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胃肠道间质瘤细胞系建立及其对C-KIT下游抑制剂敏感性研究 被引量:4

Establishment of GIST cell line and its sensitivity to C-KIT downstream inhibitors
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摘要 目的随着胃肠道间质瘤(gastrointestinal stromal tumors, GIST)诊出率不断升高,靶向抑制剂相关研究亟待开展,但国内可利用的GIST模型十分有限。本研究旨在建立人GIST细胞模型,探讨靶向抑制剂作用。方法利用手术切除的人GIST腹膜转移灶组织1例进行体外原代培养,连续传代培养>20代进行鉴定,绘制增殖周期生长曲线。免疫细胞化学检测CD117和DOG-1表达,进行染色体核型分析,PCR法检测C-KIT基因与PDGFRA基因突变,CCK8法观察C-KIT 2种通路抑制剂效果,皮下接种BABL/c-nu裸鼠检测致瘤性,PCR法进行种属鉴定及支原体检测,短串联重复序列(short tandem repeats, STR)检测鉴定细胞身份。结果可在体外连续传代的人胃肠道间质瘤细胞命名为PUMC-GIST-1,连续传代后细胞可见分枝,呈神经细胞样生长,传代后于第5天到达平台期;CD117和DOG-1表达均阳性;不同代数细胞染色体核型有变化,在38~48条;细胞C-KIT基因11号外显子5'端突变热点区有突变V559D,PDGFRA基因12号外显子CCG-CCA突变。单独用药结果显示,不同浓度GDC-0941处理48 h后,PUMC-GIST-1 (F=8.96,P<0.001)和GIST-430细胞(F=3.19,P=0.03)增殖能力均下降,差异有统计学意义;不同浓度AZD-6244处理48h后,PUMC-GIST-1细胞增殖能力整体呈现上升趋势,差异有统计学意义,F=9.78,P<0.001;GIST-430细胞增殖能力整体呈现下降趋势,差异有统计学意义,F=6.61,P<0.001。联合用药结果显示,PUMC-GIST-1增殖能力减弱,阴性对照组和5μmol/L AZD+5μmol/L GDC组吸光度(A)值分别为0.41±0.01和0.37±0.03,差异有统计学意义,F=3.30,P=0.007;GIST-430增殖能力减弱,阴性对照组和5μmol/L AZD+5μmol/L GDC组吸光度(A)值分别为2.12±0.11和0.13±0.01,差异有统计学意义,F=158.61,P<0. 001。PUMC-GIST-1较GIST-430对联合用药低敏感。当AZD与GDC浓度分别为5μmol/L时二者具有协同抑制作用,金正均Q值检验PUMC-GIST-1与GIST-430的q值分别为3.08与3.91,均>1.15。裸鼠体内接种不成瘤;PUMC-GIST-1种属鉴定为人源性,STR结果与原代一致,无支原体污染。结论建立了C-KIT突变的PUMC-GIST-1细胞系,该细胞系对C-KIT靶向药低敏感,为胃肠肿瘤研究提供了新型细胞系。 OBJECTIVE As the growing diagnose rate of gastrointestinal stromal tumor(GIST),there is an urge desire of research data on its target inhibitors.However,available GIST cell model in domestic is barely enough.The research aimed to establish a gastrointestinal stromal tumors cell model for in-depth research.METHODS Peritoneal metastatic GIST tissue was primarily cultured and subcultured up to Passage 20 in vitro before full characterization.The expressions of CD117 and DOG-1 were detected by immunocytochemistry.Chromosome karyotype along with mutations in C-KIT and PDGFRA have been analyzed.The growth inhibition rates of single and combined inhibitors for C-KIT downstream pathways on GIST were evaluated.The tumorigenicity was tested by subcutaneous inoculation in BABL/c nude mice.Mycoplasma detection and species identification were detected by PCR.STR profiling was employed for cell identity.RESULTS The cell line was consecutively passaged more than 20 times.The cell line was designated as PUMCGIST-1.Morphologically the cells arrayed like nerve cell and showed positive expression of CD117 and DOG-1.Its chromosomes ranged from 38 to 48,KIT Exon 11 V559 Dmutation and PDGFRA Exon 12 CCG-CCA mutation were detected.The results of drug alone showed that the proliferation of PUMC-GIST-1(F=8.96,P<0.001)and GIST-430 cells(F=3.19,P=0.03)decreased after 48 htreatment with different concentrations of GDC-0941.The proliferation of PUMCGIST-1 cells increased after 48 htreatment with different concentrations of AZD-6244.The difference was statistically significant(F=9.78,P<0.001).The proliferation of GIST-430 cells showed a downward trend(F=6.61,P<0.001).The results of combination therapy showed that the proliferation of PUMC-GIST-1 was weakened.The A values of the negative control group and the 5μmol/L AZD+5μmol/L GDC group were 0.41±0.01 and 0.37±0.03,respectively,and the difference was statistically significant(F=3.30,P=0.007);the proliferation of GIST-430 was weakened.The A values of the negative control group and the 5μmol/L AZD+5μmol/L GDC group were 2.12±0.11 and 0.13±0.01,respectively.The difference was statistically significant(F=158.61,P<0.001).PUMC-GIST-1 was less sensitive to GTP-430 than combination drugs.When the concentration of AZD and GDC was 5μmol/L respectively,the two had synergistic inhibition.The q values of PUMC-GIST-1 and GIST-430 were 3.08 and 3.91,respectively,which were greater than 1.15.Inoculation in nude mice was not tumorigenic;PUMC-GIST-1 species were identified as human,and STR results were consistent with the original,no mycoplasma contamination.CONCLUSION A GIST cell line with low sensitivity to C-KIT pathway inhibition was established.It would provide a base for future GIST research.
作者 周芳颖 冯海凉 卞晓翠 康维明 于健春 宗静 张丹 刘玉琴 ZHOU Fang-ying;FENG Hai-liang;BI AN Xiao-cui;KANG Wei-ming;YU Jian-chun;ZONG Jing;ZHANG Dan;LIU Yu-qin(Institute of Basic Medical Sciences/School of Basic Medicine,Chinese Academy of Medical Sciences/Peking Union Medical College,Beijing 100005,P.R.China;Institute of Basic Medical Sciences/School of Basic Medicine,Chinese Academy of Medical Sciences/Peking Union Medical College,Beijing 100005,P.R.China;Department of General Surgery,Peking Union Medical College Hospital,Beijing 100730,P.R.China)
出处 《中华肿瘤防治杂志》 CAS 北大核心 2019年第11期764-770,777,共8页 Chinese Journal of Cancer Prevention and Treatment
基金 中国医学科学院医学与健康科技创新工程(2016-12M-3-005) 国家科技资源共享平台(NST1-CR18)
关键词 胃肠道间质瘤 C-KIT 药物低敏感 细胞系 PDGFRA gastrointestinal stromal tumors C-KIT drug insensitivity cell line PDGFRA
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