CN113774024A - A pair of lung cancer cell strains with same genetic background and different metastasis potentials, and preparation method and application thereof - Google Patents
A pair of lung cancer cell strains with same genetic background and different metastasis potentials, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a pair of lung cancer cell strains with the same genetic background and different metastasis potentials, a preparation method and application thereof, and belongs to the technical field of biology. The lung cancer cell strain is a human low-metastasis large-cell lung cancer cell strain and a human high-metastasis large-cell lung cancer cell strain; they are respectively a cell strain ZQH-80 with the preservation number of CGMCC No.2832 and a cell strain ZQH-81 with the preservation number of CGMCC No.2833 which are preserved by the China general microbiological culture Collection center. The lung cancer cell strain with the same genetic background and different metastasis potentials can be used as a cell model, can also be transplanted into an animal body to construct an animal lung cancer model, is used for researching and screening molecular targets of small molecule drugs for resisting lung cancer invasion and metastasis and organ specific metastasis, develops small molecule drugs, vaccines and antibody drugs related to lung cancer invasion and metastasis, and provides a technical platform for researching lung cancer invasion and/or metastasis. Can also be used for the gene target research of the genes related to the lung cancer invasion and metastasis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a pair of lung cancer cell strains with the same genetic background and different metastasis potentials, and a preparation method and application thereof.
Background
Lung cancer is one of the most rapidly growing tumors that threaten human health and life. According to the latest report in 2020: in men, lung cancer is the first malignancy of both morbidity and mortality; in women, lung cancer is the second malignancy in the incidence and the first in the mortality. Lung cancer is the highest mortality tumor worldwide, accounting for about one-fourth of the total deaths from cancer worldwide (22.4%). Lung cancer is divided histologically into Small Cell Lung Cancer (SCLC) and Non-Small cell lung cancer (NSCLC), with NSCLC accounting for 86% of all lung cancers. Approximately 70% of NSCLC patients have been in the advanced stage at the time of initial diagnosis, with a 5-year survival rate of 4% -6%.
In recent years, with the progress and development of surgical treatment, systemic chemotherapy, radiotherapy, targeted therapy and immunotherapy, the treatment efficacy and survival time of lung cancer patients are improved to a certain extent, but the long-term prognosis of lung cancer patients is still quite poor, and the total 5-year survival rate is only about 17%. The main reasons for poor prognosis of lung cancer patients are that about 35% of lung cancer patients have distant metastasis at the time of treatment, about 50% -60% of lung cancer patients have distant metastasis during treatment, and finally 85% -90% of lung cancer patients die due to tumor metastasis. Therefore, the research and elucidation of the molecular mechanism of lung cancer metastasis, the search for new technologies and new methods for early diagnosis of lung cancer metastasis, and the research and development of molecular drugs for inhibiting or/and reversing lung cancer metastasis are the current and leading subjects in the field of lung cancer research in the future.
Lung cancer invasion and metastasis are malignant markers and biological features of lung cancer and are also leading causes of treatment failure and death in patients. At present, the biggest difficult problem in studying lung cancer metastasis is: there is a lack of natural contrast study biomaterials with the same genetic background, different transfer potential. If a pair of lung cancer cell strains with the same genetic background and different metastasis potentials can be constructed, great help is provided for the research of lung cancer metastasis.
Disclosure of Invention
The invention aims to provide a pair of lung cancer cell strains with the same genetic background and different metastasis potentials, and a preparation method and application thereof.
The invention provides a pair of lung cancer cell strains with the same genetic background and different metastasis potentials, wherein the lung cancer cell strains are human low-metastasis large-cell lung cancer cell strains and human high-metastasis large-cell lung cancer cell strains;
the human low-metastasis large cell lung cancer cell strain is a cell strain ZQH-80 with the preservation number of CGMCC No.2832 preserved by the China general microbiological culture Collection center;
the human high-metastasis large cell lung cancer cell strain is a cell strain ZQH-81 with the preservation number of CGMCC No.2833, which is preserved by the China general microbiological culture Collection center.
