JP2976230B2 - Diabetic rat - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel diabetic rat, and in particular, to a long Evans diabetic model rat established by sibling mating.

2. Description of the Related Art Diabetes has been increasing in recent years as people's lives have become more affluent, and this disease not only causes serious complications but also becomes a major social problem when it occurs in young people. I have.

At present, the cause of diabetes is considered to be insufficient action of insulin, and (1) a decrease in the absolute amount of insulin;
It is considered that (2) decreased action efficiency of insulin in target organs, and (3) existence of anti-insulin substance (Tomio Tanase, Masakazu Abe, "Diabetes-basic and clinical", Kikunori Kosaka, Seiichiro Tarui, Ide Edited by Takehiko, Asakura Shoten, Tokyo,
1975, p. 251).

In addition, the administration of an oral hypoglycemic agent or insulin injection therapy is currently used for the treatment of diabetes, but it is not considered to be sufficient yet.

Laboratory animals created by administering streptozotocin or alloxan, etc., are used for the study of diabetes, which has many unknown parts, and are used for investigating the cause and screening for drugs. It is rarely due to poisoning, and the effectiveness of the above experimental animals is questionable. The onset of diabetes is considered to be a complex intertwining of genetic factors and environmental factors. For the study of diabetes, it is desirable to use a model animal close to the onset of diabetes in humans. BB rat (infected
Immunity, inflammation, 15 (1), 28-29, 1985) has been established.

The above BB rat develops at 60-120 days after birth in both sexes,
Symptoms include hyperglycemia, polydipsia, polyphagia, polyuria, diabetes, and ketoneuria. After onset, the body loses weight rapidly and dies. the above
Symptoms of BB rats are improved by administration of insulin.

Furthermore, the diabetic rats show a decrease in lymphocytes in peripheral blood or spleen, particularly a decrease in helper T cells, suggesting that an immunological mechanism is involved in the development. However, in humans, IDDM (Insulin Dependen
Since t Diabetes Mellitus (insulin-dependent diabetes mellitus) does not exist, the emergence of an IDDM model animal without lymphopenia has been desired.

Problems to be Solved by the Invention The object of the present invention is to obtain a T
The purpose of the present invention is to establish a stable diabetic model rat without cell lymphocyte depletion and thus closer to human diabetes.

Another object of the present invention is to establish a diabetic model rat useful for screening diabetic drugs or elucidating the pathology of diabetes.

Means for Solving the Problems The above objects are achieved by a diabetes model rat established from Long Evans rats.

The present inventors arbitrarily extracted nondiabetic siblings from among diabetic siblings of Long Evans rats that had diabetic siblings, and repeated sibling mating for each of these siblings. Establish a model rat,
Here, the present invention has been completed.

The Long Evans diabetic rat of the present invention is characterized by having the following isoenzyme patterns. Testosteric acid phosphatase-2a isoenzyme- type testis esterase-6a Testicular esterase-7b Testicular esterase-8b Testicular esterase-9a Testicular esterase-10a Liver aldehyde dehydrogenase-2 b liver aldehyde dehydrogenase-ca liver glycerophosphate dehydrogenase-1 a liver hydroxyacid oxidase a erythrocyte catalase-1 b erythrocyte hemoglobin β-chain b erythrocyte rat major tissue Compatible antigen-1 (RT-1) u Plasma esterase-1 b Plasma esterase-Sia Plasma group-specific components b Small intestine esterase-1 b Small intestine esterase-3a Urinary pepsinogen in gastrointestinal mucosa -1 a Renal esterase-4b renal fumarate hydrogenase-1b renal phosphogluconate dehydrogenase b seminal cyst protein-1a The diabetic rat of the present invention also has peripheral blood or spleen lymphopenia. It is also characterized in that it is not accompanied.
However, conventionally known BB diabetic rats develop the above-mentioned lymphopenia, and in this respect, the rats of the present invention are clearly distinguished from the BB diabetic rats.

The diabetic rat of the present invention having the above characteristics is named LETL (Long Evans Tokushima Lean rat) by the present inventors, and this name will be used in the present specification.

The LETL rat of the present invention is a stable diabetes model rat close to human diabetes, and its incidence is stable.
Therefore, this is very useful for research on elucidation of the condition of diabetes, search for diabetic drugs, and the like.

Hereinafter, the method for producing (breeding) the diabetic rat of the present invention will be described in detail. The diabetic rat is subjected to successive selection mating (brother-sister mating) using an outbred long Evans rat purchased from Charles River Canada as an ancestor. It can be obtained by:

The present inventors introduced and bred outbred Long Evans rats from Charles River Canada in 1982, and developed lesions similar to human diabetes from several rats, Discovered that he died. The characteristics of this lesion are suddenly severe diarrhea, abdominal swelling, thinning of the shoulders and lower back, weight loss, etc. from around 2 to 4 months after birth, blood glucose levels in the blood rise, and unless treated with insulin He died within one month. In addition, in the rat, histopathologically, extensive lymphocyte infiltration was observed in the pancreatic islets of Langerhans, and the lymphocyte infiltration was also observed in the submandibular gland, thyroid gland and the like.

