KR101680171B1 - Diagnosis of liver diseases and screening method for drug candidates using altered expression of phospholipid and enzyme - Google Patents

Abstract

The present invention relates to a method for screening candidates for new drugs for HBV infection, diagnosis of liver disease and promotion of liver regeneration by confirming compositional change of phospholipids and expression of enzymes before and after infection of hepatitis B virus, The present invention provides a convenient and rapid screening method for new drug candidates for diagnosing liver diseases and promoting new liver regeneration, thus providing a very useful measurement method for the development of liver regeneration promoting drugs.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for screening a candidate drug candidate for liver disease diagnosis and promotion of liver regeneration using phospholipid distribution and enzyme change,

The present invention relates to a method for screening candidate drug candidates for diagnosis of HBV infection and promotion of liver regeneration by confirming the compositional change of phospholipids and expression of enzymes before and after infection of hepatitis B virus.

Lipid metabolism is one of the essential liver functions and liver metabolic abnormalities are known to cause liver diseases such as inflammation and necrosis. Hepatitis B virus (HBV) Chronic infections are known to be more than 500 million worldwide, and HBV causes a variety of liver diseases including chronic inflammation, cirrhosis and hepatocellular carcinoma (HCC). HBV infection is known to cause inhibition of lipid metabolism, but little is known about virus-mediated lipid changes.

The liver has a unique ability to regenerate through dynamic processes after injury or partial resection. This regeneration ability is essential after acute or chronic liver damage. Liver regeneration is a very complex process involving a variety of growth factors such as hepatocyte growth factor (HGF), interleukin (IL) -6, tumor necrosis factor (TNF) -α, and transforming growth factor (TGF) -β. These various factors affect the regeneration process by providing stimulatory or inhibitory signals in hepatocyte proliferation.

Liver regeneration after hepatic injury, such as hepatotoxins such as dietary toxins, pathogenic infections, and surgical incisions, is important for maintaining hepatic homeostasis. Because phospholipids are a major constituent of cell membranes, phospholipids play an important role in liver regeneration and liver metabolism. In this view, abnormal accumulation of lipids and triglycerides is observed during liver regeneration after partial hepatectomy. It is believed that the lipids accumulated during liver regeneration play a transient role for the rest of the liver by supplying the membrane components necessary for energy and cell division. Moreover, the fatty acid composition changes after partial hepatectomy, especially with increasing amounts of phospholipids. These results suggest that phospholipid metabolism is closely related to liver regeneration.

Recently, it has been reported that HBV infection can impair liver regeneration through HBV X protein (HBx), suggesting that changes in phospholipid biosynthesis are associated with liver regeneration following hepatic injury due to HBV infection . However, no studies have examined the effect of HBV on phospholipid changes during infection or liver regeneration.

Matrix-Assisted Laser Desorption Ionization (MALDI) -IMS (Imaging Mass Spectrometry) has been established as an approach to detection and localization of various endogenous molecules including drugs and their metabolites, proteins, peptides and phospholipids. MALDI-IMS is used to visualize different distribution of phospholipids at diseased and adjacent normal tissue sites in surgical tissues including breast, lung, ovarian and intrahepatic cholangiocarcinoma as well as focal cerebral ischemia.

1. Klin Lab Diagn. 2009 Jun; (6): 43-5., Impact of HBV infection and alcohol surrogates on the lipid composition of lymphocytic membranes., Makarov VK, RiasenskiDS;

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel HBV diagnostic method.

Another object of the present invention is to provide a method for screening candidate drug candidates for promoting liver regeneration, thereby providing a very useful assay method for development of liver regeneration promoting drug.

