KR101242279B1 - Culturing method of perpuetal hepatocyte cell line from rodents - Google Patents

Abstract

The method of culturing rodent permanent hepatocytes of the present invention utilizes the regenerative ability of the hepatocytes and can permanently cultivate rodent hepatocytes without producing immortalized hepatocytes through conventional transformation or using hepatocytes with tumors. have.
This makes it possible to cultivate hepatocytes in vivo and then cultivate the hepatocytes to obtain a cell line that can be cultured permanently while maintaining the unique characteristics of the hepatocytes.

Description

Culturing method of perpuetal hepatocyte cell line from rodents

The present invention relates to a method for culturing permanent hepatocytes of rodents, and more particularly, to a permanent rodent cell line of rodents that can be permanently cultured without using hepatocytes having tumors or producing immortalized hepatocytes through conventional transformation. It is to provide a culture method.

The human liver has several 500 complex functions, including liver protein, metabolism, sugar metabolism, lipid metabolism, detoxification, and synthesis of bile salts and bile pigments. It is a versatile organ. Thus, unlike the heart or kidney, a simple substitute with a pump or dialysis membrane cannot replace its function (Mito M, Artificial Organs, 10: 214-218 (1986)). On the other hand, the liver is one of the only organs that can be reproduced in our body. The liver is an organ that is almost at rest in terms of cell proliferation despite many metabolic burdens. Only 0.0012% to 0.01% of hepatocytes develop mitosis. However, a large amount of hepatocellular proliferation occurs in a short time for the recovery of functional liver after toxic liver damage and surgical resection. It is also known that hepatocytes are not limited in their ability to replicate.

Meanwhile, 205,000 patients of liver metabolism, cirrhosis, and cancer occurred in the United States alone (Leigh JP, et al., Arch Intern Med, 161: 2231-2237 (2001)). Medical expenses are estimated at about $ 9 billion. Recent advances in organ transplantation have resulted in high survival rates for patients undergoing liver transplantation, but according to the United Network for Organ Sharing (Scientific Registry) data, about 10% of people in the U.S. As only patients are receiving donated livers, the number of liver transplant recipients increases rapidly each year, and the number of patients who die in the air is increasing rapidly.

Therefore, treatments such as hemofiltration, hemodialysis, hemofusion, and plasma exchange, which focus only on the removal or dilution of toxic substances, increase the patient's ultimate survival rate. Failed. Therefore, studying the liver, a very important organ for humans, is essential for the survival of humankind.

On the other hand, hepatocytes primarily cultured for the study of hepatocytes are used for short-term studies within two weeks because the intrinsic function of hepatocytes disappears quickly, but there is a limit in observing the long-term effects. In addition, the study of cell transplantation with the hepatocytes, but limited liver cells from a single liver is ultimately limited to use for the treatment of diseases by performing hepatocyte transplantation to several individuals with a small number of hepatocytes. Conventional hepatocytes were cultured in liver cancer cells, infected with normal hepatocytes with viral oncogen, SV40 T antigen, or infected catatytic subunits of telomerase to prepare immortalized cells. In addition, AML12, a mouse hepatocyte cell line currently used in the market, prepared an immortalized cell line by culturing hepatocytes in a mouse transformed from mouse TGF-a to human TGF-a. However, hepatic cancer cell lines are not known to maintain complete normal hepatic metabolism. In addition, other immortalized cell lines have limited but hepatic cell-specific functions and are used for liver function studies such as drug metabolism and hepatotoxicity test with hepatocyte-specific protein synthesis, but limitations in cell transplantation studies due to viral oncogen infection or transformation Have

Furthermore, an indirect culturing method for culturing hepatocytes and then differentiating them into hepatocytes for culturing hepatocytes has been proposed. However, the indirect differentiation method not only has to go through complicated steps but also causes a problem that the differentiation and survival rate into hepatocytes is significantly lower than when the hepatocytes are directly cultured.

The present invention has been made to solve the above problems, the first problem to be solved of the present invention by using the regeneration capacity of the hepatocytes without the production of immortalized hepatocytes through conventional transformation, etc. To provide a permanent liver cell line culture method of rodents that can be permanently cultured.

The second problem to be solved of the present invention is to provide a permanent hepatocyte line which is cultured by the culturing method of the present invention and is expressed in a protein that is characteristically expressed in hepatocytes.

In order to achieve the first object of the present invention, (1) supplying a liver injury inducing agent to rodents in order to damage the liver of rodents, (2) extracting some or all of the liver from the rodents with liver damage and separating the liver cells And (3) culturing the isolated hepatocytes.

According to a preferred embodiment of the present invention, the rodent may be any one of rat, mouse and guinea pig.

According to another preferred embodiment of the present invention, the hepatic damage inducing agent is selected from the group consisting of 3,5-diethoxycarbonyl-1,4-dihydrocolidine (DDC), CCl ₄ and thioacetamide It can be any one or more compounds.

According to another preferred embodiment of the present invention, the supply of step (1) may be performed for 1 to 8 weeks.

According to another preferred embodiment of the present invention, the feed may include 0.01 to 10% by weight of the liver damage inducing agent.

According to another preferred embodiment of the present invention, the liver damage inducer may be supplied 1 to 100 mg / day.

According to another preferred embodiment of the present invention, the liver extraction method of step (2) can be extracted liver by a surgical method.