However, among the Long Evans diabetic rats, female individuals who have developed diabetes have no reproductive ability,
Although male individuals have a reproductive ability in the early stage of onset, repeated use causes reproductive disorders such as infertility, imbalance in sex ratio, and decrease in litters.

Therefore, in the present invention, in order to maintain the incidence of diabetes at around 30% at all times in poor breeding conditions as described above, among diabetic siblings, females who do not develop diabetes and those who do not develop diabetes The main method of mating is to repeat mating between male siblings. However, this method alone increases the incidence, making it impossible to mate siblings who do not develop diabetes. So, arbitrarily extract siblings without diabetes onset and repeat mating, but repeating this method will gradually reduce the incidence of diabetes,
The onset rate was controlled by appropriately using a male individual who developed diabetes.

Rats are bred in cages (Econ TPX cages, manufactured by CLEA Japan) in a better chip (Northeasterm product).
s), and house 1 to 2 animals per cage at a temperature of 23 ±
The feed is solid feed (CRF) in an SPF environment controlled at 2 ° C, humidity 55 ± 5%, and lighting time from 7 am to 7 pm
-1, manufactured by Oriental Yeast Co., Ltd.), and tap water was freely taken as water.

In addition, the presence or absence of diabetes during the breeding period is 1 month per month.
Rats were forced to urinate at a rate of ２2 times, and examined using a urine test paper (Ethodiastex, manufactured by Miles Sankyo). In addition, during daily breeding management, by paying attention to the wetness and smell of the chips due to polyuria and diabetes in the cage,
You may know the onset before the test.

Using the above breeding conditions and testing methods, the present inventors established 20 strains of diabetes in Long Evans rats.
By cross-breeding, a stable diabetes model rat of the present invention close to human diabetes was successfully produced.

Effect of the Invention The diabetic rat provided by the present invention is useful as a model animal for studying the elucidation of the condition of diabetes or searching for a drug for diabetes.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Crossing of rats (1) Rearing of rats Long Evans rats were purchased from Charles River Canada. Cage (econ TPX cage,
Better chips (Northeasterm produ) manufactured by CLEA Japan
cts), and housed 1-2 animals per cage, temperature 23 ± 2 ° C, humidity 55 ± 5%, lighting time was controlled from 7:00 am to 7:00 pm under SPF environment as feed. Were allowed to freely ingest solid feed (CRF-1, manufactured by Oriental Yeast Co., Ltd.) and tap water as water.

(2) Diabetes test during breeding period The presence or absence of diabetes during the breeding period is inspected once or twice a month using urine test paper (Ketodiastex, manufactured by Miles Sankyo Co., Ltd.). did. In addition, during daily breeding management, onset of diabetes may be found before the above test due to wetness of the chips due to polyuria in the cage and diabetes and urine odor peculiar to diabetes.

(3) Mating of rats a: Establishment of LETL-type diabetic rats Rats that develop diabetes after purchasing the rats are found, and rats born in each generation are numbered consecutively. Female rats (without developing diabetes) were mated with No. 6 males (with diabetes). Next, the child, female No. 49 (non-diabetic), and another parent's child, male No. 62 (diabetic), were similarly bred.

As a result, 5 out of 11 (No. 36 to No. 46) diabetic rats were born. This system was used for the LETL rats of the present invention.
Generation (F0).

Next, among the above siblings of F0, female 41 (non-diabetic)
And 5th-generation (F1) rats (4 of them develop diabetes) by crossing with a male of the 154th (diabetes onset) born to another parent. This F1 female rat No. 99 (diabetes onset) and a male No. 96 rat (diabetes onset) were mated with siblings to obtain F2 (10, of which 6 diabetic). F3 females were crossed between F69 female rats (no diabetes) and F63 male rats (non-diabetic).

F3 female rats No. 28 (non-diabetic) and male No. 23 rats (non-diabetic) were mated to obtain F4.

F4 female rats (no diabetes) and F62 male rats (diabetes) were mated to obtain F5.

F5 123 female rats (non-diabetic) and F121 male rats (non-diabetic) were mated to obtain F6.

F6 # 65 female rats (non-diabetic) and 60 male rats (non-diabetic) were mated to obtain F7.

F8 female rats of 108 (non-diabetic) and 103 male rats (non-diabetic) were mated to obtain F8.

F8 female rats of No. 102 (non-diabetic) and 97 male rats (non-diabetic) were mated to obtain F9.

F9 female rats of No. 76 (non-diabetic) and male No. 66 rats (non-diabetic) were bred to obtain F10.

F10 female rats No. 85 (non-diabetic) and male No. 81 rats (non-diabetic) were mated to obtain F11.

The F126 female rat (non-diabetic) of F11 was mated with the 120 male rat (non-diabetic) to obtain F12.

F12 female rats of No. 146 (non-diabetic) were mated with male rats of No. 140 (non-diabetic) to obtain F13.

F13 female rats (no diabetes) and F129 male rats (non-diabetic) were crossed to obtain F14.

F14 female rats with F14 (non-diabetic) and male male 236 rats (with diabetic) were mated to obtain F15.

The F15 female rat 244 (without diabetes) and the male male 239 rat (without diabetes) were mated to obtain F16.

F16 # 278 female rats (non-diabetic) and 275 male rats (non-diabetic) were mated to obtain F17.

