CN106867967A - The LM3 cell lines and its construction method of Midkine stable low-expressions - Google Patents
The LM3 cell lines and its construction method of Midkine stable low-expressions Download PDFInfo
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Abstract
The present invention relates to field of biomedical research, and in particular to a kind of construction method of the LM3 stable cell lines of Midkine low expressions.Using RNA perturbation techniques, the LM3 cell lines of Midkine stable low-expressions are built.By design of primers, amplification purpose segment, Midkine shRNA recombinant plasmids are built；Then collect and contain virulent fluid nutrient medium, co-cultured being added to after fluid nutrient medium filtering in the culture dish of inoculation LM3 cells in advance；Puromycin resistance screenings, passage, the final LM3 stable cell lines for obtaining Midkine low expressions are finally carried out to the LM3 cells after transfection.This cell line be established as studying molecular mechanism of action of the Midkine in tumour and the regulating and controlling effect to tumour energetic supersession provides new experiment material.
Description
Technical field
The present invention relates to biomedical sector, the LM3 stable cell lines and its construction method of a kind of stable low-expression Midkine albumen are related in particular to.
Background technology
Midkine albumen is a kind of important cell factor, and molecular size range is 13kDa, can be one of HBGF family member with Heparin-binding.Recent researches find that Midkine albumen is not expressed or low expression in health adult tissue, and are over-expressed in Several Kinds of Malignancy, including liver cancer, cancer of pancreas, breast cancer, intestinal cancer etc..Research finds, Midkine albumen has various biological function, such as promote cell mitogen, cell propagation, angiogenesis, inducing cell vicious transformation, anti-apoptotic isoreactivity, so that the propagation of tumour cell, differentiation and apoptosis are influenceed, one of focus as oncobiology research.Additionally, Midkine albumen also has secretory, find that Midkine expressing quantities are significantly improved in sera of patients with malignant tumors, and be nearly no detectable in normal human serum.Therefore, serum Midkine is expected to turn into a kind of new tumor markers, for the auxiliary therapies such as clinical tumor early diagnosis, Index for diagnosis, the target as Tumor biomarkers research.
Liver cancer is very high in the morbidity and mortality of China, serious harm national healthy.It is the important topic of oncobiology research to the research of onset of liver cancer mechanism.At present tumor proliferation, migration and epidermal cell interstitial (Epithelial-to-Mesenchymal Transition are occurred mainly on Midkine functional studies, the aspect such as EMT), and the biology regulatory mechanism report that Midkine albumen develops to HCC is seldom, especially Midkine is unclear with the regulatory mechanism of liver cancer energetic supersession.The present invention provides powerful to study Midkine by slow-virus transfection system construction Midkine low expressions stabilization hepatoma cell strain to the mechanism of causing a disease and biological function of liver cancer.
The content of the invention
It is an object of the invention to provide the LM3 stable cell lines and its construction method of a kind of low expression Midkine albumen, the cell line can be as cell model, for studying Midkine to the tumour especially biological function such as the regulatory mechanism of liver cancer genesis and development and influence to cell metabolism path.
A kind of LM3 stable cell lines of low expression Midkine albumen that the present invention is provided, it is using SMMC-7721 LM3 as host cell, virus-culturing fluid of the infection with Midkine shRNA genes, the stable cell lines by obtaining low expression Midkine albumen after Puromycin resistance screenings, cell expansion culture.Because Midkine genes and tGFP reporter gene fusions are expressed, the transfection efficiency of Midkine recombinant plasmids can be directly observed under fluorescence microscope.
A kind of construction method of the LM3 stable cell lines of low expression Midkine albumen that the present invention is provided, comprises the following steps：
(1) the pSM2 carriers of Midkine shRNA sequences are carried from the purchase of Open Biosystems companies.The PCR primer (as follows) of design correspondence Midkine shRNA, Midkine shRNA genes are expanded using PCR method；
Forward primer：5′-AAGCCCTTTGTACACCCTAAGCCT-3′
Reverse primer：5′-ACTGGTGAAACTCACCCAGGGATT-3′
(2) after above-mentioned PCR primer is cleaned using PCR primer cleaning agents box, OD values are determined.