According to another preferred embodiment of the present invention, the step (2) is; (Iii) crushing some or all of the extracted hepatocytes; and (ii) centrifuging the crushed hepatocytes to obtain a supernatant.

According to another preferred embodiment of the present invention, the isolated hepatocytes of step (3) may be hepatic parenchymal cells.

According to another preferred embodiment of the present invention, the step (3) is a) culturing the isolated hepatocytes in a cell proliferation medium, b) culturing the hepatocytes cultured in the cell proliferation medium in a maintenance medium And c) passaging the cultured hepatocytes.

According to another preferred embodiment of the present invention, the cell proliferation medium and the cell maintenance medium is DMEM (Dulbecco's Modified Eagle Medium) medium, RPMI 1640 medium, William medium E (Williams' medium E), IMDM (Iscove's Modified Dulbecco's Modification) ) Medium or DMEM / Ham's F-12 mixed medium.

According to another preferred embodiment of the present invention, the cell proliferation medium may comprise a cell growth factor.

According to another preferred embodiment of the present invention, the growth factor IGF-1 (insuline like growth facter-1), hepatocyte growth factor (HGF) and fibroblast growth factor-4 (fibroblast growth factor-4) 4, FGF-4) may be any one or more growth factors selected from the group consisting of.

According to another preferred embodiment of the present invention, there is provided a method of culturing permanent hepatocytes of rodents comprising the step of applying liver damage to rodents for the culture of permanent hepatocytes, in which case the liver damage is a physical stimulation or chemical stimulation May occur.

The present invention provides a permanent hepatocyte line of rodents cultured by the above-described culture method of the present invention to achieve the second object.

According to another preferred embodiment of the present invention, the permanent hepatocyte cell line (albumin), α1-antitrypsin (A1AT), tyrosine aminotransferase (TAT), glucose-6-phosphatase ( Glucose-6-phosphatase (G6P), Hepatocyte Nuclear Factor (HNF), HNF1, HNF3, HNF4, E-cadherin and N-cadherin can be expressed any one or more mRNAs or proteins selected from the group consisting of.

Hereinafter, terms used in this specification will be briefly described.

The term 'liver injury inducer' refers to a drug that chemically affects the liver of a rodent, resulting in a decrease in liver function.

The term "cell proliferation medium" is defined as a medium in which cell growth factors are included in a medium for growing animal cells commonly used in the art.

The term "cell maintenance medium" is defined as a medium in which cell growth factors are not included in a medium for growing animal cells commonly used in the art.

The method of culturing rodent permanent hepatocytes of the present invention utilizes the regenerative ability of the hepatocytes and can permanently cultivate rodent hepatocytes without producing immortalized hepatocytes through conventional transformation or using hepatocytes with tumors. have.

This makes it possible to cultivate hepatocytes in vivo and then cultivate the hepatocytes to obtain a cell line that can be permanently cultured while maintaining its unique characteristics.

In addition, the cell line of the present invention cultures cells that exhibit regenerative ability by physiological mechanisms in vivo, and thus continuously secretes physiological, natural, and hepatocyte specific proteins as compared to known immortalized hepatocytes. In addition, the culturing method has the advantage that it can be cultured in a culture medium not treated with feeder layer or gelatin and does not require additional treatment of immortalization treatment after the culture. In addition, since the cell line can be continuously cultured, it can be usefully used for drug reaction and toxicity test of hepatocytes, and there is an advantage that can be used for hepatocyte transplantation of rodents.

1 is a phase contrast micrograph of an AML12 cell line and a permanent hepatocyte line (PMH08) prepared according to a preferred embodiment of the present invention.
Figure 2 is a transmission electron micrograph of a permanent hepatocyte cell line (PMH08) prepared according to an embodiment of the present invention.
Figure 3 is an electrophoresis picture showing the mRNA expression analysis after performing RT-PCR for a permanent hepatocyte line (PMH08) prepared according to an embodiment of the present invention.
4 and 5 are electrophoresis pictures showing the expression analysis for a specific protein of the permanent hepatocytes (PMH08) prepared according to a preferred embodiment of the present invention.
Figure 6 is an immunofluorescence staining of permanent hepatocytes (PMH08) prepared in accordance with a preferred embodiment of the present invention and the positive control group humman hepatocellular carcinoma cell line (HpG2) and negative control group MC3T3-E1 (mouse preosteoblast cell line) to be.
7 is a soft agar culture photograph of the permanent hepatocyte line (PMH08) and Hep3b (humman hepatocellular carcinoma cell line) prepared according to an embodiment of the present invention.
8 is a nude mouse injection picture of the permanent hepatocyte line (PMH08) and Hep3b (humman hepatocellular carcinoma cell line) prepared according to an embodiment of the present invention.

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

Cell lines immortalized through infection or transformation by a conventional virus as described above have many limitations in experiments.

Furthermore, an indirect culturing method for culturing hepatocytes and then differentiating them into hepatocytes for culturing hepatocytes has been proposed. However, the indirect differentiation method not only has to go through complicated steps but also causes a problem that the differentiation and survival rate into hepatocytes is significantly lower than when the hepatocytes are directly cultured.