Furthermore, the lung cancer cell strain is obtained by screening the same human lung cancer cell strain by a single cell cloning limiting dilution method;
preferably, the human lung cancer cell line is a human large cell lung cancer cell line.
Furthermore, the in vitro proliferation capacity, migration capacity, invasion capacity and clone forming capacity of the human low-metastasis large-cell lung cancer cell strain ZQH-80 are all lower than those of the human high-metastasis large-cell lung cancer cell strain ZQH-81;
the in vitro apoptosis level of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is lower than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80.
Furthermore, the lung cancer metastasis suppressor gene nm23 allele structure of the human low-metastasis large-cell lung cancer cell strain ZQH-80 is normal and exists in a heterozygote form;
the mRNA transcript expression and mRNA expression level of the lung cancer metastasis suppressor gene nm23 of the human low-metastasis large-cell lung cancer cell strain ZQH-80 are normal;
the protein expression level of the lung cancer metastasis suppressor gene nm23 of the human low-metastasis large-cell lung cancer cell strain ZQH-80 is normal;
the lung cancer metastasis suppressor gene nm23 allele of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is heterozygous deletion;
the mRNA transcript expression and mRNA expression of the lung cancer metastasis suppressor gene nm23 of the human high-metastasis large-cell lung cancer cell strain ZQH-81 are deleted;
the protein expression of the lung cancer metastasis suppressor gene nm23 of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is deleted.
Furthermore, the tumor formation of the transplantation tumor of the human low-metastasis large-cell lung cancer cell strain ZQH-80 is lower than that of the transplantation tumor of the human high-metastasis large-cell lung cancer cell strain ZQH-81;
preferably, the graft tumor tumorigenicity is a nude mouse graft tumor tumorigenicity.
Furthermore, the human low-metastasis large-cell lung cancer cell strain ZQH-80 transplanted tumor does not generate distant metastasis; the human high-metastasis large-cell lung cancer cell strain ZQH-81 has the advantages that transplanted tumors generate tissue and/or organ metastasis;
preferably, the metastasis is metastasis of lymph nodes, brain, bone, liver;
more preferably, the metastasis is a mediastinal polytransphrenic lymph node metastasis.
Furthermore, the mRNA expression level of genes caldesmon-1, WNT5A, PKP2, NNMT, GLB1L3, PTPRG, SLC2A13, REPS2, GRAMD3, PRICKLE1, CXCR4, PLAU, MTS1 and KCTD4 related to lung cancer invasion and metastasis in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is higher than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80;
the mRNA expression level of genes MGST1, PRG1, SMCY, ACPP, ZFAN 6, CYorf15B, RPS4Y1, DDX3Y, SERPINB2, HGF, MMP2, MMP-14, MMP16 and TIMP2 which are related to the invasion and metastasis of lung cancer in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is lower than that of a human low-metastasis large-cell lung cancer cell strain ZQH-80;
and/or the presence of a gas in the gas,
miRNAs in the human high-metastasis large-cell lung cancer cell strain ZQH-81 related to lung cancer invasion and metastasis are 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, miR-629, hsa-miR-629, The expression level of hsa-miR-654-5p, hsa-miR-767-5p and hsa-miR-92a-1 is higher than that of a human low-metastasis large-cell lung cancer cell strain ZQH-80;
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 and hsa-miR-449a related to lung cancer invasion and metastasis in the human high-metastasis large-cell lung cancer cell strain ZQH-81 are lower than the expression level of human low-metastasis large-cell lung cancer cell strain ZQH- 80.
The invention also provides a method for preparing the lung cancer cell strain, which comprises the following steps: selecting the same human lung cancer cell strain, and screening by a single cell cloning limiting dilution method;
preferably, the screening is to establish a lung cancer nude mouse transplantation tumor model by using cell strains obtained by cloning and culturing single cells, and to screen by using an animal in-vivo imaging technology and a pathological anatomy technology.