F17 female 262 rats (non-diabetic) and male 257 rats (diabetic) were mated to obtain F18. One week later, the F17 female rat 263 (non-diabetic) and the same male rat 257 (diabetic) as described above were bred to obtain another strain of F18. The former is F18 (1), and the latter is F18 (2).

F18 (1) mated with # 260 female rat (non-diabetic) and # 256 male rat (non-diabetic) to obtain F19 (1)
Similarly, F18 (1) female rat 261 (non-diabetic) and male 257 rat (non-diabetic) of F18 (1) were bred to obtain F19 (2). Further, the F18 (2) female rat No. 275 (non-diabetic) and the 270 male rat (non-diabetic) were crossed to obtain F19 (3).

For F19 (1), its female rat 296 (non-diabetic) and male rat 294 (non-diabetic) were crossed to obtain F20 (1), and for F19 (2), its female 291 Female rats (non-diabetic) and 287 male rats (non-diabetic) were bred to obtain F20 (2) and F19 (3)
About female 269 rats (non-diabetic) and 262 female rats
Mating with a male rat (non-diabetic), F20
(3) was obtained.

Thus, the LETL rat of the present invention was established as an inbred strain after 20 generations.

The present inventors further studied the four lines from F20 (1) [mating line of 437 female rats and 431 male rats, 438 female rats and 432
Mating strain with a male rat, mating strain between a 440 female rat and a 434 male rat, and mating strain between a 441 female rat and a 435 male rat], two lines from F20 (2) [female rat 407] And the mating system between the # 401 male rat and the # 408 female rat and the # 402 male rat], and two lines from F20 (3) [
8 strains of the diabetic model rat of the present invention (LETL) strain, which are mated with a 358 male rat and a 365 male female rat with a 360 male rat. The above-mentioned LETL rats of the present invention, which are maintained and bred, are always maintained in a condition that can be distributed by the present applicant.

b: Establishment of LETO control rats At the same time as the start of the establishment of the LETL rats of the present invention,
As control rats, female rats No. 50 (non-diabetic) and male male No. 45 (non-diabetic), both littermates from the start of a. Above, were bred to obtain 12 children. Three of them developed diabetes. This is LETO (Long Evans Tokushi
ma Otsuka rat) as the 0th generation (F0) of control rats,
Mating for line establishment of non-diabetic control rats was started as follows.

That is, among the siblings of F0, the 55th female (non-diabetic) and 48
The fifth male (non-diabetic) was crossed to obtain 5 F1 rats (all without diabetes).

F1 female 68 (non-diabetic) and male 65 (non-diabetic) were mated between siblings to obtain F2.

F2 was obtained by mating F2 female 49 (non-diabetic) and male 47 (diabetes-free).

F3 female 66 (non-diabetic) and male 59 (non-diabetic) were crossed to obtain F4.

F4 females No. 150 (non-diabetic) and male Nos. 148 (non-diabetic) were mated to obtain F5.

F5 females 193 (non-diabetic) and 190 males (non-diabetic) were mated to obtain F6.

Female F6 111 (non-diabetic) and male 106 (non-diabetic) were mated to obtain F7.

F7 female 142 (non-diabetic) and male 135 (non-diabetic) were mated to obtain F8.

Female F8 No. 118 (non-diabetic) and male No. 114 (non-diabetic) were mated to obtain F9.

Female F9 No. 94 (non-diabetic) was crossed with male No. 93 (non-diabetic) to obtain F10.

F10 female 147 (non-diabetic) and male 143 (non-diabetic) of F10 were crossed to obtain F11.

The F150 female (non-diabetic) of F11 was crossed with the 147 male (non-diabetic) to obtain F12.

Female F183, female 183 (non-diabetic) and male 179 (non-diabetic) were mated to obtain F13.

As described above, none of rats born by sibling mating developed diabetes.

F13 female 166 (non-diabetic) and male 162 (non-diabetic) of F13 were crossed to obtain F14 (1).
Female 167 (non-diabetic) and male 163 (non-diabetic)
And F14 (2) were obtained.

F14 (1) female 286 (non-diabetic) and male 283 (non-diabetic) were mated to obtain F15 (1).
F15 (2) was obtained by mating the 295 female (non-diabetic) and the 291 male (non-diabetic) of (2).

F15 (1) female 306 (non-diabetic) and male 301 (non-diabetic) were mated to obtain F16 (1), and similarly, female F295 (295) 295 female (non-diabetic) And No. 291 male (non-diabetic) were crossed to obtain F16 (2).

F16 (1) female 630 (non-diabetic) and male 626 (non-diabetic) were crossed to obtain F17 (1).
F17 (2) was obtained by mating the female of No. 340 (without diabetes) and the male of No. 334 (without diabetes) of (2).

F17 (1) female 613 (non-diabetic) and male 609 (non-diabetic) were crossed to obtain F18 (1).
F18 (2) was obtained by mating the 289 female (non-diabetic) and the 286 male (non-diabetic) of (2).

F18 (1) female 618 (non-diabetic) and male 614 (non-diabetic) were crossed to obtain F19 (1). Similarly, female F292 (292) of F18 (2) (non-diabetic) And 287 male (non-diabetic) were crossed to obtain F19 (2).

F19 (1) female 607 (non-diabetic) and male 602 (non-diabetic) were mated to obtain F20 (1).