In order to accomplish the above object, the present invention provides a method for screening candidate drug candidates for diagnosis of liver disease and promotion of liver regeneration, comprising the following steps.
a) infecting the subject with HBV (hepatitis B virus);
b) treating the candidate substance with the drug candidate;
c) hepatectomy of the subject's liver tissue;
d) confirming the distribution of the phospholipid or the choline-phosphate cytidyl transferase A enzyme in the cleaved liver tissue before or after treatment with the candidate drug substance; And
e) selecting a candidate substance as a candidate substance for a change in the phospholipid or a change in the enzyme;
In one embodiment of the present invention, the phospholipid in step d) is preferably phosphatidylcholine, but is not limited thereto.
In another embodiment of the present invention, the step d) is preferably such that the ionic strength of the potassium salt phosphatidylcholine is stronger than the ionic strength of the quantized phosphatidylcholine, but is not limited thereto.
The present invention also relates to a method for hepatectomy of hepatectomy in a subject and determining the mRNA expression level of choline-phosphate cytidyltransferase A (PCYT1A) or N-methyltransferase (PEMT) after resection using the cut liver tissue Wherein the subject is determined to be infected with hepatitis B virus and regenerated in the liver when the mRNA level of the enzyme is decreased compared to the normal liver level.
In one embodiment of the invention, the method is preferably but not limited to confirming mRNA expression levels at 6 hours after resection.
In addition, the present invention relates to a method of hepatectomy for hepatectomy in a subject and measuring the content of phosphatidylcholine by using the resected hepatic tissue. When the content of the phosphatidylcholine is decreased compared to the normal level, Is judged to be degraded.
In addition, the present invention confirms the change in the distribution of phospholipids by obtaining liver tissue or liver cells in the subject, and when the amount of lysophosphatidylcholine is increased compared to the normal level, the subject is inferior to hepatitis B virus infection and liver regeneration And a determination step of determining whether or not the information is to be displayed.
In one embodiment of the present invention, the lyso-phosphatidylcholine is preferably lyso-phosphatidylcholine (22: 2), but is not limited thereto.

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Hereinafter, the present invention will be described in detail.

HBV  Characteristics of infected mice and inhibition of virus-induced liver regeneration

To confirm using MALDI-IMS that HBV infection can alter the distribution or composition of phospholipids during liver regeneration, we generated HBV-infected mouse models by injecting HBV genome hydrodynamically. The overall experimental procedure used in the present invention is shown in FIG. To determine if HBV mice express viral proteins, virus surface proteins (HBsAg) were analyzed by ELISA using mouse serum on day 3 after hydrodynamic injection. As shown in Figure 1b, both replication-competed wt HBV (HBV1.2 (wt)) and HBX-null HBV (HBV1.2 (HBx-)) both expressed HBsAg protein at similar levels in HBV infected serum.

In the present invention, in order to relate the relationship between HBV-induced liver regeneration defects and phospholipid metabolism changes, we used an experimental animal model that underwent a partial heparinization following the hydrodynamic injection of wt HBV and HBx- Phospholipid profiling and imaging were performed by -MS and MALDI-IMS analytical methods. On day 8 after partial hepatectomy, significant inhibition of liver regeneration was observed only in the wt HBV infection model (Fig. 1c, 1d). We also examined Ki67-positive cells (markers of cell proliferation) by immunohistochemical staining Were counted for hepatocyte proliferation. The hepatocyte proliferation was significantly reduced in the liver tissue of wt HBV-expressing mice as compared to the control or HBx-null liver tissues (FIG. 1e). These results indicate that the experimental animal model of the present invention is capable of inhibiting phospholipid imaging through MALDI- Suggesting that it was well established for analysis.

PCA On by IMS From mass analysis and control HBV  In liver regeneration in mice, Imaging  compare

The present inventors have confirmed whether HBV infection can change the distribution of phospholipids in liver regeneration through MALDI-IMS analysis. MALDI-IMS analysis was performed in both positive and negative models with liver samples (n = 2 per group) extracted from liver regeneration mice after partial liver resection. Figure 2a shows the results of the mean mass analysis from whole mouse liver tissue sites in the positive ion mode. The spectra show more than 80 peaks with specific m / z values with different relative intensities from other liver tissues under different experimental conditions (Mock, HBV1.2 (HBx-) and HBV1.2 (wt)). The distribution of the obtained mass analysis data was also statistically analyzed by PCA (principal component analysis) and classification method. PCA for total mass analysis was performed from each group using ClinProTools v2.2 (Bruker Daltonics). As a result, PCA provided visual features based on the detected ions according to three different experimental groups (Fig. 2b). Interestingly, PCA analysis of the mean mass analysis obtained in anion mode showed more than 55 peaks (S / N> 6) were detected, no significant changes were seen (Figure 5).

