KR100888343B1 - Hepatocellular carcinoma cell line expressing dual reporter genes including sodium iodide symporter and enhanced green fluorescence protein - Google Patents

Abstract

The present invention relates to a hepatocellular carcinoma cell line transformed with a plasmid containing a HepG2 cell by using a retroviral expression system and a plasmid comprising a sodium oxo co-transporter gene and a green fluorescent gene. Using as a monitoring gene, the effect of radionuclide treatment with I-131, Re-186, Re-188, etc. in the cells into which the sodium oxo cotransporter gene is introduced can be evaluated.

Liver cancer cell line, sodium oxo cotransporter, green fluorescent gene, reporter gene

Description

Hepatocellular carcinoma cell line expressing dual reporter genes including sodium iodide symporter and enhanced green fluorescence protein}

1 is an expression analysis of the NIS gene in liver cancer cell line according to an embodiment of the present invention (M: size marker, P: pRetroQ-PNRGW plasmid, 1: HepG2, 2: HepG2-NE),

Figure 2 is the expression analysis of EGFP gene in liver cancer cell line according to an embodiment of the present invention (A, C: bright field, 50x; B, D: fluorescence, 100x),

Figure 3 shows the radioiodine intake rate in the liver cancer cell line according to an embodiment of the present invention (□: HepG2 without perchlorate, ■: HepG2-NE without perchlorate, ▲: HepG2 with 1 mmol / L perchlorate, X: 1 mmol / HepG2-NE with L perchlorate),

Figure 4 shows the radioactive oxo outflow rate in liver cancer cell line according to an embodiment of the present invention,

5 shows image data measured using a gamma camera and a fluorescence camera of a nude mouse injected with a liver cancer cell line according to an embodiment of the present invention (A: white light image, ↗ (HepG2), ▷ (HepG2-NE) ; B: fluorescent image shown at transplanted place by HepG2-NE; C: gamma camera image of I-123; D: microPET image of I- 124, Thy: thyroid, St: stomach, Bld: bladder),

Figure 6 shows a cleavage map of the plasmid pRetroQ-PNRGW according to an embodiment of the present invention.

The present invention relates to a liver cancer cell line expressing a double reporter gene consisting of a sodium oxo cotransporter gene and a green fluorescent gene.

Hepatocellular carcinoma is the third most common in males and seventh in females in Korea and can be removed surgically if detected early. However, in advanced liver cancer, surgery, arterial embolization, ethanol injection, radiofrequency treatment, percutaneous isotope injection, etc. can be used, but it is difficult to cure. In liver cancer, immunotherapy, gene therapy, and the like have been attempted similarly to malignant tumors with other poor prognosis.

Recently, in order to treat undifferentiated thyroid cancer cells or various intractable malignancies, a method of introducing an internal radiation therapy in which a sodium iodide symporter (NIS) gene is introduced and injecting therapeutic radionuclides has been attempted.

NIS is the way in which two sodium ions enter the cell membrane and together the oxo ions enter the cell, where oxo is ingested for thyroid hormone synthesis. Carrasco et al. Published their research on the production of recombinant recombinant NIS (rNIS), and many researchers are interested in the basic and clinical applications.

Molecular imaging has the advantage of being able to image the phenomena occurring at the cellular level in the body continuously over time without sacrificing the animal's underlying phenomena or mechanisms that regulate the living body.

Introduction of molecular imaging techniques to transfer genes into living organisms, expression of endogenous genes, tracking of intracellularly administered cells, signaling, apoptosis, neovascularization of tumor tissues, and interaction between proteins and proteins Nuclear medical molecular imaging techniques using gamma cameras and PET are not only the first but also widely used methods.

Optical molecular imaging techniques using luminescent or fluorescent materials can non-invasively image tumor development, tumor growth and metastasis, and evaluation of therapeutic effects at the cellular level after using any treatment method in the field of oncology. In addition, studies using luminescence or fluorescence imaging in cells and small animals cultured with the development of in vivo optical imaging devices are used in various fields.

Recently, molecular imaging research techniques using magnetic resonance imaging (MRI) have also been actively developed. Because MRI has high spatial resolution, non-invasive continuous imaging of tumor-grafted models is possible.

