【摘要】：目的： 評(píng)估納秒級(jí)電脈沖治療綠色熒光蛋白標(biāo)記的惡性黑色素瘤細(xì)胞(A375-GFP)裸鼠皮下移植瘤的療效。 方法： 通過細(xì)胞轉(zhuǎn)染技術(shù)將帶有GFP基因的慢病毒顆粒轉(zhuǎn)染人惡性黑色素瘤A375細(xì)胞。熒光顯微鏡下篩選轉(zhuǎn)染率達(dá)100%且熒光信號(hào)穩(wěn)定的復(fù)感染指數(shù)（multiplicity of infection，MOI），建立穩(wěn)定A375-GFP細(xì)胞株，設(shè)為轉(zhuǎn)染組細(xì)胞A375-GFP。同時(shí)設(shè)立未轉(zhuǎn)染組親代細(xì)胞A375。通過四甲基偶氮唑藍(lán)（MTT）法及倍增時(shí)間檢測(cè)兩組細(xì)胞增殖能力的改變，流式細(xì)胞學(xué)檢測(cè)細(xì)胞周期分布的差異。建立裸鼠A375-GFP細(xì)胞皮下移植瘤模型，隨機(jī)分為治療組（n=6）和對(duì)照組（n=6）。納秒脈沖電場(chǎng)(脈寬200ns、場(chǎng)強(qiáng)20kV/cm、頻率5Hz，2000個(gè)脈沖,4個(gè)方位，每隔2d治療1次，，共2次)治療裸鼠皮下瘤；采用活體熒光成像技術(shù)檢測(cè)裸鼠皮下瘤治療前、治療后48h、10d皮下瘤熒光變化；組織HE染色觀察評(píng)估治療后90d局部效果；數(shù)碼相機(jī)觀察隨訪治療后局部疤痕變化。 結(jié)果： 1.成功構(gòu)建了穩(wěn)定表達(dá)GFP的A375-GFP細(xì)胞。 2.通過MTT法檢測(cè)細(xì)胞生長72h時(shí)OD值發(fā)現(xiàn)A375（1.066±0.514）及A375-GFP組細(xì)胞（1.023±0.810）比較差異沒有統(tǒng)計(jì)學(xué)意義(P＞0.05)。A375-GFP細(xì)胞的倍增時(shí)間（28.9±1.51）h與A375細(xì)胞（29.04±1.04）h差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。流式細(xì)胞學(xué)檢測(cè)示兩組細(xì)胞周期分布差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 3.穩(wěn)定建立了可視化裸鼠皮下A375-GFP細(xì)胞移植瘤模型。 4.納秒脈沖治療裸鼠皮下移植瘤后，活體熒光成像儀檢測(cè)示治療組皮下移植瘤熒光強(qiáng)度及面積于治療后48h大部分減少，至治療后10d完全消失；對(duì)照組則逐漸增加，治療組與對(duì)照組比較，差異具有統(tǒng)計(jì)學(xué)意義（F=1055.11，P0.0001）。nsPEF治療后90d組織HE染色結(jié)果證實(shí)治療組所有裸鼠皮下移植瘤完全消失。皮下移植瘤經(jīng)nsPEF治療后3-4d,治療局部區(qū)域出現(xiàn)結(jié)痂，于2周內(nèi)脫落，隨時(shí)間推移，局部疤痕逐漸變淺，且肉眼未見原位新生瘤體。 結(jié)論： 1.通過慢病毒轉(zhuǎn)染可以獲得穩(wěn)定，高效且長期表達(dá)GFP的人黑色素瘤株A375細(xì)胞。 2.慢病毒轉(zhuǎn)染的GFP對(duì)A375細(xì)胞的增殖能力未見明顯影響，可反映親代細(xì)胞的增殖能力，為后續(xù)實(shí)驗(yàn)研究奠定基礎(chǔ)。 3.成功構(gòu)建了可視化裸鼠皮下A375-GFP細(xì)胞移植瘤模型，通過電極板4個(gè)方向夾取腫瘤，并采用脈沖參數(shù)為200ns，5Hz，20kv/cm，2000個(gè)納秒級(jí)電脈沖治療皮下移植瘤2次后可以有效消融A375-GFP裸鼠皮下移植瘤，且疤痕少，操作簡(jiǎn)單，它可能成為淺表腫瘤臨床微創(chuàng)治療的一種新的途徑。
[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 鹵0.514) and A375-GFP group (1.023 鹵0.810) was determined by MTT assay. There was no significant difference in doubling time between A375-GFP cells (28.9 鹵1.51) h and A375 cells (29.04 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.5

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