CN101003792A - Constructing 9LLUC cell strain of expressing luciferase stably, and application - Google Patents

Abstract

This invention relates to a method for constructing 9LLUC cell strain capable of stably expressing firefly luciferase PGL3. This invention provides a cell strain 9LLUC, which is obtained by transfecting glioma cell 9L with lentivirus vector containing firefly luciferase PGL3 gene, and screening. Cell strain 9LLUC can be transplanted into different animals to obtain relative glioma animal models. Though stable expression of PGL3, the generation, development, infiltration, metastasis of glioma cell, and the curative effects of relative therapies can be real time observed. The glioma animal models can be used for studying the development of glioma, and screening relative drugs and therapies.

Description

The 9L of expressing luciferase stably LUCThe structure of cell strain and application

Technical field

The present invention relates to biomedical sector, specifically relate to a kind of expressing luciferase stably (PGL ₃) 9L ^LUCThe foundation of cell strain.

Background technology

Gene, cell and the animal of luciferase gene (LUC) mark make the researchist directly monitor intravital cellular activity of living body biological and gene behavior by living animal in-vivo imaging system.Particularly in various types of tumor researches, the growth and the transfer of tumour cell in the various tumor models be can directly measure apace, and can real-time monitored and assessment be carried out the variation of tumour cell in the oncotherapy.Can not have wound and quantitatively primary tumor, metastatic tumor and the spontaneous knurl of integral animal are detected and measure, even small migration also can be detected.

As one of instrument of gene transfection, lentiviral vectors can reach the Infection in Vitro Unseparated Cell in vivo because of it, and transfection is organized widely, and can be condensed to high titre and extensively favored in recent years.But the lentiviral vectors stable integration is gone into the karyomit(e) of host cell, the transgene expression long-term existence.We select the lentiviral vectors pBPLV that transforms (to equal Nature Biotechnology according to Mario Amendola, 2005, the method that 23:108-116 introduces makes up), firefly luciferase gene is cloned into this carrier, carries out the virus packing and screen stably express PGL by flow cell sorter ₃9L cell strain 9L ^LUC

The number linear dependence of intensity that luciferase is luminous in vivo and labeled cell, change luciferase or other tracer agent over to the 9L cell by adenovirus carrier, retroviral vector and other carrier in the past and can not obtain stably express, also can't set up the cell strain of stably express, can't finish for the preparation and the real-time monitored of glioma animal model.

Summary of the invention

The object of the present invention is to provide a kind of lentiviral vectors that utilizes that Photinus pyralis LUC is transfected into the 9L glioma cell, and obtained the 9L of expressing luciferase stably through screening ^LUCCell strain.

Specific embodiments of the present invention is as follows:

1.LUC segmental obtaining.By the NheI restriction enzyme (Takara company, D1162A), the HpaI restriction enzyme (Takara company, D1064A) (E1751) go up enzyme and cut and reclaim LUC fragment (Genebank accession number 6391) from the PGL3-Basic carrier by Promega company.

2. make up the slow virus expression vector pBPLV-LUC that carries goal gene.The LUC fragment that will have NheI and HpaI restriction enzyme site is inserted on the slow virus expression vector pBPLV multiple clone site, the slow virus expression vector pBPLV-LUC of goal gene is carried in acquisition, confirm that through PCR and determined dna sequence clone's target gene sequences is correct, recombinant slow virus expression vector pBPLV-LUC successfully constructs.

3. with lentiviral vectors pBPLV-LUC, packaging plasmid Virapower ^TM(Invitrogen 4975-00) is transfected into 293FT cell (Invitrogen company, article No. R 700-07) to Lentiviral Packaging Mix jointly, changed liquid in second day, continue to cultivate 48 hours, cell is collected supernatant after forming synplasm, 4 ℃, 3000rpm received malicious in centrifugal 15 minutes.

4. (ATCC CRL-2200), goes out to express the 9L of Photinus pyralis LUC by selected by flow cytometry apoptosis with the slow virus venom transfection 9L glioma cell collected ^LUCCell, and carry out large scale culturing.

5. with the 9L of expressing luciferase stably ^LUCCell carries out gradient dilution in 96 orifice plates, and the 50000cells/ hole is to the 50cells/ hole, and (Sigma company, L7538), the photon amount and the cell count of cell are proportionate PGL to add 150 μ g/ml Photinus pyralis LUC substrate D-luciferin ₃Gene stable transfection is gone into 9L ^LUCCell.