In order to easily track the transgenic cells injected or inoculated in vivo by molecular imaging techniques, the import of reporter genes with well-known expression characteristics is required. Reporter genes used for nuclear medical imaging include the mutant dopamine D2 receptor (D2R) and HSV1-tk (Herpes simplex), which use substrates of thymidine kinase enzymes involved in cell proliferation as tracers. virus 1-thymidine kinase gene and NIS gene, etc., and green fluorescence gene (GFP) and luciferase gene are used for optical imaging.

NIS-transfected cells express NIS protein in the cell membrane, and NIS-expressing cancer cells and cardiomyocytes induce radioiodine intake in cells and in vitro or in small animal imaging studies.

Since NIS gene-induced cancer cells selectively ingest radioiodine, internal radiation therapy using Re-186 and Re-188, which have similar chemical properties, may be possible in addition to I-131, which is currently used in the treatment of thyroid cancer.

However, the problem of rapid release of radioiodine ingested into cancer cells, which has been shown in many studies, is a problem to be solved for clinical use.

Each molecular imaging technique has unique advantages, but it also has inherent weaknesses that are not available in some fields, making it difficult to apply to all processes from basic to clinical research in one particular method.

Therefore, it is necessary to develop a complex molecular imaging technique that can overcome the shortcomings of one technique with another or perform two or several functions at once. The use of such a composite image reporter can be applied not only to cell-level laboratory research but also to a wide range of direct imaging studies of large animals including humans.

Even in liver cancer that does not respond to the treatments used so far, the effect of NIS gene insertion and radionuclide treatment would be highly effective. Especially, the dual reporter gene imaging technique that simultaneously expresses reporter genes used for optical molecular imaging will lead to liver cancer. It may be widely used for the progression, improvement of the treatment, and follow-up after treatment.

Therefore, the present inventors have used a retroviral vector in a liver cancer cell to carry out a cell line having a double reporter gene which simultaneously introduces an NIS gene which can be used as a therapeutic gene and an EGFP (enhanced GFP) gene for optical imaging. After the transfection of the double reporter gene into the mouse, nuclear imaging and optical images were obtained at the same time to confirm the successful in vivo imaging of the double reporter gene.

Accordingly, an object of the present invention is to provide a recombinant vector comprising a double reporter gene which is simultaneously introduced into the NIS gene for liver cancer treatment and EGFP (enhanced GFP) gene for optical imaging.

In addition, another object of the present invention is to provide a liver cancer cell line transformed with a recombination vector comprising a double reporter gene which simultaneously introduces an NIS gene for liver cancer treatment and an EGFP (enhanced GFP) gene for optical imaging. .

In order to achieve the above object, the present invention provides a liver cancer cell line transformed HepG2 cells with a plasmid containing a sodium oxo co-transporter gene and a green fluorescent gene using a retrovirus expression system.

The plasmid is plasmid pRetroQ-PNRGW having a cleavage map of Figure 6, the liver cancer cell line is preferably HepG2-NE (Accession Number: KCTC11125BP).

The present invention also provides a plasmid pRetroQ-PNRGW comprising a sodium oxo cotransporter gene and a green fluorescent gene, with the cleavage map of FIG. 6.

Hereinafter, the present invention will be described in more detail.

The method of fusion of two genes can be combined by IRES, by separating two genes by bilateral promoter, by placing two genes at different sites in the same vector, or by fusion of one function. In this case, there is a method of co-introducing viral vectors of the same titer.

In the present invention, a plasmid was prepared by introducing two genes into the same vector using a retrovirus.

More specifically, it was produced in the following manner.

Treatment of restriction enzymes of HindIII and Sal I from pRetro-CNRGW vector, followed by electrophoresis and DNA purification, NIS gene, RSV (Rous sarcoma virus) promoter, EGFP (enhanced green fluorescence protein) gene and WPRE (Woodchuck hepatitis virus post) Transcriptional regulatory element fragments were isolated and introduced into the pGEM-11Zf vector treated with the same restriction enzyme to construct a pGEM-11Zf-NRGW vector.

The vector was further treated with HindIII and Sac I to separate NIS, RSV promoter, EGFP and WPRE fragments, and then introduced into pGEM-7Zf-PGK vector, which was cloned with the same restriction enzyme-treated PGK (phosphoglycerate kinase gene) promoter. Prepare a -7Zf-PNRGW vector.