(3) double digestion, gel extraction linear carrier are carried out to Lentiviral pLenti using Mlu I and Xho I enzymes.
(4) PCR segments in step (2) are overnight connected with linear carrier pLenti in step (3) in 16 DEG C using T4DNA ligases, build pLenti-MDKshRNA recombinant plasmids.
(5) by above-mentioned coupled reaction liquid Transformed E .coli.DH5a competent cells, 37 DEG C of incubated overnights are put into more than 16 hours；Next day collects thalline, extracts recombinant plasmid pLenti-MDKshRNA from inoculum, carries out the identification of Mlu I and Xho I double digestions.
(6) cell transfection assays the previous day, 0.5 × 10 is inoculated with 100mm culture dishes6~1 × 106Cells HEK293T cell, next day cell confluency degree reaches 50~70%.
(7) cell transfection assays.Using slow-virus transfection method, the recombinant plasmid pLenti-MDKshRNA that step (5) is obtained is with virus packaging plasmid psPAX2, pVSVG according to 10：9：1 mass ratio mixes (μ g of gross mass 6), is added in 580 μ l Opti-MEM minimal mediums, is subsequently adding 12 μ l transfection reagents, mixes, is stored at room temperature 20min；HEK293T cells are taken out, culture medium, addition 5ml PBSs 1 time is removed；Remove PBS solution, addition 3ml DMEM minimal medium incubated at room temperature 20min；Then above-mentioned transfection reagent mixed liquor is added dropwise to be inoculated with advance in the 100mm culture dishes of HEK293 cells, is gently shaken up, be put into 37 DEG C, 5%CO2Cultivated in incubator；Culture dish is taken out after 6 hours, 7ml DMEM complete mediums are added, continuation is cultivated in being put into incubator.
(8) after cultivating 48 hours, collect containing above-mentioned containing virulent cell culture fluid, filter and retain filtrate using 0.45 μm low absorption sterilised membrane filter.The virus-culturing fluid can directly using or concentration after use, can also place -80 DEG C long-term and preserve.
(9) virus transfection tests the previous day, and LM3 cells are inoculated with 6 orifice plates, the inoculation 2 × 10 per hole5Cells, next day, cell confluency degree was up to 50~70%.
(10) virus transfection experiment.6 orifice plates of culture LM3 cells are taken out from cell culture incubator, cell culture fluid is removed, is added to what is obtained in step (8) in above-mentioned LM3 Tissue Culture Dish containing virulent cell culture fluid, 2.5ml is added per hole, be then placed in 37 DEG C, 5%CO2Incubator, continues to cultivate 24 hours.
(11) next day take out above-mentioned 6 orifice plate from cell culture incubator, remove and contain virulent nutrient solution, DMEM fresh medium of the addition containing 1 × P/S of the hyclone of volumetric concentration 10% and final concentration is put into cell culture incubator and cultivates 48~72 hours.
(12) after cultivating 48~72 hours, visible cell sends green fluorescence under fluorescence microscope.The Puromycin that final concentration of 2 μ g/ml are added in nutrient solution carries out positive cell line screening.Replacing in every three days once contains the DMEM fresh mediums of 2 μ g/ml Puromycin.Step sizing more than seven weeks, until visible all cells all send green fluorescence under fluorescence microscope, illustrates to obtain the positive cell line of stabilization growth.
(13) positive cell line identification.The LM3 cells after part is screened are taken out, the expression of cell inverted fluorescence microscope, RT-qPCR and Western Blot identification of M idkine genes and albumen is respectively adopted.Using the transfection method, Midkine gene silencing efficiency continues low expression up to more than 80%, it was demonstrated that Midkine low expression LM3 cell lines are successfully constructed.
