.05). Flow cytometric analysis showed that there was no significant difference in cell cycle distribution between the two groups (P > 0.05). A visualized nude mouse model of subcutaneous A375-GFP cell transplantation tumor was established stably. 4. After nanosecond pulse treatment of subcutaneous transplanted tumor in nude mice, the fluorescence intensity and area of subcutaneous transplanted tumor in the treatment group were mostly decreased after 48 hours of treatment, and disappeared completely at 10 days after treatment, while that in the control group increased gradually. Compared with the control group, the difference between the treatment group and the control group was statistically significant (FF1055.11P0.0001). The results of HE staining confirmed that all subcutaneous transplanted tumors disappeared completely in the treatment group 90 days after treatment. After treatment with nsPEF for 3 to 4 days, scab appeared in the local area of the transplanted tumor, which fell off within 2 weeks. With the passage of time, the local scar gradually became shallower, and no in situ neoplasm was found in the naked eye. Conclusion: 1. Stable, highly efficient and long term expression of GFP in human melanoma cell line A375 cells could be obtained by lentivirus transfection. The lentivirus-transfected GFP had no obvious effect on the proliferation of A375 cells, but could reflect the proliferation ability of parent cells, which laid a foundation for further experimental study. A visualized nude mouse model of subcutaneous A375-GFP cell transplantation was successfully constructed. The tumor was clamped in four directions by electrode plate. The subcutaneous transplanted tumor in A375-GFP nude mice could be ablated effectively after 2 000 nanosecond electric pulses with pulse parameters of 200ns5 Hz and 20kv / cm. With less scar and simple operation, it may be a new approach to clinical minimally invasive treatment of superficial tumors.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.5

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