Finally, the pGEM-7Zf-PNRGW vector was processed as Xba I, klenow and Sal I in order to obtain the PGK promoter, NIS, RSV promoter, EGFP and WPRE fragments, and then introduced as pSIREN-RetroQ vector which processed Bgl II, klenow and Sal I in order. This constructs the pRetroQ-PNRGW vector.

In addition, another method for producing the plasmid pRetroQ-PNRGW of the present invention is to isolate the hNIS gene from pcDNA-NIS to make a pRetro-PN vector expressed by the PGK promoter, and then pLNRGW (LTR-NeoR-RSV-EGFP-WPRE). The RSV-EGFP-WPRE fragment can be isolated to construct a pRetroQ-PNRGW vector that can finally express both NIS and EGFP genes.

In addition, the present invention produces a RetroQ-PNRGW retrovirus using the constructed plasmid, and infects HepG2 cells to produce HepG2-NE liver cancer cell line (Accession Number: KCTC11125BP).

The expression of the NIS gene of the hepatic cancer cell line was confirmed by mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR), and the expression of the EGFP gene was confirmed by observing the green fluorescence expressed by the EGFP protein through a fluorescent microscope.

As shown in FIGS. 1 and 2, NIS was expressed through RT-PCR, and expression of EGFP was confirmed by fluorescence microscopy, thereby successfully creating a HepG2 cell line having a double reporter gene of NIS and EGFP.

The function of the NIS gene among the reporter genes was confirmed by measuring the intake and outflow amount of radioiodine in the cells, wherein the radioiodine intake of NIS-expressing cells was about 9 times higher than in the control group as shown in FIG. In the radioactive oxo outflow experiment, it was confirmed that about half of oxo was leaked in about 9 minutes as shown in FIG. 4.

The cells thus obtained were transplanted into nude mice to obtain fluorescence images, gamma camera images using I-123, and PET images for small animals using I-124. As a result, transplanted hepatocellular carcinoma cell lines constructed as shown in FIG. In the obtained fluorescence image, gamma camera and small animal PET image, a clear fluorescence signal was observed and higher radioiodine intake was observed as compared with the transplantation of control cells on the opposite side.

Liver cancer cell line according to the present invention, which can be expressed by NIS and GFP reporter gene at the same time and can be combined molecular imaging technique, can be evaluated by nuclear medicine or optical method for in vitro cell experiments. It can be used as an imaging gene, and the NIS gene can be used as a therapeutic gene used for treatment with I-131.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 pRetroQ-PNRGW Vector Preparation

NIS gene, RSV promoter, EGFP gene and WPRE fragments were isolated by treating Hind and Sal restriction enzymes to pRetro-CNRGW vector and introduced into pGEM-11Zf vector to construct pGEM-11Zf-NRGW vector. It was.

In addition, Hind and Sac were re-isolated, and then introduced into the pGEM-7Zf-PKG vector where the PGK promoter was cloned, followed by Xba, klenow and Sal in order to process the PGK promoter, NIS, RSV promoter, EGFP, and WPRE. A fragment was obtained.

The pRetroQ-PNRGW vector was constructed by introducing the separated ones into the pSIREN-RetroQ vector, and the cleavage map is shown in FIG. 6.

[ Example  2] HepG2 - NE  Cell fabrication

Human liver cancer cell line HepG2 is a minimum essential medium α (MEM-α, GibcoBRL Co) supplemented with 10% fetal bovine serum (FBS, GibcoBRL Co., USA), 1% anitibiotic-antimycotic (GibcoBRL Co., USA). , USA).

Retrovirus packaging cell lines GP2-293 cells (clontech, USA) were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% FBS and 1% antibiotic-antimycotic. Each cell line was cultured in an incubator at 37 ℃, 5% CO ₂ conditions.

In order to harvest the virus, 10 μg of pRetroQ-PNRGW and pHCMV-G plasmid DNA, respectively, were used in a 1 × 10 ⁶ GP2-293 cell using a calcium phosphate transfection kit (Invitrogen, USA) to provide temporary co-operation. Co-transfection was performed.