Of the plurality of imaging data obtained using MALDI-IMS, the present inventors measured 17 ion images obtained in cationic mode showing significant differences in their ion abundance (abundance) in the ion image of three different groups of liver tissue sites (Fig. 2c, Fig. 2d). The ion images obtained from biological replicates are listed in Fig. In negative ion mode, there was no change in ion image (data not shown).

In order to identify the 17 ions selected in Figures 2c and 2d, we performed MS / MS analysis (Figure 7). These ions from the spectra show three different adduct forms (H ⁺ , Na ⁺ and K ⁺ (SMs) and two lysophosphatidyl cholines (LPCs) (Table 1).

[Table 1]

Table 1 shows the phospholipids showing changes in liver tissue during regeneration. Cn is the total chain length; Un is total unsaturation; SM is sphingomyelin; PC is phosphatidylcholine; LPC is lysophosphatidylcholine.

Due to the low strength of the m / z 776.6 ion, the structure could not be identified in this experiment. Phospholipids in 17 ion images were classified into five groups according to their level change patterns. Phospholipids belonging to groups A, B, and C were generally decreased in HBV1.2 (HBx-) and HBV1.2 (wt) compared to the control group, whereas phospholipids belonging to groups D and E Concentrations were increased in HBV1.2 (HBx-) and HBV1.2 (wt) compared to the control. Phospholipids belong to the group A is SM (42: 2) [M + K] + (m / z 851.8), SM (42: 1) [M + K] + (m / z 853.6), PC (34: 2) ^{[m + K] + (m} / z 796.6), PC (34: 1) [m + K] + (m / z 798.6) and PC (36: 2) [m + K] + (m / z824.7 ), But the ionic strength was decreased in HBV1.2 (HBx-) and HBV1.2 (wt) compared with the control group. Phospholipids belong to the group B is a PC (32: 4) [M + Na] + (m / z748.5), PC (32: 3) [M + Na] + (m / z 750.5) and m / z 776.6 ( And their ion concentrations gradually decreased in the order of the control group, HBV1.2 (HBX-) and HBV1.2 (wt). However, the phospholipid of Group D was LPC (22: 2) [M + K] ⁺ (mz / 614.2) and LPC (22: 2) [M + K] ⁺ (m / z 616.2) HBV1.2 (HBx-) and HBV1.2 (wt) showed higher ionic strength. Group E is a PC (34: 2 ([M + H] + (m / z758.7), PC (34: 1) [M + H] + (m / z760.6), PC (36: 2) [ ^{m + H] + (m /} z786.7) , and PC: inde (38 6) [m + H ] + (m / z806.6), ionic strength of these phospholipids is the control group, HBV1.2 (HBX-) and Interestingly, the phospholipids of group C, PC (32: 0) [M + K] ⁺ (m / z 772.7) and PC (34: 2) [M + Na] ⁺ (m / z 780.6) showed similar ionic strength between the control and HBV1.2 (HBx-) and was significantly higher in HBV1.2 (wt) compared to the control and HBV1.2 Showed low ionic strength.

There were no significant differences in liver histology analyzed by H & E staining in the three groups (Fig. 2e). IMS data were applied to determine the distribution of individual ions as well as lipid changes in tissues.

