JP2014087275A - Model animal of exocrine pancreatic insufficiency - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a model animal which develops chronic pancreatitis and diabetes from reduced glucose tolerance and insulin resistance after ablation of the pancreas.SOLUTION: A method for producing a model animal of exocrine pancreatic insufficiency comprising the steps of: (a) cutting a pyloric portion of the stomach and a duodenum portion into which pancreatic juice flows; (b) cutting a proximal jejunum of the anal region from the treitz ligament which is a boundary of the duodenum and the jejunum; (c) closing the anal side cut end made by the step (a), and anastomosing the anal side cut end of the jejunum made by the step (b) and the stomach side cut end of the duodenum made by the step (a) [duodenum-jejunum anastomosis]; (d) connecting and anastomosing the duodenum side cut end of the jejunum made by the step (b) to the incised rectum part [small intestine-rectum anastomosis]; and (e) applying intubation to the common bile duct, the duct connecting the gallbladder and the duodenum, to connect and anastomose to the small intestine part in the step (c) [common bile duct-jejunum anastomosis].

Description

  The present invention relates to a pancreatic exocrine dysfunction model animal, more specifically, food flows directly into the jejunum without flowing into the duodenum, pancreatic fluid flows directly into the rectum without flowing into the jejunum, and bile flows into the duodenum. The present invention relates to a model animal of exocrine pancreatic insufficiency that has undergone a surgical treatment so as to directly flow into the jejunum, a production method thereof, and use thereof.

  The gastrointestinal tract is a series of food passages from the mouth to the esophagus, stomach, duodenum, small intestine (jejunum and ileum depending on the region), large intestine (colon, rectum), and anus. The organ that produces and flows into the duodenum through the pancreatic duct is the pancreas. The pancreas is composed of pancreatic exocrine cells (pancreatic acinar cells) that are involved in food digestion and pancreatic endocrine cells (pancreatic islets of Langerhans) that maintain blood glucose levels. Pancreatic acinar cells are damaged, digestive enzyme production is reduced, food digestion ability is markedly reduced, exocrine pancreatic insufficiency, pancreatic islets of Langerhans is damaged, or reduced function is pancreatic endocrine function The latter is a condition called so-called diabetes. In addition, the bile duct is a passage of bile produced in the liver and flows into the duodenum, but before that, it travels inside the pancreas (FIG. 1A).

  Pancreaticobiliary malignancies represented by cancers that occur in the pancreas and bile ducts have a poorer prognosis than the same gastrointestinal malignancies such as gastric cancer and colon cancer. In order to completely cure pancreatic cancer and biliary tract cancer, it is absolutely necessary to remove the cancer without leaving it by surgical treatment (curative surgery). Pancreatoduodenectomy to remove the right third of the pancreas and the duodenum in order to completely remove the cancer that has occurred in the head of the pancreas (approximately 1/3 of the right side of the pancreas) and the lower bile duct (the bile duct that runs in the pancreas) Total pancreatectomy is performed to remove all of the pancreas along with the duodenum and spleen. In addition to the high difficulty of these techniques, the patient suffers a heavy burden, and loss of the pancreas causes both intra- and exocrine dysfunction. As a result, postoperative management has become difficult and has not been performed in the past.

  Insulin is a hormone that lowers blood glucose and is secreted from the islets of Langerhans. Insulin replacement therapy, which is injected from outside the body, was given to patients after pancreatectomy. It was difficult. Moreover, although the digestive adjuvant was administered with respect to the pancreatic exocrine function fall, the effect was inadequate.

  However, in recent years, in addition to improvements in surgical techniques, improvements in insulin preparations, development of high-quality exocrine pancreatic function therapeutic agents, and the like have made it possible to safely perform the above pancreatectomy. At present, these operations are being widely performed as standard treatments for pancreatobiliary malignant diseases.

  At the same time, long-term complications after pancreatectomy are becoming known. Among them, non-alcoholic fatty liver disease (NAFLD) is a condition in which a part of the disease results in non-alcoholic steatohepatitis (NASH) and a part thereof leads to cirrhosis. After pancreatoduodenectomy or total pancreatectomy This is a complication observed in about 20 to 40% [Non-Patent Document 1]. Pancreatic resection due to pancreatic exocrine dysfunction (digestive enzyme production failure) is presumed to be the cause, but details have not been elucidated yet. Therefore, establishing an animal model with non-human animals with significantly reduced pancreatic exocrine function and clarifying the physiological functions and pathological conditions after pancreatectomy is a good quality for such pancreatic complications (complications associated with pancreatic diseases). I think it is very meaningful in establishing treatment.