Therefore, the present inventors applied the excellent regenerative properties of the liver unlike other organs to induce liver regeneration by applying physical or chemical liver damage to the liver, and then cultured the liver cells to develop a permanent liver cell line culture method of rodents. Through this, it is possible to permanently culture rodent hepatocytes without using hepatocytes that have been produced or immortalized hepatocytes through conventional transformation.

To this end, the permanent hepatocyte cell culture method of rodents according to one aspect of the present invention comprises the steps of (1) supplying a liver injury inducing agent to rodents, (2) extracting some or all of the liver from rodents fed the feed, Separating, and (3) culturing the isolated hepatocytes.

First, the liver damage inducing agent is supplied to rodents as step (1). Preferably, rodents that can be used in the present invention can be used without limitation as long as it can be used for liver cell culture, more preferably may be any one of rat, mouse and guinea pig have.

According to a preferred embodiment of the present invention, the liver injury inducing agent may be used without limitation of the type so long as it can damage the liver of the rodents without feeding the rodents and killing the rodents. May be any one or more compounds selected from the group consisting of 3,5-diethoxycarbonyl-1,4-dihydrocolidine (DDC), CCl ₄ and thioacetamide, more preferably 3,5 -Diethoxycarbonyl-1,4-dihydrocolidine (DDC).

On the other hand, the liver damage inducing agent of the present invention should be supplied to live rodents, and when the liver is removed from the rodents without liver damage and cultured, permanent cell lines cannot be cultured. This indicates that mature hepatocytes in normal liver without liver damage are mostly in the G0 state of the cell cycle and only 0.0012% -0.01% participate in mitosis. In addition, mature hepatocytes are related to the characteristic morphology of mesothelial cells (stromal cells, endothelial cells, and squamous muscle cells) in the periphery of the hepatocytes by the epithelial-mesothelial cell correlation. Thus, the pharmacokinetic correlation between them is controlled by soluble signals as well as insoluble signals. Furthermore, endocrine (systemic) regulation and regulation are frequently made by the regulation of certain parts of the epithelial-mesenchymal cell correlation. As a result, if normal hepatocytes are not co-cultured with the appropriate mesothelial cells, they will not be able to maintain their proper function for long periods of time.

According to a preferred embodiment of the present invention, the method for supplying the liver damage inducer is a method of feeding the liver damage inducer to the rodents for a certain period of time (for example, DDC), injecting a rodent injectable containing a liver injury inducer It can be supplied in a variety of ways depending on the type and nature of the liver damage inducing agent, such as the method (for example, CCl ₄ ), the method of mixing with water (for example thioacetamide), but is not limited thereto.

In the case of feeding through the feed, the feed is a general term for the food to be fed to rodents, and if the feed for rodents and / or vegetables, grains, etc., which is generally used is limited to the type of food Can be used without In this case, preferably, the amount of the liver injury inducing agent added to the feed may be added in an appropriate amount depending on the size and type of rodent if the rodent is not lethal and can damage the rodent's liver. The feed may contain 0.01 to 10% by weight of liver injury inducing agent.

If the amount of the liver damage inducing agent added to the feed is less than 0.01% by weight, it is difficult to achieve the object of the invention because the effect of liver damage is insignificant. have.

In this case, preferably 1 to 100 mg / day of liver injury inducer for the rodents. According to a preferred embodiment of the present invention, the feed may be performed for 1 to 8 weeks. On the other hand, when injected into the injection can be injected at 0.01 ~ 10mg / time once a day for 1 to 8 weeks.

Next, as a step (2), a part or all of the liver is extracted from the rodent to which the liver injury inducing agent is supplied, and the liver cells are separated. Specifically, according to a preferred embodiment of the present invention, the step (2), (i) crushing some or all of the extracted hepatocytes and (ii) centrifuging the crushed hepatocytes to obtain a supernatant It may include.

First, rodents fed with the liver injury inducer for a certain period of time have liver damage. At this time, a part or all of the liver is extracted from the rodent damaged liver, and in this case, the liver may be extracted by using a surgical method. The extracted liver is crushed to a predetermined size for cell culture, preferably crushed to a size of 0.5mm, crushing means can be used without limitation as long as it can crush the normal biological tissue.

On the other hand, normal liver that does not receive liver damage is not properly cultured even when extracted and crushed. This indicates that mature hepatocytes in normal liver without liver damage are mostly in the G0 state of the cell cycle and only 0.0012% -0.01% participate in mitosis. In addition, mature hepatocytes are related to the characteristic morphology of mesothelial cells (stromal cells, endothelial cells, and squamous muscle cells) in the periphery of the hepatocytes by the epithelial-mesothelial cell correlation. Thus, the pharmacokinetic correlation between them is controlled by soluble signals as well as insoluble signals. Furthermore, endocrine (systemic) regulation and regulation are frequently made by regulation of certain parts of the epithelial-mesenchymal cell correlation. As a result, if normal hepatocytes are not co-cultured with the appropriate mesothelial cells, they will not be able to maintain their proper function for long periods of time.

In contrast, in the present invention, rodents receiving liver damage during survival enter into the cell cycle in the G0 state in response to damage to the liver parenchyma and exert tremendous regenerative capacity, and are insoluble signals and soluble signals by epithelial cell-middle cell correlation. Under the influence of liver cells, livers are extracted and cultured in an activated state, thereby enabling the cultivation of permanent hepatocytes without appropriate co-culture of the mesothelial cells or without immortalization.