In the playing liver IMS To potassium for phospholipids selected by Cationized  Adduct formation analysis

Interestingly, we observed characteristic adduct formation for the most abundant PC species, PC (34: 2), PC (34: 1), and PC (36: 2). The adduct formation of these PCs varied depending on the control and HBx expression or non-expression HBV infection (Figure 3). In the liver of the control group, the potassium (K) cationized adduct of PC showed a higher ionic strength than the protonated form, and liver regeneration was more active than in HBV liver. However, in the samples between HBV1.2 (HBx-) and HBV1.2 (wt), there was no noticeable difference in adduct formation. In the case of HBV1.2 (HBx-), the potassium cationized form of PC (34: 2) (m / z 796.6) and PC 36: 2 (m / z 824.7) High strength. In particular, PC (34: 1) (m / z 798.6) in the potassium cationized form of HBV1.2 (wt) liver showed lower intensity than the protonated form (Fig. MS / MS analysis was applied to determine the formation of protonated and potassium cationized forms of PC (34: 2), PC (34: 1) and PC (36: 2). In the MS / MS data of PCs, m / z 184 ions were mainly detected in the protonated form whereas m / z 86, m / z 163 and m / z 194 were detected primarily in the potassium cationized form. The protonation and potassium cationized PC forms ([M + H] ⁺ and [M + K] ⁺ ions) and fragment ions of the precursor ions from the spectrum showed a neutral loss of mainly C3H10N (50 Da) (FIG.

Previously (Na ⁺ + K ⁺ ) - stimulated ATPase activity was reported to be increased in hepatic plasma membranes in partially hepatectomized rats. In the present invention, high levels of potassium cationized form in PCs were observed in activated liver regeneration after partial hepatectomy. However, in HBV-infected, the level of potassium cationized form of PCs was reversed in liver regeneration.

In the playing liver PC  Kinetic analysis of major genes associated with synthesis

These results suggest that PCs are mainly regulated differently by HBV infection during liver regeneration. Thus, the inventors investigated whether gene expression known to be associated with PC biosynthesis was altered by HBV infection. PC biosynthesis is generally known to be regulated by two mechanisms, the CDP-choline and phosphatidylethanolamine N-methyltransferase (PEMT) mechanisms. The major enzymes regulating these two mechanisms are choline-phosphate cytidyl transferase A (PCYT1A) and PEMT (Fig. 4A).

In order to investigate the kinetic changes of these genes in liver regeneration, we have identified mcRNA expression levels of Pcyt1a and Pemt at several time points after partial hepatectomy. As shown in Figures 4b and 4c, expression of Pcy1ta was strongly induced at 6 hours after partial hepatectomy in the liver of control and HBx-negative HBV mice. However, in the wt HBV infection, the expression level of Pcy1ta was markedly suppressed. Interestingly, expression was slightly delayed after partial hepatectomy (24h). Expression levels of Pcyt1a protein were also delayed in wt HBV infected regenerating liver (Fig. 4d). To confirm the altered expression results of Pcyt1a enzyme, we measured the total amount of liver PCs at the initial and late time points after partial hepatectomy Respectively. As shown in FIG. 4e, the level of total PCs was significantly reduced only in wt HBV-infected mice at 48 hours after partial hepatectomy. These data suggest that PC biosynthesis is interrupted by wt HBV infection in liver regeneration, suggesting that modulation of Pcyt1a expression by HBx in the mouse liver is associated with HBV infection and viral mediator regeneration defects.

In conclusion, we analyzed the altered phospholipids using MALDI-IMS during the regeneration in an HBV mouse model. The present inventors have identified several PCs-related enzymes that show significant expression changes in HBV-infected liver and HBV-mediated regeneration defects. These results suggest that HBV infection occurs through the HBx protein, which in particular induces changes in PC expression, which are potentially related to HBV-mediated liver regeneration defects and liver disease.

As can be seen from the present invention, screening of new drug candidates for promoting liver regeneration through the compositional change of hepatic phospholipid and change of enzyme expression before and after hepatitis B virus infection, and HBV diagnosis, It provides a very useful method in terms of promptness and accuracy in the development of accelerating drugs.