The invention also provides a human high-metastasis large cell lung cancer cell strain, which is a cell strain ZQH-81 with the preservation number of CGMCC No.2833 and preserved by the common microorganism center of China Committee for culture Collection of microorganisms.
The invention also provides application of the pair of lung cancer cell lines with the same genetic background and different metastasis potentials or the human high-metastasis large-cell lung cancer cell line in preparation of a lung cancer model.
The invention successfully obtains a pair of two human high and low metastatic potential large cell lung cancer cell strains 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 path conduction. The pair of cell strains can be used as a cell model and can also be transplanted into an animal body to construct an animal lung cancer model, and the cell strains are used for researching and screening molecular targets of small molecule drugs for resisting lung cancer invasion and metastasis and organ specificity metastasis, developing small molecule drugs, vaccines and antibody drugs related to lung cancer invasion and metastasis, and providing a technical platform for researching lung cancer invasion and/or metastasis. Can also be used for the gene target research of the genes related to the lung cancer invasion and metastasis.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
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FIG. 1 shows the results of CCK8 comparing the in vitro proliferation capacity of human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
FIG. 2 shows the results of the comparison of the in vitro 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.
FIG. 3 shows the results of the comparison of in vitro invasion abilities of human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
FIG. 4 shows the results of the in vitro cloning formation of human high metastatic large cell lung cancer cell line ZQH-81 and human low metastatic large cell lung cancer cell line ZQH-80.
FIG. 5 shows the result of flow cytometry for detecting the level of apoptosis 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.
FIG. 6 shows the results of TUNEL method for detecting and comparing the apoptosis level between human high-metastatic large cell lung cancer cell line ZQH-81 and human low-metastatic large cell lung cancer cell line ZQH-80.
FIG. 7 shows the Southern blot detection results of the polymorphism of nm23-H1 gene in human lung cancer cell lines ZQH-81 and ZQH-80 with different metastatic potentials.
FIG. 8 shows the comparison result of mRNA transcripts of 23-H1 genes between ZQH-81 and ZQH-80 of human lung cancer cell strains with different metastatic potentials, wherein: 1 is DNA Ladder Marker; 2 is beta-actin; 3 is ZQH-81; 4 is ZQH-80; 5 is SpacA-1; 6 is A2.
FIG. 9 shows the results of comparison of 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.
FIG. 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 potentials.
FIG. 11 shows the results of in vivo animal imaging comparing distant metastasis of nude mouse transplantable tumor 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 B corresponding to ZQH-81 are two sets of tests of ZQH-81, respectively, and A and B corresponding to ZQH-80 are two sets of tests of ZQH-80, respectively.
FIG. 12 is the pathological anatomy result comparing the tumor formation of nude mouse transplanted tumor between human high-metastasis large-cell lung cancer cell strain ZQH-81 and human low-metastasis large-cell lung cancer cell strain ZQH-80.
FIG. 13 is a diagram of cluster analysis 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.
FIG. 14 is a clustering analysis chart of miRNAs differentially expressed between a human high-metastatic large-cell lung cancer cell strain ZQH-81 and a human low-metastatic large-cell lung cancer cell strain ZQH-80.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
In the invention, the human low-metastasis large-cell lung cancer cell strain ZQH-80 and the human high-metastasis large-cell lung cancer cell strain ZQH-81 are prepared by a new single-cell limiting 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 clinically separated lung cancer tissue cells are cloned by adopting a limiting dilution method, and lung cancer cell strains with the same genetic background and different metastatic potentials are screened.
