CN104001188B - Centrifugal force and the shear stress response gene 1 function and application in fatty liver and diabetes - Google Patents

Abstract

The present invention discloses a kind of RECS1 function and application in fatty liver, diabetes, belongs to function and the application of gene.The present invention is with RECS1 knock out mice and wild type C57 mouse as experimental subjects, the fatty liver induced by high fat diet, diabetes model, result shows and the contrast of wild type C57 mouse, the carbohydrate metabolism disturbance of RECS1 knock out mice, fatty live lesions is serious, and accumulation of lipid increases notable.This explanation RECS1, to maintaining glycometabolism stable state to have ability of regulation and control, has the ability that liver lipids is removed and AFL is formed.Thus find RECS1 function in fatty liver, diabetes, it is mainly reflected in it and there is the effect that can improve fatty liver, diabetes.For the above-mentioned functions of RECS1, RECS1 can be used at preparation treatment fatty liver and the medicine of diabetes.

Description

Centrifugal force and the shear stress response gene 1 function in fatty liver and diabetes and Application

Technical field

The invention belongs to function and the application of gene, particularly to a kind of centrifugal force and shear stress response gene 1 (responsive to centrifugal force and shear stress gene 1, RECS1) is in fatty liver diabetes Function and application in disease.

Background technology

Diabetes are the chronic metabolic disease of a kind of serious harm human health, it is contemplated that to the quantity of patient in 2030 Will be added to 4.39 hundred million, it means that the whole world 20-79 year adult in will to have 7.7% be diabetic.In the past two In 30 years, diabetic's quantity is the most double, and the health to global human is a threat the most serious.Current youngster In child, teenager and Young Patients, diabetes B and the diabetes B early stage incidence of disease increase severely, and this also means that following diabetes Morbidity crowd will be enlarged by, difficulty of prevention and cure will increase [1].

Liver is as the biochemical plant of human body, and three big energetic supersession materials are both needed to process in this factory process just can transfer to Used by body, reflect liver critical role in metabolism.Three big metabolisms are disorderly, and especially sugar and fat metabolic disturbance are 2 Patients with type Ⅰ DM morbidity and progress play promotion and deterioration effect.Along with the prolongation of diabetic duration, there is the danger of hepatic disease Dangerous and lesion degree is consequently increased.Diabetic keratopathy hepatic injury refers to that liver histological that diabetes cause and function become Changing, be a kind of chronic complicating diseases of diabetes, persistence hyperglycemia state can cause hepatic insufficiency.And on the other hand, liver function Can extremely may result in glucose metabolism disorders, aggravate diabetes.

Due to the most clear to the cause of disease and the pathogenesis of diabetes, lack of the treatment of the cause of disease.Emphasize at present Early treatment, long-term treatment, complex treatment and the principle for the treatment of individuation.IDF (IDF) proposes diabetes Five main points of modern treatment, respectively: diet control, kinesiatrics, blood sugar monitoring, drug therapy and diabetes education.By In acute complicationses such as the seldom spontaneous generation DKAs of diabetes B patient, most of patients need not insulin treatment Sustain life, (unless in some stage, it may be necessary to insulin controls metabolic disorder), so OHA is at 2 type glycosurias Sick drug therapy occupies sizable ratio.The hypoglycemic medicine that the current Application comparison of oral drug therapy aspect is many mainly has Four classes: Drugs Promoting Insulin Secretion (sulfonylureas and meglitinide), biguanides, alpha-glucosidase inhibitor class and insulin sensitizer Class.These medicines and insulin action are in each metabolic organ of whole body (except alpha-glucosidase inhibitor), so correcting blood Also can produce a lot of side effects [2], such as put on weight (except biguanides) while sugar, other side effects are specifically shown in down:

1) promote that the bad reaction of insulin secretion agent is mainly hypoglycemia, there will be after taking medicine nervous, perspire, have famine Starving sense symptom, severe patient even there will be the disturbance of consciousness, the most also there will be the allergic reactions such as fash and liver and kidney dysfunction.