Thus, LETO rats were established. This system is still being bred by the present inventor, and all of them are growing stably without developing diabetes.

Example 2 Diabetes test 1) Measurement of plasma glucose Blood was collected from rat tail vein 1 to 2 weeks after onset of diabetes, centrifuged at 3000 rpm for 10 minutes, and the supernatant was used as a sample for measuring blood glucose. Blood glucose measurement is glucose B
-Performed using Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

 The results are shown in Table 1 below.

As shown in Table 1 above, the diabetic rat of the present invention (LETL)
Shows that the average value of the blood sugar is 632 mg / d, which is about 5 times or more compared to 121 mg / d of normal rats (LETO).

2) Measurement of Blood Insulin Blood insulin was measured using the rat plasma obtained in the same manner as in 1) above, using a rat insulin measurement kit of Dynabot.

Radioimmunoassay (RIA) for insulin measurement
The procedure was performed as follows according to the law. That is, a borate buffer (0.1 ml) containing 0.1 ml of guinea pig anti-insulin serum (supplied in the above kit) and 0.25% of bovine serum albumin (BSA, manufactured by Sigma) was added to 0.05 ml of the sample obtained in the same manner as above.
M, pH 8.5) and reacted at 4 ° C. for 24 hours. Then, 0.1 ml of ¹²⁵ Ip Insulin (a solution diluted twice in BSA borate buffer (supplied in the above kit)) was added and reacted for further 72 hours at 4 ° C. To this reaction solution, 0.5 ml of rabbit anti-guinea pig serum was added. In addition, after standing at room temperature for 25 minutes,
Centrifugation was performed at 3000 rpm for 30 minutes. The supernatant is decanted and the radioactivity of both bound and unbound reactants (LS5000TD, Beckman)
Was measured for 2 minutes, and the insulin level in the blood was calculated from the standard curve created at the same time.

 The results obtained are shown in Table 2 below.

Is shown. The average value below the measurement limit was calculated by substituting 49.

As is clear from Table 2 above, normal rats (LETO)
Has a blood insulin level of 4545 pg / d,
In the diabetic rat (LETL) of the present invention, 6 out of 9 rats measured were below the measurement limit, and a clear decrease in insulin was observed.

Example 3 Assay of isoenzyme type Measurement of acid phosphatase-2 (Acp-2) The assay of Acp-2 isoenzyme type in testis was performed according to the method of Vendor et al. [Beuder, K., Bissbort, S., Kuhn, A ., Na
gel, M. and Gunther, E., Biochem. Genet., 24, 1-11, (19
85)].

That is, as a sample, 0.5 g of testis was added to 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron),
After centrifugation at 13000 rpm for 10 minutes at 4 ° C., the supernatant was used.

The above sample was coated on a cellulose acetate membrane (TITAN III-LIP
O, manufactured by Helena Co., Ltd.) and electrophoresed using a Tris-histidine (pH 5.9) buffer solution at 120 V for 60 minutes in the direction from + to-.

The plate after electrophoresis was immersed in the following staining solution at 37 ° C. for 1 hour for staining, fixed with 5% acetic acid, and the type of isoenzyme in the sample was determined from the migration pattern according to the above-mentioned literature.

[Staining solution composition]: α- or β-naphthyl phosphate 10 mg Fast violet B salt 10 mg 1 M citrate buffer (pH 4.5) 50 μ distilled water (hereinafter referred to as DW) Make the total amount 10 ml and aldehyde dehydrogenase- 2 (Ahd
-2) and aldehyde dehydrogenase-c (Ahd
-C) Measurement The liver isotypes of Ahd-2 and Ahd-c were assayed by the method of Adams et al. [Adams, M., Bav.
erstock, PR, Watts, CHS, and Gutman, GA, Boichem.
Genet., 22 , 611-629 (1984)]. That is, as a sample, 0.5 g of liver was added to 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron), and the mixture was heated at 4 ° C and 1300 ° C.
After centrifugation at 0 rpm for 10 minutes, the supernatant was used.

The above sample was applied to a cellulose acetate membrane of Titan III-Lipo (manufactured by Helena) in an amount of 0.3 μm, and tris-glycine (pH 8.6) buffer was used at 4 ° C. and 200 V for 30 minutes at a temperature of − to + for 30 minutes. Was electrophoresed.

After the above electrophoresis, the cellulose acetate membrane was stained with the following staining solution (37 ° C., 1 hour), and fixed with 5% acetic acid.
From the migration pattern, the type of isoenzyme in the sample was determined according to the literature.

[Staining solution composition]: 1 M Tris-HCl (pH 8.0) 0.1 ml acetaldehyde 0.5 ml NAD (nicotinamide adenine dinucleotide) 12.5 mg 1 M KCl 1 ml sodium pyruvate 25 mg pyrazole 25 mg MTT (3- (4,5-dimethylthia) Zolyl-2) -2,5-
Diphenyltetrazolium bromide) 3 mg PMS (phenazine methosulfate metasulfate) 1 mg DW Total amount of 10 ml Measurement of erythrocyte catalase-1 (Cs-1) Rats were laparotomized under ether anesthesia, and blood was collected from the inferior vena cava. . The collected blood was centrifuged at 4 ° C. and 3000 rpm for 10 minutes, and then red blood cells were washed by adding 5 ml of 0.8% NaCl aqueous solution. After repeating the washing operation twice, centrifugation was performed under the same conditions to prepare a red blood cell pack (RBC pack). After adding 1 volume of water to 1 volume of the RBC pack to cause hemolysis, centrifugation was performed at 4 ° C. and 13000 rpm for 10 minutes, and the supernatant was used as a sample, and the method of Yamada et al. [Yamada, J.,
Nikaido, H. and Kondo, Y., Jpn. J. Genet., 56, 447-455 (1
981)], the isoenzyme type of Cs-1 was assayed.