Figure 1 shows the characteristics of the HBV mouse model and the inhibition of virus-mediated liver regeneration. a. Figure showing the overall experimental process of the experiment related to the invention. be. FIG. 5 shows the identification and characteristics of the HBV-infected mouse model used in the present invention. b. Figure 3 shows HBsAg levels in mouse serum on the third day after hydrodynamic injection. c. Figure 8 shows the inhibition of liver regeneration by HBx in the HBV mouse model. Figure 8 shows the result of visual examination of the regenerating liver of the HBV or HBx mouse model on the eighth day after partial hepatectomy. d. Figure 8 shows a box plot of liver weight / weight on day 8 after partial hepatectomy. e. The expression of Ki 67 (marker of cell proliferation) in formalin-fixed liver tissue obtained 48 hours after partial hepatectomy. Data are presented as means ± SD and are representative of three independent experiments ***, P <0.001 (n = 5-6 mice per group) compared to control group and HBV1.2 (HBx-)
Figure 2 shows the PCA analysis of the mean MS spectra of phospholipids from liver tissue sections obtained in three different conditions of positive ionization mode and the comparison of MALDI images of phospholipids in regenerating liver tissue after partial liver resection. a. Figure shows a typical MS spectrum of liver tissue sites in three different conditions obtained from imaging mass spectrometry. b. PCA analysis of IMS data obtained in the cationization mode. c, d. Figure 1 shows a representative MALDI image analysis of molecular ion showing the level of HBV infection according to HBV infection. e. Figure 3 shows the H & E stained liver tissue observed 24 hours after partial hepatectomy.
Figure 3 shows IMS and MS / MS analysis results for phospholipids selected in regenerating liver tissue. a. A comparison of the images of the H ⁺ and K ⁺ adducts of the three phosphatidylcholines in regenerating liver tissue. b. MS / MS analysis of selected PCs [M + H] ⁺ and [M + K] ⁺ ions.
Figure 4 is a kinetic analysis of the phosphatidylcholine (PCs) synthesis-related gene and the results of measurement of total PCs in liver tissue regenerated after partial hepatectomy. a. Figure showing the mechanism of PC biosynthesis. PCYT1A, choline-phosphate cytidyl transferase A; PE, phosphatidylethanolamine; PMME, triolein-phosphatidyl N-monomethylethanolamine; PDME, triolein-phosphatidyl-N, N-dimethylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase. bd. Results of kinetic analysis of Pcyt1a and Pemt expression in liver tissues after partial hepatectomy and mRNA levels (b) and real-time PCR results of specific genes analyzed by semi-quantitative RT-PCR at specific time intervals after liver resection c) and Western blot analysis (d). e. Figure 1 shows the results of quantitative analysis of liver PCs after partial hepatectomy. Data are presented as mean ± standard deviation and are representative of three independent experiments. **. P &lt;0.01; ***, P <0.001 (n = 3 to 4 mice per group) compared to the control vector and HBV1.2 (HBx-)
Figure 5. PCA analysis of the mean MS spectra of phospholipids from liver tissue sites obtained from three different conditions in negative ion mode. a. Figure shows a typical MS spectrum of liver tissue sites in three different conditions obtained from imaging mass spectrometry. b. Figure shows PCA analysis of IMS data obtained in positive ionization mode.
Figure 6. MALDI image analysis of phospholipids from biological equivalents in regenerating liver tissue after partial hepatectomy.
Figure 7. MS / MS data of the 17 phospholipid ions listed in Table 1.

The present invention will now be described in more detail by way of non-limiting examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  One. HBV  Mouse model for infection

Briefly, male BALB / c mice (n = 3 per group) from 6 weeks to 7 weeks developed hydrodynamic 25 (PGEM-4z, pHBV1.2 (wt), pHBV1.2 (HBx-)) was injected into the tail vein with the same volume of PBS (phosphate-buffered saline) equivalent to 10% of the mouse body weight. The total volume of DNA was injected intravenously at high pressure within 4-6 seconds (hydrodynamic vivo transfection). All procedures including laboratory animals were approved by Konkuk University Animal Protection Committee.