2) biguanides is mainly gastrointestinal reaction (patient has nauseating, the phenomenon of poor appetite after taking medicine) and breast Acid acid poisoning (after taking phenformin, patient there will be the symptom that weak, the disturbance of consciousness is even gone into a coma), some patient Have liver and kidney dysfunction and allergic reaction and macrocytic anemia reaction.

3) alpha-glucosidase inhibitor is the absorption by delay glucose thus reduces the purpose of blood sugar, due to self Not absorbing, the side effect to whole body is more relatively small, and its topmost side effect is gastrointestinal reaction, and patient has after taking medicine Abdominal distension, suffer from abdominal pain, suffer from diarrhoea and the intestines exhaust phenomenon such as too much.

4) insulin sensitizer works by activating PPAR-, and the side effect of its maximum is hepatic lesion and increasing Add blood volume, thus increase the weight of heart burden.

Owing to diabetes patient needs to take medicine all the life, the side effect of Long-term taking medicine is compared worry by a lot of patients, and these are secondary The control of effect also tends to one of key factor becoming glucose-lowering treatment success or failure.

Tumor necrosis factor-alpha (tumor necrosis factor-α, TNF-α) is a multifunctional cytokine, it It it is the main regulatory factors of Apoptosis, inflammation and immunity.TNF-α misregistration signal disease a series of with the mankind is closely related, bag Include [3] such as septicemia, diabetes, cancer, osteoporosis and atherosclerotics.Therefore, TNF-α is the most often listed in rush glycosuria Cause of disease.Insulin is internal unique hypoglycemic hormone, and the biological action of insulin is to be carried out by insulin receptor Signal conducts.On the one hand TNF-α mechanism of action in causing insulin resistance and 2 patients with type Ⅰ DM is to directly act on insulin Signal transduction system, interference insulin receptor after signal transduction and change the sensitiveness [4] of insulin；On the other hand by stimulating Other cell or tissues produce metabolism benefit indirectly-acting in insulin signal transduction system or the work that affects more internal enzymes Property and disturb glycometabolism [5].TNF-α is by playing a role from target cell membrane two kinds of different receptor-specifics combinations. One class is tumour necrosis factor receptor-1 (TNFR1), and molecular weight is 55KD(rodent, p55), the 60KD(mankind, p60).Separately One class is tumor necrosis factor receptor 2 (TNFR2), and molecular weight is 75KD(rodent, p75), the 80KD(mankind, p80).It is subject to Body is all the glycosylation albumen of single cross-film, and two kinds of acceptors are different in different Tissue distribution ratios.Up to the present, recognize Mainly be responsible for most of biological effect of TNF-α for TNFR1, as mediated apoptosis, break up, propagation etc., and TNFR2 is mainly responsible for Signal conducts, and TNFR2 can exciting transcription factor NF-KB, Apoptosis [6] can be regulated under given conditions.

RECS1(responsive to centrifugal force and shear stress gene 1) it is blood Shearing force response protein.The transcriptional expression of blood shear stress induction RECS1 is reported in Nojima laboratory [7] the earliest, and clones CDNA and amino acid sequence to RECS1.People RECS1 is 1 has 311 amino acid whose 7 transmembrane proteins, with Lifeguard With glutamate binding proteins, there is the highest homology.Have been reported that the l cell pair showing stable expression RECS1 Agonistic antibody special for TNFR2 shows certain tolerance, and display RECS1 may participate in the regulation and control [8] of TNF-α signal. Recent study result shows, RECS1 combines TNFR1, and suppresses the nuclear transcription factor-kappa B (NF-that overexpression TNFR1 induces κ B) activation [9], thus negative regulation TNF-α signal.Thus speculate that the expression of RECS1 gene is likely via regulation TNF-α letter Number, affect the function of insulin.

[bibliography]

1, S.Wild, G.Roglic, A.Green, R.Sicree, H.King, Diabetes Care 27,1047 (May,

2004).

2, D.M.Nathan, N.Engl.J.Med.347,1342 (Oct, 2002).

3, J.I.Barzilay, L.Abraham, S.R.Heckbert, Diabetes.50,2384 (Oct, 2001)

4、G.S.Hotamisligil, D.L.Murray, L.N.Choy, Proc Natl Acad Sci USA 91, 4854(May,1994).