(1) Taking clinically separated lung cancer tissues, preparing lung cancer cell suspension, counting living cells, diluting according to 100 cells per milliliter of liquid, then sucking the cell suspension by using a 1ml syringe, dripping the cell suspension into a 96-hole plate culture dish when shaking the syringe, dripping only one drop of the cell suspension into each hole, standing for 5min, observing under an inverted microscope, commonly confirming that only one lung cancer cell exists in each hole by 3 persons, adding a culture solution (RPMI1640+ 30% calf serum, pH6.5) into each hole, and adding 5% CO at 37 ℃ and 5% CO2And (5) incubating in an incubator. When the cells in the hole grow and proliferate and most of the bottom of the hole of the culture plate is paved, repeatedly blowing and washing the cells by using a suction pipe to suspend the cells, sucking the cells, transferring the cells into a culture bottle for culture, and freezing and storing the cells;
(2) through repeated screening, 124 cell strains from single cell clone culture are obtained in total;
(3) respectively establishing a lung cancer nude mouse transplantation tumor model by the 124 cell strains obtained from the single cell clone culture;
(4) repeatedly applying animal living body imaging technology and pathological dissection technology, screening out a lung cancer cell strain which only forms tumor in a nude mouse but does not generate distant metastasis, and another lung cancer cell strain which not only forms tumor in the nude mouse but also generates distant multi-organ and multi-focus metastasis. The lung cancer cell strain which only forms tumor in a nude mouse and does not generate distant metastasis is named as: ZQH-80, the lung cancer cell strain which not only forms tumor in nude mice, but also generates distant multi-organ and multi-focus metastasis is named as: ZQH-81.
By the method, a pair of human large cell lung cancer cell strains with the same genetic background and different metastasis potentials is prepared.
Wherein, the human low-metastasis large cell lung cancer cell strain ZQH-80 is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is as follows: the preservation date of the microorganism research institute of Chinese academy of sciences, the great Tutun of the sunward area in Beijing, 2008, 12 months and 26 days, the preservation number is CGMCC No.2832, and the classification and the naming are as follows: a cell line.
The human high-metastasis large-cell lung cancer cell strain ZQH-81 is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is as follows: the preservation date of the microorganism research institute of Chinese academy of sciences, the great Tutun of the sunward area in Beijing, 2008, 12 months and 26 days, the preservation number is CGMCC No.2833, and the classification and the naming are as follows: a cell line.
Example 2 comparative experiment on characteristics of ZQH-80 and ZQH-81 Lung cancer cell lines
In vitro proliferation ability
1. Experimental methods
The invention uses CCK-8 method to detect and compare the characteristics and difference of the in vitro proliferation capacity of ZQH-80 and ZQH-81 lung cancer cell strains.
2. Results of the experiment
The results of the CKK-8 experiment are shown in FIG. 1, and FIG. 1 shows: the OD values of 1 st, 2 nd, 3 th, 4 th and 5 th days of the human high-metastasis large-cell lung cancer cell strain ZQH-81 are all obviously higher than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80(P is less than 0.001); on the 6 th day, the OD value of the human high-metastasis large-cell lung cancer cell strain ZQH-81 has no significant difference (P is more than 0.05) compared with the OD value of the human low-metastasis large-cell lung cancer cell strain ZQH-80.
The experimental results show that: the in vitro proliferation capacity of the ZQH-81 lung cancer cell strain is obviously higher than that of the ZQH-80 lung cancer cell strain.
Second, in vitro migration ability
1. Experimental methods
The invention uses scratch test method to detect and compare the characteristics and difference of the in vitro migration ability of the human high-metastasis large-cell lung cancer cell strain ZQH-81 and the human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The scratch test results are shown in fig. 2, and fig. 2 shows: the in vitro migration capacity of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is obviously higher than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80(P is less than 0.01).
Third, in vitro invasion ability
1. Experimental methods
The invention applies an in vitro invasion experiment (improved Boyden method) to detect and compare the characteristics and the difference of the in vitro invasion capacities of a human high-metastasis large-cell lung cancer cell strain ZQH-81 and a human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The results of the in vitro invasion assay (modified Boyden method) are shown in fig. 3, and fig. 3 shows: the in vitro invasion capacity of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is obviously higher than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80(P is less than 0.01).