According to a preferred embodiment of the present invention, the crushed hepatocytes may be centrifuged to obtain a lower supernatant. Specifically, when the crushed hepatocytes are centrifuged, hepatic parenchyma cells are formed in the lower layer of centrifugation, and non-stromal cells are formed in the upper layer. In this case, since most hepatic constituent cells constitute non-epileptic cells and hepatocytes make up hepatic parenchymal cells, a hepatic parenchymal cell-integrated supernatant must be obtained to obtain a permanent hepatocyte cell line without a complicated process such as a differentiation process. Can be cultured. Therefore, the isolated hepatocytes of step (3) may be hepatic parenchymal cells.

Next, the step (3) comprises culturing the isolated hepatocytes.

According to a preferred embodiment of the present invention, step (3) comprises the steps of a) culturing the isolated hepatocytes in a cell growth medium, b) culturing the hepatocytes cultured in the cell growth medium in a maintenance medium and c) It may comprise the step of subcultured the cultured hepatocytes.

Specifically, in step a), the isolated hepatocytes are cultured in a cell proliferation medium. Specifically, the step a) serves to fix the isolated hepatocytes in the incubator (culture plate), wherein the cell proliferation medium that can be used is a medium commonly used for culturing animal cells in the animal cell culture art. Means, commercially available medium for animal cell culture, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α-MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc.Exp. Bio.Med , 73: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)) , A mixture of DMEM and F12 (Barnes, D. et al. Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc.Exp. Biol. Med 100: 115 (1959)) and the MCDB series (Ham , RG et al., In Vitro 14:11 (1978)) and the like. Preferably, it is DMEM, F12, a mixture of DMEM and F12 and RPMI1640, and most preferably DMEM / F12 may be used, but is not limited thereto.

In this case, preferably, the cell proliferation medium is insuline like growth facter-1 (IGF-1), hepatocyte growth factor (HGF) and fibroblast growth factor-4 for growth of hepatocytes. , FGF-4) may include any one or more growth factors selected from the group consisting of, more preferably IGF-1.

According to a preferred embodiment of the present invention, the culture in the cell proliferation medium of step a) may be performed for 1 to 4 weeks. If less than one week, primary cultured cells can stably settle and fail to proliferate.If more than four weeks, confluence in the medium is covered with a single cell on the bottom of the incubator. ) May exceed 100%.

Next, as the step b), hepatocytes cultured in the cell proliferation medium are cultured in the maintenance medium. Specifically, step b) is a process in which subcultures are performed, which can be seen as a process that can be proliferated by the release of soluble signal factors from the hepatocytes without providing cell growth factors from the outside. According to a preferred embodiment of the present invention, the medium that can be used at this time may be used a medium commonly used for culturing the animal cells described above, in which case the growth factor that may be included in the cell proliferation medium may not be included. have.

On the other hand, according to a preferred embodiment of the present invention, the culture in the maintenance medium of step b) may be cultured for 4 to 8 weeks, but is not limited thereto.

Hepatocytes cultured in maintenance medium can then be cultured into permanent hepatocytes via conventional subcultures.

On the other hand, the contents of chemical damage to the liver of rodents for the culture of permanent hepatocytes of rodents were described, but the present invention is not limited thereto, and the regeneration of the liver after injuring the liver by applying a physical shock including surgical resection of the rodents It is also possible to use rodents to culture rodent hepatocytes.

According to another feature of the present invention, there is provided a permanent liver cell line of rodents cultured by the above-described culture method of the present invention. In the present invention, rodents receiving liver damage during survival enter tremendous regenerative capacity in the G0 state in response to damage to the liver parenchyma, and exhibit the effects of insoluble and soluble signals due to epithelial cell-middle cell correlation. As liver cells are extracted and cultured while the hepatocytes are activated, it is possible to culture permanent hepatocytes without co-culture of appropriate mesothelial cells or without immortalization.

According to a preferred embodiment of the present invention, the permanent hepatocytes of the present invention have the morphological characteristics of ordinary hepatocytes (see FIGS. 1 and 2), and the permanent hepatocytes are albumin (albumin), α1-1-expressing hepatocytes in general. Antitrypsin (α1-antitrypsin (A1AT), tyrosine aminotransferase (TAT), glucose-6-phosphatase (G6P), Hepatocyte Nuclear Factor (HNF), HNF1, HNF3, HNF4, EN4 Any one or more genes (mRNA) or proteins selected from the group consisting of -cadherin and N-cadherin may be normally expressed (see FIGS. 4 and 5).

According to a preferred embodiment of the present invention, the permanent hepatocytes may be normal hepatocytes, not tumor cells, or even permanent hepatocytes even without undergoing a separate transformation (see FIG. 6).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Induction of Liver Damage through DDC Feed

8-week-old male FVB / n species (CORETECH, Korea) were purchased and fed a normal diet for 2 days, and then 3,5-diethoxycarbonyl, a kind of porphyrinogenic liver damage inducer, was used to induce liver damage. Feeds containing 0.1% of -1,4-dihydrocolidine (DDC; BioServe, Frenchtown, NJ) were fed for three weeks and fed with sufficient water and feed.