5, R.J.Grigsby, R.T.Dobrowsky, Biochem Biophys Res Commun 287,1121(Oct, 2001).

6、L.Tartaglia, P.Pennica, D.Goeddel, J Biol Chem 268, 18542(Sep,1993) .

7, H.Yoshisue, K.Suzuki, A.Kawabatal, Atherosclerosis 162,323 (Jun, 2002).

8, Cai Cifeng, Wu Mingjiang, Liao Zhiyong. Chinese biological chemistry and molecular biosciences journal 26,36(Jan, 2010)

9, Liao Zhiyong. Chinese biological chemistry and molecular biosciences journal 27,412(May, 2011).

Summary of the invention

In view of problem present in above-mentioned existing glucose-lowering treatment technology, it is an object of the invention to the expression determining RECS1 with Correlation between fatty liver, diabetes, it is provided that a kind of RECS1 treats fatty liver, diabetes as drug targets in screening Medicine in application, it is provided that one for treating the new application of target gene RECS1 of fatty liver, diabetes.

The purpose of the present invention is realized by following technical scheme:

The present invention, with wild type C57 mouse and RECS1 knock out mice as experimental subjects, is induced by high fat diet Mice model of obesity (diet induced obesity, DIO) model, result shows to contrast with wild type WT mouse, RECS1 base Because knock-out mice shows obesity, its body weight is apparently higher than the WT mouse of diet of the same race, and RECS1 knock out mice Fasting blood-glucose, Diagnostic Value of Fasting Serum insulin level are above control group WT mouse, are found by lumbar injection glucose tolerance test The tolerance of glucose is substantially weakened by RECS1 knock out mice.From mouse liver general appearance, liver weight and liver Dirty/body weight ratio and pathological staining, liver organization lipid components quantitative analysis etc. all illustrates HFD group (Highfat diet, high fat Diet) RECS1 KO mouse adipose liver pathological changes serious, accumulation of lipid increases notable.This shows that RECS1 gene knockout can aggravate Fatty liver, the generation of diabetes, RECS1 has the function maintaining glycometabolism stable state with improving fatty liver, diabetes.

For the above-mentioned functions of RECS1, RECS1 can in preparation prevention, alleviate and/treatment fatty liver, diabetes conditions Application in medicine.

For the above-mentioned functions of RECS1, RECS1 can apply in terms for the treatment of the medicine of diabetes in preparation.

A kind of medicine treating Fatty Liver Disease, comprises RECS1.

A kind of medicine treating diabetes, comprises RECS1.

The present invention has such advantages as relative to prior art and effect:

1. present invention discover that the New function of RECS1 gene, i.e. RECS1 gene can improve the effect of fatty liver, diabetes.

2. RECS1 effect in improving fatty liver, diabetes, preparation is used for treating fatty liver and diabetes medicament side Face is applied.

Accompanying drawing explanation

Fig. 1 is the body weight of WT and RECS1-KO mouse, blood sugar, insulin result figure.

A is Mouse Weight result figure；

B is fasting blood glucose level statistical chart；

C figure is serum Fasting insulin level statistical chart.

Fig. 2 is that WT and RECS1-KO mouse is by lumbar injection glucose tolerance result figure.

A is by different time points mouse blood sugar level statistic figure after lumbar injection glucose；

B is each group of glucose tolerance in mice TG-AUC (area under the curve, AUC) comparison diagram.

Fig. 3 is the liver general appearance result figure of RECS1-KO and WT mouse.

A is liver substantially result figure；

B is liver weight, liver weight and mouse weight ratio Data-Statistics block diagram own.

Fig. 4 is liver organization lipid components and the lipid-metabolism situation result figure of RECS1-KO and WT mouse.A figure is HE With oil red O colored graph；

B figure is the content quantitative analytic statistics block diagram of triglycerides, T-CHOL and free fatty.

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit In this.