The above sample was treated with 0.015M Tris-HCl buffer (pH 8.8) for 100
After diluting to 200-fold and applying 0.6 μl to a cellulose acetate membrane of Titan III-Lipo (manufactured by Helena), 0.02 ME
Tris boric acid containing DTA (200 V, 30
And electrophoresed in the-to + direction.

After the above electrophoresis, the gel was washed with 1 mM hydrogen peroxide twice a minute, further washed with water, and then with 5 ml of 0.05 M ferric chloride.
It was immersed in a mixed solution of 5 ml of 0.05 M potassium ferricyanide, immediately stopped with 5% acetic acid, and the isoenzyme type in the sample was determined from the transfer pattern according to the literature.

Esterase-1 (Es-1) and Esterase-
Si (Es-Si) and small intestinal esterase-1 (Es-1)
And Measurement of Esterase-3 (Es-3) Rats were laparotomized under ether anesthesia, and blood and small intestine were collected from the abdominal inferior vena cava. Blood collected at 4 ℃, 3000rpm
For 10 minutes to obtain plasma.

In addition, 0.5 g of the small intestine was added to 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron), and then 4 ° C., 13 ° C.
Centrifugation was performed at 000 rpm for 10 minutes, and the supernatant was used as a sample.

The above sample was diluted with a diluent IV A [0.5 M Tris-HCl buffer (pH
6.8) 25 ml, glycerin 40 ml, 0.01% BPB 20 ml and water 15 m
l], and 5 μl of the solution is diluted with 10% polyacrylamide gel [the following solutions IA5ml, IB5ml, IIC1.25ml, IIDO.
5ml and DW8.25ml, total 20ml], 4 ° C, 10mA using Tris-glycine (pH8.6) buffer
For 30 minutes and then at 15 mA for 30 minutes (for plasma esterase) or 15 mA for 65 minutes (for small intestinal esterase), respectively.
Was electrophoresed in the + direction.

[Solution IA: polyacrylamide 39.0 g bis 1.0 g glycerin 20.0 ml H ₂ O total amount is 100.0 ml Solution IB: Tris 9.15 g concentrated hydrochloric acid 3.0 3.0 ml H ₂ O total amount is 100.0 ml Solution II C: ammonium persulfate 0.4 g The total amount of H ₂ O is 100.0 ml. Solution II D: TEMED 2.0 ml The total amount of H ₂ O is 100.0 ml.] After the above electrophoresis, esterase is stained with the following staining solution, and then fixed with 5% acetic acid The isoenzyme type in the sample was determined from the distance traveled according to the following literature.

[Staining solution composition]: α- or β-naphthyl butyrate 10 µ fast violet salt 50 mg acetone 100 µ 1 M phosphate buffer (pH 6.8) 100 µ DW Make the total amount 10 ml [Isoenzyme type assay literature]: Womack, JE, J .Hered., 63 , 41-42 (1972) Womack, JE, Biochem. Genet., 9, 13-24 (1973) Matsumoto, K., Syuto, B., Miyake, Y-I., Matsuhashi, A.
and Aizawa, M., Biochem. Genet., 17 , 1121-1129 (1979) Nikaido, H., Hayakawa, J., Kondo, Y., and Yamada, J., J.
Hared. 75 , 419-420 (1984) Katoh, H., Shoji, Y., Yoshimura, Y., and Esaki, K., Lab.
Anim., 20 , 158-161 (1986) Hedrich, HJ, von Deimling, O. and Kluge, R., Bioche.
m.Genet., 25, 79-93 (1987) Womack, JE, Experimentia, 28 , 1372 (1972) Mizuno, M. and Suzuki, K., Jpn. Genet., 53 , 137-142 (1
978) Measurement of renal esterase-4 (Es-4) Rats were laparotomized under ether anesthesia, and kidneys were collected.
To 0.5 g of the collected kidney, 1 ml of distilled water was added, and homogenized (using a homogenizer manufactured by Polytron).
After centrifugation at 13000 rpm for 10 minutes, the supernatant was used as a sample, and 5 µ of the sample was applied to the same 10% polyacrylamide gel as used above in
Using Tris-glycine (pH 8.6) buffer, 4 ° C, 15m
A, Electrophoresis was performed in the-to + direction for 65 minutes.

After the electrophoresis, esterase-4 was stained using the same staining solution as described above, and then fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the migration distance according to the literature [Woma
ck, JE, Biochem. Genet., 9, 13-24 (1973)].

