This patent application is assigned to Stelic Institute Of Regenerative Medicine,
The following quote was obtained by the news editors from the background information supplied by the inventors: "Previously, non-alcoholic fatty liver disease was believed to be a benign disease that does not progress. However, it was revealed that even non-drinkers develop inflammation similar to alcoholic hepatitis and show hepatic fibrosis histology, and now the non-alcoholic fatty liver disease is known as a disease with poor prognosis. In particular, metabolic syndromes due to obesity, diabetes, or the like have been drawing attention in recent years. It is becoming a common view that nonalcoholic steatohepatitis (NASH) is one of such syndromes. However, the mechanism remains unclear, and effective methods and/or agents for treating NASH have not been established. This is partly because NASH is due to human lifestyle-related diseases, and thus appropriate experimental animals have not been established.
"For the development of effective methods and agents for treating NASH, it is essential to elucidate the pathological condition of NASH, which progresses to lethal diseases such as liver cirrhosis and liver cancer. However, experimental animals that are currently used as a NASH model mouse in research include single-gene modified mice such as leptin receptor-deficient mice (Non-patent Document 1), hepatocyte-specific Pten-deficient mice (Non-patent Document 2), and retinoic acid receptor a dominant-negative transgenic mice (Non-patent Document 3), and mice induced with a special diet such as a methionine/choline-deficient diet (Non-patent Document 4). However, the human pathogenesis differs from that in the genetically-modified mice in which a single-gene mutation is responsible for the development and progression of the pathological condition, and is unlikely to be due to only the intake of a particular nutrient. Furthermore, insulin resistance and hepatic fibrosis cannot be simultaneously monitored in these mice. Furthermore, the ALT, an index in serobiochemical analysis, is only slightly elevated in mice that develop fibrosis, and therefore several mouse tissue slices are required to assess their pathological condition. Conversely, the pathological condition is assumed to be different from that of human because ALT is elevated to a markedly high level. Furthermore, the pathological condition recovers spontaneously after treatment. This makes it difficult to test and assess drugs for their efficacy. Thus, for developing methods and agents for treating NASH, it is desirable to establish an experimental animal model that is compatible with the human clinical condition.
"Although various studies have been conducted, there is no experimental animal exhibiting pathological conditions similar to those of human. Thus, under the current circumstances, it is difficult to conduct detailed screening to elucidate the pathogenesis or to establish therapeutic methods."
In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "Problems to be Solved by the Invention
"An objective of the present invention is to provide steatohepatitis model animals and liver cancer model animals that exhibit pathological findings similar to those of humans, and uses thereof. More specifically, an objective of the present invention is to provide techniques for producing model animals that develop into fatty liver, steatohepatitis, hepatic fibrosis, liver cirrhosis, and liver cancer from insulin resistance.
"Means for Solving the Problems
"The present inventors conducted dedicated studies to achieve the above-described objectives. The present inventors induced insulin resistance by administering an agent for inducing organ inflammation, and induced fatty liver in mice by feeding them with a high fat diet. As a result, the present inventors successfully developed steatohepatitis in mice. A detailed observation of the mice revealed the following pathological findings: (1) macrovesicular fat deposition in liver cells and liver cell ballooning; (2) infiltration of inflammatory cells; and (3) fibrosis around mainly the central vein.
"The above-described pathological findings are characteristic of human nonalcoholic steatohepatitis (NASH). Specifically, by using the above-described method, the present inventors for the first time successfully produced mice that exhibit similar pathological findings as those of human NASH.
"The NASH animal model produced by the present inventors is different from conventional animal models in the following points: (1) similarly to that observed in the histophathological findings of human, fatty degeneration and fibrosis of liver cells progress mainly around the central vein instead of the portal vein; and (2) the histophathological images of human 'burned-out NASH' are observed, in which only hepatic fibrosis is seen, although fat deposition and loss of inflammatory cells are observed as the pathological condition progresses.
"The animal model of the present invention is characteristic in that it is produced without genetic modification. Furthermore, the model animals of the present invention unfailingly (100%) develop a pathological condition similar to the progression and prognosis of human NASH at a constant time course, and are the first model animals that exhibit the same course of progression of pathological condition in human. In addition, the model animals of the present invention have a remarkably beneficial effect in that all of insulin resistance, fatty liver, steatohepatitis, hepatic fibrosis, and liver cirrhosis can be observed at the same time.
