WO2021249441A1 - 一对具有相同遗传背景和不同转移潜能的肺癌细胞株及其制备方法和用途 - Google Patents

一对具有相同遗传背景和不同转移潜能的肺癌细胞株及其制备方法和用途 Download PDF

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WO2021249441A1
WO2021249441A1 PCT/CN2021/099216 CN2021099216W WO2021249441A1 WO 2021249441 A1 WO2021249441 A1 WO 2021249441A1 CN 2021099216 W CN2021099216 W CN 2021099216W WO 2021249441 A1 WO2021249441 A1 WO 2021249441A1
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lung cancer
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周清华
陈晓峰
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周清华
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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a pair of lung cancer cell lines with the same genetic background and different metastatic potentials, and a preparation method and application thereof.
  • Lung cancer is one of the tumors with the fastest increase in morbidity and mortality, and the greatest threat to the health and life of the population. According to the latest report in 2020, among men, lung cancer is the malignant tumor with the highest morbidity and mortality; among women, lung cancer is the second malignant tumor with the second morbidity and the first mortality. Lung cancer is the tumor with the highest mortality rate in the world, accounting for about a quarter (22.4%) of the total cancer deaths worldwide. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) according to histological types. NSCLC accounts for 86% of all lung cancers. About 70% of NSCLC patients are in the advanced stage at the time of initial diagnosis, and their 5-year survival rate is 4%-6%.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • Lung cancer invasion and metastasis are the malignant signs and biological characteristics of lung cancer, and they are also the main cause of treatment failure and death of patients.
  • the biggest problem in studying lung cancer metastasis is the lack of natural comparative research biomaterials with the same genetic background and different metastatic potentials. If a pair of lung cancer cell lines with the same genetic background and different metastatic potential can be constructed, it will greatly help the study of lung cancer metastasis.
  • the purpose of the present invention is to provide a pair of lung cancer cell lines with the same genetic background and different metastatic potentials, and a preparation method and application thereof.
  • the present invention provides a pair of lung cancer cell lines with the same genetic background and different metastatic potentials, and the lung cancer cell lines are human low-metastatic large cell lung cancer cell lines and human high-metastatic large cell lung cancer cell lines;
  • the human low-metastatic large-cell lung cancer cell line is the cell line ZQH-80 with the deposit number CGMCC No.2832 deposited by the General Microbiology Center of the China Microbial Culture Collection Management Committee;
  • the human high-metastatic large cell lung cancer cell line is the cell line ZQH-81 with the deposit number CGMCC No. 2833 deposited by the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the lung cancer cell line is obtained by screening the same human lung cancer cell line using a single-cell clone limiting dilution method
  • the human lung cancer cell line is a human large cell lung cancer cell line.
  • the in vitro proliferation ability, migration ability, invasion ability and clone formation ability of the human low-metastatic large cell lung cancer cell line ZQH-80 are lower than that of the human high-metastatic large cell lung cancer cell line ZQH-81;
  • the in vitro apoptosis level of the human high metastatic large cell lung cancer cell line ZQH-81 is lower than that of the human low metastatic large cell lung cancer cell line ZQH-80.
  • the allele of the lung cancer metastasis suppressor gene nm23 of the human low-metastatic large cell lung cancer cell line ZQH-80 has a normal structure and exists in a heterozygous form
  • the mRNA transcript expression and mRNA expression level of the lung cancer metastasis suppressor gene nm23 of the human low-metastatic large cell lung cancer cell line ZQH-80 are normal;
  • the protein expression level of the lung cancer metastasis suppressor gene nm23 of the human low-metastatic large cell lung cancer cell line ZQH-80 is normal;
  • the allele of the lung cancer metastasis suppressor gene nm23 of the human highly metastatic large cell lung cancer cell line ZQH-81 is a loss of heterozygosity
  • the mRNA transcript expression and mRNA expression of the lung cancer metastasis suppressor gene nm23 of the human highly metastatic large cell lung cancer cell line ZQH-81 are missing;
  • the protein expression of the lung cancer metastasis suppressor gene nm23 of the human high metastatic large cell lung cancer cell line ZQH-81 is missing.
  • the tumorigenicity of the transplanted tumor of the human low-metastatic large cell lung cancer cell line ZQH-80 is lower than that of the human high-metastatic large cell lung cancer cell line ZQH-81;
  • the tumorigenicity of the transplanted tumor is the tumorigenicity of the transplanted tumor in nude mice.
  • transplanted tumor of the human low-metastasis large cell lung cancer cell line ZQH-80 does not metastasize;
  • transplanted tumor of the human high-metastasis large cell lung cancer cell line ZQH-81 undergoes tissue and/or organ metastasis;
  • the metastasis is lymph node, brain, bone, and liver metastasis;
  • the metastasis is multiple mediastinal lymph node metastasis.
  • the mRNA expression level of MTS1 and KCTD4 is higher than that of human low-metastatic large cell lung cancer cell line ZQH-80;
  • the mRNA expression level of is lower than that of human low-metastatic large cell lung cancer cell line ZQH-80;
  • the present invention also provides a method for preparing the aforementioned lung cancer cell line, which includes the steps of: taking the same human lung cancer cell line and selecting it by single-cell clone limited dilution method;
  • the screening is to separately establish lung cancer transplanted tumor models in nude mice with the obtained cell lines cultured by single cell clones, and screen them by using animal in vivo imaging technology and pathological anatomy technology.
  • the present invention also provides a human high metastatic large cell lung cancer cell line, which is a cell line ZQH-81 with a deposit number of CGMCC No. 2833 deposited by the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the present invention also provides the use of the aforementioned pair of lung cancer cell lines with the same genetic background and different metastatic potential or the aforementioned human high-metastatic large cell lung cancer cell lines in preparing lung cancer models.
  • the present invention successfully obtains a pair of human large cells with high and low metastatic potential that have the same genetic background, different metastatic potentials, different cell biological behaviors, different molecular biological behaviors, different metastasis-related gene structures and functions, and different signal pathways.
  • Lung cancer cell line This pair of cell lines themselves can be used as cell models, or they can be transplanted into animals to construct animal lung cancer models for the research and screening of molecular targets of small molecule drugs against lung cancer invasion and metastasis, organ-specific metastasis, and development of treatment for lung cancer invasion and metastasis.