  Intrapancreatic / exocrine pancreas that progresses after pancreatectomy required for the treatment of lethal diseases such as pancreatobiliary malignancies and is the cause of serious complications such as nonalcoholic fatty liver disease and diabetes and chronic pancreatitis Elucidation of the pathophysiology of dysfunction is essential for the development of effective therapies and therapeutic drugs. As an animal model for studying pancreatic diseases, a streptozotocin administration method that specifically eliminates pancreatic islets of Langerhans has already been established for a pancreatic endocrine dysfunction (diabetes) model [Non-patent Document 2]. Has also been reported as a method for producing NASH model animals [Patent Document 1]. On the other hand, chronic pancreatitis is raised as a disease state in which the exocrine pancreatic function is lowered. As an animal model thereof, in addition to the method by alcohol administration [Non-patent Document 3], a spontaneously-occurring animal model has also been reported [Non-patent document 3]. Patent Document 4]. Moreover, a pancreatic resection model is mentioned as a model in which the pancreatic endocrine / exocrine function is reduced. This is an animal model obtained by excising 60-90% of the pancreas, and is mainly used for the study of pancreatic regeneration. There are reported examples in mice [Non-Patent Document 5], rats [Non-Patent Document 6], dogs [Non-Patent Document 7], and the like. However, there are no rodent total pancreatectomy models such as mice and rats, and there is only one report that 99.5% of pancreatectomy rats have been successfully produced [Non-patent Document 8].

  Total pancreatectomy for small animals is difficult to preserve the duodenum, and is too invasive, making it very difficult to create a viable model after surgery. In addition, when considering a case that developed as a complication of pancreatic resection in a NASH model animal, in the NASH model animal by streptozotocin administration described in Patent Document 1, antigens and β-cells on the pancreatic islets of Langerhans were administered by streptozotocin administration. Against the drug-induced autoimmunity in which the β-cells of the islets of Langerhans are destroyed by the attack of cytotoxic T cells by the activated immune system, resulting in the inability to secrete insulin It is unlikely that the onset mechanism resulting from the same progress as the onset progression due to the physical invasion of pancreatic resection. Therefore, the course of pancreatic endocrine / exocrine dysfunction as a complication of pancreatectomy in humans cannot be observed using the model animals listed in the above literature. Based on the above, at present, there are no animal models in which the pancreatic exocrine function is significantly reduced and the duodenum is preserved. For the treatment of pancreatic exocrine dysfunction and the development of therapeutic agents, human clinical pathologies Development of experimental model animals in accordance with the above is desired.

  Although various studies are currently underway, there is no experimental animal model that is similar to the post-panectomy condition in humans, and there is no surviving experimental animal model. It is difficult to screen for treatments and drugs.

JP 2009-178143 A

Kato H et al., J. Hepatobiliary. Pancreat. Sci., (2010) 17, p296-304. Sakata N et al., Transplantation. (2010) 89, p686-693. Lugea A et al., Gastroenterology. (2011) 140, p987-997. Yuzawa Sakaguchi et al., Japanese Journal of Gastroenterology. (2009) 106, A172 Wang Y et al., Horm. Metab. Res. (2012) 44, p33-40. Kwon DY et al., J. Pharmacol. Sci. (2009) 111, p361-71. Hayakawa H et al., J. Endocrinol. (1996) 149, p259-267. Taghizadeh A et al., Br. J. Exp.Pathol. (1969) 50, p605-611.

  An object of the present invention is to provide a model animal of pancreatic exocrine dysfunction showing a pathological finding and biochemical examination similar to humans necessary for the development of therapeutic methods and therapeutic drugs for pancreatic exocrine dysfunction, and a method of using the animal Is to provide.

  The present inventors have intensively studied to solve the above problems. In that process, instead of letting pancreatic juice (pancreatic digestive enzyme) flow into the original duodenum-small intestine, we examined focusing on placing a gastrointestinal bypass that flows directly into the rectum that is not involved in the digestion and absorption of food. After trial and error, we succeeded in producing a model animal of an exocrine pancreatic insufficiency rat that does not depend on total pancreatectomy and can survive after treatment. As a result, glucose uptake in muscle and adipose tissue was suppressed due to significant digestion and absorption disturbance due to exocrine pancreatic dysfunction and decreased insulin secretion, and as a result, the rat succeeded in inducing fatty liver. . A detailed examination of the rat 1 month after the operation revealed a balloon-like enlargement of hepatocytes showing fat deposition in the liver tissue.

  The above pathological findings are characteristic of liver tissue and hepatocytes accompanying pancreatic exocrine function decline. That is, the present inventor succeeded for the first time in creating a model animal showing pathological findings similar to human pancreatic exocrine dysfunction by the above method.