Example  2. Partial liver resection

In order to induce physiological liver regeneration in mice, a 70% partial hepatectomy model was used as previously described (Park et al., Ahn SH et al., 2013; Mitchell C, Willenbring H., 2008) , About 70% of the total liver weight was removed after intraperitoneal injection of xylazine (10 μg / kg) and zoletil (10 mg / kg) on the fourth day after hydrodynamic injection. After anesthesia, the left and middle lobe sections of each mouse were removed through abdominal central incision. After sealing the abdomen, the animals were allowed to eat the food at random after returning to the cage. The degree of liver regeneration was evaluated by the ratio of body weight to liver weight on the eighth day after partial hepatectomy.

Example  3. Proliferation of Mouse Hepatocytes

To evaluate the proliferative state of mouse hepatocytes after partial hepatectomy, a Ki67-labeling index was observed as a proliferation marker. Ki67-stained hepatocytes were identified in a 4 [mu] m tissue section of liver samples embedded in paraffin according to the manufacturer's instructions (Amersham Life Science, Amersham, UK).

Example  4. MALDI  Organizational processing for analysis

Liver tissues (n = 2) obtained from the test mice were stored at -80 ° C. until they were used after rapid cooling in liquid nitrogen. The 10 mu m thick tissue area was cut using a cryostat (Leica CM-1850, Leica Microsystems Inc., Solms, Germany) at a temperature range of -20 ° C to -25 ° C. For MALDI-IMS analysis, the tissue sites began to dissolve on glass slides coated with indium-tin-oxygen (ITO) (HST Global, Inc., Hampton, VA, USA). Tissues were then frozen and incubated at 4 ° C and room temperature. Finally, the tissue was dried in a desiccator. For MALDI-MS profiling analysis, a binary matrix solution was prepared by dissolving 3.5 mg of DHB and CHCA, respectively, in 1 ml of 70% methanol containing 0.1% TFA, and ImagePrep (Bruker Daltonics, Billerica , MA, USA). A biological replica was produced from two mice randomly selected for each group.

Example  5. MALDI Imaging  Mass spectrometry

Mass spectrometry was performed using an Autoflex Ⅲ mass spectrometer (Nd: YAG, 355 nm; Bruker Daltonics Inc.) equipped with smart beam laser technology. Imaging experiments were performed using both positive and negative ions from a single tissue site and all ions with a mass to charge (m / z) ratio in the range of 500-1200 at 200 Hz laser frequency (3,000 consecutive laser shots) were used in reflectron mode Was detected. For image analysis, data were acquired using a spatial resolution of 150 μm with 500 consecutive laser shots per pixel under the control of the Flex software suite (FlexControl 3.0, FlexImaging 2.1, FlexAnalysis 3.3, FlexAnalysis 3.3, Bruker Daltonics) 50 laser shots per work). Prior to each data collection, external calibration was performed using phospholipid-mixed calibration standards in the mass range 674-834 Da (cation mode) and 564-906 Da (negative ion mode). MALDI LIFT (MS / MS) analysis was performed directly on tissues after MALDI MS. Other phospholipid classes were identified by using LIFT data and a lipid database (www.lipidmaps.org).

Clinpro Tools (version 2.2, Bruker Daltonics) and flexImaging software were used for MALDI imaging data processing and all spectral intensities were separated by the total number of ion values obtained. Ion intensities were evaluated after standardization by comparison of liver sites and ion pairs in different experimental groups. A Top Hat baseline with a 10% minimum baseline width was used for baseline subtraction. Data reduction was performed with a factor of 4.

Example  6. Real - time -PCR ( RT - PCR )