  • the small molecule drugs, vaccines, and antibody drugs of the company provide a technical platform for the study of lung cancer invasion and/or metastasis. It can also be used for gene target research of genes related to lung cancer invasion and metastasis.
  • Figure 1 shows the results of CCK8 comparing the in vitro proliferation ability of human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 2 shows the comparison of the migration ability of the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80 in vitro.
  • Figure 3 shows the comparison of the in vitro invasiveness of the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 4 shows the comparison results of in vitro clone formation ability between human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 5 shows the results of flow cytometry detecting the level of apoptosis between human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 6 shows the results of the TUNEL method detecting and comparing the level of apoptosis between human high-metastatic large cell lung cancer cell line ZQH-81 and human low-metastatic large cell lung cancer cell line ZQH-80.
  • Figure 7 shows the Southern blot detection results of nm23-H1 gene polymorphisms in human lung cancer cell lines ZQH-81 and ZQH-80 with different metastatic potential.
  • Figure 8 shows the comparison of nm23-H1 mRNA transcript expression between human lung cancer cell lines ZQH-81 and ZQH-80 with different metastatic potentials.
  • 1 is DNA Ladder Marker
  • 2 is ⁇ -actin
  • 3 is ZQH-81
  • 4 is ZQH-80
  • 5 is SpacA-1
  • 6 is A2.
  • Figure 9 shows the comparison of the protein expression of the lung cancer metastasis suppressor gene nm23-H1 between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 10 shows the comparison of the mRNA expression levels of the lung cancer metastasis suppressor gene nm23-H1 between human lung cancer cell lines ZQH-81 and ZQH-80 with different metastatic potential.
  • Figure 11 shows the results of animal live imaging comparing the distant metastasis of transplanted tumors in nude mice between human high-metastatic large cell lung cancer cell line ZQH-81 and human low-metastatic large cell lung cancer cell line ZQH-80; in the figure, A and ZQH-81 correspond to B is the two sets of tests of ZQH-81, respectively, and ZQH-80 corresponds to A and B are the two sets of tests of ZQH-80 respectively.
  • Figure 12 shows the results of pathological anatomy comparing the tumorigenicity of transplanted tumors in nude mice between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 13 is a cluster analysis diagram of differentially expressed genes between human high-metastatic large cell lung cancer cell line ZQH-81 and human low-metastatic large cell lung cancer cell line ZQH-80.
  • Figure 14 is a cluster analysis diagram of the differentially expressed miRNAs between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • the raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.
  • both the human low-metastasis large cell lung cancer cell line ZQH-80 and the human high-metastasis large cell lung cancer cell line ZQH-81 are prepared using a new single-cell limited dilution cloning technology.
  • Example 1 Preparation of human low-metastatic large cell lung cancer cell line ZQH-80 and human high-metastatic large cell lung cancer cell line ZQH-81
  • the cells of the clinically isolated lung cancer tissues were cloned by the limiting dilution method, and lung cancer cell lines with the same genetic background and different metastatic potential were screened.
  • the human low-metastatic large cell lung cancer cell line ZQH-80 was deposited by the General Microbiology Center of China Microbial Culture Collection Management Committee.
  • the deposit number was CGMCC No. 2832, and the classification was named: cell strain.
  • the human high metastatic large cell lung cancer cell line ZQH-81 was deposited by the General Microbiology Center of the China Microbial Culture Collection Management Committee. The deposit address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing. The deposit date is December 26, 2008 , The preservation number is CGMCC No. 2833, and the classification designation: cell strain.
  • the invention uses the CCK-8 method to detect and compare the characteristics and differences of the in vitro proliferation ability of the ZQH-80 and ZQH-81 lung cancer cell lines.
  • the experimental results show that the in vitro proliferation ability of the ZQH-81 lung cancer cell line is significantly higher than that of the ZQH-80 lung cancer cell line.
  • the present invention uses the scratch test method to detect and compare the characteristics and differences of the migration ability in vitro between the human high-metastasis large cell lung cancer cell line ZQH-81 and the human low-metastasis large cell lung cancer cell line ZQH-80.
  • the invention uses an in vitro invasion experiment (modified Boyden method) to detect and compare the characteristics and differences of the in vitro invasion ability of the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • Figure 3 shows that the in vitro invasion ability of the human high metastatic large cell lung cancer cell line ZQH-81 is significantly higher than that of the human low metastatic large cell lung cancer cell line ZQH-80(P ⁇ 0.01).
  • the invention uses the in vitro plate clone formation experiment to detect the characteristics and differences of the in vitro clone formation ability of the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • the present invention uses flow cytometry and TUNEL method to detect and compare the apoptosis characteristics and differences between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • the invention uses the Southern blot method to detect and compare the characteristics and differences of nm23-H1 allele polymorphisms between the human high-metastasis large cell lung cancer cell line ZQH-81 and the low-metastatic large cell lung cancer cell line ZQH-80.
  • nm23-H1 gene exists in the form of heterozygotes (4.6 kb or 2.4 kb and 2.2 kb) in the human low-metastatic large cell lung cancer cell line ZQH-80, but in the human high-metastatic large cell lung cancer cell line ZQH-80
  • the cell lung cancer cell line ZQH-81 lacks both 4.6kb and 2.4kb. Therefore, the nm23-H1 gene polymorphism of the human high metastatic large cell lung cancer cell line ZQH-81 is a loss of heterozygosity.
  • the experimental results indicate that the alleles of the lung cancer metastasis suppressor gene nm23 of the ZQH-80 lung cancer cell line are normal in structure and exist in heterozygous form; the lung cancer metastasis suppressor gene nm23-H1 allele polymorphism of the ZQH-81 lung cancer cell line is heterozygous. Lack of fit.
  • the invention uses Northern blot hybridization technology to detect and compare the expression characteristics and differences of the mRNA transcripts of the lung cancer metastasis suppressor gene nm23-H1 between the human high-metastasis large cell lung cancer cell line ZQH-81 and the human low-metastasis large cell lung cancer cell line ZQH-80.
  • the experimental results show that the expression of the mRNA transcript of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-80 lung cancer cell line is normal; the expression of the mRNA transcript of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-81 lung cancer cell line is missing.
