CN103966170A - Highly metastatic hepatoma cell line with GFP (Green Fluorescent Protein)-labeled PNC and application of highly metastatic hepatoma cell line - Google Patents
Highly metastatic hepatoma cell line with GFP (Green Fluorescent Protein)-labeled PNC and application of highly metastatic hepatoma cell line Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and relates to a highly metastatic hepatoma cell line with GFP (Green Fluorescent Protein)-labeled PNC, a construction method and an application of the highly metastatic hepatoma cell line, a screening model of an anti-metastasis traditional Chinese medicine monomer based on tumor invasiveness new target PNC and an application of the screening model. The highly metastatic hepatoma cell line with the GFP-labeled PNC is constructed by transfecting a highly metastatic hepatoma cell line by a GFP-PTB fusion protein expression plasmid. The highly metastatic hepatoma cell line has the advantages that a highly metastatic hepatoma cell line model with the GFP (Green Fluorescent Protein)-labeled PNC does not need other labels in vitro, special reagents and consumptive materials, can be directly used for screening anti-metastasis traditional Chinese medicine monomer, is simple in screening step, is rapid in method, is low in cost, is easy to operate and is good in stability.
Description
Technical field
The invention belongs to biological technical field, relate to high secondary liver cancer cell strain and construction process and application with GFP mark PNC, and screening model and the application thereof of anti-metastasis traditional Chinese medicine monomer based on tumor invasiveness new target drone PNC.
Background technology
It is the first that China's onset of liver cancer rate occupies the whole world, and liver cancer case fatality rate is always high.For early stage focal liver cancer, generally take clinically the methods for the treatment of of operation in conjunction with chemicotherapy.But conventional chemotherapy medicine, such as Zorubicin, daunorubicin, mitomycin, efficacy of floxuridine acid etc., in killing tumor cell, also cause serious injury to organism normal cell, has obvious toxic-side effects.Particularly later period of hepatocarcinoma patient, the toxic side effect of chemotherapy can cause larger injury to the original weak health of patient.In addition, later period of hepatocarcinoma is often found blood or lymphatic metastasis, causes cancer cells diffusion, and there is no at present clear and definite effectively anti-metastasis drug.Therefore explore the novel targets of oncotherapy, and develop the chemotherapy new drug that toxic side effect is lower, significant for the targeting of raising oncotherapy and the side reaction of minimizing chemotherapy of tumors.
Aggressive (infiltrate-shift), as one of important symbol of malignant tumour, is to cause cancer patients's main causes of death.And PTS, especially anti-metastasis drug are for a long time in the market requirement " state of hungering and thirst ".Recently research discovery, PNC (perinucleolar compartment) is one and the closely-related nucleus substructure of tumor invasiveness, it is mainly made up of rna binding proteins such as non-coding RNA and PTB, CUG-BP1 such as MRP.Because PNC ratio in neoplastic cell nuclei and its aggressive are proportionate, and closely related with patient's prognosis.Therefore taking PNC as probe, the screening model of setting up the PTS with inhibiting effect on tumor metastasis has potential advantages.
The Chinese medicine that China is traditional, such as glossy ganoderma, Radix Angelicae Sinensis, the Radix Astragali, pseudo-ginseng etc., because of its anti-tumour effect, chemicotherapy sensitization and alleviate chemicotherapy poison secondary aspect unique effect be more and more subject to people's attention.But Chinese medicine is still not clear as its concrete mechanism of action of a complex component, at present, along with to Chinese medical extract, especially deepening continuously of the antitumor mechanism research of traditional Chinese medicine monomer, the prospect of traditional Chinese medicine monomer in oncotherapy receives publicity day by day.Have the effect of traditional Chinese medicine monomer in liver cancer treatment that studies show that, but in the face of huge traditional Chinese medicine monomer storehouse, how selecting simple and effective and have the traditional Chinese medicine monomer that reduces liver cancer aggressive potential, is again a major issue urgently to be resolved hurrily, most important for the selectivity exploitation of medicine.Therefore the foundation of the high secondary liver cancer cell model based on novel tumor transfer of molecules marker PNC, for excavating and there is the traditional Chinese medicine monomer of anti-hepatoma Metastasis effect from traditional Chinese medicine, or significant as the exploitation of the structural modification medicine of lead compound.
