摘要
[目的]优化人非小细胞肺癌组织获取单细胞的方法,提高单细胞的活率、回收率,减小抗原表位的影响。[方法]获取临床肺癌组织样本。比较4种不同浓度的Ⅳ型胶原酶(0.2 mg/m L、0.5 mg/m L、1.0 mg/m L、2.0 mg/m L)37℃消化30 min,血球计数板计数用7-氨基放线菌素(7-AAD)、CD45、CD3染色各组消化后的细胞,流式细胞仪检测细胞活率和抗原表位表达。[结果]各组细胞消化后细胞数量为:0.2 mg/m L组1.27±0.18×10^(6)个,0.5 mg/m L组2.15±0.21×10^(6)个,1.0 mg/m L组1.04±0.14×10^(6)个,2.0 mg/m L组0.25±0.03×10^(6)个;细胞活率分别为:0.2 mg/m L组79.62±4.13%,0.5 mg/m L组80.17±7.20%,1.0 mg/m L组79.34±8.53%,2.0 mg/m L组5.08±4.91%;CD45-比例分别为:0.2 mg/m L组70.09±7.60%,0.5 mg/m L组72.71±3.05%,1.0 mg/m L组85.43±5.29%,2.0mg/m L组82.37±9.07%;CD45+比例分别为:0.2 mg/m L组24.23±8.12%,0.5 mg/m L组23.51±3.20%,1.0 mg/m L组11.93±6.81%,2.0 mg/m L组14.24±7.92%;CD3+比例分别为:0.2 mg/m L组48.2±5.1%,0.5 mg/m L组49.5±6.2%,1.0 mg/m L组47.0±3.8%,2.0 mg/m L组15.2±2.9%。[结论] 0.5 mg/m L组Ⅳ型胶原酶消化细胞后,单细胞数量最多,细胞活率和CD45、CD3抗原表位较高。
[Objective]Optimize the method of obtaining single cells from human non-small cell lung cancer tissues,improve the viability and recovery rate of single cells,and reduce the impact of epitopes. [Method] Obtain clinical lung cancer tissue samples. Compare four different concentrations of Type Ⅳ collagenase( 0. 2 mg/m L,0. 5 mg/m L,1. 0 mg/m L,2. 0 mg/m L)digestion at 37 ℃ for 30 min,and use 7-aminoactinomycin( 7-AAD) for Hemocytometer counting. The digested cells of each group were sta Ined for CD45,CD3 and cell viability and epitope expression were detected by flow cytometry.[Result]The number of cells in each group after digestion was: 0. 2 mg/m L group 1. 27 ± 0. 18 × 10^(6) cells,0. 5 mg/m L group 2. 15 ± 0. 21 ×10^(6) cells,1. 0 mg/m L group 1. 04 ± 0. 14 × 10^(6) cells,2. 0 mg/m L group 0. 25 ± 0. 03 × 10^(6) cells;cell viability rates are:0. 2 mg/m L group 79. 62 ± 4. 13%,0. 5 mg/m L group 80. 17 ± 7. 20%,1. 0 mg/m L group 79. 34 ± 8. 53%,2. 0 mg/m L 5. 08± 4. 91%;CD45-ratios are: 0. 2 mg/m L group 70. 09 ± 7. 60%,0. 5 mg/m L group 72. 71 ± 3. 05%,1. 0 mg/m L group85. 43 ± 5. 29%,2. 0 mg/m L group 82. 37 ± 9. 07%;CD45+ratios are: 0. 2 mg/m L group 24. 23 ± 8. 12%,0. 5 mg/m L group 23. 51 ± 3. 20%,1. 0 mg/m L group 11. 93 ± 6. 81%,2. 0 mg/m L group 14. 24 ± 7. 92%;CD3+ratios are: 0. 2 mg/m L group 48. 2 ± 5. 1%,0. 5 mg/m L group 49. 5 ± 6. 2%,1. 0 mg/m L group 47. 0 ± 3. 8%,2. 0 mg/m L group 15. 2 ± 2. 9%.[Conclusion]0. 5 mg/m L Type Ⅳ collagenase digested cells,the number of cells is the largest,the viability and CD45,CD3 epitopes are higher.
作者
郭婷婷
孟文彤
GUO Ting-ting;MENG Wen-tong(West China Hospital,Sichuan University,Chengdu 610041,China)
出处
《生物技术》
CAS
2021年第1期46-49,15,共5页
Biotechnology
基金
国家自然科学基金资助项目(81972911)。