  • the present invention uses Western blot to detect and compare the characteristics and differences of the protein expression level of the lung cancer metastasis suppressor gene nm23-H1 between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • the present invention uses RT-PCR to detect and compare the characteristics and differences of the mRNA expression level of the lung cancer metastasis suppressor gene nm23-H1 between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • the RT-PCR results showed ( Figure 10): The mRNA expression level of the lung cancer metastasis suppressor gene nm23-H1 in the human high metastatic large cell lung cancer cell line ZQH-81 was significantly lower than that in the human low metastatic large cell lung cancer cell line ZQH-80. The mRNA expression level of the suppressor gene nm23-H1 (P ⁇ 0.01).
  • the experimental results show that the mRNA expression level of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-80 lung cancer cell line is significantly higher than the mRNA expression level of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-81 lung cancer cell line.
  • the present invention applies ZQH-81 and ZQH-80 lung cancer cell lines to inoculate nude mice, establishes a nude mouse lung cancer transplantation tumor model, uses animal live imaging technology and pathological anatomy technology to study and compare human high metastatic large cell lung cancer cell line ZQH-81 with human Low metastatic large cell lung cancer cell line ZQH-80 tumorigenesis, number of distant metastatic tumor lesions and metastasis rate.
  • the experimental results indicate that the transplanted tumors of the ZQH-80 lung cancer cell line in nude mice do not have distant metastasis; the transplanted tumors of the ZQH-81 lung cancer cell line in nude mice have extensive metastasis to lymph nodes, brain, bone, liver and other organs.
  • the invention uses gene expression profile chip and RT-PCR technology to detect and screen the differentially expressed genes related to lung cancer invasion and metastasis between human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
  • the invention uses the mi-RNA chip to detect and screen the differentially expressed mi-RNAs related to lung cancer invasion and metastasis between the human high metastatic large cell lung cancer cell line ZQH-81 and the human low metastatic large cell lung cancer cell line ZQH-80.
  • the mi-RNA chip results show ( Figure 14): Compared with the human large cell lung cancer cell line ZQH-80 with low metastatic potential, the miRNAs related to lung cancer invasion and metastasis in the human high metastatic large cell lung cancer cell line ZQH-81: hsa-let- 7e, hsa-miR-1185, hsa-miR-137, has-miR-148a, hsa-miR-181a-2, hsa-miR-194, hsa-miR-449, hsa-miR-299-3p, hsa- miR-301b, hsa-miR-30a, hsa-miR-363, hsa-miR-369-3p, hsa-miR-376a, hsa-miR-377, hsa-miR-505, hsa-miR-574-3p, The expression levels of hsa
  • the human low-metastatic large cell lung cancer cell line ZQH-80 and the human high-metastatic large cell lung cancer cell line ZQH-81 prepared by the present invention are a pair with the same genetic background, different metastatic potential, different cell biological behavior, Two human large cell lung cancer cell lines with different molecular biological behaviors, different structures and functions of metastasis-related genes, and different signal pathways, with high and low metastatic potential, and natural contrast.