Example 2 Liver Extraction and Hepatocyte Separation from Mice

Mice were anesthetized with ketamine and xylene to remove liver from the mice, and then surgically opened via median incision. Insert a 24-gauge intravenous catheter into the inferior vena cava and perfused with 10 ml PBS (contains 0.05% trypsin, no calcium) through the catheter and a two-step collagenase digestion [collagenase mixture; Liver was extracted after perfusion of 0.1% collagenase A + D, 0.1% pronase, 0.01% DNase, and 3 mM CaCl ₂ ] containing PBS solution containing 0.05% trypsin (hereinafter referred to as collagenase solution). The extracted liver was pulverized finely and placed in a collagenase solution to obtain a single cell suspension, followed by agitation for 37 to 30 minutes with vibration, and then passed through a 100um mesh filter. Centrifuge the solution through 50g for 5 minutes using a centrifuge to separate the liver parenchymal cells and non-epileptic cells, wash the hepatic parenchyma cells in the lower layer (lower solution) three times with a culture solution and trypan blue exclusion method. The number of living cells was checked through.

Example 3 Culture of Hepatocytes

Isolated hepatocytes were cultured 5 x 10 ⁶ viable cells per ml in a 60mm culture dish. Culture medium for cell proliferation was DMEM / F12 culture (10% FBS, 1000 ul / L insulin) containing 10 ng / ml dexamethasone and 100 ul / L of IGF-1 at 5% CO ₂ at 37 ° C. Cultures were conducted in collaborative research (ITS), containing transferrin and selenium. After incubation for 16 hours, the cells that did not adhere to the culture dish were discarded with the culture medium and the culture was continued while exchanging for the same culture solution. Cultures were exchanged once every 3-4 days. After the cells settled on the culture plate and started to proliferate, the culture was performed in the same manner as the culture medium for cell proliferation without the addition of IGF-1 in the maintenance medium.

After culturing for one week through the culture solution, the first subculture was performed with 100 mm culture medium using trypsin EDTA and was called passage 1 (p1). The cultures were passaged for various periods for about 2 months until passage 4 (p4), and then, when 80-90% proliferated, they were passaged regularly every 3-4 days to culture permanent hepatocytes derived from mice. (Named 'PMH08') it was passaged up to passage 100 (p100).

Example 4 Confirmation of Hepatocyte Cell Lines by Phase Phase Microscopy

The PMH08 passaged in Example 3 was analyzed using a phase contrast microscope (Axiovert 200) (Carl Zeiss, Gottingen, German).

Figure 1a is a phase contrast micrograph observing the progression from the primary culture to the characteristic hepatocytes. Specifically, a is a photograph after primary culture, b is passage 1 (p1), c is passage 2 (p2), and d is passage 3 (p3), and a hepatocellular polygonal form was observed.

FIG. 1B is a phase contrast micrograph for PMH08 passaged, a and b are images of a conventional AML12 cell line, c, d is a phase difference micrograph of the PMH08 hepatocyte line of the present invention (200x). This resulted in a polygonal shape, a characteristic finding of hepatocytes, and a shape similar to that of AML12 cell line (human TGFa-transgenic mice, ATCC, VA, USA).

Example 5 Identification of Hepatocyte Cell Lines by Transmission Electron Microscopy Analysis

In order to confirm the morphological findings of hepatocytes, hepatocytes of Example 3 were fixed in 2.5% glutaraldehyde / PBS. Ultra-thin slices were made using ultrathin slices, stained with uranyl acetate and lead citrate, and analyzed by transmission electron microscopy (TEM, JEM-1200EXII, JEOL, Japan).

2A and 2B are transmission electron micrographs of PMH08 cell line. Specifically, the mitochondria, the peroxide body, and the bile ducts between the cells were observed in the PMH08 cell line as in general hepatocytes.

Example 6 RT-PCR Analysis

RT-PCR analysis was performed to confirm that the PMH08 cell line expresses proteins that are normally expressed in hepatocytes.

(1) total RNA extraction

Into the PMH08 cell line prepared in Example 3, 1ml of Trizol solution per 10mm medium was treated for 5 minutes at room temperature, scraped the cells with Trizol solution from the medium with a cell scraper, and placed in a dube, followed by 200ul chloroform. After addition, the dive was shaken vigorously. After waiting 3 minutes at room temperature, centrifugation was performed at 12,000g for 15 minutes at 4 ° C. After immersing the supernatant in RNAase free tubes, 0.5 ml of isopropanol was added per 1 ml of Trizol, and the tubes were vigorously shaken. After waiting 10 minutes at room temperature again, centrifugation was performed at 12,000g for 15 minutes at 4 ° C. The supernatant except the pellet (pellet) was discarded and the pellet was washed with 75% ethanol and centrifuged again at 12,000 g for 5 minutes at room temperature. After centrifugation, the supernatant was discarded and the pellet dried. The pellet was dissolved in DEPC treated water and placed at 55 ° C. for 5-10 minutes to obtain total RNA.

(2) cDNA synthesis

The isolated total RNA 2ug, random hexamer (Invitrogen) 1ul, 2.5mM dNTP mixture (Invitrogen) 1ul, 5x RT buffer 2ul, 0.1M dTT 1ul, RNA out 0.4ul and MMLV reverse transcriptase 0.5ul (Invitrogen) is added DEPC treated DW was added to the final volume to 10 ul and stirred. After reacting the cDNA synthesis reaction at room temperature for 10 minutes, the reaction was performed with a thermocycler (Applied Biosystems (GeneAmp PCR System 2700), MJ Research Inc. (PTC-100)) at 42 ° C. for 60 minutes and 95 ° C. for 10 minutes. Was synthesized.