"Moreover, the present inventors newly found that the above-described NASH model animals develop liver cancer following liver cirrhosis as they continue the rearing. This is the same change of pathological condition observed in humans. Thus, the animals prepared by the methods of the present invention are very useful as model animals of human liver cancer.
"Human liver cancer causes the liver surface to bulge. However, conventional model animals for liver cancer, which are prepared by administering a chemical substance, develop liver cancer that does not cause bulging of the liver surface. Meanwhile, model animals of the present invention, which are prepared without genetic modification and drug administration, exhibit bulging of the liver surface in a fashion similar to the case of human liver cancer. Thus, the model of the present invention is a model much closer to human liver cancer. Furthermore, liver cirrhosis is not developed by administering chemical substances, while model animals of the present invention develop massive type cord-like liver cell carcinoma. Furthermore, infiltration of inflammatory cells and development of liver cancer caused by cirrhosis to displace normal liver cells are observed in the model animals of the present invention. In the model, the origin of liver cancer is macrovesicular fatty liver which shows a pathological condition very similar to that of human NASH. The liver cancer develops from hepatic fibrosis and liver cirrhosis. Thus, the animal model of the present invention is very useful, as it has not been reported previously.
"As described above, the present inventors successfully produced model animals for steatohepatitis and liver cancer which show similar pathological findings to those of human, and thereby completed the present invention. By using these model animals, it is possible to efficiently screen for substances for treating or preventing diseases, and effectively evaluate the efficacy of medicinal substances.
"The present invention relates to model animals which develop into fatty liver, steatohepatitis, hepatic fibrosis, cirrhosis, and liver cancer from insulin resistance, and more specifically, the present invention provides:  A non-human animal model for steatohepatitis produced by administering an agent for inducing organ inflammation;  The non-human animal of , wherein the steatohepatitis is a non-alcoholic steatohepatitis;  a non-human animal model for diabetes produced by administering an agent for inducing organ inflammation;  the non-human animal of any one of to , wherein the agent for inducing organ inflammation is an N-acetyl-.beta.-D-glucosaminidase inhibitor;  the non-human animal of any one of  to , which comprises the step of inducing fatty liver by administering an agent for inducing organ inflammation to the animal and rearing the animal with a high-fat diet;  the non-human animal of any one of  to , wherein the non-human animal is a mouse;  a method of producing a non-human animal model of steatohepatitis, which comprises the step of inducing inflammation in an organ of the non-human animal;  a method of screening for a substance for treating or preventing steatohepatitis, which comprises the steps of: (a) administering a test substance to the non-human animal model of steatohepatitis of ; and (b) evaluating an ameliorating effect on steatohepatitis;  a method of evaluating a medicinal substance for efficacy against steatohepatitis amelioration, which comprises the steps of: (a) administering a test medicinal substance to the non-human animal model of steatohepatitis of ; and (b) evaluating an ameliorating effect on steatohepatitis;  a method of screening for a substance for treating or preventing a diabetic disorder, which comprises the steps of: (a) administering a test substance to the non-human animal model for a diabetic disorder of ; and (b) evaluating an ameliorative effect on diabetic disorder;  a method of evaluating the side effects risks of a pharmaceutical agent for treating or preventing a diabetic disorder, which comprises the steps of: (a) administering a test pharmaceutical agent to the non-human animal model for a diabetic disorder of ; and (b) evaluating the pharmaceutical agent for treating or preventing diabetic disorder for side effects;  a non-human animal model for liver cancer, which is produced by further rearing the non-human animal of any one of  to ;  the non-human animal of , which is structurally characterized by the following pathological morphology: (a) massive type cord-like liver cell carcinoma; (b) infiltration of inflammatory cells; or © liver cancer caused by cirrhosis developed such that it displaces normal liver cells;  a method of screening for a substance for treating or preventing liver cancer, which comprises the steps of: (a) administering a test substance to the non-human animal model for liver cancer of  or ; and (b) evaluating a therapeutic effect on liver cancer; and  a method of evaluating a medicinal substance for efficacy against liver cancer treatment, which comprises the steps of: (a) administering a test medicinal substance to the non-human animal model of liver cancer of  or ; and (b) evaluating a therapeutic effect on liver cancer.