Summary of the invention
The object of the invention is by setting up vitro Drug screening model based on novel tumor transfer of molecules marker PNC, for the selectivity exploitation of the traditional Chinese medicine monomer of inhibiting effect on tumor metastasis provides technology platform.
First, the invention provides a kind of high secondary liver cancer cell strain and construction process thereof with GFP mark PNC.
With the high secondary liver cancer cell strain of GFP mark PNC, build by the high secondary liver cancer cell strain of GFP-PTB fusion protein expression plasmid transfection.
The construction process of the high secondary liver cancer cell strain with GFP mark PNC: comprise the steps:
1) obtain high metastatic hepatoma cell strain HepG2M by tumor cell line vitro culture;
2) build PTB expression plasmid GFP-PTB with GFP mark: by high-fidelity DNA polymerase with PCR method amplification people PTB cDNA sequence, segment after amplification is inserted GFP expression plasmid pEGFP-C1 at HindIII and BamH1 site, obtains the PTB fusion protein expression plasmid with GFP mark;
3) step 2) GFP-PTB fusion protein expression plasmid transfection step 1) the metastatic hepatoma cell strain of height, obtain the high secondary liver cancer cell strain with GFP mark PNC, and screen and obtain stable cell strain HepG2M-GFP-PTB by G418.
Further, described in step 1) for to obtain metastatic cancer cell strain-in-situ inoculating again by tumor cell line in-situ inoculating-separation Metastatic Lymph Nodes-vitro culture, repeat 5 times, obtain high metastatic hepatoma cell strain HepG2M.
In addition, the present invention also provides the application of the high secondary liver cancer cell strain with GFP mark PNC.
High secondary liver cancer cell strain with GFP mark PNC is for the preparation of diagnosing cancer of liver reagent.
High secondary liver cancer cell strain with GFP mark PNC is for the preparation of liver-cancer medicine screening model.
The present invention also provides a kind of screening model and structure and using method of anti-metastasis traditional Chinese medicine monomer.
The screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC, described screening model adopts the high secondary liver cancer cell strain with GFP mark PNC, by traditional Chinese medicine monomer, the PNC inhibiting rate of the high secondary liver cancer cell strain with GFP mark PNC is screened.
The construction process of the screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC, comprises the steps:
1) obtain metastatic cancer cell strain-in-situ inoculating again by tumor cell line in-situ inoculating-separation Metastatic Lymph Nodes-vitro culture, so repeat 5 times, obtain high metastatic hepatoma cell strain HepG2M;
2) by the immunofluorescence experiment of PTB antibody, detect the above-mentioned high PNC ratio shifting in hepatoma cell strain, the PNC ratio of 3 strain cells is all more than 95%;
3) structure of the PTB expression plasmid (GFP-PTB) with GFP mark: by high-fidelity DNA polymerase with PCR method amplification people PTB cDNA sequence, segment after amplification is inserted GFP expression plasmid pEGFP-C1 at HindIII and BamH1 site, obtains the PTB fusion protein expression plasmid with GFP mark;
4) above-mentioned GFP-PTB fusion protein expression plasmid passes through
2000 3 strain cells in transfection steps 1 respectively, and screen 2-3 week acquisition by G418 and surely turn hepatoma cell strain; HepG2M-GFP-PTB;
5) adopt fluorescent microscope to verify the PNC ratio in above-mentioned stable cell strain, consistent with the PTB immunofluorescence detected result of step 2, obtain can be used for the high metastatic cancer cell model with GFP mark PNC that anti-metastasis traditional Chinese medicine monomer is screened.