  • the present invention has established a pair of human lung cancer cell lines with the same genetic background but completely different metastatic potentials.
  • the pair of cell lines can be used as cell models themselves, or they can be implanted into the body to be constructed as animal models. Testing and screening drugs to confirm whether the drugs can effectively inhibit the metastasis of lung cancer cells.
  • the human low-metastatic large cell lung cancer cell line ZQH-80 and the human high-metastatic large cell lung cancer cell line ZQH-81 of Example 1 were taken as a set of cell models.
  • ZQH-81 and ZQH-80 lung cancer cell lines were respectively inoculated into nude mice to establish nude mice lung cancer transplantation tumor models, and a set of animal lung cancer models were obtained.
  • the present invention has successfully obtained a pair of human high and low metastases with the same genetic background, different metastatic potentials, different cell biological behaviors, different molecular biological behaviors, different metastasis-related gene structures and functions, and different signal pathways.
  • Potential large cell lung cancer cell line This pair of cell lines can be used as cell models themselves, or they can be implanted into the body to construct animal lung cancer models, used for detection and screening of drugs, and provide a technical platform for the study of lung cancer invasion and/or metastasis. It can also be used for gene target research of genes related to lung cancer invasion and metastasis.

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Abstract

一对具有相同遗传背景和不同转移潜能的肺癌细胞株及其制备方法和用途。所述肺癌细胞株为人低转移大细胞肺癌细胞株和人高转移大细胞肺癌细胞株；它们分别是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2832的细胞株ZQH-80和保藏编号为CGMCC No.2833的细胞株ZQH-81。该具有相同遗传背景、不同转移潜能的肺癌细胞株可以作为细胞模型，也可以移植入动物体内构建成为动物肺癌模型，用于研究筛选抗肺癌侵袭转移、器官特异性转移的小分子药物的分子靶点，开发治肺癌侵袭转移相关的小分子药物、疫苗、抗体药物，为研究肺癌侵袭和/或转移提供技术平台。还可以用于与肺癌侵袭转移相关基因的基因靶点研究。

Description

一对具有相同遗传背景和不同转移潜能的肺癌细胞株及其制备方法和用途 技术领域
本发明属于生物技术领域，具体涉及一对具有相同遗传背景和不同转移潜能的肺癌细胞株及其制备方法和用途。
背景技术
肺癌是发病率和死亡率增长最快，对人群健康和生命威胁最大的肿瘤之一。根据2020年的最新报道显示：男性中，肺癌是发病率和死亡率均例第一的恶性肿瘤；在女性中，肺癌是发病率例第二，死亡率例第一的恶性肿瘤。肺癌是全球死亡率最高的肿瘤，约占全球癌症总死亡人数的四分之一(22.4％)。肺癌按组织学类型分为小细胞肺癌(Small cell lung cancer，SCLC)和非小细胞肺癌(Non-small cell lung cancer，NSCLC)，NSCLC占全部肺癌的86％。约占70％的NSCLC患者在初诊断时已为晚期阶段，其5年存活率4％-6％。
近年来，随着外科手术治疗、全身化疗、放射治疗、靶向治疗、免疫治疗的进步及发展，使肺癌患者的治疗疗效及生存时间在一定程度上得到了改善，但肺癌患者的长期预后仍十分不良，总的5年生存率仅17％左右。导致肺癌患者预后不良的主要原因是约35％左右的肺癌患者在就诊时就已有远处转移，约50％-60％的肺癌患者在治疗过程中发生远处转移，最终有85％-90％的肺癌患者死亡是由于肿瘤转移所致。因此，研究和阐明肺癌转移发生的分子机制，寻找早期诊断肺癌转移的新技术、新方法，研发抑制或/和逆转肺癌转移的分子药物是目前和未来肺癌研究领域的方向及前沿课题。
肺癌侵袭和转移是肺癌的恶性标志和生物学特征，也是导致患者治疗失败和死亡的主要原因。目前，研究肺癌转移的最大难题是：缺乏具有相同遗传背景，不同转移潜能的天然对比研究生物材料。如果能够构建一对具有相同遗传背景，不同转移潜能的肺癌细胞株，将对于肺癌转移的研究提供极大帮助。
发明内容
本发明的目的是提供一对具有相同遗传背景和不同转移潜能的肺癌细胞株及其制备方法和用途。
本发明提供了一对具有相同遗传背景和不同转移潜能的肺癌细胞株，所述肺癌细胞株为人低转移大细胞肺癌细胞株和人高转移大细胞肺癌细胞株；
所述人低转移大细胞肺癌细胞株是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2832的细胞株ZQH-80；
所述人高转移大细胞肺癌细胞株是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2833的细胞株ZQH-81。
进一步地，所述肺癌细胞株由同一株人肺癌细胞株，采用单细胞克隆有限稀释法筛选而得；
优选地，所述人肺癌细胞株为人大细胞肺癌细胞株。
进一步地，所述人低转移大细胞肺癌细胞株ZQH-80的体外增殖能力、迁移能力、侵袭能力和克隆形成能力均低于人高转移大细胞肺癌细胞株ZQH-81；
所述人高转移大细胞肺癌细胞株ZQH-81的体外凋亡水平低于人低转移大细胞肺癌细胞株ZQH-80。
进一步地，所述人低转移大细胞肺癌细胞株ZQH-80的肺癌转移抑制基因nm23等位基因结构正常，为杂合子形式存在；
所述人低转移大细胞肺癌细胞株ZQH-80的肺癌转移抑制基因nm23的mRNA转录本表达和mRNA表达水平正常；
所述人低转移大细胞肺癌细胞株ZQH-80的肺癌转移抑制基因nm23的蛋白表达水平正常；
所述人高转移大细胞肺癌细胞株ZQH-81的肺癌转移抑制基因nm23等位基因为杂合性缺失；
所述人高转移大细胞肺癌细胞株ZQH-81的肺癌转移抑制基因nm23的mRNA转录本表达和mRNA表达缺失；