After 8 hours of incubation at 37 ° C. and 5% CO ₂ , washed twice with PBS, exchanged with fresh culture medium, and harvested retrovirus after 48 hours.

The produced retrovirus was infected with HepG2 cells by the addition of 5 μg / ml polybrene, and each colony was inoculated into a 24 well plate to select colonies with superior GFP expression. Selected colonies were passaged to secure sufficient cell numbers and used in the next experiment. The selected HepG2 cells were named HepG2-NE cells (Accession Number: KCTC11125BP).

Example 3 Expression Analysis of NIS and EGFP Genes

Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to confirm the expression of NIS genes in HepG2-NE cells. First, total RNA was extracted from the established cell lines using the Trizol (Invitrogen, USA) method, followed by RT-PCR using the Access RT-PCR System (Promega, USA).

The reverse strand reaction was performed at 48 ° C. for 45 minutes to synthesize first strand cDNA, followed by denaturation at 94 ° C. for 2 minutes, and then PCR reactions were performed at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute.

The primers of the NIS gene used for reverse transcriptase polymerase chain reaction were upstream primers (SEQ ID NO: 1; 5'-CCTTCTACACGGCTGTGGG-3 ') and downstream primers (SEQ ID NO: 2; 5'-TGGATGCTGTGCTGAGGGTG-3'), which were used as positive controls. GAPDH (Glyceraldehyde 3 phosphate dehydrogenase) primers were upstream primers (SEQ ID NO: 3; 5'-AAATCAAGTGGGGCGATGCT-3 ') and downstream primers (SEQ ID NO: 4; 5'-AGCTTCCCGTTCAGCTCAGG-3').

As a result, as shown in Figure 1, it was confirmed that the NIS gene is expressed in HepG2-NE cells, HepG2 cells without the pRetroQ-PNRGW gene was not expressed NIS.

In addition, as shown in FIG. 2, HepG2-NE cells produced after transfection were identified by fluorescence microscopy, and as a result, more than 50% of the cells fluoresced.

Example 4 Measurement of Radioiodine Intake Rate

To measure hourly radioiodine uptake, 1 × 10 ⁵ HepG2 and HepG2-NE cells were inoculated into a well in a 24 well plate. After incubation for 24 hours, 0.5 ml of Hanks balanced salt solution (HBSS, Invitrogen, USA) containing 3.7 kBq / 0.5 ml of radioiodine (I-125) and 10 mM sodium iodide (NaI) was added to each well.

Incubated for 5, 10, 15, 30, 45, 60, 90, 120 minutes in a 5% CO ₂ , 37 ℃ incubator. Each time, the cells were washed twice with cold 2 ml HBSS and lysed with 2% SDS, and then radioactivity ingested into cells was measured with a gamma ray meter (Packard, IL, USA).

BCA protein quantification kit (Pierce Co., USA) was used to measure the protein amount of each ingested cell.

The function of the NIS gene was evaluated by adding 50 mM KClO ₄ to determine the intake rate of I-125 and inhibiting the intake of oxo by ClO ₄ ⁻ . The intake rate of radioactive oxo was evaluated in terms of pmol / mg protein corrected by the amount of membrane protein.

The result is as shown in FIG.

The radioiodine uptake of HepG2-NE cells was 130 pmol / mg protein at 10 minutes and 207 pmol / mg protein at 30 minutes, and the highest intake at 120 minutes was 248 pmol / mg protein.

The intake rate of HepG2 cells was 23.4 pmol / mg protein, and the intake rate of HepG2-NE cells was about 9 times higher than that of HepG2 cells. When HepG2-NE cells were treated with ClO ₄ ⁻ , the radioisotope uptake of 30 minutes was reduced to about 19 pmol / mg protein.

Example 5 Radioiodine Outflow Measurement

The amount of radioactive iodine ingested from HepG2-NE cells into which the NIS gene was introduced was measured.

HBSS containing 7.4 kBq / mL I-125 and 10 mM NaI was added and incubated for 30 minutes at 37 ° C. After washing twice with cold HBSS, HBSS was added and 0, 3 HBSS was recovered at 6, 9, 12, 15, 21, and 27 minutes, and radioactivity of the radioactive oxo leaked from the recovered HBSS at each time was measured by a gamma ray meter (Packard, IL, USA).