6. with 9L ^LUCCell inoculation is in Fischer rat encephalic, and the result showed that tumour increased gradually by the detection of living animal detector in 0,7,14,21 days in the injection back, and the photon amount significantly increases.

The present invention changes the 9L glioma cell by the slow virus expression vector over to Photinus pyralis LUC is stable, sets up stably express PGL ₃9L ^LUCCell strain.The stably express PGL that utilizes present method to set up ₃9L ^LUCCell strain can prepare corresponding glioma animal model in migrating to different animal bodies, pass through PGL ₃Expression can real-time monitored and the result of treatment of generation, development, infiltration, transfer and the corresponding treatment measure of assessment glioma cell, and reach the purpose of spike in the living animal body.Use the glioma animal model of this cell strain preparation and in the research of generation development mechanism, medicine screening and the screening of different treatment plan of glioma, have splendid application prospect.

Description of drawings

Fig. 1 slow virus expression plasmid pBPLV-LUC makes up schema.

The plastidogenetic synplasm of the common transfection 293FT of Fig. 2 slow virus expression vector and packaging plasmid.(fluorescent microscope, * 100)

Figure 39 L ^LUCCell strain.(fluorescent microscope, * 100)

Figure 49 L ^LUCThe linear relevant indicators of cell fluorescence photon amount and cell count.

Figure 59 L ^LUCGlioma model live body detected result.

Embodiment

The structure of embodiment 1, slow virus expression plasmid pBPLV-LUC.

Slow virus expression vector pBPLV is according to (Nature Biotechnology, 2005, the method structures of 23:108-116) introducing such as Mario Amendola.With the MA1 expression plasmid behind PstI restriction enzyme and SalI digestion with restriction enzyme, adding multiple clone site sequence (5 '-GGCTAGCATGCTCTAGAGCGCTG-3 ', 3 '-ACGTCCGATCGTACGAGATCTCGCGACAGCT-5 ') structure slow virus expression plasmid pBPLV.

Photinus pyralis LUC PGL ₃-Basic plasmid is available from Promega company, by the NheI restriction enzyme (Takara company, D1162A), HpaI restriction enzyme (Takara company, D1064A) each 2 μ l, PGL ₃-Basic plasmid 30 μ l, 10 * T Buffer, 5 μ l are hatched the 2h enzyme in 37 ℃ and are cut back to close the LUC gene fragment, and clip size is 1881bp; Slow virus expression vector pBPLV 30 μ l, NheI restriction enzyme 2 μ l, Aor51 HI restriction enzyme (Takara company, D1118A) 2 μ l, 10 * M Buffer, 5 μ l are hatched the 2h enzyme in 37 ℃ and are cut, and reclaim test kit by gel and reclaim and purifying slow virus expression vector.Fetch respectively and receive slow virus expression vector 4 μ l and reclaim Luc ^PGL3Fragment 6 μ l connect in 16 ℃ of incubation 30min, transform, and obtain to carry the slow virus expression vector pBPLV-LUC of goal gene.Identify through PCR, meet with expected results; It is carried out forward and reverse order-checking and analysis revealed, frameshit does not take place change.Show that the target gene sequences that we clone is correct, slow virus expression vector pBPLV-LUC successfully constructs (the results are shown in Figure 1).

The packing of embodiment 2,293FT cell

The 293FT cell is cultivated with the high sugared DMEM that contains 10% foetal calf serum (FBS).In an aseptic 5ml pipe, add 1.5ml serum-free DMEM substratum, add packaging plasmid mixture 4.2 μ g pLP1,2 μ gpLP2,2.8 μ g pLP/VSVG (Virapower successively ^TMLentiviral Packaging Mix, Invitrogen company is 4975-00) with 5 μ g slow virus expression plasmid, mixings gently.In another aseptic 5ml pipe, with 42 μ l Lipofectamine 2000 (Invitrogen 11668-027) is diluted in the 1.5ml serum-free DMEM substratum, mixing gently, room temperature is placed 5min.Mix above 2 pipe liquid, mixings gently.Incubated at room 20min is to form DNA-Lipofectamine 2000 mixtures.In DNA-lipid mixture forming process, with 0.25% tryptic digestion 293FT cell, counting is resuspended in cell among the DMEM of the high sugar that contains 10% serum, and making its density is 1.2 * 10 ⁶Individual/ml.DNA-Lipofectamine 2000 mixtures are joined in the 10cm Tissue Culture Dish that contains the 5ml growth medium, with 5ml 293FT cell suspension (totally 6 * 10 ⁶Individual cell) joins in the 10cm Tissue Culture Dish that contains DNA-Lipofectamine 2000 mixtures and growth medium, move forward and backward the culture dish mixing lightly.Cell is placed 37 ℃, 5%CO ₂Overnight incubation in the incubator.Changed the DMEM of liquid height sugar in second day, continue to cultivate 48 hours, cell is collected supernatant after forming synplasm, and 4 ℃, 3000rpm, centrifugal 15 minutes receipts supernatant liquors (the results are shown in Figure 2).