The using method of the screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC, comprises the steps:
1) adopt CCK8 method to analyze the growth-inhibiting situation of alternative traditional Chinese medicine monomer to the high secondary liver cancer cell strain with GFP mark PNC, calculate the drug level that 48h time point cell growth suppresses to reach 50% (GI50%) and 99% (GI99%);
2) after spending the night with the high secondary liver cancer cell strain creep plate of GFP mark PNC, add respectively the traditional Chinese medicine monomer effect of GI50% and GI99% concentration after 24 hours, fix with 4% paraformaldehyde, and wash after mounting in Microscopic observation;
3) adopt fluorescent microscope to detect the PNC ratio after traditional Chinese medicine monomer effect, count 100 cells at every turn, average after counting 3 times; Or adopt after laser confocal microscope imaging, coordinate the tumour cell PNC ratio after the effect of StereoInvestigator software counting traditional Chinese medicine monomer;
4) calculate PNC inhibiting rate: PNC ratio × 100% of PNC inhibiting rate=(the PNC ratio of PNC ratio-traditional Chinese medicine monomer group of solubility promoter DMSO group)/solubility promoter DMSO group, then row statistical study;
5) using the PNC inhibiting rate of traditional Chinese medicine monomer as screening foundation: in the time of GI50% concentration, reach 30% above PNC inhibiting rate as preferentially selecting, reach selecting as second of 30% above PNC inhibiting rate using GI99% concentration.
PTS, especially anti-metastasis drug are for a long time in the market requirement " state of hungering and thirst ".In recent years, the prospect of traditional Chinese medicine monomer in liver cancer treatment receives publicity day by day.Having but how simple and effective ground filters out from huge traditional Chinese medicine monomer storehouse the traditional Chinese medicine monomer that reduces tumor invasiveness potential, is again a major issue urgently to be resolved hurrily.And PNC ratio and its aggressive are proportionate in liver cancer cell core, be therefore expected to as novel metastases molecular marked compound and for the screening of anti-metastasis new drug.Therefore the foundation of the high secondary liver cancer cell model based on PNC mark, the traditional Chinese medicine monomer for excavation from traditional Chinese medicine with inhibiting effect on tumor metastasis is significant.
In addition, PTB is as one of chief component albumen of PNC, can clearly show location and the size of PNC in nucleus, the tumour cell that surely turns GFP mark PTB can intuitively reflect the PNC ratio in cell, therefore in high secondary liver cancer cell, imports GFP-PTB and can be used as the Effective selection model of anti-metastasis traditional Chinese medicine monomer.The screening model of the anti-metastasis traditional Chinese medicine monomer based on such cell provided by the present invention, most important for the selectivity exploitation of medicine.
The invention has the advantages that:
1, the high secondary liver cancer cell model with GFP mark PNC, without other external marks and special reagent and consumptive material, can be directly used in the screening of anti-metastasis traditional Chinese medicine monomer, and screening step is easy, and method is quick, and expense is low, and easy handling, good stability;
2, Chinese medicine has the advantage of low toxic side effect than conventional chemotherapy medicine, and the therefore foundation of the high secondary liver cancer cell model based on PNC pointer is extremely closed for high flux screening and the selectivity exploitation of the traditional Chinese medicine monomer with inhibiting effect on tumor metastasis;
3, also can be used for zooperal living imaging analysis with the high secondary liver cancer cell model of GFP mark, for checking in the body of anti-metastasis traditional Chinese medicine monomer provides good hepatoma model.
Brief description of the drawings
Fig. 1 is the PNC Subcellular Localization figure in HepG2M-GFP-PTB (the high hepatoma cell strain that shifts), and the bright spot of entoblast periphery, is PNC structure.
Fig. 2 is the PNC ratio histogram after traditional Chinese medicine monomer Quercetin, isoliquiritigenin, curcumine processing HepG2M-GFP-PTB cell: wherein, PNC ratio significantly reduces after traditional Chinese medicine monomer is processed, and is dose-dependently.
Specific embodiment
Further set forth the present invention below in conjunction with specific embodiment, should be understood that following examples are only not used in and limit the scope of the invention for the present invention is described.
In the embodiment of the present invention, needed material, reagent all can be buied in market.