Whole mouse liver tissue was lysed using Trizol reagent (Invitrogen / Gibco BRL, Carlsbad, Calif., USA) according to the manufacturer's instructions and total RNA was isolated. The reverse transcription reaction was performed at a final concentration of 20 占 퐇 using 2 占 퐂 total RNA and M-MLV reverse transcriptase (iNtRON, Seoul, Korea). The resulting cDNA was PCR amplified under the following conditions: initial denaturation was carried out at 94 ° C for 5 minutes and 25-30 cycles at 94 ° C (30 seconds), 58 ° C (30 seconds) and 72 ° C (30 seconds) Followed by final extension at 72 [deg.] C for 5 minutes. The primer sequences for amplification are as follows: Phosphorylated cytidyl transferase 1, choline, alpha isoform (Pcyt1a), forward 5'-AGGCTACTGTGACCGAGGTA-3 '. Reverse 5'-AGTCACCCTGACATAGGGCT-3 '; Phosphatidylethanolamine N-methyltransferase (Pemt), forward 5'-GAGAACTCGGAAGCTGAGCA-3 ', reverse 5'-CAGCACAAACACGAATCCCC-3'. Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and PCR amplification was performed using an Applied Biosystems (ABI7500) real-time PCR machine. Relative quantitative analysis was performed using the comparative DELTA Ct method. The results are shown as n-fold difference relative to the calibrator (RQ = 2 - ^{DELTA Ct} )

Example  7. Western Blat  analysis

Each liver tissue was homogenized in RIPA buffer solution. 50 μg of protein was isolated on a 10-12% SDS-PAGE gel and applied to Western blot analysis. The primary antibodies used were anti-PCyt1a (N-20, Santa Cruz Biotechnology, CA, USA) and anti-beta-actin (Sigma Aldrich). As the secondary antibody, a suitable peroxidase (Santa Cruz Biotechnology, CA, USA) conjugated with the secondary antibody was used. Chemiluminescence detection assays were performed with ECL Detection Reagent (GE Healthcare, Buckinghamshire, UK) and visualized with a LAS-4000 Luminescent Image Analyzer (LAS-4000, Fuji, Tokyo, Japan).

Example  8. Total PC Quantitative analysis of quantities

Mice were sacrificed at specific time points after partial hepatectomy. The total amount of PC was measured using a phosphatidylcholine assay kit (Cell biolabs Inc, San Diego, Calif.) According to the manufacturer's instructions. At least three mice were used for analysis by each group.

Example  9. Data Analysis

Data were obtained by at least 3 independent experiments and the values are expressed as mean ± standard deviation (SD). For MALDI-IMS analysis, two liver tissue samples per group were analyzed independently. Group differences were tested for statistical significance using Student's t-test or variance analysis. Statistical significance was set at p <0.05.

Claims (8)

A method for screening a candidate for a drug for the diagnosis of liver disease and promoting liver regeneration, comprising the steps of:
a) infecting an experimental subject other than a human with HBV (hepatitis B virus);
b) treating the candidate substance of the new drug to an experimental subject other than the human;
c) hepatectomy of the liver tissue of the subject except for the human;
d) confirming the distribution of the phospholipid or the choline-phosphate cytidyl transferase A enzyme in the cleaved liver tissue before or after treatment with the candidate drug substance; And
e) selecting a candidate substance as a candidate substance for a change in the phospholipid or a change in the enzyme;
The phospholipid in step d) is phosphatidylcholine,
Wherein the step d) is characterized in that the ionic strength of the potassium salt phosphatidylcholine is stronger than the ionic strength of the quantized phosphatidylcholine. delete delete Hepatectomy was performed in subjects other than humans, and the level of mRNA expression of choline-phosphate cytidyltransferase A (PCYT1A) or N-methyltransferase (PEMT) was determined after excision using the cut liver tissue Wherein when the mRNA level of the enzyme is decreased compared to the normal liver level, the subject other than the human is determined to be infected with hepatitis B virus and regenerated in the liver. 5. The method of claim 4, wherein the mRNA expression level is confirmed at 6 hours after the resection. The content of phosphatidylcholine is decreased by the hepatectomy in the subject excluding the human and the content of the phosphatidylcholine is decreased by using the cut liver tissue and the content of the phosphatidylcholine is decreased compared to the normal level. And judges that liver regeneration is degraded. When the amount of lysophosphatidylcholine is increased compared with the normal level by confirming the change in the distribution of the phospholipid by obtaining liver tissue or liver cells in a subject other than a human, the subject other than the human is infected with hepatitis B virus And judges that the information is being processed. 8. The method of claim 7, wherein the lyso-phosphatidylcholine is lyso-phosphatidylcholine (22: 2).

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