Animal for research and raising:

Animal used as test: C57BL/6 mouse and RECS1 KO mouse, male, 8 week old.C57BL/6 mouse is purchased from Beijing China Fukang bio tech ltd (WT, the certification of fitness number: 949431)；(RECS1-KO is purchased from RECS1 knock out mice RIKEN company of Japan, article No.: RBRC01772).

Animal used as test feed formula: high lipid food (HFD) is (purchased from Fukang bio tech ltd of Beijing China, article No. D12942): percent of calories: protein: 20%；Carbohydrate: 20%；Fat: 60%, total thermal mass ratio: 5.24kcal/g.Low fat feed (NC) (purchased from Fukang bio tech ltd of Beijing China, article No. D12450B): heat percentage Ratio: protein: 20%；Carbohydrate: 70%；Fat: 10%, total thermal mass ratio: 3.85kcal/g.

Animal feeding and environmental condition: all of experiment mice is all raised in angiocardiopathy institute of Wuhan University SPF level Animal House (credit number: SYXK(Hubei Province): 2009-0053).Alternately illumination in every 12 hours, temperature 24 ± 2 DEG C, humidity 40%-70%, Mouse freely drinks water feed.

[embodiment 1] mouse adipose liver, diabetes model (DIO) obtain

1. animal used as test packet: select 8 week old, male, WT mouse and RECS1 KO mouse, give respectively two kinds special Feed D12942 high lipid food (Highfat diet, HFD) and D12450B low fat feed (Normal chow, NC) are raised, i.e. WT NC group, KO NC group, WT HFD group, KO HFD group totally 4 groups.

2. high lipid food guidance model operating process:

Use WT and KO mouse, set up DIO model, carry out phenotype correlation analysis, specify RECS1 gene pairs metabolic disease Morbidity plays a significant role.Select 8 week old, male, WT mouse and RECS1 KO mouse, give two kinds of special feeds respectively D12942 high lipid food (Highfat diet, HFD) and D12450B low fat feed (Normal chow, NC) are raised, i.e. WT NC group, KO NC group, WT HFD group, KO HFD group totally 4 groups.Record mouse food ration, mouse empty body weight the most in detail Detect 1 time every 4 weeks with fasting blood-glucose.In experiment 0 week and the 24th week, mouse ether inhalation anesthesia, through socket of the eye venous blood collection, divide Insulin content in serum, detection serum.Test the 22nd week, carry out lumbar injection glucose experiment (IPGTT), to evaluate Mouse body is to glucose tolerance.Within 24th week, draw materials in whole end, takes out mouse liver and takes pictures, and then to be placed in formaldehyde molten for a part Liquid is fixed or O.C.T frozen section embedding medium (Tissue Freezing Medium) embeds as pathological analysis use, another It is partially placed in liquid nitrogen, is placed on-80 DEG C of Refrigerator stores.

[embodiment 2] fatty liver, diabetes model (DIO) Mouse Weight, blood sugar and insulin level measure

1. mouse empty body weight, appetite detects, and socket of the eye venous sinus is taken a blood sample.

1) body weight detection

1. fasting: 8:00 will treat experiment mice fasting (can't help water) in the morning, and afternoon, 2:00 started experimental implementation.

2. weigh: weighed at 0 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks respectively, a plastics keg is placed on dynamically On electronic balance, pick up mouse, put in weighing keg, measure body weight record data.Forage volume detects: waiting to weigh has operated Cheng Hou, adds feed to mouse, and records the forage volume of mouse on dynamic electron balance.

2) socket of the eye venous sinus blood sampling after ether inhalation anesthesia, separates serum.

1. anesthesia: will dip ether cotton balls and put in anesthesia cylinder, and be put into by mouse rapidly simultaneously, build lid, ether is inhaled Enter method general anesthesia.

2. conventional crawl mouse, is placed on blood collecting table, and left hand thumb and forefinger catch the skin between mouse two ear to make mouse solid Fixed, and compressing neck both sides gently, hindering venous return, make the abundant evagination of eyeball, prompting retroorbital venous clump is congested.

3. the right hand holds heparin tube, by the most advanced and sophisticated angle at 45 ° with mouse face, after in inserting along endocanthion, eye enters sliding eyeball Side, thrusts to direction, eyeground, rotates light heparin tube when feeling to have resistance to cut quiet clump, and blood i.e. flows into heparin tube.