Measurement of testis esterase-6 (Es-6) Testes were collected from rats under ether anesthesia. 0.5 g of the collected testes was added to 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron), and then 4 ° C. and 13000 rpm
Centrifugation for 10 minutes at, use the supernatant as a sample, dilute the sample 2-fold with diluent IVA, and
% Polyacrylamide gel [4 ml of solution IA, 5 ml of IB, II C
1.25 ml, adjusted from 0.5 ml of II D and 9.25 ml of DW, total 20 ml], and using 0.0125 M Tris-glycine (pH 8.6) buffer at 4 ° C., 10 mA for 30 minutes, and then 15 mA for 30 minutes. ,
Electrophoresis was performed in the + to + direction. After the electrophoresis, esterase-6 was stained with the following staining solution, and then fixed with 5% acetic acid, and the isoenzyme type in the sample was determined based on the migration distance [Bender, K., Nagel, M., Muller, CRand Gunt
her, E .., Transplant. Proceed., 11 , 1653-1656 (1979):
Bender, K., Nagel, M. and Gunther, E .., Biochem. Genet., 2
0 , 221-228 (1982)).

[Staining solution composition]: 1 M phosphate buffer (pH 6.8) 1 ml Potassium phyllocyanide 6 mg Potassium pherocyanide 8 mg 5-bromoindoxyacetic acid (in DMSO) 8 mg DW The total amount is 10 ml, and testis esterase-7 ( Es−
7), esterase-8 (Es-8), esterase-9
Measurement of (Es-9) and Esterase-10 (Es-10) Testes were collected from rats under ether anesthesia, and 0.5 g of the collected testes was added with 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron). 4 ℃, 13000rpm
Centrifuge for 10 minutes, use the supernatant as a sample, apply 10 μl of this sample to the same 8% polyacrylamide gel as above, and use 0.0125 M Tris-glycine (pH 8.6) buffer, Electrophoresis was performed at 4 ° C., 10 mA for 30 minutes, then at 15 mA for 30 minutes, in the − to + direction. After the electrophoresis, the esterase was stained using the following staining solution, then fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the migration based on the following literature.

[Staining solution composition]: α- or β-naphthyl butyrate 10 µ Fast violet salt 50 mg Acetone 100 µ 1 M phosphate buffer (pH 6.8) 100 µ DW Make the total volume 10 mL [Isoenzyme type assay literature]: Moutier, R., Toyama, K.and Charrier, MF, Biochem.Ge
net., 8 , 321-328 (1973) Moutier, R., Toyama, K. and Charrier, MF, Biochem. Ge
net., 9 , 109-115 (1973) Matsumoto, K., Biochem. Genet., 18,879-887 (1980) Measurement of renal fumarate hydrogenase (Fh-1) Rats were laparotomized under ether anesthesia. The kidney was collected, 0.5 ml of the collected kidney was added with 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron), and then 4 ° C., 1300
Centrifuge at 0 rpm for 10 minutes, use the supernatant as a sample, dilute the sample 2-fold with buffer to
Was applied to a cellulose acetate membrane, and 0.1 M Tris
Using sodium hydrogen phosphate buffer (pH 8.1)
Electrophoresis was performed in the-to + direction at 200 V for 90 minutes.

After the electrophoresis, Fh-1 was stained using the following staining solution, and then fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the distance traveled by the literature [Carleer, J. and Ansay, M.,
7 , 565-566 (1976): Cramer, DV, Blankert, JJand Paul, LC, 23 , 623-629.
(1985)).

[Staining solution composition]: fumaric acid 6 mg NAD (25 mg / ml) 0.08 ml Na pyruvate (50 mg / ml) 0.04 ml MTT (6 mg / ml) 0.08 ml PMS (2 mg / ml) 0.25 ml 0.2 M Tris-HCl buffer (PH 8.0) 2ml Maleate dihydrogenase 60IU DW Total amount of 10ml Measurement of rat major histocompatibility antigen (RT-1) of erythrocytes A small amount of blood was collected from the tail vein of a rat, and 0.15M was added to a Spitz tube. Take 15 ml of a solution containing 0.01 M EDTA in NaCl + 0.02 M HEPES (pH 7.2), put 300 μ of the above blood into it,
Immediately cover with parafilm and stir well. This is then centrifuged at 1000 rpm for 10 minutes and washed. EDTA the sediment
3 times with the above buffer solution containing no
m, pack by centrifugation for 10 minutes.

Next, 4 volumes of 0.15M NaCl + 0.02M HEPES (pH 7.2) buffer solution were added to 1 volume of dextran 70 (MW = 70,000 average) 6 w / v%, glucose (Dextran D “Otsuka”, Otsuka Pharmaceutical Co., Ltd.), FC
The above-mentioned washed blood is added with a solution containing S at a final concentration of 1% to prepare a 1% blood cell suspension.

Into each well of a disposable U plate (manufactured by Sanko Junyaku Co., Ltd.), 25 μl of the buffer containing Dextran and FCS is added. Next, the antiserum, which had been previously diluted to × 5 and × 10, was further serially diluted and 25 μl was added to each well, and 25 μl of a 1% blood cell suspension was added thereto.
The plate is shaken with a micromixer (manufactured by Taiyo Kagaku Kogyo KK) and left at 37 ° C. for 1 hour.