(14) compared with prior art, the present invention obtains stable low-expression Midkine cell lines by adjusting the conditions such as slow-virus transfection time, rotaring redyeing system, and the method has transfection efficiency high, can special, lasting, stabilization low expression Midkine advantages.Mechanism of action of the present invention for research Midkine albumen in liver cancer provides powerful with tumour energetic supersession.
Brief description of the drawings
Fig. 1：The cleavage map of pLenti-MDKshRNA recombinant plasmids；Wherein M represents the digest of λ-Hind III, and swimming lane 1 represents recombinant plasmid, and swimming lane 2 represents recombinant plasmid cleavage map.
Fig. 2：Cell fluorescence qualification figure of the LM3 stable cell lines of low expression people's Midkine genes in inverted fluorescence microscope (100 times).
Fig. 3：The LM3 stable cell lines RT-qPCR qualification figures of low expression people's Midkine genes.
Fig. 4：The LM3 stable cell lines Western Blot qualification figures of low expression people's Midkine genes.Swimming lane 1 is control, and swimming lane 2 is stable cell lines, and antibody is Rabbit Anti-Midkine Antibody.
Specific embodiment
Embodiment 1, recombinant expression carrier pLenti-MDKshRNA build
(1) from Open Biosystems companies pSM2 carrier of the purchase commercialization with Midkine shRNA sequences.Midkine shRNA genes are expanded using PCR method, primer sequence is as follows：
MDKshRNA Forward Primer:5′-AAGCCCTTTGTACACCCTAAGCCT-3′
MDKshRNA Reverse primer:5′-ACCTGGTGAAACTCACCCAGGGATT-3′
Pcr amplification reaction system is as follows：
PCR reaction conditions：
(2) above-mentioned PCR primer is carried out by cleaning recovery using treasured biotech firm TaKaRa MiniBEST DNA Fragment Purification Kit, and is dissolved in 110 μ l sterilized waters.Operating procedure is with reference to specification.
(3) PCR primer of recovery is carried out into Mlu I and Xho I double digestions, endonuclease reaction system is as follows：50 μ l digestion systems, add 5 μ l PCR primers, 10U Mlu I, 10U Xho I, 5 μ l 10 × H buffer respectively, and remaining is supplied with dH2O, after mixing, are put into 37 DEG C of water-baths and are incubated 1 hour.
(4) by digestion products on 1.2%~1.5% (g/ml) Ago-Gel electrophoresis, and carry out gel extraction.Using precious biotech firm's gel reclaims kit TaKaRa MiniBEST Agarose Gel DNA Extraction Kit, with reference to product description operation.Midkine shRNA fragment lengths are 345bp, are finally dissolved in 50 μ l sterilized waters.
(5) Midkine shRNA segment concentrations are determined using NanoDrop1000.
(6) slow virus carrier plenti is prepared.Double digestion is carried out to recombinant plasmid pLenti using Mlu I and Xho I restriction enzymes, is incubated 1 hour in 37 DEG C of water-baths；Then in 0.8% agarose gel electrophoresis, and gel extraction linear carrier.
(7) plenti linear carriers and Midkine shRNA segments are overnight connected in 16 DEG C using T4DNA ligases.
(8) use thermal conversion process, by above-mentioned coupled reaction liquid Transformed E .coli.DH5a competent cells after, in the LB culture medium incubated overnights containing 100 μ g/ml Ampicillin, next day extracts pLenti-MDKshRNA recombinant plasmids.Plasmid extraction kit uses TaKaRa MiniBEST Plasmid Purification Kit, and concrete operation step is with reference to specification.
(9) Mlu I and Xho I double digestions are carried out to the pLenti-MDKshRNA recombinant plasmids for extracting to identify (see Fig. 1).Endonuclease reaction system is as follows：50 μ l digestion systems, pLenti-MDKshRNA recombinant plasmids, 10U Mlu I, 10U Xho I containing 1 μ g, 5 μ l 10 × H buffer, remaining is supplied with dH2O, after mixing, is put into 37 DEG C of water-baths and is incubated 1 hour；Then detected in 1% (g/ml) agarose gel electrophoresis.