The angle the most slowly adjusting heparin tube is allowed to level or slopes slightly downward, and 0.5mLEP pipe is placed in blood sampling by auxiliary Blood is collected in the lower section of pipe end, and puts on ear tag number on 0.5mLEP pipe.After blood sampling terminates, extract heparin tube, loosen a left side Hand, uses sterile gauze block to oppress eye socket gently and helps hemostasis.

5. serum is separated: the EP pipe standing 1-2h at room temperature collecting blood makes blood natural coagulation.4 DEG C, 4000rpm/ Min, centrifugal 30min, be sufficiently separated serum.Draw serum respectively with micropipettor to put in aseptic EP pipe, EP pipe is marked Note, is stored in-80 DEG C of refrigerators subsequently.

2. fasting blood-glucose and serum insulin level test experience

Opened needing mouse fasting (can't help water), i.e. fasting between morning 8:00 to afternoon 2:00 of experiment after 6 hours Beginning experimental implementation.

1) blood sugar is surveyed

1. blood glucose meter prepares: check blood glucose meter (Johnson Co., ONETOUCH) battery, by right-side switch, by test paper Being properly placed left side slot, screen shows the numeral with blood sugar test paper bar respective code, shows pattern of bleeding subsequently, points out blood sugar Instrument enters state to be measured.

2. fixing mouse: the right hand grabs rat-tail, left hand holds one piece of towel, by towel doubling, pinches towel with thumb and forefinger Fold position, wraps into mouse head and health the towel in palm, thumb and forefinger and is being fixed by rat-tail root.

3. tail is cut: eye scissors is cutting rat-tail rapidly at rat-tail end 0.1-0.2cm, treats that drop of blood flows out voluntarily.

4. blood sugar test: blood glucose meter test paper edge touches drop of blood, blood immerses test paper, blood glucose meter countdown display in 5 seconds Reading.

2) ELISA method detection mouse islets element

1. reagent and consumptive material prepare:

Sample (frost serum), deionized water one bottle (1000mL), mouse islets element ELISA kit (Millipore, Article No. EZRMI-13K) insulin-containing ELISA ELISA Plate (1 piece, be stored in 2-8 DEG C), ELISA Plate sealed membrane (1), 10 × HRP elution buffer (2 bottles, every bottle of 50 mL, use before use deionized water dilute 10 times), insulin standards (concentration is: 0.2,0.5,1,2,5,10ng/ mL, every kind of amount is for 0.25mL), insulin quality controls buffer(0.25 mL), matrix solution (0.5 mL), analysis buffer (20 mL), insulin detection antibody (10 mL), enzyme solutions (12 mL), substrate (12 mL), end Only solution (12 mL).

2. experimental procedure:

A. confirm that incubator is opened, be familiar with ELIASA operation, serum specimen to be detected is found out from-80 DEG C of refrigerators；

B. melt at room temperature multiple for serum to be detected to liquid, prepare detection；

C. 10 × HRP elution buffer, every bottle of use 450 mL deionized water dilution are diluted；

D. the ELIAS strip taken off is installed to 300 μ LTBS elution buffer washing 3 on an empty ELISA Plate frame Secondary.Washing and complete tip upside down on blotting paper by ELISA Plate, clap several times gently, being exhausted by raffinate (notes: before carrying out next step Avoid ELISA Plate to be dried, untapped ELIAS strip is sealed in the sack of dress ELISA Plate, 2-8 DEG C of preservation)；

E. 10 μ L analysis buffer are added non-specific pore (NSB) and all of sample well；

F. 10 μ L matrix solutions are added in NSB hole, gauge orifice and control wells；

G. in default gauge orifice, add 10 μ L insulin standards；

H. in default quality controls hole, add insulin quality controls buffer1 and 2 each 10 μ L；

I. in each sample well, add 10 μ L sample；

J. (all above load procedure completed within 1 hour, room to add 80 μ L insulin detection antibody in every hole Temperature is hatched 2 hours, and during this period by ELISA Plate as on ELISA Plate oscillator, adjusting rotating speed is 400-500rpm concussion)；