Four, in vitro cloning and forming ability
1. Experimental methods
The invention uses in vitro plate clone formation experiment to detect the characteristics and the difference of the in vitro clone formation capacity of the human high-metastasis large-cell lung cancer cell strain ZQH-81 and the human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The results of the in vitro plate clone formation experiments are shown in FIG. 4, where FIG. 4 shows: the in vitro cloning forming ability of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is obviously higher than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80(P is less than 0.01).
Level of apoptosis in vitro
1. Experimental methods
The invention uses flow cytometry and TUNEL method to detect and compare the apoptosis characteristics and difference between the human high-metastasis large-cell lung cancer cell strain ZQH-81 and the human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The flow cytometry (fig. 5) and TUNEL (fig. 6) measurements show: the apoptosis level of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is obviously lower than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80(P is less than 0.01 and P is less than 0.01).
Sixthly, polymorphism of allele nm23-H1 of lung cancer metastasis suppressor gene
1. Experimental methods
The invention uses Southern blotting method to detect and compare the polymorphism characteristics and differences of alleles 23-H1 between ZQH-81 and ZQH-80 of human high-metastasis large-cell lung cancer cell strain.
2. Results of the experiment
The results of Southern blot experiments showed (FIG. 7): the nm23-H1 gene exists in the form of heterozygote (4.6kb or 2.4kb and 2.2kb) in the human low-metastatic large-cell lung cancer cell strain ZQH-80, and both 4.6kb and 2.4kb are lacked in the human high-metastatic large-cell lung cancer cell strain ZQH-81. Therefore, the nm23-H1 gene polymorphism of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is heterozygous deletion.
The experimental results show that: the lung cancer metastasis suppressor gene nm23 allele structure of the ZQH-80 lung cancer cell line is normal and exists in a heterozygote form; the polymorphism of the lung cancer metastasis suppressor gene nm23-H1 allele of the ZQH-81 lung cancer cell line is loss of heterozygosity.
Seventhly, mRNA transcript expression of lung cancer metastasis suppressor gene nm23-H1
1. Experimental methods
The invention uses Northern blot hybridization technology to detect and compare the expression characteristics and the difference of mRNA transcripts of human high-metastasis large-cell lung cancer cell strain ZQH-81 and human low-metastasis large-cell lung cancer cell strain ZQH-80 lung cancer metastasis suppressor gene nm 23-H1.
2. Results of the experiment
Northern blot hybridization results showed (fig. 8): the mRNA transcript expression of the lung cancer metastasis suppressor gene nm23-H1 in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is deleted, while the mRNA transcript expression of the lung cancer metastasis suppressor gene nm23-H1 in the human low-metastasis large-cell lung cancer cell strain ZQH-80, the human lung adenocarcinoma cell SpacA-1 and the human lung adenocarcinoma cell A2 is normal.
The experimental results show that: the mRNA transcript of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-80 lung cancer cell strain is expressed normally; the mRNA transcript expression of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-81 lung cancer cell strain is deleted.
Eighthly, protein expression of lung cancer metastasis suppressor gene nm23-H1
1. Experimental methods
The invention applies Western blot to detect and compare the protein expression level characteristics and differences of the lung cancer metastasis suppressor gene nm23-H1 between a human high-metastasis large-cell lung cancer cell strain ZQH-81 and a human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The Western blot experiment results show (fig. 9): the protein expression of the lung cancer metastasis suppressor gene nm23-H1 in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is deleted, and the protein expression of the lung cancer metastasis suppressor gene nm23-H1 in the human low-metastasis large-cell lung cancer cell strain is normal.
Jiu, mRNA expression of lung cancer metastasis suppressor gene nm23-H1
1. Experimental methods
The invention applies RT-PCR to detect and compare the mRNA expression level characteristics and differences of the lung cancer metastasis suppressor gene nm23-H1 between the human high-metastasis large-cell lung cancer cell strain ZQH-81 and the human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The RT-PCR results show (FIG. 10): the mRNA expression level of the lung cancer metastasis suppressor gene nm23-H1 in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is obviously lower than that of the lung cancer metastasis suppressor gene nm23-H1 in the human low-metastasis large-cell lung cancer cell strain ZQH-80(P is less than 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 obviously higher than that of the lung cancer metastasis suppressor gene nm23-H1 of the ZQH-81 lung cancer cell line.