That is, the present invention
[1] Gastric pyloric part and jejunum, or part of duodenum continuous to gastric pyloric part and jejunum are anastomosed, and food flows directly into the jejunum without flowing into the duodenum; duodenum and rectum, or duodenum A portion of the jejunum that is connected to the rectum is anastomosed, and the pancreatic juice flows directly into the rectum without flowing into the jejunum; and the common bile duct and jejunum are anastomosed through a small-diameter tube, and bile enters the duodenum. Without influx, it flows directly into the jejunum; a model animal of exocrine pancreatic dysfunction characterized by comprising non-human animals,
[2] A portion of the duodenum continuous to the stomach pylorus and the jejunum are anastomosed, a portion of the jejunum continuous to the duodenum and the rectum are anastomosed, and the common bile duct and jejunum are anastomosed via a small-diameter tube The animal model of exocrine pancreatic dysfunction according to the above [1], characterized in that,
[3] The animal model of exocrine pancreatic dysfunction according to [1] or [2] above, wherein the non-human animal is a rat,
[4] (a) cutting the duodenum between the stomach pylorus and the duodenum into which pancreatic juice flows;
(B) cutting the proximal proximal jejunum from the Tritz ligament, which is the boundary between the duodenum and the jejunum;
(C) anastomosing the stump of the duodenum continuous to the gastric pyloric region produced by the step (a) and the jejunal stump produced by the step (b);
(D) closing the mouth side stump of the duodenum produced by the step (a) and anastomosing the jejunal stump continuous with the duodenum produced by the step (b) to the rectal incision;
(E) inserting one end of the small-diameter tube into the mouth side of the jejunum formed in the step (c) and anastomosing the other end of the small-diameter tube to the cut common bile duct on the liver side;
The present invention relates to a method for producing a model animal of exocrine pancreatic dysfunction characterized by applying a surgical treatment with a non-human animal.

The present invention also provides
[5] (a) a step of administering a test substance to the pancreatic exocrine dysfunction model animal according to any one of [1] to [3];
(B) a step of evaluating an improvement effect on chronic pancreatitis;
A method for screening a therapeutic and / or prophylactic agent for chronic pancreatitis characterized by comprising:
[6] (a) a step of administering a test substance to the pancreatic exocrine dysfunction model animal according to any one of [1] to [3];
(B) a step of evaluating the improvement effect on diabetes;
A method of screening for a therapeutic and / or prophylactic agent for diabetes characterized by comprising:
[7] (a) a step of administering a test drug to the pancreatic exocrine dysfunction model animal according to any one of [1] to [3];
(B) a step of evaluating an improvement effect on chronic pancreatitis;
A method for evaluating the efficacy of a drug for improving chronic pancreatitis,
[8] (a) a step of administering a test drug to the pancreatic exocrine dysfunction model animal according to any one of [1] to [3];
(B) a step of evaluating the improvement effect on diabetes;
A drug efficacy evaluation method for improving diabetes of a drug characterized by comprising,
[9] (a) Transplanting cells or organs for pancreatic regeneration therapy into the pancreatic exocrine dysfunction model animal according to any one of [1] to [3];
(B) a step of evaluating an improvement effect on pancreatic exocrine dysfunction;
The present invention relates to a method for evaluating pancreatic regeneration therapy for exocrine dysfunction characterized by comprising:

  According to the present invention, it is possible not only to evaluate the pathological condition in which the exocrine pancreatic function is remarkably reduced, that is, the pathological condition such as advanced chronic pancreatitis and total pancreatectomy, and to pursue the mechanism of the disease. It can be diverted to development of therapeutic methods for improving diseases, that is, development of cell transplantation therapy, research on regenerative medicine, development of therapeutic drugs, and the like.

It is the figure which showed typically the preparation method of a pancreatic exocrine dysfunction rat. A. Normal dissection within the abdominal cavity of rats. B. Duodenum and jejunostomy. C. Small intestinal rectal anastomosis, duodenal jejunostomy, common bile duct jejunostomy. It is a figure of a laparotomy finding one month after an operation. A. Small intestinal rectal anastomosis. B. Duodenojejunostomy and common bile duct jejunostomy. It is the figure which showed the serum lipase density | concentration (A), fecal lipase density | concentration (B), serum trypsin density | concentration (C), and fecal trypsin density | concentration (D) one month after an operation | movement. Serum lipase, fecal lipase, serum trypsin, and fecal trypsin were all markedly lower than normal. It is the figure which showed the liver tissue findings 1 month after an operation. A. A hepatocyte balloon-like swelling (solid arrow) showing fat deposition was observed in the liver tissue of rats with exocrine pancreatic function abolished. B. No such findings were observed in the control group rats. The staining conditions are hematoxylin and eosin staining, and the magnification is 200 times.

  Examples of pancreatic exocrine dysfunction model animals of the present invention (hereinafter sometimes referred to as “model animals”) include a gastric pyloric region (boundary between the stomach and duodenum) and jejunum, or a part of the duodenum continuous to the gastric pyloric region. The jejunum is anastomosed and food flows directly into the jejunum without flowing into the duodenum; the duodenum and rectum, or a portion of the jejunum that continues to the duodenum and the rectum is anastomosed, and pancreatic juice does not flow into the jejunum And the common bile duct and jejunum are anastomosed via a small-diameter tube, and bile flows directly into the jejunum without flowing into the duodenum; As used herein, anastomosis means joining two parts of a luminal organ by surgery. Moreover, the pancreatic exocrine dysfunction includes a decrease in pancreatic exocrine function.

