经体外培养的肝癌组织人源性异种移植小鼠模型的建立
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摘要
目的通过添加人工基质胶,对原代肝癌组织进行体外培养,进而建立肝癌组织人源性异种移植(PDX)小鼠模型。方法收集中山大学肿瘤防治中心肝胆科共5份肝癌组织,经清洗、冻存、复苏等处理,添加人工基质胶,进行为期50～80 d的体外培养;免疫缺陷小鼠肾包膜下接种建立肝癌组织人源性异种移植模型;建模小鼠饲养80～120 d后,取移植瘤进行病理学检测,苏木精-伊红(HE)染色观察组织形态,免疫组化染色检测甲胎蛋白(AFP)和磷脂酰肌醇蛋白聚糖3(GPC3)的表达情况,每张玻片取3个视野统计阳性细胞,计算阳性率。结果 5例PDX模型中成功2例,建模成功率为40%。其中001号建模小鼠移植侧肾脏萎缩、质地变硬,移植瘤生长,肾脏周围附着大量附属物;004号建模小鼠腹腔大量腹水,且移植侧肾脏可见黄色肝癌病灶。病理HE染色可见移植瘤组织细胞异型性明显,核分裂增多,失去正常的结构;免疫组化法检测移植瘤组织AFP和GPC3,均呈阳性表达,平均阳性率分别为88%、93%;表明移植瘤组织均保留了原代肝癌组织特性及相应蛋白表达。结论经长期体外培养的原代肝癌组织成功构建人源性异种移植小鼠模型,实现PDX模型构建技术的突破,为进一步的肝癌药物筛选奠定基础。
Objective To establish patient-derived tumor xenograft(PDX)mice model of hepatocellular carcinoma after longterm culture in vitro by using Matrigel. Methods A total of 5 hepatocellular carcinoma tissues were obtained from the hepatobiliary surgery department of the Sun Yat-sen University Cancer Center. After washing,cryopreservation and resuscitation,tissues were cultured in vitro by using Matrigel for 50～80 days,then these tissues were transplanted into the subrenal capsule of immunodeficient mice. Feeding the mice for 80～120 days,the transplanted tumor tissues were collected for pathological examination. Morphology was observed by hematoxylin-eosin(HE)staining,and the expressions of alpha fetoprotein(AFP)and glypican 3(GPC3)were detected by immunohistochemical staining. Three visual fields were taken from each slide for counting positive cells. Results Two of the five PDX models were successful,reaching 40% success rate. The transplanted side kidney of 001 mouse was atrophic and hard,the transplanted tumor grew with numerous attachments around the kidney;A large amount of ascites was observed in the abdominal cavity of 004 mouse,and yellow transplanted tumor tissue can be seen in the kidney of the transplanted side of 004 mouse. HE staining showed obvious cell atypia,multinuclear cells in the transplanted tumor tissues;AFP and GPC3 proteins were both positively expressed,with a positive rate of 88% and 93%,respectively. Pathological examination showed that the characteristics and the corresponding protein expressions of primary liver cancer were retained in the transplanted tumor tissues. Conclusion We established patient-derived tumor tissue xenograft mice model of hepatocellular carcinoma after long-term culture in vitro. This is a breakthrough in PDX model construction and lays solid foundation for further drug screening.
Objective To establish patient-derived tumor xenograft(PDX)mice model of hepatocellular carcinoma after longterm culture in vitro by using Matrigel. Methods A total of 5 hepatocellular carcinoma tissues were obtained from the hepatobiliary surgery department of the Sun Yat-sen University Cancer Center. After washing,cryopreservation and resuscitation,tissues were cultured in vitro by using Matrigel for 50～80 days,then these tissues were transplanted into the subrenal capsule of immunodeficient mice. Feeding the mice for 80～120 days,the transplanted tumor tissues were collected for pathological examination. Morphology was observed by hematoxylin-eosin(HE)staining,and the expressions of alpha fetoprotein(AFP)and glypican 3(GPC3)were detected by immunohistochemical staining. Three visual fields were taken from each slide for counting positive cells. Results Two of the five PDX models were successful,reaching 40% success rate. The transplanted side kidney of 001 mouse was atrophic and hard,the transplanted tumor grew with numerous attachments around the kidney;A large amount of ascites was observed in the abdominal cavity of 004 mouse,and yellow transplanted tumor tissue can be seen in the kidney of the transplanted side of 004 mouse. HE staining showed obvious cell atypia,multinuclear cells in the transplanted tumor tissues;AFP and GPC3 proteins were both positively expressed,with a positive rate of 88% and 93%,respectively. Pathological examination showed that the characteristics and the corresponding protein expressions of primary liver cancer were retained in the transplanted tumor tissues. Conclusion We established patient-derived tumor tissue xenograft mice model of hepatocellular carcinoma after long-term culture in vitro. This is a breakthrough in PDX model construction and lays solid foundation for further drug screening.