Ten, nude mouse transplantation tumor formation and metastasis
1. Experimental methods
The invention uses ZQH-81 and ZQH-80 lung cancer cell strains to inoculate a nude mouse, establishes a nude mouse lung cancer transplantation tumor model, and uses animal living body imaging technology and pathological anatomy technology to research and compare the transplantation tumor tumorigenicity, the distant metastasis tumor focus number and the metastasis rate between the human high-metastasis large cell lung cancer cell strain ZQH-81 and the human low-metastasis large cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The in vivo imaging results of the nude mice with transplanted tumor showed (fig. 11): the in vivo imaging fluorescence intensity of the human high-metastasis large-cell lung cancer cell strain ZQH-81 at week 9 is obviously higher than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80; in vivo imaging results also show that the nude mice of the human high-metastasis large-cell lung cancer cell strain ZQH-81 group have systemic multi-organ and multi-site and multiple extensive tumor metastasis; and the group of the human low-metastasis large-cell lung cancer cell strain ZQH-80 does not have distant metastasis.
Pathological anatomy results show (fig. 12): the transplantation tumor volume and the tumor of the human high-metastasis large-cell lung cancer cell strain ZQH-81 are obviously higher than those of the human low-metastasis large-cell lung cancer cell strain ZQH-80 at week 9, pathological anatomy also finds that the transplantation tumor of the human high-metastasis large-cell lung cancer cell strain ZQH-81 group has multiple and wide metastasis of lung, mediastinal lymph node, bone and brain, and the distant organ metastasis rate is 100%; and no tumor metastasis is found in the lung, mediastinal lymph node, bone and brain of the human low-metastasis large-cell lung cancer cell strain ZQH-80 group, and the distant organ metastasis rate is 0%.
The experimental results show that: ZQH-80 lung cancer cell strain nude mouse transplantation tumor does not generate distant metastasis; ZQH-81 lung cancer cell strain nude mouse transplantation tumor has extensive metastasis of lymph node, brain, bone, liver and other organs.
Eleven, genes related to lung cancer invasion and metastasis
1. Experimental methods
The invention applies gene expression spectrum chip and RT-PCR technology to detect and screen differential expression gene related to lung cancer invasion and metastasis between human high-metastasis large-cell lung cancer cell strain ZQH-81 and human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
Gene expression profiling chip and RT-PCR experimental results show (FIG. 13): compared with a human low metastatic potential large cell lung cancer cell strain ZQH-80, the mRNA expression level of genes caldesmon-1, WNT5A, PKP2, NNMT, GLB1L3, PTPRG, SLC2A13, REPS2, GRAMD3, PRICKLE1, CXCR4, PLAU, MTS1 and KCTD4 related to lung cancer invasion and metastasis in the human high metastatic large cell lung cancer cell strain ZQH-81 is obviously increased on average (P is less than 0.01); and the mRNA expression levels of MGST1, PRG1, SMCY, ACPP, ZFAND6, CYorf15B, RPS4Y1, DDX3Y, SERPINB2, HGF, MMP2, MMP-14, MMP16 and TIMP2 genes are all obviously reduced (P is less than 0.01).
Twelve miRNAs related to lung cancer invasion and metastasis
1. Experimental methods
The invention uses mi-RNA chip to detect and screen the difference expression mi-RNAs related to the invasion and metastasis of lung cancer between the human high-metastasis large-cell lung cancer cell strain ZQH-81 and the human low-metastasis large-cell lung cancer cell strain ZQH-80.