The foundation of embodiment 2, low expression Midkine stable cell lines, specific implementation step is as follows：
(1) after pLenti-MDKshRNA recombinant plasmids are largely extracted, OD values are determined.
(2) 1 × 106 HEK293T cell is inoculated with 100mm culture dishes, addition 10ml contains the DMEM culture mediums of 10% hyclone, 1 × P/S, is put into 37 DEG C, 5%CO2Cultivated in incubator, next day cell confluency degree about 50~70%.
(3) cell transfection assays.By recombinant plasmid pLenti-MDKshRNA and virus packaging plasmid psPAX2, pVSVG according to 10：9：1 mass ratio is mixed into Opti-MEM basic culture solutions, and concrete operations are as follows：3 μ g pLenti-MDKshRNA recombinant plasmids, 2.7 μ g psPAX2 and 0.3 μ g pVSVG packaging plasmids are added in 580 μ l Opti-MEM basic culture solutions respectively, it is well mixed, then 12 μ l Transfect Reagent (ThermoFisher) transfection reagents are added, is mixed, is stored at room temperature 20 minutes；HEK293T cells are taken out, nutrient solution is removed, addition 5ml PBSs are once；Remove PBS solution, addition 3ml DMEM basic culture solutions room temperature and place 20min；Then the above-mentioned nutrient solution containing transfection reagent is added dropwise in HEK293T Tissue Culture Dish, is gently shaken up, be put into 37 DEG C, 5%CO2Cultivated in incubator；After culture 6 hours, the DMEM culture mediums containing 1 × P/S of 10% hyclone and final concentration are added, continue to cultivate 48 hours.
(4) after cultivating 48 hours, collect and contain virulent cell culture fluid, virulent cell culture fluid is contained using 0.45 μm of membrane filtration, retain and contain virulent filter liquor.The virus-culturing fluid can directly using or concentration after use, it is also possible to be placed on -80 DEG C and save backup for a long time.
(5) virus transfection tests the previous day, and LM3 cells are inoculated with 6 orifice plates, and 2 × 105cells is accessed per hole, and addition contains 10% hyclone, the DMEM culture mediums of 1 × P/S, is put into 37 DEG C, 5%CO2Cultivated in incubator, next day cell confluency degree about 50~70%.
(6) virus transfection experimental day, take out 6 orifice plates of inoculation LM3 cells in advance, remove cell culture fluid, 2ml PBSs are added per hole once, PBS solution is removed, the virus-culturing fluid for then obtaining step (3) is added in 6 orifice plates according to 250 μ l/ holes, gently mix, be put into CO2Incubator, cultivates 24 hours.
(7) 6 orifice plates of next day taking-up, remove virus-culturing fluid, and per hole, addition 2.5ml contains the DMEM fresh cultures of 10% hyclone, 1 × P/S, continues culture 48~72 hours.Whether period observation of cell under fluorescence microscope sends green fluorescence.
(8) positive cell line screening.After culture 48~72 hours, visible cell sends green fluorescence under fluorescence microscope, and final concentration of 2 μ g/ml Puromycin screenings positive cell is then added in cell culture fluid；Change once containing the DMEM complete mediums of 2 μ g/ml Puromycin within every three days, step sizing more than seven weeks, until observing (see Fig. 2) untill nearly all cell all sends green fluorescence；When cell confluency degree reaches 80~90%, passed on according to 1/5 ratio, while add the DMEM complete mediums containing 2 μ g/ml Puromycin being screened；1cell/ holes can also be diluted to using culture medium in picking positive cell cluster under fluorescence microscope, positive monoclonal cell line is screened using 96 orifice plates.