  In addition, the method for producing the model animal of the present invention includes (a) a step of cutting the duodenum between the gastric pylorus and the duodenum into which pancreatic fluid flows; (b) from the Triz ligament which is the boundary between the duodenum and the jejunum Cutting the proximal jejunum on the anal side; (c) anastomosing the stump of the duodenum continuous to the gastric pyloric region generated by the step (a) and the stump of the jejunum generated by the step (b) (D) closing the mouth side stump of the duodenum generated by the step (a) and anastomosing the jejunal stump continuous with the duodenum generated by the step (b) to the rectal incision; A surgical step comprising: inserting one end of a small-diameter tube into the mouth side of the jejunum formed in the step (c) and anastomosing the other end of the small-diameter tube into the cut common bile duct on the liver side There is no particular limitation as long as it is a method of applying treatment, The part of the duodenum that continues to the stomach pylorus and the jejunum are anastomosed, the part of the jejunum that continues to the duodenum and the rectum are anastomosed, and the common bile duct and the jejunum are anastomosed via a thin tube. The model animal of the present invention can be produced.

  The non-human animal is not particularly limited as long as it is an animal generally used as a laboratory animal, but is preferably a non-human vertebrate, more preferably a non-human mammal, specifically, a rat or mouse. , Rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc., and rodents such as rats, mice, rabbits, etc. can be preferably exemplified. Rats can be mentioned as rodents most suitable for this model because of their ease of use. Moreover, it is preferable that the non-human animal used for preparation of a model animal uses the age of the physical strength which can endure surgery, and in the case of a rat, about 14 weeks old after birth is preferable.

  The genetic background of the animal used for the production of the model animal of the present invention is not particularly limited, and an animal having any genetic background can be used, and the ability to reject transplanted tissue as a non-human animal to be used. It is also possible to use immunodeficient animals lacking. Usually, wild-type animals can be preferably used.

  In the production of the model animal of the present invention, it is preferable to carry out the method according to the operation in actual clinical practice for the purpose of preventing surgical death and postoperative infection. That is, general anesthesia can be suitably exemplified as an anesthesia method. More preferably, general anesthesia is performed by inhalation anesthesia using an anesthesia apparatus. Examples of anesthetics include diethyl ether, halothane, enflurane, isoflurane, methoxyflurane, sevoflurane, desflurane and the like.

  The hair of the model animal is removed with an animal clipper or hair remover, or a combination of animal clippers and a hair remover, to prevent contamination of the body hair into the abdominal cavity. The dosage form of the hair remover is not particularly limited, and commercially available products for humans can be used. As an example, Epirat (registered trademark) hair removal cream (Kracie) can be mentioned.

  The surgical field (skin at the surgical site) is disinfected with a common disinfectant solution used in human surgery. Specific examples of the disinfectant include popidone iodine (registered trademark: isodine), poloxamer iodine, ethanol for disinfection, chlorhexidine (registered trademark: hibiten), and the like.

  The surgical thread used for the gastrointestinal anastomosis (surgical suture) is preferably a sterilized absorbent thread. The type of absorbent thread is not particularly limited. As a specific material, Polyglactin 910), dexone (polyglycolic acid), monocryl and the like are exemplified, and monofilaments include PDS (polydioxanone), maxon and the like. The diameter of the surgical thread can be defined by the size of the model animal to be produced. In the case of a rat, USP-sized 5-0 thread can be preferably mentioned.

  Instruments used for surgery are preferably sterilized by methods used in human surgery. The sterilization method is not particularly limited, and the high-pressure steam sterilization method can be suitably used for instruments that can withstand heat and water. In addition, ethylene oxide gas, hydrogen peroxide low-temperature gas plasma sterilization, formaldehyde sterilization, etc. It can be illustrated. As for the small-diameter tube used for the common bile duct jejunostomy, a sterilized tube is used for indwelling in the body, and as a sterilization method, gas sterilization with ethylene gas can be preferably mentioned.

  The small diameter tube used for common bile duct jejunostomy is a sterilizable tube that is not destroyed when placed in the body of an animal, is non-toxic to animals, and has a small diameter that is flexible. There is no particular limitation. Examples of the material include silicon, polyurethane, polyethylene, polytetrafluoroethylene, and the like, and a polyethylene tube can be preferably cited from the advantages of easy insertion and low plasticity.

  The model animal is preferably administered with electrolyte / glucose solution (infusion) after surgery to prevent perioperative death due to surgical depletion and postprandial food loss. The administration route is not particularly limited, and subcutaneous, intraperitoneal, and intravenous routes can be exemplified. About the kind of infusion, if the said component is contained, it will not restrict | limit in particular, The 4th liquid (registered trademark: Solita) etc. of a postoperative recovery liquid can be mentioned suitably.