所述人高转移大细胞肺癌细胞株ZQH-81的肺癌转移抑制基因nm23的蛋白表达缺失。
进一步地，所述人低转移大细胞肺癌细胞株ZQH-80的移植瘤成瘤性低于人高转移大细胞肺癌细胞株ZQH-81的移植瘤成瘤性；
优选地，所述移植瘤成瘤性为裸鼠移植瘤成瘤性。
进一步地，所述人低转移大细胞肺癌细胞株ZQH-80移植瘤不发生远处转移；所述人高转移大细胞肺癌细胞株ZQH-81移植瘤发生组织和/或器官转移；
优选地，所述转移为淋巴结、脑、骨、肝脏的转移；
更优选地，所述转移为多发性纵膈淋巴结转移。
进一步地，所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的基因caldesmon-1、WNT5A、PKP2、NNMT、GLB1L3、PTPRG、SLC2A13、REPS2、GRAMD3、PRICKLE1、CXCR4、PLAU、MTS1和KCTD4的mRNA表达水平高于人低转移大细胞肺癌细胞株ZQH-80；
所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的基因MGST1、PRG1、SMCY、ACPP、ZFAND6、CYorf15B、RPS4Y1、DDX3Y、 SERPINB2、HGF、MMP2、MMP-14、MMP16和TIMP2的mRNA表达水平低于人低转移大细胞肺癌细胞株ZQH-80；
和/或，
所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的miRNAs hsa-let-7e、hsa-miR-1185、hsa-miR-137、has-miR-148a、hsa-miR-181a-2、hsa-miR-194、hsa-miR-449、hsa-miR-299-3p、hsa-miR-301b、hsa-miR-30a、hsa-miR-363、hsa-miR-369-3p、hsa-miR-376a、hsa-miR-376a、hsa-miR-377、hsa-miR-505、hsa-miR-574-3p、hsa-miR-584、hsa-miR-629、hsa-miR-629、hsa-miR-654-5p、hsa-miR-767-5p、hsa-miR-92a-1的表达水平高于人低转移大细胞肺癌细胞株ZQH-80；
所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的miRNAs hsa-miR-212、hsa-miR-892b、hsa-miR-630、hsa-miR-548d-5p、hsa-miR-500、hsa-miR-486-5p、hsa-miR-1207-5p、hsa-miR-1224-5p、hsa-miR-1288、hsa-miR-129-5p、hsa-miR-132、hsa-miR-135a、hsa-miR-150、hsa-miR-188-5p、hsa-miR-210、hsa-miR-338-3p、hsa-miR-339-5p、hsa-miR-34a、hsa-miR-449a的表达水平低于人低转移大细胞肺癌细胞株ZQH-80。
本发明还提供了一种制备前述的肺癌细胞株的方法，它包括如下步骤：取同一株人肺癌细胞株，采用单细胞克隆有限稀释法筛选而得；
优选地，所述筛选为将获得的单个细胞克隆培养的细胞株分别建立肺癌裸鼠移植瘤模型，应用动物活体成像技术和病理解剖技术筛选。
本发明还提供了一种人高转移大细胞肺癌细胞株，它是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2833的细胞株ZQH-81。
本发明还提供了前述的一对具有相同遗传背景和不同转移潜能的肺癌细胞株或前述的人高转移大细胞肺癌细胞株在制备肺癌模型中的用途。
本发明成功获得一对具有相同遗传背景、不同转移潜能、不同细胞生物学行为、不同分子生物学行为、转移相关基因结构和功能不同、信号通路传导不同的两株人高、低转移潜能大细胞肺癌细胞株。这对细胞株本身可以作为细胞模型，也可以移植入动物体内构建成为动物肺癌模型，用于研究筛选抗肺癌侵袭转移、器官特异性转移的小分子药物的分子靶点，开发治肺癌侵袭转移相关的小分子药物、疫苗、抗体药物，为研究肺癌侵袭和/或转移提供技术平台。还可以用于与肺癌侵袭转移相关基因的基因靶点研究。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外增殖能力比较的CCK8结果。
图2为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外迁移能力比较结果。
图3为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外侵袭能力比较结果。
图4为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外克隆形成能力比较结果。
图5为流式细胞术检测人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间细胞凋亡水平结果。
图6为TUNEL法检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间细胞凋亡水平结果。
图7为不同转移潜能人肺癌细胞株ZQH-81和ZQH-80中nm23-H1基因多态性Southern印记检测结果。
图8为不同转移潜能人肺癌细胞株ZQH-81和ZQH-80间nm23-H1基因mRNA转录本表达比较结果，图中：1为DNA Ladder Marker；2为β-actin；3为ZQH-81；4为ZQH-80；5为SpacA-1；6为A2。
图9为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间肺癌转移抑制基因nm23-H1的蛋白表达比较结果。
图10为不同转移潜能人肺癌细胞株ZQH-81和ZQH-80间肺癌转移抑制基因nm23-H1的mRNA表达水平比较结果。
图11为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间裸鼠移植瘤远处转移比较的动物活体成像结果；图中，ZQH-81对应的A和B分别为ZQH-81的两组试验，ZQH-80对应的A和B分别ZQH-80的两组试验。
图12为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间裸鼠移植瘤成瘤性比较的病理解剖结果。
图13为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间差异表达基因聚类分析图。
图14为人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间差异表达miRNAs聚类分析图。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品，通过购买市售产品获得。
本发明中，人低转移大细胞肺癌细胞株ZQH-80和人高转移大细胞肺癌细胞株ZQH-81均应用单细胞有限稀释克隆新技术制备。
实施例1、人低转移大细胞肺癌细胞株ZQH-80和人高转移大细胞肺癌细胞株ZQH-81的制备
采用有限稀释法对该临床分离的肺癌组织的细胞进行克隆化，筛选具有相同遗传背景、不同转移潜能的肺癌细胞株。
(1)取临床分离的肺癌组织，制备肺癌细胞悬液，进行活细胞计数，按每毫升液体100个细胞进行稀释，再用1ml注射器吸取细胞悬液，在振摇注射器时，将细胞悬液滴入96孔板培养皿，每孔只滴一滴细胞悬液，静置5min后在倒置显微镜下观察，经3人共同确认该孔内仅有一个肺癌细胞后，即对该孔添加培养液(RPMI1640+30％小牛血清，pH6.5)，在37℃，5％CO 2培养箱内孵育。待见该孔细胞生长增殖并将培养板孔底大部分铺盖时，即用吸管反复吹洗，使细胞悬浮，将之吸取再移入培养瓶内培养，冻存；
(2)经过反复筛选，共获得124个来自单个细胞克隆培养的细胞株；
(3)分别将上述124个来自单个细胞克隆培养的细胞株，分别建立肺癌裸鼠移植瘤模型；