(3) polymerase reaction

The composition of the RT-PCR reactants is 2x Master Mix (Promega) and 0.5ul (10 pmole) of the respective front and back primers. 1ul of synthesized cDNA was added thereto, and the final volume was 12ul. The base sequence of each primer added is shown in [Table 1, 2].

RT-PCR reaction (Applied Biosystems (GeneAmp PCR System 2700), MJ Research Inc. (PTC-100)) was performed once at a denaturation temperature of 94 ° C for 3 minutes, 1 minute at denaturation temperature of 94 ° C, and annealing temperature of 55 ° C. The cycle was repeated 25-35 times for 1 minute at 1 minute, elongation temperature at 72 ° C. and finally the final extension reaction was carried out at 72 ° C. for 10 minutes. RT-PCR products were subjected to electrophoresis on 1.2% agarose gel and measured using LAS-3000 (FujiFilm, Tokyo, Japan).

Gene name Primer sequence Alpha1-antitrypsin (AAT) 5'-AATGGAAGAAGCCATTCGAT-3 '(front) 5'-AAGACTGTAGCTGCTGCAGC-3 '(rear) Albumin (ALB) 5'-CGAGAAGCT TGGAGAATATGG-3 '(front) 5'-GTCAGAGCAGAGAAGCATGG-3 '(rear) Tyrosin Amino Transferase (TAT) 5’-AGCGACTCATGAGCCCTTTA-3 ’(front) 5'-TGTGTCAGCTCGAGGAAATG-3 (Rear) Glucose -6-phosphatase (G6P) 5'-TGCATTCCTGTATGGTAGTGG-3 '(front) 5'-GAATGAGAGCTCTTGGCTGG-3 (Rear) Hepatocyte Nuclear Factor 1 (HNF1) 5'-GTAAGGTCCACGGTGTACGGTA-3 '(forward) 5'-GAGGCTGCTGATAGGAGGGAT-3 '(rear)

Gene name Primer sequence Hepatocyte Nuclear Factor 1 (HNF3b) 5'-ACCTGAGTCCGAGTCTGACC-3 '(front) 5'-GGCA CCTTGAGAAAGCAGTC-3 '(rear) Hepatocyte Nuclear Factor 1 (HNF4a) 5’-ACACGTCCCCATCTG AAG-3 ’(front) 5'-CTTCCTTCTTCATGCCAG-3 '(rear) Tryptophan-2,3-dioxygenase (TO) 5’-GCTCAAGGTGATAGCTCGGA-3 ’(front) 5'-GGAACTCTGCCATCTGTTCC-3 (Rear) E-cadherin 5’-GCCACAGATGATGGTTCACC-3 ’(front) 5'-CTTCATGCAGTTGTTGACCG-3 '(rear) β-actin 5'-GGTGGG AATGGGTCAGAAGG-3 '(front) 5'-AGGAAGAGGATGCGGCAGTG-3 '(rear)

RT-PCR products were analyzed by electrophoresis on 1.2% agarose gel. 3 shows the results of electrophoresis of RT-PCR. As a result, the PMH08 cell line of the present invention is characterized in albumin, a1-antitrypsin (A1AT), tyrosine aminotransferase (TAT), Glucose-6-phosphatase (G6P), Hepatocyte Nuclear Factor (HNF): HNF1, HNF3, HNF4 MRNA was expressed similarly to the AML12 cell line (human TGFa-transgenic mice, ATCC, VA, USA).

Meanwhile, in FIG. 3, lane 1 shows AML12 (human TGFα-trnasgenic mice), lane 2 shows a cell line of the present invention (PHC08 early passage), and lane 3 shows a cell line of the present invention (PHC08 intermediate passage), lanes. 4 is the cell line (PHC08 Late passage) of the present invention, lane 5 is adult liver tissue.

Example 7 Western Blot Evaluation

(1) protein extraction

After washing the PMH08 cell line prepared in Example 3 with cold PBS 2-3 times, the Pro-prep protein extraction solution (1.0mM PMSF, 1.0mM) together with the protease inhibitor and the phosphase-1 inhibitor in 100mm culture vessel. EDTA, 1uM Pepstatin A, 1uM Leupeptin, 1uM Aprotinini, NtRON biotechnology) were treated with 500ul. The cell lysate was left on ice for 15 minutes and then centrifuged at 3000 g at 4 ° C. for 20 minutes to remove cell membrane components. The protein concentration was bovine serum albumin (BSA) as a compatibilizer and Bio-Rad Protein Assay Kit ( Bio-Rad, Hercules, CA, USA).

(2) Western blot

The 15 ug protein isolated by the above method was denatured with 10% mini gel (Bio-Rad, Hercules, CA, USA) SDS-PAGE (poly acrylamide gel electrophoresis), and immun-Blot PVDF membrane (Bio-Rad , Hercules, CA, USA) was electrically transferred to 100 V for 1 hour. The blocking of mambrane was performed for 1 hour at room temperature with TBS-T (TBS, 0.1% Tween 20) solution containing 5% of skim milk. To measure protein expression, primary antibodies were diluted in TBS solution at the ratio of [Table 3, primary antibodies used in Western blot experiments], over-night reaction at 4, and washed three times with TBST. As a secondary antibody, anti-mouse IgG, anti-goat IgG, or anti-rabbit IgG (Assay designs Ann Arbor, MI, USA) combined with horse radish peroxidas (HRP) was diluted 1: 2,500 and reacted at room temperature for 1 hour. . After washing three times with TBS and reacted with Chemiluminescent HRP substrate (Milipore Corporation, Billerica, MA, USA) for 10-60 seconds, it was measured using LAS-3000 (FujiFilm, Tokyo, Japan).

