| 段永娟,兰 洋,易美慧,毕 杨,陈晓燕,刘立鹏,张傲利,张陆阳,宗苏玉,郑佳睿,张英驰,竺晓凡.HES4抑制急性T淋巴细胞白血病细胞系Jurkat对阿糖胞苷的敏感性[J].现代生物医学进展英文版,2021,(11):2001-2006. |
| HES4抑制急性T淋巴细胞白血病细胞系Jurkat对阿糖胞苷的敏感性 |
| HES4 Inhibits Chemosensitivity of T-cell Line Jurkat to Cytarabine |
| Received:December 31, 2020 Revised:January 23, 2021 |
| DOI：10.13241/j.cnki.pmb.2021.11.001 |
| 中文关键词: HES4 过表达 敲除 Jurkat细胞 Ara-C |
| 英文关键词: HES4 Overexpression Knockout Jurkat cell Ara-C |
| 基金项目:国家自然科学基金面上项目（81770175） |
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| 中文摘要: |
| 摘要 目的：研究hes家族bHLH转录因子4（HES4）与急性T淋巴细胞白血病（T-ALL）细胞系Jurkat对化疗药物阿糖胞苷（Ara-C）的敏感性作用。方法：构建Lenti-pCDH-HES4-puro质粒，包装慢病毒感染Jurkat细胞，感染空载体Lenti-pCDH-puro为对照组，puromycin筛选阳性细胞，Realtime-PCR检测转录水平HES4过表达情况，CCK8法检测Jurkat对Ara-C敏感性，Ara-C终浓度为0.1、1、10、100、1000、10000 nmol?L-1；设计3对针对于HES4的特异sgRNA序列，构建LentiGuide-sgRNA1-puro、LentiGuide-sgRNA2-Puro、LentiGuide-sgRNA3-puro质粒，LentiGuide-puro空载体为对照组，包装慢病毒感染Jurkat细胞，puromycin筛选，提取基因组DNA，PCR扩增sgRNA的靶序列，一代测序检测HES4的敲除效率，CCK8法检测敲除HES4后Jurkat对Ara-C敏感性，Ara-C终浓度为0.1、1、10、100、1000、10000 nmol?L-1。结果：与对照组相比，HES4在Jurkat中过表达（2.37 ± 0.09）倍（P < 0.001），在Ara-C为1 nmol?L-1、100 nmol?L-1 浓度下，过表达HES4使Jurkat对Ara-C的药物敏感性降低（P值分别小于0.01、0.05）； sgRNA1、sgRNA 2、sgRNA 3敲除分值分别为83、71、63，其中sgRNA1的敲除效果最佳，在Ara-C为1000 nmol?L-1浓度下，敲除HES4促进Jurkat对Ara-C的敏感性（P < 0.05），3条sgRNAs之间无明显区别（P > 0.05）。结论：HES4抑制T-ALL细胞系Jurkat对Ara-C的敏感性，为T-ALL化疗耐受的机制研究提供指导。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the relationship between hes family bHLH transcription factor 4 (HES4) and sensitivity of T-cell acute lymphoblastic leukemia (T-ALL) cell line Jurkat to cytarabine (Ara-C), a chemotherapeutic drug. Methods: Lentiviral vector Lenti-pCDH-HES4-puro was constructed and packaged to infect Jurkat cells, and empty vector Lenti-pCDH-puro was used as control group. Puromycin was used to screen positive cells. Real-time PCR was used to detect HES4 overexpression. CCK8 method was used to detect Jurkat's sensitivity to Ara-C. The final concentration of Ara-C was 0.1, 1, 10, 100, 1000, 10000 nmol?L-1. Three pairs of specific sgRNA sequences targeting HES4 were designed to construct the recombinant plasmids LentiGuide-sgRNA1-puro, LentiGuide- sgRNA2-puro and LentiGuide-sgRNA3-puro, and LentiGuide-puro was used as control group. Then the packaging lentivirus infected Jurkat cells was screened by puromycin and genomic DNA was extracted. The target sequence of sgRNA was amplified by PCR, and then the knockout efficiency of HES4 was detected by DNA sequencing. The sensitivity of Jurkat to Ara-C was detected by CCK8 method. The final concentration of Ara-C was 0.1, 1, 10, 100, 1000, 10000 nmol?L-1. Results: Compared with the control group, HES4 overexpression in Jurkat was (2.37±0.09) times (P < 0.001). Overexpression of HES4 can inhibit Jurkat's drug sensitivity to Ara-C. When Ara-C was 1 nmol?L-1 and 100 nmol?L-1, overexpression of HES4 decreased the drug sensitivity of Jurkat to Ara-C (P < 0.01 and 0.05, respectively); the knockout score of sgRNA1, sgRNA 2 and sgRNA 3 was 83, 71 and 63, respectively, and the knockout effect of sgRNA1 was the best. When Ara-C was 1000 nmol?L-1, HES4 knockout promoted the sensitivity of Jurkat to Ara-C (P < 0.05), but there was no significant difference among the three sgRNAs (P > 0.05). Conclusion: HES4 inhibits the sensitivity of Jurkat to Ara-C, which provides guidance for the study of the mechanism of chemotherapy tolerance in T-ALL. |
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