  The method for producing the model animal of the present invention will be described in more detail below. The surgical position is supine (surgical position with the abdomen up and back down), and the abdominal wall is opened through a midline incision. In order to allow the pancreatic juice to flow directly into the rectum, which is not involved in digestion and absorption, the duodenum, which is the influx route of the pancreatic fluid through the pancreatic duct by exocrine pancreas, is separated from the pyloric region of the stomach at the anal side [duodenotomy], and the separated jejunum The cut end of the duodenum on the continuous side (oral stump of the duodenum) is closed (closed). With regard to the position to be separated, when a rat is used, a 1 mm anal side from the pyloric part of the stomach can be exemplified as a suitable position. Further, the above duodenal resection can be performed at the pyloric site of the stomach instead of the separation at the position of the stomach from the pyloric region to the anal side. Examples of the surgical procedure for the closing and the end-side anastomosis include full-layer continuous sutures.

Next, the jejunum is dissected from the duodenum-jejunum boundary (Triz ligament) at the anal side [jejunostomy], and the cut end of the jejunum connected to an end different from the closed end of the closed duodenum is An end-to-side anastomosis is made to the rectum which has been incised on the opposite side of the membrane [small intestine rectal anastomosis]. With regard to the position to be dissected, when a rat is used, the 1 cm anal side of the Tritz ligament can be exemplified as a suitable position, and the size of the incision in the rectum can be about 2 mm. The above jejunostomy can be replaced with a duodenum-jejunum boundary or a duodenum-jejunum boundary from the duodenum-jejunum boundary to the mouth-side position in place of the duodenum-jejunum boundary. Examples of the surgical technique for the end-side anastomosis include full-layer continuous sutures.

  The gastropyloric part or the cut end of the duodenum connected to the gastric pyloric part by the duodenotomy is anastomosed with the cut end of the jejunum adhering to the large intestine-rectum by the jejunostomy [duodenal jejunostomy]. By such duodenal jejunostomy, food that is taken orally and passes through the stomach passes through the gastrointestinal tract downstream from the jejunum without passing through the duodenum where pancreatic juice is secreted. An anastomosis surgical technique may include full-layer continuous anastomosis.

  By the small intestinal rectal anastomosis, the pancreatic juice flows directly into the rectum without flowing into the jejunum, but the bile does not flow directly into the rectum together with the pancreatic juice, that is, the pancreatic juice remains flowing into the rectum. In order to divert only bile to the jejunum, first, a flexible thin plastic tube is inserted into the intestinal lumen from the opposite side of the mesentery of the anal jejunum from the duodenojejunostomy, and then the same as the insertion It penetrates the small intestinal wall on the side and leads out of the intestine. The tip of a flexible small-diameter tube passing through the small intestine lumen is inserted into the separated common bile duct on the liver side and ligated with an absorbent thread. A flexible small-diameter tube in the common bile duct and the small intestine wall penetration is fixed [common bile duct jejunum anastomosis]. When using a rat, as the position for inserting the flexible tube, the anal side can be exemplified by about 3 cm from the duodenojejunostomy, and the position for penetrating the flexible small diameter tube outside the intestinal tract is as follows: The mouth side can be raised about 1 cm from the insertion part. As a method for fixing the polyethylene tube in the small intestine wall penetration portion, a purse string suture can be mentioned.

  The non-human animal produced by the above technique exhibits symptoms of pancreatic exocrine dysfunction and is useful as a model animal for pancreatic exocrine dysfunction. The model animal of the present invention is characterized in that it simultaneously exhibits decreased serum lipase and serum trypsin concentrations, fatty liver and increased blood glucose level (lower glucose tolerance). That is, the model animal of the present invention is characterized by being able to observe the pathological conditions of chronic pancreatitis and diabetic diseases simultaneously with the symptoms of markedly reduced pancreatic exocrine function.

Moreover, the model animal of the present invention exhibits the following examination findings.
(1) The stool is muddy.
(2) Serum lipase and serum trypsin concentrations are lower than in the healthy control group.
(3) Fecal lipase and fecal trypsin concentrations are lower than in the healthy control group.
(4) The blood glucose level is higher than that in the healthy control group.
(5) Fat deposition and balloon-like swelling are observed in hepatocytes.

  Since the model animal of the present invention has a feature that it is not naturally cured due to its physical structure, screening for drugs for treating and / or preventing chronic pancreatitis and diabetes, determination of drug efficacy for chronic pancreatitis and diabetes, It can be suitably used for evaluation in regenerative medicine.

  As a screening method for a therapeutic and / or prophylactic agent for chronic pancreatitis or diabetes according to the present invention, there are a step of administering a test substance to the model animal of the present invention and a step of evaluating an improvement effect on chronic pancreatitis or diabetes. If it is the method provided, it will not restrict | limit in particular, There is no restriction | limiting in particular as said test substance. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts And plant extracts.

  The method for evaluating the efficacy of the present invention for improving chronic pancreatitis and diabetes is a method comprising the steps of administering a test drug to the model animal of the present invention and evaluating the improving effect on chronic pancreatitis and diabetes. The test drug is not particularly limited, and examples of the test drug include a newly developed digestive enzyme preparation for improving pancreatic exocrine function and a therapeutic drug for diabetes that lowers blood glucose level.