(4)反复应用动物活体成像技术和病理解剖技术，筛选到一株肺癌细胞株只在裸鼠体内成瘤，但不发生远处转移，另外一株肺癌细胞株不但在裸鼠体内成瘤，而且发生远处多器官、多病灶转移。将只在裸鼠体内成瘤，不发生远处转移的肺癌细胞株命名为：ZQH-80，将不但在裸鼠体内成瘤，而且发生远处多器官、多病灶转移的肺癌细胞株命名为：ZQH-81。
通过前述方法，制备得到了一对具有相同遗传背景、不同转移潜能的的人大学细胞肺癌细胞株。
其中，人低转移大细胞肺癌细胞株ZQH-80由中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏地址：北京市朝阳区大屯路，中国科学院微生物研究所，保藏日期为2008年12月26日，保藏编号为CGMCC No.2832，分类命名：细胞株。
人高转移大细胞肺癌细胞株ZQH-81由中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏地址：北京市朝阳区大屯路，中国科学院微生物研究所，保藏日期为2008年12月26日，保藏编号为CGMCC No.2833，分类命名：细胞株。
实施例2、ZQH-80肺癌细胞株和ZQH-81肺癌细胞株的特性比较实验
一、体外增殖能力
1、实验方法
本发明应用CCK-8法检测和比较ZQH-80和ZQH-81肺癌细胞株体外增殖能力特点及差异。
2、实验结果
CKK-8实验结果如图1所示，图1显示：人高转移大细胞肺癌细胞株ZQH-81的第1、2、3，4和5天的OD值均显著高于人低转移大细胞肺癌细胞株ZQH-80(P＜0.001)；第6天，人高转移大细胞肺癌细胞株ZQH-81的OD值与人低转移大细胞肺癌细胞株ZQH-80间比较无显著性差异(P＞0.05)。
实验结果说明：ZQH-81肺癌细胞株体外增殖能力显著高于ZQH-80肺癌细胞株。
二、体外迁移能力
1、实验方法
本发明应用划痕实验法检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外迁移能力的特点及差异。
2、实验结果
划痕实验结果如图2所示，图2显示：人高转移大细胞肺癌细胞株ZQH-81的体外迁移能力显著高于人低转移大细胞肺癌细胞株ZQH-80(P＜0.01)。
三、体外侵袭能力
1、实验方法
本发明应用体外侵袭实验(改良Boyden法)检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外侵袭能力特点及差异。
2、实验结果
体外侵袭实验(改良Boyden法)结果如图3所示，图3显示：人高转移大细胞肺癌细胞株ZQH-81的体外侵袭能力显著高于人低转移大细胞肺癌细胞株ZQH-80(P＜0.01)。
四、体外克隆形成能力
1、实验方法
本发明应用体外平板克隆形成实验检测人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80体外克隆形成能力特点及差异。
2、实验结果
体外平板克隆形成实验结果如图4所示，图4显示：人高转移大细胞肺癌细胞株ZQH-81体外克隆形成能力显著高于人低转移大细胞肺癌细胞株ZQH-80(P＜0.01)。
五、体外凋亡水平
1、实验方法
本发明应用流式细胞术和TUNEL法检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间细胞凋亡特点及差异。
2、实验结果
流式细胞术(图5)和TUNEL法(图6)检测结果显示：人高转移大细胞肺癌细胞株ZQH-81细胞凋亡水平显著低于人低转移大细胞肺癌细胞株ZQH-80的细胞凋亡水平(P＜0.01，P＜0.01)。
六、肺癌转移抑制基因nm23-H1等位基因多态性
1、实验方法
本发明应用Southern印记法检测和比较人高转移大细胞肺癌细胞株ZQH-81与低转移大细胞肺癌细胞株ZQH-80间nm23-H1等位基因多态性特点及差异。
2、实验结果
Southern印记法实验结果显示(图7)：nm23-H1基因在人低转移大细胞肺癌细胞株ZQH-80中以杂合子(4.6kb或2.4kb及2.2kb)形式存在，而在人高转移大细胞肺癌细胞株ZQH-81中4.6kb和2.4kb均缺少。因此，人高转移大细胞肺癌细胞株ZQH-81的nm23-H1基因多态性为杂合性缺失。
实验结果说明：ZQH-80肺癌细胞株的肺癌转移抑制基因nm23等位基因结构正常，为杂合子形式存在；ZQH-81肺癌细胞株的肺癌转移抑制基因nm23-H1等位基因多态性为杂合性缺失。
七、肺癌转移抑制基因nm23-H1的mRNA转录本表达
1、实验方法
本发明应用Northern印记杂交技术检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间肺癌转移抑制基因nm23-H1的mRNA转录本表达特点及差异。
2、实验结果
Northern印记杂交结果显示(图8)：人高转移大细胞肺癌细胞株ZQH-81中肺癌转移抑制基因nm23-H1的mRNA转录本表达缺失，而人低转移大细胞肺癌细胞株ZQH-80，人肺腺癌细胞SpacA-1和人肺腺癌细胞A2中肺癌转移抑制基因nm23-H1的mRNA转录本表达正常。
实验结果说明：ZQH-80肺癌细胞株的肺癌转移抑制基因nm23-H1的 mRNA转录本表达正常；ZQH-81肺癌细胞株的肺癌转移抑制基因nm23-H1的mRNA转录本表达缺失。
八、肺癌转移抑制基因nm23-H1的蛋白表达
1、实验方法
本发明应用Western blot检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间肺癌转移抑制基因nm23-H1的蛋白表达水平特点及差异。
2、实验结果
Western blot实验结果显示(图9)：人高转移大细胞肺癌细胞株ZQH-81中肺癌转移抑制基因nm23-H1的蛋白表达缺失，而人低转移大细胞肺癌细胞株中肺癌转移抑制基因nm23-H1的蛋白表达正常。
九、肺癌转移抑制基因nm23-H1的mRNA表达
1、实验方法
本发明应用RT-PCR检测和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间肺癌转移抑制基因nm23-H1的mRNA表达水平特点及差异。
2、实验结果
RT-PCR结果显示(图10)：人高转移大细胞肺癌细胞株ZQH-81中肺癌转移抑制基因nm23-H1的mRNA表达水平显著低于人低转移大细胞肺癌细胞株ZQH-80中肺癌转移抑制基因nm23-H1的mRNA表达水平(P＜0.01)。
实验结果说明：ZQH-80肺癌细胞株的肺癌转移抑制基因nm23-H1的mRNA表达水平显著高于ZQH-81肺癌细胞株的肺癌转移抑制基因nm23-H1的mRNA表达水平。
十、裸鼠移植瘤成瘤与转移
1、实验方法
本发明应用ZQH-81和ZQH-80肺癌细胞株接种裸鼠，建立裸鼠肺癌移植瘤模型，应用动物活体成像技术和病理解剖技术研究和比较人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间移植瘤成瘤性、远处转移瘤病灶数及转移率。
2、实验结果
移植瘤裸鼠活体成像结果显示(图11)：第9周时人高转移大细胞肺癌细胞株ZQH-81活体成像荧光强度显著高于人低转移大细胞肺癌细胞株ZQH-80；活体成像结果亦显示人高转移大细胞肺癌细胞株ZQH-81组裸鼠发生全身多器官、多部位，多发性广泛肿瘤转移；而人低转移大细胞肺癌细胞株ZQH-80组没有发生远处转移。
病理解剖结果显示(图12)：第9周时人高转移大细胞肺癌细胞株ZQH-81移植瘤体积和肿瘤均显著高于人低转移大细胞肺癌细胞株ZQH-80，病理解剖还发现人高转移大细胞肺癌细胞株ZQH-81组移植瘤发生肺部、纵膈淋巴结、骨和大脑的多发性、广泛转移，远处器官转移率为100％；而人低转移大细胞肺癌细胞株ZQH-80组的肺部、纵膈淋巴结、骨和大脑均没有发现肿瘤转移，远处器官转移率为0％。
实验结果说明：ZQH-80肺癌细胞株裸鼠移植瘤不发生远处转移；ZQH-81肺癌细胞株裸鼠移植瘤发生淋巴结、脑、骨、肝脏等器官广泛转移。
十一、与肺癌侵袭转移相关的基因