(9) low expression Midkine genetic tests.According to the exon sequence of people's Midkine genes, a pair of specific primers (as follows) are designed.Positive cell line total serum IgE is extracted, RT-qPCR detections, electrophoresis detection (result such as Fig. 3) is carried out.Nucleic acid extracting reagent and qPCR detection reagents are respectively adopted precious biotech firm RNAiso Plus and One Step SYBR PrimeScrip RT-PCR Kit, and operating procedure is with reference to product description.Primer sequence is as follows：
MDK Forward Primer：5’-TGCCCTGCAACTGGAAGAA-3’
MDK Reverse Primer：5’-CTTGGTGACGCGGATGGT-3’
(10) transfection efficiency detection.Extract stable cell line total protein, protein quantification is carried out using BCA methods, then carry out Western Blot and detect Midkine expressing quantities (see Fig. 4).Concrete operation method is as follows：By in the LM3 passages of Midkine low expressions to 100mm culture dishes, the PBS of addition 8ml precoolings 1 time, abandon supernatant, the RIPA cell pyrolysis liquids (ThermoFisher) of 350 μ l precoolings are added in above-mentioned culture dish, culture dish is placed in cell lysis 30min on ice, period gently mixes several times；Then cell is quickly scraped using the cell spatula of precooling, and is transferred in 1.5ml centrifuge tubes；Then 12000rpm, 4 DEG C of centrifugation 30min, careful Aspirate supernatant is transferred in new 1.5ml centrifuge tubes.Protein concentration (ThermoFisher), operating method reference product specification are determined using BCA methods.20 μ g protein extracts are drawn, 4 × SDS PAGE Loading Buffer of final volume 1/5 are added, 5min is denatured in 100 DEG C, carry out 10%SDS- polyacrylamide gel electrophoresises, constant voltage 80V；Then albumen electricity is gone into pvdf membrane, constant current 250mA；After transferring film, the skimmed milk power (being prepared with TBST solution) that pvdf membrane mass volume ratio is 5% is closed 1 hour, be subsequently adding TBST solution and wash film 3 times, each 10min adds rabbit anti-MDK antibody, in 4 DEG C of night incubations.In morning next day, primary antibody is reclaimed, addition TBST washes film 3 times, and each 10min adds mouse secondary antibody, is incubated at room temperature 1 hour, and TBST washes film 3 times, each 10min.Imaging analysis are carried out after adding ECL chemical illuminating reagents.As shown in figure 4, the visible Midkine protein bands of compared with control cells, and the expressing quantity is very low after transfecting Midkine shRNA recombinant plasmids, is hardly visible band, silence efficiency shows that Midkine low expression LM3 cell lines are successfully constructed more than more than 80%.
(11) the LM3 stable cell lines of the low expression Midkine for obtaining step (8), after Amplification Culture, are placed in liquid nitrogen and preserve for a long time.
Claims (5)
- The construction method of the LM3 stable cell lines of 1.Midkine low expressions, it is characterised in that including Following steps：(1) the shRNA segments of correspondence Midkine genes are expanded from carrier pSM2, is cloned Onto the slow virus carrier pLenti containing tGFP reporter genes, pLenti-MDKshRNA weights are built Group plasmid；(2) HEK293T cells are inoculated with 100mm culture dishes；Using slow-virus transfection system, The recombinant plasmid that step (1) is obtained is with slow virus packaging plasmid psPAX2, pVSVG according to 10： 9：1 mass ratio is added in Opti-MEM minimal mediums, is then added to transfection reagent above-mentioned In culture medium, all it is added in HEK293T cell culture mediums after being well mixed, the cell is put into Cultivated in incubator；After 48 hours, the cell culture fluid containing MDK shRNA viruses is collected, It is sterile filtered and collects containing virulent filter liquor；Above-mentioned viral filter liquor is added to inoculation LM3 in advance Co-cultured in the culture dish of cell；Puromycin antibiotic-screening positive cell lines are finally added, finally Obtain the LM3 cell lines of Midkine stable low-expressions.