As the antiserum, an anti-W / Ms antibody against inbred rat F344 / Ms maintained at the National Institute of Genetics (Ms) was used. The inbred rat RT-1 has already been typed, and the F344 used above is
Type 1 of l of antiserum, W / Ms is classified into Type 2, and Type 1 is distinguished by the alphabet of l, Type 2 is u, Type 3 is n, and Type 4 is a.
By immunizing F344 / Ms with W / Ms splenocytes, an antiserum having an anti-u antibody is produced. If the anti-u antibody is absorbed by a u-type rat, its titer is lost. That is, if it is absorbed by W / Ms red blood cells, all the titers will be lost.

Measurement of plasma group-specific component (GC) Rats were laparotomized under ether anesthesia, blood was collected from the inferior vena cava, and the collected blood was centrifuged at 3000 rpm for 10 minutes at 4 ° C to obtain plasma. .

The plasma was diluted IV A [0.5 M Tris-HCl buffer (pH 6.
8) 25 ml, glycerin 40 ml, 0.01% BPB 20 ml and water 15 ml]
And dilute it 2 times, and use this as a sample, and
The sample was applied to the same 10% polyacrylamide gel as used in the above, and subjected to electrophoresis in a −125 direction at −15 mA at 4 ° C. using 0.0125 M Tris-glycine (pH 6.8) buffer at 65 ° C.

After the electrophoresis, the GC was stained with Ponceau S (Helena), followed by decolorization and fixation with 5% acetic acid.
utier, R., Biochem. Genet., 8, 321-328 (1973)).

Measurement of liver glycerophosphate dehydrogenase 1 (Gdc-1) Gdc-1 was measured by the method of Ericsson [Eriksson, K., Her.
edity., 37 , 341-349 (1976)].

That is, 0.5 g of the liver was added to 1 ml of distilled water, and homogenized (using a homogenizer manufactured by Polytron) at 4 ° C., 1300
Perform centrifugation at 0 rpm for 10 minutes, use the supernatant, and use 1 volume of the supernatant as a sample buffer [0.068 g of EDTA2Na in 10 ml of 0.0068 M tris, 100 ml of 0.0011 M citric acid, and 10 μ of 2-mercaptoethanol] One volume was added to obtain a sample, and the sample was applied to a cellulose acetate membrane of Titan III-Lipo (manufactured by Helena) at a thickness of 0.6 μm, and was subjected to 180 V using 0.137 M Tris and 0.02 M citrate buffer (pH 8.3). 75 minutes, 10 ° C
Then, electrophoresis was performed in the + to + direction.

After the electrophoresis, the cellulose acetate membrane was stained with the following staining solution (37 ° C., 30 minutes), fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the migration pattern according to the literature.

[Staining solution composition]: 0.2 M Tris-HCl (pH 9.0) 1.6 ml PMS (2.4 mg / ml) 0.2 ml MTT (10 mg / ml) 0.2 ml NAD (50 mg / ml) 0.4 ml DL-α-glycerophosphate ( 100 mg / ml) 0.1 ml Sodium pyruvate 20 mg DW Make the total amount 10 ml Liver hydroxyacid oxidase-1
Measurement of (Hao-1) Hao-1 was measured by the method of Kramer [Cramer, DV, Bioch.
em. Genet., 24 , 217-227 (1986)]. That is, as a sample, 0.5 g of liver was added to 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron), and the mixture was heated at 4 ° C. and 1300
After centrifugation at 0 rpm for 10 minutes, the supernatant was used.

The above sample was applied to a cellulose acetate membrane of Titan III-Lipo (manufactured by Helena) at a concentration of 0.9 μl, and a Tris-citrate buffer (0.034 M Tris, 0.005 M citric acid, pH 8.3) was applied.
Was used to perform electrophoresis in the − to + direction at 200 V for 30 minutes at 4 ° C.

After the electrophoresis, the cellulose acetate membrane was stained with the following staining solution (37 ° C., 30 minutes), fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the migration pattern according to the literature.

[Staining solution composition]: 1 M Tris-HCl (pH 7.4) 0.02 ml DL-α-hydroxyisocaproic acid 13 mg o-dianisidine 5 mg peroxidase 11 mg DW Measurement of hemoglobin β-chain (Hbb) of erythrocytes with a total volume of 10 ml The rats were laparotomized under ether anesthesia, blood was collected from the inferior vena cava, and the collected blood was centrifuged at 3000 rpm for 10 minutes at 4 ° C., and erythrocytes were added to 5 ml of 0.8% aqueous NaCl and washed. After repeating the washing operation twice, centrifugation was performed under the same conditions to prepare a red blood cell pack (RBC pack). One volume of this erythrocyte pack contains Tris-borate buffer (0.06
After hemolysis by adding 4 volumes of 5M Tris, 0.024M boric acid, pH 9.0), centrifugation was performed at 4 ° C. and 13000 rpm for 10 minutes, and the supernatant was used as a sample, and the literature [Brdicka, R., Folia Bio
logica (Praque), 11, 328-329 (1965)], and the Hbb isoenzyme type was determined.

The above sample was applied to a cellulose acetate membrane (Helena).
Tris containing 0.3μ applied and containing 0.0016M EDTA-2Na
Using a borate buffer (pH 8.4), 350V, 30 minutes, 4 ° C
Then, electrophoresis was performed in the + to + direction.