1、实验方法
本发明应用基因表达谱芯片和RT-PCR技术检测、筛选人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间与肺癌侵袭转移相关的差异表达基因。
2、实验结果
基因表达谱芯片和RT-PCR实验结果显示(图13)：与人低转移潜能大细胞肺癌细胞株ZQH-80比较，人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的基因caldesmon-1、WNT5A、PKP2、NNMT、GLB1L3、PTPRG、SLC2A13、REPS2、GRAMD3、PRICKLE1、CXCR4、PLAU、MTS1和KCTD4基因mRNA表达水平均显著上调(P＜0.01)；而MGST1、PRG1、SMCY、ACPP、ZFAND6、CYorf15B、RPS4Y1、DDX3Y、SERPINB2、HGF、MMP2、MMP-14、MMP16和TIMP2基因mRNA表达水平则均显著下调(P＜0.01)。
十二、与肺癌侵袭转移相关的miRNAs
1、实验方法
本发明应用mi-RNA芯片检测和筛选人高转移大细胞肺癌细胞株ZQH-81与人低转移大细胞肺癌细胞株ZQH-80间与肺癌侵袭转移相关的差异表达mi-RNAs。
2、实验结果
mi-RNA芯片结果显示(图14)：与人低转移潜能大细胞肺癌细胞株ZQH-80比较，人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的miRNAs：hsa-let-7e、hsa-miR-1185、hsa-miR-137、has-miR-148a、hsa-miR-181a-2、hsa-miR-194、hsa-miR-449、hsa-miR-299-3p、hsa-miR-301b、hsa-miR-30a、hsa-miR-363、hsa-miR-369-3p、hsa-miR-376a、hsa-miR-377、hsa-miR-505、hsa-miR-574-3p、hsa-miR-584、hsa-miR-629、hsa-miR-654-5p、hsa-miR-767-5p、hsa-miR-92a-1表达水平显著上调(P＜0.01)；而hsa-miR-212、 hsa-miR-892b、hsa-miR-630、hsa-miR-548d-5p、hsa-miR-500、hsa-miR-486-5p、hsa-miR-1207-5p、hsa-miR-1224-5p、hsa-miR-1288、hsa-miR-129-5p、hsa-miR-132、hsa-miR-135a、hsa-miR-150、hsa-miR-188-5p、hsa-miR-210、hsa-miR-338-3p、hsa-miR-339-5p、hsa-miR-34a、hsa-miR-449a表达水平则显著下调(P＜0.01)。
由上述实施例可知本发明制备的人低转移大细胞肺癌细胞株ZQH-80和人高转移大细胞肺癌细胞株ZQH-81是一对具有相同遗传背景、不同转移潜能、不同细胞生物学行为、不同分子生物学行为、转移相关基因结构和功能不同、信号通路传导不同的两株人高、低转移潜能，具有天然对比作用的人大细胞肺癌细胞株。
根据前述结果可以知道，本发明建立了一对具有相同遗传背景、但是转移潜能完全不同的人肺癌细胞株，这对细胞株本身可以作为细胞模型，也可以植入体内构建成为动物模型，用于检测、筛选药物，确认药物是否可以有效抑制肺癌细胞的转移。
实施例3、人低转移大细胞肺癌细胞株ZQH-80和人高转移大细胞肺癌细胞株ZQH-81的应用
一、作为细胞模型
1、建立细胞模型
取实施例1的人低转移大细胞肺癌细胞株ZQH-80、人高转移大细胞肺癌细胞株ZQH-81，作为一组细胞模型。
2、筛选药物
a、取步骤1建立的肺癌模型；
b、取候选药物，分别作用于前述一对肺癌模型；
c、观察候选药物对肺癌以及肺癌转移的量化指标的影响情况，并进行评分，评价潜在治疗肺癌以寄抑制肺癌转移的药物。
二、构建动物模型
1、构建动物肺癌模型
分别将ZQH-81和ZQH-80肺癌细胞株接种裸鼠，建立裸鼠肺癌移植瘤模型，得到一组动物肺癌模型。
2、筛选药物
a、取步骤1建立的动物肺癌模型；
b、取候选药物，分别作用于前述一对动物肺癌模型；
c、观察候选药物对肺癌以及肺癌转移的量化指标的影响情况，并进行评分，评价潜在治疗肺癌以寄抑制肺癌转移的药物。
综上，本发明成功获得一对具有相同遗传背景、不同转移潜能、不同细胞生物学行为、不同分子生物学行为、转移相关基因结构和功能不同、信号通路传导不同的两株人高、低转移潜能大细胞肺癌细胞株。这对细胞株本身可以作为细胞模型，也可以植入体内构建成为动物肺癌模型，用于检测、筛选药物，为研究肺癌侵袭和/或转移提供技术平台。还可以用于与肺癌侵袭转移相关基因的基因靶点研究。

Claims (10)

  1. 一对具有相同遗传背景和不同转移潜能的肺癌细胞株，其特征在于：所述肺癌细胞株为人低转移大细胞肺癌细胞株和人高转移大细胞肺癌细胞株；
    所述人低转移大细胞肺癌细胞株是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2832的细胞株ZQH-80；
    所述人高转移大细胞肺癌细胞株是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2833的细胞株ZQH-81。
  2. 根据权利要求1所述的肺癌细胞株，其特征在于：所述肺癌细胞株由同一株人肺癌细胞株，采用单细胞克隆有限稀释法筛选而得；
    优选地，所述人肺癌细胞株为人大细胞肺癌细胞株。
  3. 根据权利要求1或2所述的肺癌细胞株，其特征在于：所述人低转移大细胞肺癌细胞株ZQH-80的体外增殖能力、迁移能力、侵袭能力和克隆形成能力均低于人高转移大细胞肺癌细胞株ZQH-81；
    所述人高转移大细胞肺癌细胞株ZQH-81的体外凋亡水平低于人低转移大细胞肺癌细胞株ZQH-80。
  4. 根据权利要求1或2所述的肺癌细胞株，其特征在于：所述人低转移大细胞肺癌细胞株ZQH-80的肺癌转移抑制基因nm23等位基因结构正常，为杂合子形式存在；
    所述人低转移大细胞肺癌细胞株ZQH-80的肺癌转移抑制基因nm23的mRNA转录本表达和mRNA表达水平正常；
    所述人低转移大细胞肺癌细胞株ZQH-80的肺癌转移抑制基因nm23的蛋白表达水平正常；
    所述人高转移大细胞肺癌细胞株ZQH-81的肺癌转移抑制基因nm23等位基因为杂合性缺失；
    所述人高转移大细胞肺癌细胞株ZQH-81的肺癌转移抑制基因nm23的mRNA转录本表达和mRNA表达缺失；
    所述人高转移大细胞肺癌细胞株ZQH-81的肺癌转移抑制基因nm23的蛋白表达缺失。
  5. 根据权利要求1或2所述的肺癌细胞株，其特征在于：所述人低转移大细胞肺癌细胞株ZQH-80的移植瘤成瘤性低于人高转移大细胞肺癌细胞株ZQH-81的移植瘤成瘤性；
    优选地，所述移植瘤成瘤性为裸鼠移植瘤成瘤性。
  6. 根据权利要求1或2所述的肺癌细胞株，其特征在于：所述人低转移大细胞肺癌细胞株ZQH-80移植瘤不发生远处转移；所述人高转移大细胞肺癌细胞株ZQH-81移植瘤发生组织和/或器官转移；
    优选地，所述转移为淋巴结、脑、骨、肝脏的转移；
    更优选地，所述转移为多发性纵膈淋巴结转移。
  7. 根据权利要求1或2所述的肺癌细胞株，其特征在于：所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的基因caldesmon-1、WNT5A、PKP2、NNMT、GLB1L3、PTPRG、SLC2A13、REPS2、GRAMD3、PRICKLE1、CXCR4、PLAU、MTS1和KCTD4的mRNA表达水平高于人低转移大细胞肺癌细胞株ZQH-80；
    所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的基因MGST1、PRG1、SMCY、ACPP、ZFAND6、CYorf15B、RPS4Y1、DDX3Y、SERPINB2、HGF、MMP2、MMP-14、MMP16和TIMP2的mRNA表达水平低于人低转移大细胞肺癌细胞株ZQH-80；