Embodiment 1
One, by HepG2 cell (purchasing to ATCC), obtain secondary liver cancer cell-liver in-situ inoculating (repeating 5 times) again in liver in-situ inoculating-separation Metastatic Lymph Nodes-vitro culture, obtain high invasive hepatoma cell strain HepG2M;
Two, adopt PTB antibody (purchasing to Invitrogen 1:1000 dilution) to carry out immunofluorescence detection, the PNC ratio in HepG2M cell is 97.5%;
Three, build the PTB expression plasmid (GFP-PTB) with GFP mark: by high-fidelity DNA polymerase pcr amplification people PTB cDNA sequence, segment after amplification is inserted GFP expression plasmid pEGFP-C1 (purchasing to Shanghai He Yuan Bioisystech Co., Ltd) at HindIII and BamH1 site, obtains the PTB fusion protein expression plasmid with GFP mark;
Four, above-mentioned GFP-PTB fusion protein expression plasmid passes through
2000 (purchasing to Invitrogen) transfection HepG2M cell, and screen and within approximately 3 weeks, obtain stable cell strain HepG2M-GFP-PTB by G418.Basically identical in PNC ratio in this cell and HepG2M cell is 97.8%.Obtain can be used for the high secondary liver cancer cell model with GFP mark PNC of anti-metastasis traditional Chinese medicine monomer screening, five, HepG2M-GFP-PTB cell (by 5000/hole) inoculation 96 orifice plates, after adherent spending the night, add the traditional Chinese medicine monomer (Quercetin of gradient concentration, isoliquiritigenin, curcumine, naringenin, kaempferol etc., all purchase to Rui Fensi bio tech ltd, Chengdu, with DMSO hydrotropy), continue to cultivate after 48 hours, adopt CCK8 method to analyze the growth-inhibiting situation of traditional Chinese medicine monomer to HepG2M-GFP-PTB cell, and drug level while adopting GraphPadPrism5 computed in software cell growth inhibition to reach 50% (GI50%) and 99% (GI99%), see the following form:
| GI50％ | GI99％ | |
| Quercetin | 7.9±1.8μg/ml | 27.5±4.9μg/ml |
| Isoliquiritigenin | 8.2±2.5μg/ml | 24.3±6.1μg/ml |
| Curcumine | 9.1±2.7μg/ml | 32.3±8.7μg/ml |
| Naringenin | 21.8±5.8μg/ml | 70.1±13.8μg/ml |
| Kaempferol | 5.4±1.5μg/ml | 18.4±3.6μg/ml |
Six, HepG2M-GFP-PTB cell is after in 24 orifice plates, creep plate spends the night, and adds respectively the above-mentioned traditional Chinese medicine monomer effect 24 hours of GI50% and GI99% concentration, with add with the hole of GI99% concentration equal proportion DMSO in contrast; Seven, take out cell climbing sheet, and fix after 20 minutes by 4% paraformaldehyde room temperature, PBS washes 2 times, ultrapure washing 1 time, each 3 minutes.Then every cell climbing sheet adds the anti-fluorescent quenching mountant of 10 μ l and carries out mounting; Eight, adopt after laser confocal microscope imaging, coordinate PNC ratio (also can directly count, and get average) in the HepG2M-GFP-PTB cell after the effect of Stereo Investigator software counting traditional Chinese medicine monomer under high power field, see the following form:
| DMSO group PNC ratio | GI50% group PNC ratio | GI99% group PNC ratio | |
| Quercetin | 97.4％ | 83.2％ | 64.1％ |
| Isoliquiritigenin | 97.2％ | 65.2％ | 25.3％ |
| Curcumine | 97.1％ | 72.5％ | 35.1％ |
| Naringenin | 96.8％ | 88.4％ | 76.9％ |
| Kaempferol | 97.4％ | 89.7％ | 79.2％ |
Nine, calculate PNC inhibiting rate: PNC ratio × 100% of PNC inhibiting rate=(the PNC ratio of PNC ratio-traditional Chinese medicine monomer group of solubility promoter DMSO group)/solubility promoter DMSO group.Result is taking isoliquiritigenin as best, its PNC inhibiting rate to HepG2M-GFP-PTB cell in the time of GI50% concentration reaches more than 30%, secondly, their PNC inhibiting rates to HepG2M-GFP-PTB cell in the time of GI99% concentration reach more than 30%, specifically see the following form for curcumine, Quercetin:
| GI50% group PNC inhibiting rate | GI99% group PNC inhibiting rate | |
| Quercetin | 14.6％ | 34.2％ |
| Isoliquiritigenin | 32.9％ | 74％ |
| Curcumine | 25.3％ | 63.9％ |
| Naringenin | 8.7％ | 20.6％ |
| Kaempferol | 7.9％ | 18.7％ |
