收稿日期: 2023-06-09
网络出版日期: 2023-11-22
基金资助
广东省自然科学基金项目(2020A1515010246);深圳市科技计划项目(JCYJ20210324135414039)
Construction of chronic myeloid leukemia K562 cell line with overexpression of human TCRP1 gene based on lentivirus vector and its biological function detection
Received date: 2023-06-09
Online published: 2023-11-22
目的 利用慢病毒载体构建过表达人舌癌耐药相关基因（TCRP1）的慢性髓系白血病（CML）K562细胞系并检测其生物学功能。 方法 将TCRP1的重组质粒与包装质粒共转染293T细胞，收集病毒液测定滴度后感染K562细胞，使用嘌呤霉素筛选TCRP1过表达的细胞株K562/TCRP1，荧光定量PCR和Western blot方法检测TCRP1的表达，采用连续细胞计数法和CCK-8法分别对K562/TCRP1及其对照细胞株的增殖情况进行检测，并分析2个细胞株对不同浓度的伊马替尼（IM）的药物敏感性。 结果 TCRP1慢病毒表达载体成功转染进入K562细胞，荧光定量PCR和Western blot结果显示K562/TCRP1细胞株的TCRP1表达在mRNA水平和蛋白水平均显著高于对照组，连续细胞计数法和CCK-8法结果表明K562/TCRP1细胞的增殖能力和细胞活力增强，IM处理K562/TCRP1细胞的IC50值显著高于其对照细胞（P < 0.05）。 结论 利用慢病毒载体成功构建了TCRP1过表达的K562细胞株，并且发现TCRP1的过表达可能增强K562细胞的增殖能力和IM耐药能力，为进一步探讨TCRP1在慢性髓系白血病发病机制中的可能作用提供基础。
刘孝荣 , 何悦 , 陈妍 , 辛泽锋 , 邓栩文 , 钟惠锋 , 陈运生 . 利用慢病毒载体构建过表达人TCRP1基因的慢性髓系白血病K562细胞系及其生物学功能检测[J]. 实用医学杂志, 2023 , 39(19) : 2456 -2460 . DOI: 10.3969/j.issn.1006-5725.2023.19.008
Objective To construct the chronic myeloid leukemia （CML） K562 cell line with overexpression of human tongue cancer drug resistance related gene （TCRP1） by lentivirus vector and to test its biological function. Methods The recombinant plasmid of TCRP1 and the packaging plasmid were co-transfected into 293T cells， and the K562 cells were infected after the titer of the virus was determined. Puromycin was used to screen TCRP1 overexpression cell line K562/TCRP1， and the expression of TCRP1 was detected by fluorescence quantitative PCR and Western blot. The proliferation of K562/TCRP1 and its control cell line was detected by continuous cell counting method and CCK-8 method respectively. The drug sensitivity of the two cell lines to different concentrations of imatinib （IM） was analyzed. Results The TCRP1 lentivirus expression vector was successfully transfected into K562 cells. The results of fluorescence quantitative PCR and Western blot showed that the expression of TCRP1 in K562/TCRP1 cell line was significantly higher than that in the control group at the level of mRNA and protein. The results of continuous cell counting and CCK-8 methods showed that the proliferation of K562/TCRP1 cells were enhanced， and the IC50 value of IM-treated K562/TCRP1 cells was significantly higher than that of the control cells （P < 0.05）. Conclusion The K562 cell line with overexpression of TCRP1 was successfully constructed using lentivirus vector， and we found that the overexpression of TCRP1 might enhance the proliferation and IM resistance of K562 cells， which provided a basis for further exploring the possible role of TCRP1 in the pathogenesis of chronic myeloid leukemia.
| 1 | JABBOUR E， KANTARJIAN H. Chronic myeloid leukemia： 2022 update on diagnosis， therapy， and monitoring［J］. Am J Hematol， 2022， 97（9）：1236-1256. |
| 2 | MINCIACCHI V R， KUMAR R， KRAUSE D S. Chronic Myeloid Leukemia： A Model Disease of the Past， Present and Future［J］. Cells， 2021， 10（1）：117. |
| 3 | CORTES J， PAVLOVSKY C， SAUSSELE S. Chronic myeloid leukaemia［J］. Lancet， 2021， 398（10314）：1914-1926. |
| 4 | LIU H， JIA X， LUO K， et al. TCRP1 activated by mutant p53 promotes NSCLC proliferation via inhibiting FOXO3a［J］. Oncogenesis，2022， 11（1）：19. |
| 5 | WANG C， LIU H， QIU Q， et al. TCRP1 promotes NIH/3T3 cell transformation by over-activating PDK1 and AKT1［J］. Oncogenesis， 2017， 6（4）：e323. |
| 6 | FORD M， MAURO M， AFTANDILIAN C， et al. Management of Chronic Myeloid Leukemia in Children and Young Adults［J］. Curr Hematol Malig Rep， 2022， 17（5）：121-126. |
| 7 | ABDULMAWJOOD B， COSTA B， ROMA-RODRIGUES C， et al. Genetic Biomarkers in Chronic Myeloid Leukemia： What Have We Learned So Far？ ［J］ .Int J Mol Sci， 2021， 22（22）：12516. |
| 8 | SHANMUGANATHAN N， BRANFORD S. The Hidden Pathogenesis of CML： Is BCR-ABL1 the First Event？［J］. Curr Hematol Malig Rep， 2019， 14（6）：501-506. |
| 9 | LIU X， WANG C， GU Y， et al. TCRP1 contributes to cisplatin resistance by preventing Pol beta degradation in lung cancer cells［J］. Mol Cell Biochem， 2015， 398（1/2）：175-183. |
| 10 | GU Y， FAN S， LIU B， et al. TCRP1 promotes radioresistance of oral squamous cell carcinoma cells via Akt signal pathway［J］. Mol Cell Biochem， 2011， 357（1/2）：107-113. |
| 11 | 刘孝荣，文飞球，马东礼，等.TCRP1在慢性髓系白血病细胞中的表达及其对细胞增殖的影响［J］. 中国医师杂志， 2017，19（5）：692-696. |
| 12 | LIN S C， HAGA K， ZENG X L， et al. Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning［J］. Nat Protoc， 2022， 17（4）：1004-1027. |
| 13 | ALVES R， GONCALVES A C， RUTELLA S， et al. Resistance to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia-From Molecular Mechanisms to Clinical Relevance［J］. Cancers （Basel）， 2021， 13（19）.4820. |
| 14 | ZHAO S， LI X， YIN L， et al. TCRP1 induces tamoxifen resistance by promoting the activation of SGK1 in MCF-7 cells［J］.Oncol Rep， 2020， 43（6）：2017-2027. |
| 15 | GU Y， ZHANG Z， YIN J， et al. Epigenetic silencing of miR-493 increases the resistance to cisplatin in lung cancer by targeting tongue cancer resistance-related protein 1（TCRP1） ［J］. J Exp Clin Cancer Res， 2017， 36（1）：114. |
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