K. taking off sealed membrane, discard liquid, pat gently on blotting paper, exhaust raffinate；

L. elution buffer is used to wash plate 3 times, each 300 μ L, wash that plate is complete all sucks raffinate with blotting paper every time；

M. every hole adds enzyme solutions 100 μ L, and after sealed membrane sealing, under room temperature, ELISA Plate oscillator shakes 30 minutes, Taking off sealed membrane, discard solution, beating is blotted；

N. elution buffer is used to wash plate 6 times, each 300 μ L, wash that plate is complete all to be sucked raffinate with blotting paper and add every time Enter 100 μ L substrate solutions, ELISA Plate oscillator shakes about 15 minutes.Now it can be seen that depth gradual change occurs in gauge orifice Blue.(note: 15 minutes may be significantly faster than that in this step, blue appearance and progress, it is also possible to considerably slower than 15 minutes, Depend on ambient temperature.The time hatched please is judged by vision.Or using ELIASA 370nm wavelength detecting, reading exists Between 1.2 to 1.8, then it is suitable incubation time)

O. adding 100 μ L stop baths, concussion ELISA Plate guarantees sufficiently to mix, and now blueness becomes yellow.5 minutes Light absorption value is read respectively at inherent 450nm and 590nm.According to the calibration curve measured, converse concentration by light absorption value.

The evaluation index of the diabetic lesions order of severity mainly includes body weight, blood sugar and insulin level, and these indexs are equal With diabetes order of severity positive correlation.Body weight, blood sugar and insulin result of variations are as shown in Figure 1, it has been found that WT mouse is being given With the body weight after HFD diet apparently higher than NC feed group, give the RECS1 KO mouse HFD feed of 24 weeks and NC feed is raised After Yanging, from the RECS1 KO Mouse Weight of the 4th week beginning HFD group apparently higher than the WT Mouse Weight of HFD group, it is continued until 24th week (see Figure 1A), shows that RECS1 may play an important role in the diabetes that high fat diet induction causes.

Find the mouse of HFD group from the beginning of 4 weeks through fasting blood-glucose detection, fasting blood glucose level the most corresponding NC comparison Group raises, and the RECS1 KO mouse fasting blood glucose level of HFD group is higher (see figure than the WT mouse fasting blood glucose level of HFD group 1B).The serum Fasting insulin level respectively organizing mouse compares, and the WT mouse of HFD group and RECS1 KO mouse were at 24 weeks Significantly raising time all than 0 week, RECS1 KO group is substantially higher in WT mouse (see Fig. 1 C).This just shows to knock out after RECS1 gene Significantly affects mouse glycometabolism stable state under HFD raising state, and create significant insulin resistance.RECS1 is described Gene can significantly improve the glycometabolism stable state of mouse, the beneficially performance of insulin function.

[embodiment 3] glucose tolerance test (intraperitoneal glucose tolerance test, IPGTT)

Test the 22nd week, carry out lumbar injection glucose experiment (IPGTT), to evaluate mouse body to sugar tolerance.

1. before surveying blood sugar, first measure the empty body weight of mouse, calculate the volume injected of glucose according to 10 μ L/g.

Fasting blood-glucose when being the most first 0 minute before the injection of detection glucose sugar, rapidly through lumbar injection grape after detection Liquid glucose.

3. lumbar injection method of operating: (1) fixes mouse；Pick up mouse, the little finger of toe of left hand and the nameless tail grabbing mouse Bar, another three fingers are caught the neck of mouse, are made the head of mouse downwards, fully exposed by mouse web portion.(2) inserting needle location and note Penetrate: from the belly side right hand syringes of inserting needle, by angle at 45 ° with mouse web portion for tip, inserting needle, pumpback, inject hour hands Head walks a small distance in subcutaneous abdomen, enters abdominal cavity at belly opposite side, after having injected medicine, slowly after ventrimeson Extract syringe needle, and slight rotating needle, prevent leakage.