Ten, the anti-metastasis effect of the above-mentioned traditional Chinese medicine monomer of checking in BD BioCoat Matrigel invasion and attack cells (8 μ m aperture transwell): in the upper indoor HepG2M-GFP-PTB cell that adds of transwell, and add respectively the traditional Chinese medicine monomer of GI50% concentration, with DMSO in contrast; In the lower indoor ln (laminin, LN) that adds.Cultivate after 24h, cotton swab is wiped the non-invasion and attack cell of chamber surface, then removes transwell cell and is inverted air-dryly, then after 30 minutes, takes out PBS cleaning with 37 DEG C of dyeing of 0.1% Viola crystallina.Observation experiment result under inverted microscope, counts the cell in 4 visuals field, averages.As a result, the every high power field of the cell of isoliquiritigenin group is on average worn film number and is starkly lower than other each group, is secondly curcumine and Quercetin, and naringenin and kaempferol (getting rid of after the inhibition of cell growth) anti-invasion effect not obvious.
As can be seen here, according to the traditional Chinese medicine monomer of this screening model screening gained, high secondary liver cancer cell strain is had to obvious anti-invasion effect, and consistent with classical anti-invasion analytical results; And it is than classical way, without special examination consumptive material and reagent such as BioCoat Matrigel invasion and attack cell, lns, thereby in cost performance very advantageous.The selectivity exploitation that can be liver cancer anti-metastasis traditional Chinese medicine monomer provides foundation.
Claims (9)
1. the high secondary liver cancer cell strain with GFP mark PNC, is characterized in that: build by the high secondary liver cancer cell strain of GFP-PTB fusion protein expression plasmid transfection.
2. the construction process of the high secondary liver cancer cell strain with GFP mark PNC claimed in claim 1: comprise the steps:
1) prepare high metastatic hepatoma cell strain HepG2M;
2) build PTB expression plasmid GFP-PTB with GFP mark: by high-fidelity DNA polymerase with PCR method amplification people PTB cDNA sequence, segment after amplification is inserted GFP expression plasmid pEGFP-C1 at HindIII and BamH1 site, obtains the PTB fusion protein expression plasmid with GFP mark;
3) step 2) GFP-PTB fusion protein expression plasmid transfection step 1) the metastatic hepatoma cell strain of height, obtain the high secondary liver cancer cell strain with GFP mark PNC, and screen and obtain stable cell strain HepG2M-GFP-PTB by G418.
3. the construction process of the high secondary liver cancer cell strain with GFP mark PNC according to claim 2, it is characterized in that: described in step 1) obtain metastatic cancer cell strain-in-situ inoculating again for hepatoma cell strain in-situ inoculating-separation Metastatic Lymph Nodes-vitro culture, repeat 5 times, obtain high metastatic hepatoma cell strain HepG2M.
4. the high secondary liver cancer cell strain with GFP mark PNC claimed in claim 1 is for the preparation of diagnosing cancer of liver reagent.
5. the high secondary liver cancer cell strain with GFP mark PNC claimed in claim 1 is for the preparation of liver-cancer medicine screening model.
6. the construction process of the screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC, comprises the steps:
1) obtain metastatic cancer cell strain-in-situ inoculating again by tumor cell line in-situ inoculating-separation Metastatic Lymph Nodes-vitro culture, so repeat 5 times, obtain high metastatic hepatoma cell strain HepG2M;
2) by the immunofluorescence experiment of PTB antibody, detect the above-mentioned high PNC ratio shifting in hepatoma cell strain, the PNC ratio of 3 strain cells is all more than 95%;
3) structure of the PTB expression plasmid (GFP-PTB) with GFP mark: by high-fidelity DNA polymerase with PCR method amplification people PTB cDNA sequence, segment after amplification is inserted GFP expression plasmid pEGFP-C1 at HindIII and BamH1 site, obtains the PTB fusion protein expression plasmid with GFP mark;
4) above-mentioned GFP-PTB fusion protein expression plasmid passes through
2000 3 strain cells in transfection steps 1 respectively, and screen 2-3 week acquisition by G418 and surely turn hepatoma cell strain; HepG2M-GFP-PTB;
5) adopt fluorescent microscope to verify the PNC ratio in above-mentioned stable cell strain, consistent with the PTB immunofluorescence detected result of step 2, obtain can be used for the high metastatic cancer cell model with GFP mark PNC that anti-metastasis traditional Chinese medicine monomer is screened.