  The administration method of the test substance or the test drug is not particularly limited, and for example, it can be performed orally or by injection, but a preferable administration method includes oral ingestion from the oral cavity. Further, when the test substance is a protein, for example, a viral vector having a gene encoding the protein is constructed, and the gene is introduced into the model animal of the present invention using its infectivity. Is possible.

  Evaluation of the improvement effect on chronic pancreatitis is based on biochemical test values such as serum lipase concentration, serum trypsin concentration, fecal lipase concentration, fecal trypsin concentration, etc. in model animal-derived samples, and PFD test (exocrine pancreatic function test) test values. The comparison with the test value of the sample collected from the control animal can be used as an index. In addition, as a method for evaluating the improvement effect on diabetes, a method of determining biochemical test values such as blood glucose level and HbA1c value in a sample derived from a model animal with comparison with a test value of a sample collected from a control animal as an index Can be mentioned. The improvement using the test value as an index means that the test value of the model animal-derived sample approaches the test value of the test sample collected from the control animal.

  Since the improvement effect on chronic pancreatitis and diabetes can be referred to as the improvement effect on exocrine pancreatic dysfunction, the evaluation of the improvement effect on pancreatic exocrine dysfunction, that is, the evaluation of the improvement effect on chronic pancreatitis and diabetes, By examining the presenting pathological findings, it is possible to determine whether or not fatty liver has been improved by using an index. The pathological findings of fatty liver can illustrate, for example, balloon-like swelling of hepatocytes that show fat deposition in liver tissue. The improvement here means that the symptoms of fatty liver are restored to normal or the symptoms are alleviated. A person skilled in the art can determine whether or not the fatty liver symptoms have been appropriately improved for the model animal using the pathological findings described in the present specification as an index.

  The method for evaluating pancreatic regeneration therapy for exocrine dysfunction according to the present invention includes the step of transplanting cells or organs for the purpose of pancreatic regeneration therapy to the model animal of the present invention and the step of evaluating the improvement effect on pancreatic exocrine dysfunction And the cells and organs include pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, Examples thereof include an artificial organ differentiated from such pluripotent stem cells into the pancreas. Examples of a method for evaluating the improvement effect on exocrine pancreatic dysfunction include a method for evaluating the improvement effect on chronic pancreatitis and diabetes and a method for evaluating the improvement effect on exocrine pancreatic dysfunction.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

1. Preparation of Pancreatic Exocrine Deficient Rat 1-1 Preparation Animal A Wistar rat (CLEA Japan, Tokyo, Japan) weighing 14 weeks and weighing 350 g was operated by the following method to prepare a model animal.

1-2 Anesthesia, disinfection, and laparotomy Rats were subjected to general anesthesia by continuous inhalation of isoflurane 1.5-2% using an experimental animal anesthesia apparatus (Shinano Manufacturing Co., Ltd., Tokyo, Japan). The surgical field was depilated with animal hair clippers and hair removal cream and disinfected with 5% Hibiten (registered trademark) solution (Dainippon Sumitomo Pharma, Osaka, Japan). The surgical position was supine (surgical position with the abdomen up and back down), and the abdominal wall was opened through a midline incision.

1-3 Surgical process First, the surgical operation was performed by cutting the duodenum about 1 mm from the pylorus (the boundary between the stomach and the duodenum) perpendicularly to the intestinal axis (FIG. 1B). Next, the proximal jejunum on the anal side about 1 cm was dissected from the Tritz ligament (between the duodenum and jejunum) (FIG. 1B). By this operation, the duodenum and the digestive tract from the mouth side or the anal side were disconnected. The cut end of the duodenum is continuously stitched with 5-0 absorbent thread (the digestive tract is formed with a layered structure of mucous membrane, muscle layer, and serosa. And the intestinal tract wall is continuously closed using the single thread.

  The cut end of the anal jejunum, which is cut about 1 cm on the anal side of the Tritz ligament, that is, the cut end of the gastrointestinal tract continuous with the small intestine, large intestine, and rectum is raised on the ventral side of the colon, and cut about 1 mm from the pylorus. The cut end of the duodenum on the stomach side (the end of the duodenum continuous to the pyloric part of the stomach) and all-layer continuous anastomosis with 5-0 absorbent thread (the yarn is applied to all layers of the digestive tract at once, The joining method was performed (FIG. 1C). As a result of this operation, food that is taken orally and passes through the mouth, esophagus, and stomach flows into the jejunum through the duodenum where bile and pancreatic juice are secreted, and then passes through the ileum, colon, rectum, and anus. It was.

  An end-to-side anastomosis between the anal cut end of the duodenum isolated from the passage of food (the jejunal stump continuous to the duodenum) and the rectum with an incision of about 2 mm across the mesentery was performed. All layers were continuously sutured using an absorbent thread (FIG. 1C). As a result of this operation, bile and pancreatic juice that were originally secreted into the duodenum and involved in digestion and absorption in the small intestine flowed into the rectum that was not involved in digestion and absorption of food.