After the above electrophoresis, Hbb was analyzed using Ponceau S (manufactured by Helena).
Was stained and then decolored and fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the migration pattern according to the literature.

Gastrointestinal mucosa winery pepsinogen-1 (Pg
-1) Measurement Rats were laparotomized under ether anesthesia, the gastric mucosa was removed, and 0.5 g of the gastric mucosa was added to 1 ml of distilled water and homogenized (using a homogenizer manufactured by Polytron) at 4 ° C and 13,000 rpm.
Centrifugation was performed for 10 minutes, and the supernatant was used as a sample.

The sample is diluted with diluent IV A [0.5 M Tris HCl buffer (pH 6.8)
25 ml, glycerin 40 ml, 0.01% BPB 20 ml and water 15 ml], and 10 μl thereof is the same as that used in the above.
Apply to a 10% polyacrylamide gel and use Tris-glycine buffer (pH 8.6) at 10 mA for 30 minutes, then 1
Electrophoresis was performed at 5 mA for 25 minutes at 4 ° C. in the − to + direction.

After electrophoresis, add 1 ml of 0.1N NCl containing 0.7 g of bovine hemoglobin.
Infiltration was for 5 minutes, followed by an additional 60 minutes with 0.1 N HCl. Then, 0.05% Coomassie Brilliant Blue (30
% Methanol, 10% acetone).
Decolorize with 30% methanol and 10% acetone, and determine the isoenzyme type in the above sample from the literature [Cramer,
DV, Immunogenet., 13 , 555-558 (1981)].

Measurement of Kidney Phosphogluconate Dehydrogenase (Pgd) Rats were laparotomized under ether anesthesia, kidneys were collected, and 0.5 g of the collected kidney was homogenized by adding 1 ml of distilled water (using a homogenizer manufactured by Polytron). 4 ℃, 13000rpm
For 10 minutes, and the supernatant was used as a sample.

0.6 μ of the sample was applied to a cellulose acetate membrane Lipo (manufactured by Helena), and 0.1 M phosphate buffer (pH 7.0) 100
Using 2 mg of NADP per ml, 200 V, 30
Electrophoresed in the-to + direction for minutes.

After electrophoresis, Pgd is stained using the following staining solution,
After fixing with 5% acetic acid, the isoenzyme type in the above sample was determined by the literature [Carter, ND, Nature 224, 1214 based on the moving distance.
(1969)].

[Staining solution composition]: 1 M Tris-HCl (pH 8.0) 1 ml Trisodium 6-phosphogluconate (1 mg / 5 μ) 10 μ 1 M magnesium chloride 0.2 ml nicotinamide adenine dinucleotide phosphate (NAD)
P) 1 mg 3- (4,5-dimethylthiazolyl 2) -2,5-diphenyltetrazolium bromide (MTT) 1 mg PMS 1 mg DW Make the total amount 10 ml Measurement of sperm gland protein (svp-1) Under anaesthesia, the laparotomy is performed, the sperm gland is taken out, and the sperm fluid is squeezed out into 1 ml of distilled water. After thoroughly stirring, the mixture was centrifuged at 13000 rpm at 4 ° C. for 10 minutes, and the supernatant was used as a sample.

The sample was applied to a cellulose acetate membrane (manufactured by Helena).
6 μ was applied, and electrophoresis was performed at 4 ° C. and 400 V for 30 minutes in the − to + direction.

After the electrophoresis, Svp-1 was stained with Ponceau S (manufactured by Helena), decolorized and fixed with 5% acetic acid, and the isoenzyme type in the sample was determined from the distance traveled according to literature [Gasser, DLB].
iochem. Genet, 6, 61-63 (1972)].

Inventive diabetic rat (LETL) and control rat (LETO)
The results of the above isoenzyme type measurements are shown in Table 3 below.

<Measurement of peripheral blood lymphocyte subset in LETL and LETO> Diabetic rat of the present invention (LETL) and control rat (LETO)
BB / s rats for comparison (diabetic animals, vol. 2, 19
1988, pp. 30-37, `` La Shimaitis in BB rats and NK activity of peripheral blood lymphocytes '', Hiroaki Maruyama, Yoshihito Kasahara, Yukichi Suzuki,
The percentage of peripheral blood lymphocyte subsets was determined for each of the samples (see Subaru Taniguchi) according to the method described in the above literature.

 The results obtained are shown in Table 4 below.

From Table 4 above, there was no significant difference in the lymphocyte subset between the diabetic rat of the present invention (LETL) and its normal control rat (LETO), but the known diabetic rat BB / s showed ox19 (PanT ) And ox19 + / ox8- lymphocytes are remarkably reduced, suggesting that lymphopenia has developed.

────────────────────────────────────────────────── ─── Continued on the front page (56) Reference Infection, inflammation, and immunity, Vol. 15, No. 1, p. 28-39 (58) Field surveyed (Int.Cl. ⁶ , DB name) A01K 67/027 JICST file (JOIS) BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A diabetic disease model rat which is a LETL rat which is established from the Long Evans strain and has a trait that lymphocyte infiltration is observed in the islets of Langerhans. 2. The diabetic rat according to claim 1, which is free from peripheral blood or spleen lymphopenia.