7. the screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC, it is characterized in that: described screening model adopts the high secondary liver cancer cell strain with GFP mark PNC, by traditional Chinese medicine monomer, the PNC inhibiting rate of the high secondary liver cancer cell strain with GFP mark PNC is screened.
8. the using method of the screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC claimed in claim 7, is characterized in that: comprise the steps:
1) adopt CCK8 method to analyze the growth-inhibiting situation of alternative traditional Chinese medicine monomer to the high secondary liver cancer cell strain with GFP mark PNC, calculate GI50% and the GI99% drug level of 48h time point;
2), after spending the night with the high secondary liver cancer cell strain creep plate of GFP mark PNC, add respectively the traditional Chinese medicine monomer effect 24 hours of GI50% and GI99% concentration;
3) fix with 4% paraformaldehyde, and wash mounting;
4) adopt fluorescent microscope to detect the PNC ratio after traditional Chinese medicine monomer effect, count 100 cells at every turn, average after counting 3 times; Or adopt after laser confocal microscope imaging, coordinate the tumour cell PNC ratio after the effect of StereoInvestigator software counting traditional Chinese medicine monomer;
5) calculate PNC inhibiting rate: PNC ratio × 100% of PNC inhibiting rate=(the PNC ratio of PNC ratio-traditional Chinese medicine monomer group of solubility promoter DMSO group)/solubility promoter DMSO group, then row statistical study;
6) using the PNC inhibiting rate of traditional Chinese medicine monomer as screening foundation: in the time of GI50% concentration, reach 30% above PNC inhibiting rate as preferentially selecting, reach selecting as second of 30% above PNC inhibiting rate using GI99% concentration.
9. the using method of the screening model of the anti-metastasis traditional Chinese medicine monomer based on PNC according to claim 8, it is characterized in that: step 4) be specially: cell climbing sheet 4% paraformaldehyde room temperature was fixed after 20 minutes, PBS washes 2 times, ultrapure washing 1 time, each 3 minutes.Then every cell climbing sheet adds the anti-fluorescent quenching mountant of 10 μ l and carries out mounting.
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Citations (3)
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| US20050084852A1 (en) * | 2002-03-29 | 2005-04-21 | Sui Huang | Perinucleolar compartment as a cancer marker |
| CN1202243C (en) * | 2001-09-20 | 2005-05-18 | 复旦大学附属中山医院 | Human liver cancer cell line |
| CN103353527A (en) * | 2013-07-01 | 2013-10-16 | 浙江大学 | PNC detection kit for detecting tumor invasion and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202243C (en) * | 2001-09-20 | 2005-05-18 | 复旦大学附属中山医院 | Human liver cancer cell line |
| US20050084852A1 (en) * | 2002-03-29 | 2005-04-21 | Sui Huang | Perinucleolar compartment as a cancer marker |
| CN103353527A (en) * | 2013-07-01 | 2013-10-16 | 浙江大学 | PNC detection kit for detecting tumor invasion and application thereof |
Non-Patent Citations (3)
| Title |
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| SUI HUANG等: "The Dynamic Organization of the Perinucleolar Compartment in the Cell Nucleus", 《THE JOURNAL OF CELL BIOLOGY》, vol. 137, no. 5, 2 June 1997 (1997-06-02), XP003010741, DOI: doi:10.1083/jcb.137.5.965 * |
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| 文翼平: "动物细胞PNC结构中新发现的RNA-蛋白质间的多种相互作用", 《中国博士学位论文全文数据库农业科技辑》, no. 3, 15 March 2014 (2014-03-15) * |
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