  Subsequently, in order to allow bile to flow into the small intestine, the common bile duct and jejunum were anastomosed as follows. Insert a polyethylene tube (PE50, Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) into the intestinal lumen from the opposite side of the mesentery of the small intestine, approximately 3 cm from the duodenojejunostomy. The small intestinal wall on the opposite side of the mesentery on the 1 cm mouth side was penetrated and guided outside the intestinal tract. The common bile duct was identified in the liver and duodenum, and it was cut off, and the tip of a polyethylene tube that passed through the small intestinal lumen was inserted about 1 cm into the liver. About 5 mm from the site where the polyethylene tube was inserted, the common bile duct on the liver side was ligated with 5-0 absorbent thread, and the polyethylene tube in the common bile duct was fixed. Using the absorbent thread that was used for fixation as it is, purse-string sutures (hang the thread around the penetration part like a purse string) at the small intestinal wall penetration part of the polyethylene tube and ligate the thread for fixation of the common bile duct As a result, a common bile duct jejunostomy was performed (FIG. 1C). The small intestine wall defect part of the puncture part of the polyethylene tube was closed with a nodule suture. By this operation, the bile secreted from the liver flows into the small intestine without going through the duodenum, and is involved in the digestion and absorption of food. In addition, pancreatic juice secreted from the pancreas into the duodenum did not pass through the small intestine and flowed into the rectum without coming into contact with food taken orally.

  Since the rectum has no digestive and absorption function of food, and the rectal contents are excreted from the anus as feces, the food taken orally is excreted without digestion and absorption by pancreatic juice, and the exocrine pancreatic function is abolished It becomes a model that shows the physiological function. As a comparison control group, rats of the same age with only a single laparotomy were used.

2. 2. Evaluation of pancreatic exocrine function 2-1 Sample collection Rats were general anesthetized one month after the operation, and 200 μL of blood sample was collected from the tail vein. Thereafter, the abdomen was opened through a midline incision, and the pancreas and liver were removed and sacrificed. At that time, it was confirmed that the duodenal rectal anastomosis, gastrojejunostomy, and common bile duct jejunostomy were maintained. The blood was centrifuged at 6000 rpm for 20 minutes after adding 2 μL of heparin, and the serum was collected and used for lipase and trypsin measurement. The pancreas and liver were placed in 10% formalin solution and fixed at 4 ° C. for 24 hours. After fixation, it is embedded in paraffin and a section slide is prepared. Tissue specimens were stained with hematoxylin-eosin and evaluated for histological findings.

2-2 Evaluation items Evaluation of the exocrine pancreatic function of the prepared animal model is based on the presence of weight loss and diarrhea due to dyspepsia one month after surgery, serum lipase / trypsin concentration as a marker reflecting pancreatic exocrine function, pancreas Liver tissue findings associated with decreased exocrine function were performed.

2-3 Intra-abdominal findings 1 month after surgery Rats were laparotomized under general anesthesia 1 month after surgery, and intra-abdominal findings were evaluated. The small intestinal rectal anastomosis (FIG. 2A), duodenal jejunostomy and common bile duct jejunostomy (FIG. 2B) were all maintained without failure. Although there was mild intestinal adhesion due to surgical operation, no intestinal obstruction was observed (a state in which the intestinal contents were not excreted), and no abscess (abscess) formation was suspected to be present.

2-4 Change in body weight Preoperative body weight was reduced to 295 g compared to 350 g. The body weight of the same age-old rat is 396-440g, and it is a remarkable weight loss.

2-5 Stool characteristics Feces of rats with reduced pancreatic exocrine function were mud. On the other hand, the stool of the rats in the control group was normal hard stool.

2-6 Serum Lipase and Trypsin Concentration Lipase is a fat digestive enzyme contained in pancreatic juice, and trypsin is a protein digestive enzyme also contained only in pancreatic juice, both of which are good indicators of exocrine pancreatic function. Serum lipase and trypsin concentrations were measured with lipase kit S (DS Pharma Biomedical, Osaka, Japan) and trypsin (E) [S] (TFB, Tokyo, Japan), respectively. As a result, serum lipase concentration was 11.5 IU / L (international unit), serum trypsin concentration was 9.3 ng / mL in rats with reduced pancreatic exocrine function, and serum lipase concentration was 19.1-30.3 in control group rats. IU / L and serum trypsin concentration of 14.5 ng / mL showed a tendency to decrease in rats with exocrine pancreatic dysfunction (FIGS. 3A and 3B). Intestinal fluid content of the small intestine (where pancreatic fluid does not flow in rats with exocrine pancreatic insufficiency) has a lipase concentration of 76.4 IU / L and a trypsin concentration of 92.6 ng / mL in rats with reduced pancreatic exocrine function. In contrast, the control group rats had a lipase concentration of 1749.3-1827.2 IU / L and a trypsin concentration of 406.6-1080.6 ng / mL (FIGS. 3C and D). The rat model produced by this technique had a markedly reduced pancreatic exocrine function.