Primary antibody Catalog number Company Dilution a-fetoprotein (AFP) sc-8108 Santa Crize, CA, USA 1: 200 Albumin A90-134A Bethyl, TX, USA 1: 500 β-actin A5441 Sigma, Oi, USA 1: 5,000 Cytokeratin 19  ab15463 abcam, Cambridge, UK 1: 200 E-cadherin # 3195 Cell Signaling, MA, USA 1: 1,000 Glutamine Synthetase 610518 BD Bioscience, NJ, USA 1: 5,000 N-Cadherin  ab18203 abcam, Cambridge, UK 1: 500

4 and 5, albumin and the epithelial proteins E-cadherin and N-cadherin, which are proteins expressed in hepatocytes, were expressed in PMH08 cell line along with AML12 and hepatic progenitor cells. ) And CK-19 expressed in biliary cells were not expressed in PMH08 cell line compared to the control group (HepG2: humman hepatocellular carcinoma cell line, NK Cell Line, seoul, Korea), and expressed in progeniter cells. AFP (alpha-fetoprotein) was also not expressed.

On the other hand, in Figures 4 and 5, lane 1 shows AML12 (human TGFα-trnasgenic mice), lane 2 shows a cell line of the present invention (PHC08 early passage), and lane 3 shows a cell line of the present invention (PHC08 intermediate passage). , Lane 4 is the cell line (PHC08 Late passage) of the present invention, lane 5 is HepG2 hepatocyte line.

Example 8 Immunofluorescence Staining Evaluation

(1) Cell preparation

AML12 cell line (mouse hepatocyte: human TGFa-transgenic mice, ATCC, VA, USA), HepG2 (humman hepatoblastoma cell line, Korea Cell Line Bank, Seoul, Korea) and MC3T3- E1 (mouse preosteoblast cell line, ATCC, VA, USA) was used and the cell lines were cultured in 500 cells per chamber on a chamber slide (Lage Nunc International, IL, USA) with four chambers.

(2) immunofluorescence staining

When the cells proliferated about 70-80%, the culture medium was removed from the chamber slide and washed three times with PBS. After washing it was fixed for 5 minutes with cold 95% ethanol. Again washed three times with PBS and treated with protein block solution (Golden Bribrgr International, WA, USA) for 10 minutes. These cells were diluted in PBS at a ratio of [Table 4, primary antibody used for immunotype and staining] in order to see the fluorescence staining, and washed three times with PBS after over-night reaction at 4 ℃. As a secondary antibody, Rhodamine Red-bound Donkey anti-rabbit IgG or Fluorescin (FITC) -bound Donkey anti-goat IgG (Jackson ImmunoResearch, PA, USA) was diluted 1: 200 and reacted at room temperature for 1 hour. After the secondary antibiotic reaction, the cells were washed three times with PBS. 4 ', 6'-diamidino-2-phenylindole (DAPI,) (Molecular probes, CA, USA) was counterstained at 0.5ug / ml to confirm the location and preservation of the nucleus. Was performed for 3 minutes. Stained cells were washed three times with PBS and observed through a fluorescence microscope (Axioskop 2 plus) (Carl Zeiss, Gottingen, German).

Primary antibody Catalog number Company Dilution a-fetoprotein (AFP) sc-8108 Santa Crize, CA, USA 1: 100 Albumin A90-134A Bethyl, TX, USA 1: 300 Cytokeratin 18 E431-1 Epitomics, CA, USA 1: 100 Cytokeratin 19  ab15463 abcam, Cambridge, UK 1: 200 E-cadherin # 3195 Cell Signaling, MA, USA 1: 100

As can be seen in Figure 6, it was confirmed that the PMH08 cell line expressed in the normal hepatocytes albumin and CK18 are expressed in the same compared to the positive control group (HepG2: humman hepatocellular carcinoma cell line), hepatic progeniter cells and biliary tract CK-19 expressed in the cells was not expressed compared to the control group (HepG2: humman hepatocellular carcinoma cell line), and also did not express AFP (alpha-fetoprotein) expressed in progeniter cells and hepatocellular carcinoma. In addition, all the negative control group (MC3T3-E1: mouse preosteoblast cell line) was not expressed.

Example 9 Cancer Induced Test

In order to confirm whether the PMH08 cell line expresses cancer-causing factors, we performed soft agar assay and nude mouse injection.