- 2. construction method according to claim 1, pLenti-MDKshRNA construction of recombinant plasmid Process comprises the following steps：(1) with pSM2 as template, Midkine shRNA gene segments are expanded using PCR method, PCR primer sequence is,Forward primer：5'-AAGCCCTTTGTACACCCTAAGCCT-3'Reverse primer：5'-ACTGGTGAAACTCACCCAGGGATT-3'(2) by step (1) gained PCR primer electrophoresis detection, purifying, OD values are determined；(3) double digestion, gel extraction are carried out to slow virus carrier pLenti using Mlu I and Xho I enzymes Linear carrier；(4) will be linear in PCR segments in step (2) and step (3) using T4 DNA ligases Carrier pLenti is overnight connected in 16 DEG C；(5) thermal conversion process is used, by above-mentioned coupled reaction liquid Transformed E .coli.DH5a competent cells, In 37 DEG C of incubated overnights more than 16 hours；Next day collects thalline, extracts plasmid from inoculum PLenti-MDKshRNA, carries out the identification of Mlu I and Xho I double digestions.
- 3. construction method as claimed in claim 1, the LM3 stable cell lines of low expression Midkine Structure, it is characterised in that：(1) cell transfection assays the previous day, 0.5 × 10 is inoculated with 100mm culture dishes6~1 × 106 Cells HEK293T cell, next day cell confluency degree reaches 50~70%；(2) cell transfection assays, using slow-virus transfection method, by what is built PLenti-MDKshRNA recombinant plasmids are with slow virus packaging plasmid psPAX2, pVSVG according to 10： 9：1 mass ratio mixes (all μ g of plasmid quality summation 6), is added to 580 μ l Opti-MEM bases In basal culture medium, then add 12 μ l transfection reagents in above-mentioned culture medium, mixing, room temperature static 20 After minute, the above-mentioned culture medium containing transfection reagent is added dropwise to be inoculated with the training of HEK293T cells Support in ware, be put into culture in incubator；Supplement contains the hyclone of volumetric concentration 10% and end after 6 hours The DMEM culture mediums of 1 × P/S of concentration (Penicillin-Streptomycin), are then placed in incubator In continue cultivate 48 hours；(3) virus transfection tests the previous day, and LM3 cells are inoculated with 6 orifice plates, 1×105~2 × 105Cells/ holes, by LM3 cell culture containing the hyclone of volumetric concentration 10% and The DMEM culture mediums of 1 × P/S of final concentration (Penicillin-Streptomycin), next day, LM3 was thin Born of the same parents' degree of converging reaches 50~70%；(4) what is obtained in virus transfection experimental day, collection step (2) contains Midkine shRNA The cell culture fluid of virus, is filtered and is retained filtrate using 0.45 μm low absorption sterilised membrane filter；Remove LM3 cell culture fluids, co-culture being added in LM3 Tissue Culture Dish containing virulent filtrate；Training After supporting 24 hours, remove and contain virulent nutrient solution, change and contain 10% hyclone and final concentration The DMEM nutrient solutions of 1 × P/S, are put into cell culture incubator and cultivate 48~72 hours；(5) positive cell line screening；After above-mentioned cell culture 48~72 hours, in cell culture fluid Adding the Puromycin of final concentration of 2 μ g/ml carries out positive cell line screening, changes once within every three days Fresh culture containing final concentration of 2 μ g/ml Puromycin；Treat that cell confluency degree reaches 80~90% Shi Chuandai；When cell was reached after the 7th generation, all sent out in fluorescence microscopy Microscopic observation almost all cell Go out green fluorescence, take out part cell and identified；By the later cell expansion culture of eighth generation, and freeze Cell is deposited, it is final to obtain positive colony cell line.
- 4. construction method as claimed in claim 3, it is characterised in that：The sun that above-mentioned steps are obtained Property cell line, take out part cell extraction total serum IgE, using RT-qPCR methods detect Midkine bases Because of expression quantity；Another part cell extraction albumen is taken out, Midkine is detected using Western Blot methods Expressing quantity.
- 5. the Midkine stable low-expressions that a kind of any construction method of Claims 1 to 4 is obtained LM3 cell lines.
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| CN111254175B (en) * | 2018-11-30 | 2023-04-18 | 中国科学院大连化学物理研究所 | Method for collecting Midkine protein in vitro and using Midkine protein for treating cells |
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Application publication date: 20170620 |