诊断学理论与实践

诊断学理论与实践 ›› 2022, Vol. 21 ›› Issue (05): 567-574.doi: 10.16150/j.1671-2870.2022.05.004

• 论著 • 上一篇    下一篇

消化道肿瘤单细胞转录组测序研究中生物样本前处理改良的探索

杨蕊馨, 杜宇童, 燕然林, 朱正纲, 李琛, 于颖彦()   

  1. 上海交通大学医学院附属瑞金医院普外科，上海消化外科研究所，上海 200025
  • 收稿日期:2022-09-13 出版日期:2022-10-25 发布日期:2023-01-29
  • 通讯作者: 于颖彦 E-mail:yingyan3y@sjtu.edu.cn
  • 基金资助:
    国家自然科学基金(82072602);国家自然科学基金(82173222);上海市科学技术委员会基金(20DZ2201900);上海市科学技术委员会基金(18411953100);上海市转化医学协同创新中心基金(TM202001);教育部·上海市生物医药临床研究与转化协同创新中心(CCTS-2022202)

Improving exploration of biological sample pretreatment in single-cell transcriptome sequencing of gastrointestinal tumors

YANG Ruixin, DU Yutong, YAN Ranlin, ZHU Zhenggang, LI Chen, YU Yingyan()   

  1. Department of General Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Received:2022-09-13 Online:2022-10-25 Published:2023-01-29
  • Contact: YU Yingyan E-mail:yingyan3y@sjtu.edu.cn

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摘要：

目的：探索在消化道上皮源性肿瘤单细胞转录组测序(简称单细胞测序)中提高组织单细胞悬液中上皮细胞占比的方法，以改善检测样本质量。方法：分析GEO公共数据库内常见消化道上皮源性肿瘤（胃癌、结肠直肠癌及胰腺癌）单细胞转录组测序数据集中的上皮细胞组分占比。比较传统酶消化法以及酶消化后辅以上机前机械性物理吹打法（改良法）处理后单细胞悬液中上皮细胞占比和细胞活力差异。结果：分析公共数据库内数据显示，4份正常胃上皮及胃癌样本的上皮细胞占比为17.05%、6.11%、8.93%和14.66%; 2份结肠直肠癌上皮细胞占比为3.86%和16.60%；2份胰腺癌上皮细胞占比为3.22%和21.37%。对2份新鲜采集的胃癌样本，采用传统酶消化法制备单细胞悬液的细胞结团率为56.26%±1.98%和38.34%±1.26%，细胞活力检测提示活细胞占比分别为98.43%±0.56%和97.24%±0.48%；而对传统酶消化法处理后的单细胞悬液在上机前用0.33 mm规格的胰岛素注射器施以机械性吹打操作30 s，可使单细胞悬液的上皮结团率降至22.78%±1.38%和14.46%±0.92%（P均<0.000 1）；活细胞占比分别为95.16%±0.42%和93.52%±0.82%（P<0.05）。结论：在采用传统酶消化后辅以机械性物理吹打法的组织样本前处理操作，可减少上皮细胞结团，从而提高单细胞悬液中的上皮细胞占比，活细胞占比虽有小幅下降，但可满足上机要求。

关键词: 单细胞转录组测序, 胃癌, 结直肠癌, 胰腺癌, 样本前处理, 细胞结团率

Abstract:

Objective: To explore the method of increasing the proportion of epithelial cells in the single-cell suspension of tissue in single-cell transcriptome sequencing in gastrointestinal epithelial tumors for improving the quality of test samples. Methods: The proportion of epithelial cell in the single-cell transcriptome sequencing data set of common gastrointestinal epithelial tumors (gastric cancer, colorectal cancer and pancreatic cancer) in GEO public database was analyzed. The difference in proportion and activity of epithelial cells in single-cell suspension between traditional enzyme digestion method and mechanical physical blowing method (improved method). Results: The data analysis in public database showed that the proportion of epithelial cells in 4 samples of normal gastric epithelium or gastric cancer was 17.05%, 6.11%, 8.93% and 14.66% respectively. The proportion of epithelial cells in 2 samples of colorectal cancer was 3.86% and 16.60%. The proportion of epithelial cells in 2 samples of pancreatic cancer was 3.22% and 21.37%. For two freshly collected gastric cancer samples, the cell agglomeration rate of single-cell suspension prepared by traditional enzyme digestion method was 56.26%±1.98% and 38.34%±1.26%, and the percentage of living cells in cell activity test was 98.43%±0.56% and 97.24%±0.48%, respectively. However, in the single-cell suspension treated by traditional enzyme digestion and mechanically blown with 0.33 mm insulin syringe for 30 seconds before being put on the machine, the epithelial cell agglomeration rates in 2 test samples were reduced to 22.78%±1.38% and 14.46±0.92%(P<0.000 1), and the proportion of living cells was 95.16%±0.42% and 93.52%±0.82% respectively(P<0.05). Conclusions: The pretreatment of tissue samples with traditional enzyme digestion and mechanical physical blowing could reduce epithelial cell agglomeration and increase the proportion of epithelial cells in single-cell suspension. The proportion of living cells decreased slightly, while it could meet the requirements of subsequent sequencing.

Key words: Single-cell RNA-seqencing, Gastric cancer, Colorectal cancer, Pancreatic cancer, Biospecimen pretreatment, Cell agglomeration rate

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