(1) Soft agar assay

Hep3b (humman hepatocellular carcinoma cell line, Korea Cell Line Bank, seoul, Korea) was used as the PMH08 cell line and the positive control cell line prepared in Example 3. 1.5 ml of 0.5% base agar was added to 35 mm medium and waited until it solidified at room temperature. Each 5 × 10 ⁴ cells were mixed in 1.5 ml of 0.3% top agar and added for 0.5% base agas solidified in 35 mm medium. The negative control group included no cells, and the positive control group was Hep3b (humman hepatocellular carcinoma cell line, Korea Cell Line Bank, seoul, Korea). Each culture medium was incubated at 37 ° C. in 5% CO ₂ . Cultures were exchanged once every 3-4 days and culture continued for 30 days. After 30 days, colonies were identified by staining with 0.5% crystal violet solution. As shown in FIG. 7, the colony was formed in the positive control group Hep3b, but the colony was not formed in the PMH08 cell line.

7 is a soft agar culture photograph of PMH08 cell line and Hep3b (humman hepatocellular carcinoma cell line), FIG. 7A is Hep3b, FIG. 7B is PM32 cell line of p32, and FIG. 7C is PM128 cell line of p128.

(2) Nude mouse injection

Hep3b (humman hepatocellular carcinoma cell line, Korea Cell Line Bank, seoul, Korea) was used as the PMH08 cell line and the positive control cell line prepared in Example 3. Male nude mice (CD1 nude, Oriental Bio, Seongnam, Korea) were used for 4-5 weeks, and the nude mice were anesthetized by intraperitoneal injection of a mixture of ketamine (Yuhan Corporation) and romfun (Bay Korea) at 100ul per 10g of mouse. Was implemented. The cells to be injected were mixed with 5 x 10 ⁶ cells in 50 μl PBS and 2 x 10 ⁶ cells in the Hep3b cell line, diluted in 50 ul matrigel (BD Biosciences, MA, USA) and then 100 ul per mouse wing. Subcutaneous injection was carried out in the locus. The PMH08 cell line was injected into 5 nude mice and the positive control Hep3b cell line was injected into 4 nude mice. Nude mice were examined twice a week and positive control Hep3b developed tumors at the injection site at 4 weeks, and tumor size was measured at 5 and 10 weeks. One of the positive controls died when the tumor suddenly grew on 5 weeks and 6 days. Nude mice injected with PNH08 cell line did not develop tumors during the observation for 7 months.

Number Note Tumor size at 5 weeks Tumor size at 10 weeks One 10.0 x 9.7 mm Dead 2 7.0 x 7.8 mm 17.3 x 13.5 mm 3 9.0 x 8.7mm 20.0 x 17.5 mm 4 No 12.0 x 13.4mm

FIG. 8A is a nude mouse injection picture of the 8 PMH08 cell line and the positive control group Hep3b (humman hepatocellular carcinoma cell line). FIG. 8A is 7 months after the PMH08 cell line injection, and FIG. 8B is the 10th week of the injection of the positive control Hep3b cell line.

Rodent stem cell culture method of the rodent of the present invention is very useful in the bio-industry because it is possible to permanently cultivate rodent hepatocytes without the production of immortalized hepatocytes through conventional transformation, etc. Can be utilized.

Claims (19)

(1) comprising at least one compound selected from the group consisting of 3,5-diethoxycarbonyl-1,4-dihydrocolidine (DDC) and thioacetamide in rodents for damaging livers of rodents Supplying a liver injury inducer;
(2) extracting some or all of the liver and isolating hepatic parenchymal cells in rodents with liver damage;
(3) disrupting some or all of the extracted hepatic parenchymal cells;
(4) centrifuging the crushed hepatic parenchymal cells to obtain a supernatant;
(5) The isolated hepatic parenchymal cells were obtained by IGF-1 (insuline like growth facter-1), hepatic parenchymal growth factor (HGF) and fibroblast growth factor-4 (fibroblast growth factor-4, FGF- 4) Dulbecco's Modified Eagle Medium (DMEM) medium, RPMI 1640 medium, William's Modified Dulbecco's Modification (IMDM) medium, and any one or more cell growth factors selected from the group consisting of Culturing in any of the cell proliferation medium of DMEM / Ham's F-12 mixed medium;
(6) hepatic parenchymal cells cultured in the cell proliferation medium were DMEM (Dulbecco's Modified Eagle Medium) medium, RPMI 1640 medium, William medium E (Williams' medium E), IMDM (Iscove's Modified Dulbecco's Modification) medium and DMEM / Ham's F -Culturing in any one of the cell maintenance medium of the mixed medium; And
(7) passaging the cultured hepatic parenchymal cells;
Permanent liver cell line culture method of rodent comprising a. The method of claim 1,
The rodent is a permanent rodent cell culture method of a rodent, characterized in that any one of rat (mouse) and mouse (guinea pig). delete The method of claim 1,
Method of culturing permanent hepatocytes of rodents, characterized in that the supply of step (1) is performed for 1 to 8 weeks. The method of claim 1,
The feeding of step (1) is a method of culturing a permanent liver cell line of rodents, characterized in that the method of feeding the liver damage inducer after mixing the feed or water or injecting the liver injury inducer into the rodent. The method of claim 5,
The feed is a permanent liver cell line culture method of rodent, characterized in that the liver damage inducing agent is contained 0.01 to 10% by weight. The method of claim 1,
Permanent liver cell line culture method of rodent, characterized in that for supplying liver damage inducer 1 ~ 100 mg / day. The method of claim 1,
Liver extraction method of the step (2) is the permanent liver cell line culture method of rodent, characterized in that to extract the liver by a surgical method. delete delete delete delete delete delete delete delete delete delete delete

Images