2. Results of the experiment
The mi-RNA chip results show (FIG. 14): compared with the human low metastatic potential large cell lung cancer cell strain ZQH-80, miRNAs related to lung cancer invasion and metastasis in the human high metastatic potential large cell lung cancer cell strain 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 and hsa-miR-363, the expression levels of 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 and hsa-miR-92a-1 are obviously up-regulated (P is less than 0.01); and the expression levels of 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 and hsa-miR-449a are obviously reduced (P < 0.01).
From the above examples, it can be seen that 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 invention are a pair of human large cell lung cancer cell lines with the same genetic background, different metastatic potentials, different cell biological behaviors, different molecular biological behaviors, different metastasis related gene structures and functions, different signal path conduction high and low metastatic potentials of two human strains, and natural contrast effect.
According to the results, the invention establishes a pair of human lung cancer cell strains with the same genetic background but completely different metastasis potentials, and the pair of cell strains can be used as cell models and can also be implanted into the body to establish animal models for detecting and screening drugs and confirming whether the drugs can effectively inhibit the metastasis of lung cancer cells.
Example 3 application of human Low-metastatic large cell lung cancer cell line ZQH-80 and human high-metastatic large cell lung cancer cell line ZQH-81
One, as a cell model
1. Establishing a cell model
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 used as a set of cell models.
2. Screening for drugs
a. Taking the lung cancer model established in the step 1;
b. taking candidate drugs to act on the pair of lung cancer models respectively;
c. and observing the influence of the candidate drug on the lung cancer and the quantitative index of lung cancer metastasis, grading, and evaluating the potential drug for treating the lung cancer to inhibit lung cancer metastasis.
Second, construct animal model
1. Construction of animal Lung cancer model
Respectively inoculating ZQH-81 and ZQH-80 lung cancer cell strains to a nude mouse, and establishing a nude mouse lung cancer transplantation tumor model to obtain a group of animal lung cancer models.
2. Screening for drugs
a. Taking the animal lung cancer model established in the step 1;
b. taking candidate drugs, and respectively acting on the pair of animal lung cancer models;
c. and observing the influence of the candidate drug on the lung cancer and the quantitative index of lung cancer metastasis, grading, and evaluating the potential drug for treating the lung cancer to inhibit lung cancer metastasis.
In conclusion, the invention successfully obtains a pair of two human high and low metastatic potential large cell lung cancer cell strains 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 path conduction. The pair of cell strains can be used as cell models, can also be implanted into the body to construct animal lung cancer models, is used for detecting and screening medicaments, and provides a technical platform for researching lung cancer invasion and/or metastasis. Can also be used for the gene target research of the genes related to the lung cancer invasion and metastasis.
Claims (10)
1. A pair of lung cancer cell strains with the same genetic background and different metastatic potentials is characterized in that: the lung cancer cell strain is a human low-metastasis large-cell lung cancer cell strain and a human high-metastasis large-cell lung cancer cell strain;
the human low-metastasis large cell lung cancer cell strain is a cell strain ZQH-80 with the preservation number of CGMCC No.2832 preserved by the China general microbiological culture Collection center;
the human high-metastasis large cell lung cancer cell strain is a cell strain ZQH-81 with the preservation number of CGMCC No.2833, which is preserved by the China general microbiological culture Collection center.
2. The lung cancer cell line of claim 1, wherein: the lung cancer cell strain is obtained by screening the same human lung cancer cell strain by a single cell cloning limiting dilution method;
preferably, the human lung cancer cell line is a human large cell lung cancer cell line.
3. The lung cancer cell line according to claim 1 or 2, wherein: the in vitro proliferation capacity, migration capacity, invasion capacity and clone forming capacity of the human low-metastasis large-cell lung cancer cell strain ZQH-80 are all lower than those of a human high-metastasis large-cell lung cancer cell strain ZQH-81;
the in vitro apoptosis level of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is lower than that of the human low-metastasis large-cell lung cancer cell strain ZQH-80.