Embodiment 3,9L ^LUCThe foundation of cell strain.

The good 9L cell routine of growth conditions is cultivated 0.25% tryptic digestion behind the 24h, slow virus liquid 1ml with preparation, Polybrene (final concentration is 8 μ g/ml) adds in the culture dish jointly, conventional overnight incubation, change fresh growth medium, every 3d changes liquid once, cultivates after 14 days, cell dissociation is prepared into single cell suspension, goes out 9L with green fluorescent protein by selected by flow cytometry apoptosis ^LUCCell, after being cultured to 80-90% and merging, 1:2 go down to posterity (the results are shown in Figure 3).

Embodiment 4,9L ^LUCThe detection of luciferase.With 9L ^LUCCell carries out gradient dilution in 96 orifice plates, is respectively 50000,40000,20000,10000,5000,2000,1000,500,200,100,50, the media/ hole, adding Photinus pyralis LUC substrate D-luciferin (150 μ g/ml, Sigma company, L7538), place living animal detector camera bellows to detect its fluorescence intensity flat board, the photon amount and the cell count of showed cell are proportionate as a result, show PGL ₃Gene stable transfection is gone into 9L ^LUCCell (the results are shown in Figure 4).

Embodiment 5, be that example prepares the cerebral glioma animal model with the Fischer rat.

Bull Fischer rat (220-250g), after abdominal injection 3.5% Chloral Hydrate (10ml/1kg) anesthesia, the prostrate stereo brain orienting instrument that is fixed in, head unhairing, routine disinfection.Vertically cut scalp along median line, expose bregma.1.0mm behind the bregma mid point, side, the sagittal suture right side is opened 3.0mm and is holed with small-sized dental burr.Extract 10 μ l 9 with 10 μ l microsyringes ^LUCThe glioma cell suspension is fixed in the three-dimensional instrument that manually advances, and perpendicular to lamina externa cranii inserting needle 5mm, rollback 0.5mm slowly injects the back that finishes let the acupuncture needle remain at a certain point 5min with the speed of 1 μ l/min, slowly pulls out pin through the cranium hole, and the scalp wound is sewed up in bone wax sealing bone hole.In the equal aseptic technique of art process.After animal is clear-headed, sends Experimental Animal Center back to and raise.Respectively at detecting the growth of tumor situation by the living animal detector in 0,7,14,21 days behind the tumor cell transplantation.(L7538) back 5-7min after abdominal injection 3.5% chloral hydrate anesthesia, places the detector camera bellows to observe to animal abdominal injection D-luciferin for 150mg/kg, Sigma company.The result shows that tumour increases gradually, and the photon amount significantly increases.Show animal model success (the results are shown in Figure 5).

Claims (6)

1, makes up the 9L of expressing luciferase stably ^LUCCell strain is characterized in that using lentiviral vectors Photinus pyralis LUC is transfected into the 9L glioma cell, and obtains the 9L of expressing luciferase stably through screening ^LUCCell strain.

2, the 9L of expressing luciferase stably ^LUCThe application of cell strain is characterized in that the 9L of the expressing luciferase stably that will make up ^LUCCell strain is implanted into can set up different glioma animal models in the different animal bodies, these animal models can be applicable to the medicine screening of glioma.

3,9L according to claim 2 ^LUCThe application of cell strain is characterized in that using 9L ^LUCThe glioma animal model that cell strain makes up can be used for the screening of different treatment plans.

4,9L according to claim 2 ^LUCThe application of cell strain is characterized in that using 9L ^LUCThe glioma animal model that cell strain makes up can be used for the generation development mechanism research of glioma.

5, according to claim 2 or 3 or 4 described 9L ^LUCThe application of cell strain is characterized in that used animal comprises rodents such as the rat, nude mice of different strains.

6, according to claim 2 or 3 or 4 or 5 described 9L ^LUCThe application of cell strain is characterized in that used different strain rats comprise Wister, Fischer etc.

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