引文
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[16]傅佳琦,滕理送,王浩浩.患者来源肿瘤异种移植模型的建立与应用[J].浙江医学,2019,41(4):385-388,392.
[17]Wang Y,Wang JX,Xue H,et al.Subrenal capsule grafting technology in human cancer modeling and translational cancer research[J].Differentiation,2016,91(4-5):15-19.
[18]Wang Y,Revelo MP,Sudilovsky D,et al.Development and characterization of efficient xenograft models for benign and malignant human prostate tissue[J].Prostate,2005,64(2):149-159.
[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]Llovet JM,Zucman-Rossi J,Pikarsky E,et al.Hepatocellular carcinoma[J].Nat Rev Dis Primers,2016,2:16018.
[4]Llovet JM,Villanueva A,Lachenmayer A,et al.Advances in targeted therapies for hepatocellular carcinoma in the genomic era[J].Nat Rev Clin Oncol,2015,12(7):408-424.
[5]Cheng AL,Kang YK,Chen Z,et al.Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma:a phase III randomised,doubleblind,placebo-controlled trial[J].Lancet Oncol,2009,10(1):25-34.
[6]Johnson JI,Decker S,Zaharevitz D,et al.Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials[J].Br J Cancer,2001,84(10):1424-1431.
[7]Zhai W,Lim TK,Zhang T,et al.The spatial organization of intra-tumour heterogeneity and evolutionary trajectories of metastases in hepatocellular carcinoma[J].Nat Commun,2017,8:14565.
[8]Choy E,Yelensky R,Bonakdar S,et al.Genetic analysis of human traits in vitro:drug response and gene expression in lymphoblastoid cell lines[J].PLoS Genet,2008,4(11):e1000287.
[9]Jin K,Teng L,Shen Y,et al,Patient-derived human tumour tissue xenografts in immunodeficient mice:a systematic review[J].Clin Transl Oncol,2010,12(7):473-480.
[10]Broutier L,Mastrogiovanni G,Verstegen MM,et al.Human primary liver cancer-derived organoid cultures for disease modeling and drug screening[J].Nat Med,2017,23(12):1424-1435.
[11]Papapetrou EP.Patient-derived induced pluripotent stem cells in cancer research and precision oncology[J].Nat Med,2016,22(12):1392-1401.
[12]Doungpunta J,Santhi A,Sathanawongs A,et al.Fivefold increase in derivation rates of mouse embryonic stem cells after supplementation of the media with multiple factors[J].Theriogenology,2009,72(2):232-242.
[13]Taylor-Papadimitriou J,Purkis P,Fentiman IS.Cholera toxin567and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells[J].J Cell Physiol,1980,102(3):317-321.
[14]Jin K,He K,Han N,et al.Establishment of a PDTT xenograft model of gastric carcinoma and its application in personalized therapeutic regimen selection[J].Hepatogastroenterology,2011,58(110-111):1814-1822.
[15]Siolas D,Hannon GJ.Patient-derived tumor xenografts:transforming clinical samples into mouse models[J].Cancer Res,2013,73(17):5315-5319.
[16]傅佳琦,滕理送,王浩浩.患者来源肿瘤异种移植模型的建立与应用[J].浙江医学,2019,41(4):385-388,392.
[17]Wang Y,Wang JX,Xue H,et al.Subrenal capsule grafting technology in human cancer modeling and translational cancer research[J].Differentiation,2016,91(4-5):15-19.
[18]Wang Y,Revelo MP,Sudilovsky D,et al.Development and characterization of efficient xenograft models for benign and malignant human prostate tissue[J].Prostate,2005,64(2):149-159.