    和/或，
    所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的miRNAs hsa-let-7e、hsa-miR-1185、hsa-miR-137、has-miR-148a、hsa-miR-181a-2、hsa-miR-194、hsa-miR-449、hsa-miR-299-3p、hsa-miR-301b、hsa-miR-30a、hsa-miR-363、hsa-miR-369-3p、hsa-miR-376a、hsa-miR-376a、hsa-miR-377、hsa-miR-505、hsa-miR-574-3p、hsa-miR-584、hsa-miR-629、hsa-miR-629、hsa-miR-654-5p、hsa-miR-767-5p、hsa-miR-92a-1的表达水平高于人低转移大细胞肺癌细胞株ZQH-80；
    所述人高转移大细胞肺癌细胞株ZQH-81中与肺癌侵袭转移相关的miRNAs hsa-miR-212、hsa-miR-892b、hsa-miR-630、hsa-miR-548d-5p、hsa-miR-500、hsa-miR-486-5p、hsa-miR-1207-5p、hsa-miR-1224-5p、hsa-miR-1288、hsa-miR-129-5p、hsa-miR-132、hsa-miR-135a、hsa-miR-150、hsa-miR-188-5p、hsa-miR-210、hsa-miR-338-3p、hsa-miR-339-5p、hsa-miR-34a、hsa-miR-449a的表达水平低于人低转移大细胞肺癌细胞株ZQH-80。
  8. 一种制备权利要求1～7任一项所述的肺癌细胞株的方法，其特征在于：它包括如下步骤：取同一株人肺癌细胞株，采用单细胞克隆有限稀释法筛选而得；
    优选地，所述筛选为将获得的单个细胞克隆培养的细胞株分别建立肺癌裸鼠移植瘤模型，应用动物活体成像技术和病理解剖技术筛选。
  9. 一种人高转移大细胞肺癌细胞株，其特征在于：它是由中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCC No.2833的细胞株ZQH-81。
  10. 权利要求1～7任一项所述的肺癌细胞株或权利要求9所述的肺癌细胞株在制备肺癌模型中的用途。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115197915A (zh) * 2022-08-01 2022-10-18 首都医科大学附属北京胸科医院 恶性表型大细胞肺癌细胞株及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103131669A (zh) * 2011-11-26 2013-06-05 上海市第六人民医院 具有高骨转移潜能的人大细胞肺癌细胞株
CN111187753A (zh) * 2020-01-16 2020-05-22 杭州市第一人民医院 一种高潜能脑转移肺癌细胞株及其应用、构建方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010011281A2 (en) * 2008-07-22 2010-01-28 The Research Foundation Of State University Of New York Methods and compositions for the diagnosis and treatment of cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103131669A (zh) * 2011-11-26 2013-06-05 上海市第六人民医院 具有高骨转移潜能的人大细胞肺癌细胞株
CN111187753A (zh) * 2020-01-16 2020-05-22 杭州市第一人民医院 一种高潜能脑转移肺癌细胞株及其应用、构建方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
LI CHUN-YANG, TAN LI, ZHANG GUO-JING, LI PING, TONG CHANG, FAN JING, ZHU YUN-SONG: "Transcriptional Regulation of Urokinase Receptor in High- (95D) and Low-metastatic (95C) Human Lung Cancer Cells", ACTA BIOCHIMICA ET BIOPHYSICA SINICA, vol. 36, no. 6, 1 June 2004 (2004-06-01), pages 405 - 411, XP055879400, DOI: 10.1093/abbs/36.6.405 *
LIU XU, TANG YALI, LI DIANJUN, WANG WURU: "Establishment of An in Vivo Brain, Bone and Muscle Metastasis Model of Human Lung Adenocarcinoma Cells in Nude Mice", CHINESE JOURNAL OF LABORATORY ANIMAL SCIENCE, vol. 4, no. 1, 31 March 1994 (1994-03-31), XP055879388 *
LU, YINGLIN ET AL.: "Spontaneous Metastatic Properties on Human Giant Cell Carcinoma of the Lung PLA-801 Maternal Cell Line and its Cloned Cell Lines (A, C, D, E) after Subcutaneous Implantation in Nude Mice", ZHONGHUA ZHONGLIU ZAZHI - CHINESE JOURNAL OF ONCOLOGY, vol. 11, no. 1, 31 December 1989 (1989-12-31), CN , pages 3 - 7, XP009532731, ISSN: 0253-3766 *
WU WEIXIN, WANG GUAN-JUN, CHEN XIAO, ET AL.: "Restudy the in vivo spontaneous metastasis of human pulmonary giant cell carcinoma cell sub lines PLA-801C and PLA-801D", CHINESE JOURNAL OF LABORATORY DIAGNOSIS / ZHONG GUO SHI YAN ZHEN DUAN XUE, vol. 9, no. 2, 30 April 2005 (2005-04-30), pages 194 - 197, XP055879390, ISSN: 1007-4287 *
ZHU WEIYONG, ZHENG JIE;FANG WEIGANG, ET AL : "Isolation and Characterization of Human Lung Cancer Cell Subline with Different Metastatic Petential", CHINESE JOURNAL OF PATHOLOGY, vol. 24, no. 3, 30 June 1995 (1995-06-30), pages 136 - 138, XP055879385 *

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