The measured radioactive oxo was evaluated in terms of percentage of dose.

As a result, as shown in FIG. 4, 22% of oxo was leaked in HepG2-NE cells after 30 minutes of radioactive oxo intake, and 36.7% in 15 minutes of oxo stored in the cells. , 27 minutes, 9.4%. The oxo outflow half-life T _1/2 in the cells was 9 minutes.

Example 6 Animal Experiment

Nude mice (Balb / c nu / nu, male, 5 weeks old) were used, and all animals were bred normally in accordance with the ethical guidelines for the use of experimental animals in the animal laboratory of Kyungpook National University Hospital.

HepG2 hepatocellular carcinoma cells and HepG2-NE cells into which NIS and GFP were introduced were made into 3 × 10 ⁷ and 6 × 10 ⁷ cells, and then injected subcutaneously into the shoulders and thighs of nude mice to form a mass. Animals used for small animal PET imaging were injected with 6 × 10 ⁷ cells only in the thigh.

In order to measure the expression of NIS protein of cells transplanted in vivo with gamma camera and small animal PET images, I-123 5.6 MBq was injected intraperitoneally to obtain gamma camera images. I-124 (12.95 MBq) was injected through the tail vein.

Two hours after radioisotope injection, whole body images were obtained using a gamma camera equipped with a needle hole aimer (Basicam, Siemens, USA) and a small animal PET (Concorde MicroPET R4, Concorde Microsystems Inc., Knoxville, Tennessee).

In addition, in order to measure the image of EGFP, 16 hours after cell transplantation, an image was acquired by using a fluorescence camera (KODAK imaging station, Japan). Gamma and fluorescence camera images were obtained using pentobarbital (2 mg / 200 μl saline), and PET images for small animals were obtained under anesthesia using isoflurane.

The result is as shown in FIG.

That is, HepG2 cells showed no fluorescence signal in the fluorescence camera image, whereas HepG2-NE cells showed a clear fluorescence signal. The shoulder tumor graft site, which had fewer cells transplanted, had a smaller fluorescence signal than the femoral area where more cells were inserted.

In addition, in the gamma camera and small animal PET images, the HepG2-NE cell injection site where the NIS gene was introduced showed a higher concentration of radiotracer than the HepG2 cell injection site.

As a result of comparing the total radioactivity of the region of interest in the PET image for small animals, the HepG2-NE cell injection site showed 10-fold difference of 10 KBq (274 nCi) and HepG2 cell injection site of 1 KBq (27 nCi).

From these results, it was confirmed that the NIS gene is also active in the body.

As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

As described above, the hepatocellular carcinoma cell line according to the present invention can increase the therapeutic effect by introducing the NIS gene and performing radionuclide treatment in liver cancer that does not respond to the therapy used so far, and in particular, a reporter used for optical molecular imaging By using a dual reporter gene imaging technique that simultaneously expresses genes, it can be widely used for progression, improvement and follow-up of liver cancer.

<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Hepatocellular carcinoma cell line expressing dual reporter gene          including sodium iodide symporter and enhanced green fluorescence          protein <130> DP20070044 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for NIS gene <400> 1 ccttctacac ggctgtggg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for NIS gene <400> 2 tggatgctgt gctgagggtg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for GAPDH <400> 3 aaatcaagtg gggcgatgct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for GAPDH <400> 4 agcttcccgt tcagctcagg 20  

Claims (4)

delete In a hepatocellular carcinoma cell line in which HepG2 cells were transformed with a plasmid containing a sodium oxo cotransporter gene and a green fluorescent gene using a retrovirus expression system, the plasmid is plasmid pRetroQ-PNRGW having a cleavage map of FIG. 6. Liver cancer cell line. In a hepatocarcinoma cell line in which a HepG2 cell is transformed with a plasmid containing a sodium oxo cotransporter gene and a green fluorescent gene using a retrovirus expression system, the hepatocarcinoma cell line is HepG2-NE (Accession Number: KCTC11125BP). Liver cancer cell line. A plasmid pRetroQ-PNRGW containing a sodium oxo cotransporter gene and a green fluorescent gene, having the cleavage map of FIG. 6.

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