4. The lung cancer cell line according to claim 1 or 2, wherein: the lung cancer metastasis suppressor gene nm23 allele structure of the human low-metastasis large-cell lung cancer cell strain ZQH-80 is normal and exists in a heterozygote form;
the mRNA transcript expression and mRNA expression level of the lung cancer metastasis suppressor gene nm23 of the human low-metastasis large-cell lung cancer cell strain ZQH-80 are normal;
the protein expression level of the lung cancer metastasis suppressor gene nm23 of the human low-metastasis large-cell lung cancer cell strain ZQH-80 is normal;
the lung cancer metastasis suppressor gene nm23 allele of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is heterozygous deletion;
the mRNA transcript expression and mRNA expression of the lung cancer metastasis suppressor gene nm23 of the human high-metastasis large-cell lung cancer cell strain ZQH-81 are deleted;
the protein expression of the lung cancer metastasis suppressor gene nm23 of the human high-metastasis large-cell lung cancer cell strain ZQH-81 is deleted.
5. The lung cancer cell line according to claim 1 or 2, wherein: the tumor formation of the transplantation tumor of the human low-metastasis large-cell lung cancer cell strain ZQH-80 is lower than that of the transplantation tumor of the human high-metastasis large-cell lung cancer cell strain ZQH-81;
preferably, the graft tumor tumorigenicity is a nude mouse graft tumor tumorigenicity.
6. The lung cancer cell line according to claim 1 or 2, wherein: the human low-metastasis large-cell lung cancer cell strain ZQH-80 transplanted tumor does not generate distant metastasis; the human high-metastasis large-cell lung cancer cell strain ZQH-81 has the advantages that transplanted tumors generate tissue and/or organ metastasis;
preferably, the metastasis is metastasis of lymph nodes, brain, bone, liver;
more preferably, the metastasis is a mediastinal polytransphrenic lymph node metastasis.
7. The lung cancer cell line according to claim 1 or 2, wherein: the mRNA expression level of genes caldesmon-1, WNT5A, PKP2, NNMT, GLB1L3, PTPRG, SLC2A13, REPS2, GRAMD3, PRICKLE1, CXCR4, PLAU, MTS1 and KCTD4 related to lung cancer invasion and metastasis in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is higher than that of a human low-metastasis large-cell lung cancer cell strain ZQH-80;
the mRNA expression level of genes MGST1, PRG1, SMCY, ACPP, ZFAN 6, CYorf15B, RPS4Y1, DDX3Y, SERPINB2, HGF, MMP2, MMP-14, MMP16 and TIMP2 which are related to the invasion and metastasis of lung cancer in the human high-metastasis large-cell lung cancer cell strain ZQH-81 is lower than that of a human low-metastasis large-cell lung cancer cell strain ZQH-80;
and/or the presence of a gas in the gas,
miRNAs in the human high-metastasis large-cell lung cancer cell strain ZQH-81 related to lung cancer invasion and metastasis are 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, miR-629, hsa-miR-629, The expression level of hsa-miR-654-5p, hsa-miR-767-5p and hsa-miR-92a-1 is higher than that of a human low-metastasis large-cell lung cancer cell strain ZQH-80;
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 and hsa-miR-449a related to lung cancer invasion and metastasis in the human high-metastasis large-cell lung cancer cell strain ZQH-81 are lower than the expression level of human low-metastasis large-cell lung cancer cell strain ZQH- 80.
8. A method for producing the lung cancer cell line according to any one of claims 1 to 7, wherein: it comprises the following steps: selecting the same human lung cancer cell strain, and screening by a single cell cloning limiting dilution method;
preferably, the screening is to establish a lung cancer nude mouse transplantation tumor model by using cell strains obtained by cloning and culturing single cells, and to screen by using an animal in-vivo imaging technology and a pathological anatomy technology.
9. A human high-metastasis large cell lung cancer cell strain is characterized in that: it is a cell strain ZQH-81 with the preservation number of CGMCC No.2833 preserved by the China general microbiological culture Collection center.
10. Use of the lung cancer cell line of any one of claims 1 to 7 or the lung cancer cell line of claim 9 in the preparation of a lung cancer model.
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