"Effects of the Invention
"To produce experimental animals that develop pathological conditions similar to those of humans, insulin resistance was induced in mice, and fatty liver was induced by feeding them with a high fat diet.
"Mice of different ages were sacrificed and each organ, mainly liver, was analyzed histopathologically (HE staining, fat staining, immunostaining for macrophages and fibroblasts). NAFLD Activity Score (NAS; reference: 'Kleiner D E et al., Hepatology. 2005 June; 41(6): 1313-21') was calculated to assess the pathological features in detail. Model animals of the present invention can also be assessed for NASH by using the same NAFLD Activity Score as for human. Thus, the model animals of the present invention are very useful as NASH model animals.
"Furthermore, serobiochemical tests were carried out using FUJIFILM DRI-CHEM. Gene expression analysis was performed using Real-Time RT-PCR (
"As described above, the present inventors successfully produced steatohepatitis model animals (for example, NASH model animals) and liver cancer model animals that show pathological findings similar to those of humans.
"The present invention provides simple techniques for stably producing animals that develop at an early stage a pathological condition similar to human NASH, which leads to fatty liver, steatohepatitis, hepatic fibrosis, and liver cirrhosis, followed by spontaneous development of liver cancer as a result of progression of the pathological condition, by inducing insulin resistance in experimental animals such as mice, and loading them with a high fat diet.
"Furthermore, diabetic disorders (diabetic nephritis, retinopathy, hyperlipidemia, and arteriosclerosis) can also be simultaneously observed in the animals. Thus, the present invention also provides techniques for producing experimental animals that enable the pathological conditions of metabolic syndrome to be observed at the same time.
"Meanwhile, in ob/ob mice and db/db mice which are commonly used as an NASH model, the pathological condition does not develop uniformly with aging. It is necessary to monitor the pathological condition of the mice to accurately assess the disease state of NASH, which makes the experiments cumbersome and complicated. Furthermore, the pathological lesions are not always irreversible. This has made the efficacy assessment difficult (Horie Y et al., J din Invest 113: 1774-1783, 2004; Yanagitani A et al., Hepatology 40: 366-375, 2004; Anstee Q M et al., Int J Exp Path 87: 1-16, 2006). Meanwhile, in the model animals of the present invention, the period leading to the mature pathological condition is constant and its progression is irreversible. Thus, the model animals of the present invention can be used to solve the above-described problems.
"The model animals of the present invention can be used in preclinical tests for various therapeutic agents, and are very useful in developing agents and searching for therapeutic targets.
BRIEF DESCRIPTION OF THE DRAWINGS
"FIG. 1 shows a result of serobiochemical test in mice of the present invention.
"FIG. 2 shows in photographs a result of fat staining of liver from a mouse of the present invention.
"FIG. 3 shows in photographs a result of immunostaining of liver from a mouse of the present invention.
"FIG. 4 shows in photographs a result of HE staining of liver from a 20-week-old mouse of the present invention.
"FIG. 5 shows a result of HE staining of liver from C57BL/6J in photographs and the NAFLD Activity Score in a graph.
"FIG. 6 shows in photographs a result of F4/80 immunostaining of pancreas and adipose tissue from C57BL/6J.
"FIG. 7 shows in photographs a result of immunostaining of F4/80 and ER-TR7 of 8-week-old BALB/c and C3H/HeN mice.
"FIG. 8 shows photographs of liver cancer in 20-week-old C3H/HeN.
"FIG. 9 shows in photographs reproducible results of NASH model mice reared with other high fat diets.
"FIG. 10 shows in photographs and graphs results of pharmacological tests using the NASH model mice. 1 shows the result of histological analysis in photographs. 2 shows the result of gene expression analysis in photographs.
"FIG. 11 shows the onset of diabetic complications in photographs. 1 shows diabetic nephropathy in photographs, while 2 shows diabetic retinopathy (immunostaining images of new blood vessels; CD31) in photographs."
URL and more information on this patent application, see:
Keywords for this news article include: Biomedical Engineering, Biomedicine, Diabetes, Genetics, Oncology, Hepatology, Proinsulin, Retinopathy, Inflammation, Therapeutics, Endocrinology, Liver Surface, Ophthalmology, Bioengineering, Hyperinsulinism, Gastroenterology, Hepatic Fibrosis, Machine Learning, Peptide Hormones, Insulin Resistance, Cancer Gene Therapy.
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