2-7 Evaluation of glucose tolerance Although not directly related to exocrine pancreatic function, blood glucose level, which is an index of pancreatic endocrine function, was also measured 1 month after surgery as an evaluation item of pancreatic function. A blood sample was collected from the tail vein and measured with Asensia Breeze 2 (Bayer Yakuhin, Tokyo, Japan). The blood glucose level of the exocrine pancreatic dysfunction rat was 153 mg / dL, and that of the control group rat was 115-119 mg / dL.

2-8 Histological examination The hepatic histological findings associated with a decrease in pancreatic exocrine function indicate fat deposition in hepatocytes and balloon-like swelling of hepatocytes. Although it was a histological finding of a liver image of a rat with exocrine pancreatic insufficiency, hepatocyte balloon swelling was observed (FIG. 4A). On the other hand, such a finding was not observed in the control group rats (FIG. 4B). As for the pancreas, the pancreatic tissue was well maintained in both groups, and no fibrosis or loss of pancreatic islets was observed.

  The present invention is expected to provide a model animal of exocrine pancreatic dysfunction in which various pathological conditions such as diabetes and fatty liver progress, and is a completely novel therapeutic method / therapeutic agent for pancreatic exocrine function decline after pancreatectomy It is also expected to contribute to the development of biomarkers and pancreatic transplantation methods.

1 Common bile duct 2 Common bile duct amputation 3 Duodenal excision 4 Jejunostomy 5 Duodenal jejunostomy 6 Common bile duct jejunostomy 7 Intestinal rectal anastomosis

Claims (9)

Gastric pylorus and jejunum, or part of the duodenum continuous to the gastropylorus and the jejunum are anastomosed, and food flows directly into the jejunum without flowing into the duodenum; continuous into the duodenum and rectum or duodenum A portion of the jejunum and the rectum are anastomosed, and pancreatic juice flows directly into the rectum without flowing into the jejunum; and the common bile duct and jejunum are anastomosed through a small-diameter tube and bile flows into the duodenum A pancreatic exocrine dysfunction model animal characterized by consisting of a non-human animal. A portion of the duodenum continuous to the gastropyloric region and the jejunum are anastomosed, a portion of the jejunum continuous to the duodenum and the rectum are anastomosed, and the common bile duct and jejunum are anastomosed via a small-diameter tube. The animal model of pancreatic exocrine dysfunction according to claim 1. The model animal of pancreatic exocrine dysfunction according to claim 1 or 2, wherein the non-human animal is a rat. A method for producing a model animal of exocrine pancreatic insufficiency, wherein a surgical treatment comprising the following steps (a) to (e) is applied to a non-human animal.
(A) cutting the duodenum between the stomach pylorus and the duodenum into which pancreatic juice flows;
(B) cutting the proximal proximal jejunum from the Tritz ligament, which is the boundary between the duodenum and the jejunum;
(C) anastomosing the stump of the duodenum continuous to the gastric pyloric region produced by the step (a) and the jejunal stump produced by the step (b);
(D) closing the mouth side stump of the duodenum produced by the step (a) and anastomosing the jejunal stump continuous with the duodenum produced by the step (b) to the rectal incision;
(E) inserting one end of the small-diameter tube into the mouth side of the jejunum formed in the step (c) and anastomosing the other end of the small-diameter tube to the cut common bile duct on the liver side; A method for screening a therapeutic and / or prophylactic agent for chronic pancreatitis, comprising the following steps (a) and (b):
(A) a step of administering a test substance to an exocrine pancreatic dysfunction model animal according to any one of claims 1 to 3;
(B) a step of evaluating an improvement effect on chronic pancreatitis; A screening method for a therapeutic and / or prophylactic agent for diabetes comprising the following steps (a) and (b).
(A) a step of administering a test substance to an exocrine pancreatic dysfunction model animal according to any one of claims 1 to 3;
(B) a step of evaluating the improvement effect on diabetes; A drug efficacy evaluation method for improving chronic pancreatitis of a drug characterized by comprising the following steps (a) and (b).
(A) administering a test drug to the pancreatic exocrine dysfunction model animal according to any one of claims 1 to 3;
(B) a step of evaluating an improvement effect on chronic pancreatitis; A method for evaluating the efficacy of a drug for improving diabetes, comprising the following steps (a) and (b).
(A) administering a test drug to the pancreatic exocrine dysfunction model animal according to any one of claims 1 to 3;
(B) a step of evaluating the improvement effect on diabetes; A method for evaluating pancreatic regeneration therapy for exocrine dysfunction characterized by comprising the following steps (a) and (b):
(A) transplanting cells or organs for the purpose of pancreatic regeneration therapy to the pancreatic exocrine dysfunction model animal according to any one of claims 1 to 3;
(B) a step of evaluating an improvement effect on pancreatic exocrine dysfunction;