4. after lumbar injection 15 points, 30 points, 60 points, 120 minutes points are cut tail and are surveyed mouse blood sugar value, and remember Record blood sugar numerical value and detection time.

We are further by lumbar injection glucose tolerance test (intraperitoneal glucose Tolerancetests, IPGTT) assess each group of mouse tolerance to glucose, experiment the 22nd week, by injection After the glucose of 1.0g/kg body weight, WT mouse and the RECS1 KO mouse blood sugar level of HFD group increase severely at 15 minutes points Reaching peak value, elapse over time to injecting latter 60 minutes, two groups of mouse blood sugar levels somewhat decline, but still in higher than on an empty stomach Blood sugar level (blood sugar when 0 minute), little recovers to fasting blood glucose level constantly 2, and RECS1 KO mouse blood sugar level is from 0 The blood sugar level (Fig. 2 A) minute being constantly in higher than WT mouse to 2 hours.Each group mouse blood sugar TG-AUC (area Under the curve, AUC) compare, it has been found that the AUC of WT mouse HFD group is significantly higher than NC group, RECS1 KO HFD group AUC be noticeably greater than AUC(Fig. 2 B of WT HFD group), show that RECS1 is to maintaining glycometabolism stable state to have powerful ability of regulation and control.

[embodiment 4] liver general appearance, liver organization lipid components and lipid-metabolism situation measure

1. end liver organization is drawn materials eventually

1., after mouse weights, the most de-neck is put to death.Lie on the back fixing mouse, mouse chest, belly hair is moistened with distilled water Wet.

2. with a tweezers clamp mouse web portion center skin, skin is cut off under xiphoid-process along belly center to head, to tail End cuts off skin, successively exposes subcutaneous fascia, muscle etc., opens abdominal cavity, fully exposes each internal organs.

3. quickly find and take off the liver of mouse, the liver specimens taken off is placed on sterile gauze, wipe dry liver table Remained blood on face, is placed in liver in sterile petri dish, takes pictures rapidly, weigh.

4. paraffin specimen: be placed in 10% neutral formalin fixing.Frost sample: cut partial liver, be placed in OCT Tinfoil mould in embed, be placed on cryofixation on dry ice.

2. liver organization processes and pathological staining related experiment

1) liver dehydration, transparent, waxdip

Cut the part lobe of the liver fixed in 10% neutral formalin to be organized in the embedding frame of mark, at low discharge stream Water rinses more than 30 minutes.Following procedure is set on machine according to below scheme, is 1. dehydrated: 75% alcohol (45 minutes) → 75% Alcohol (45 minutes) → 85% alcohol (45 minutes) → 85% alcohol (45 minutes) → 95% alcohol (45 minutes) → 95% alcohol (45 points Clock) → absolute alcohol (1 hour) → absolute alcohol (1 hour)；The most transparent: dimethylbenzene (1 hour) → dimethylbenzene (1 hour)；③ Soak cured (65 DEG C): paraffin (1 hour) → paraffin (1 hour).After treating that tissue rinses, the embedding frame comprising tissue is put into machine In device basketry, start said procedure.After said procedure completes, take out organization embedding frame and send pathology room investing tissue, clean simultaneously Machine is standby.

2) liver tissue slices

This laboratory microtome (slice thickness 5um)

3) liver organization hematoxylin-eosin (HE) dyeing

Liver organization paraffin section is put into (5 minutes × 3 times) → 100% in 65 DEG C of baking ovens (30 minutes) → dimethylbenzene Alcohol (1 minute) → 90% alcohol (1 minute) → 70% alcohol (1 minute) → distillation washing → haematoxylin (5 minutes) → running water Wash away loose colour → 1% hydrochloride alcohol (1 to 3 second) in section → wash from the beginning several under → Scott promotees blue liquid (sodium acid carbonate 0.35g, magnesium sulfate 2g, distilled water 100ml) under (1 minute) → wash from the beginning is several → Yihong (1 minute) → distilled water washes away section On loose colour → 70% alcohol once → 90% alcohol once → 100% alcohol (30 seconds × 3 times) → dimethylbenzene (2 minutes × 3 times) → when dimethylbenzene is the most dry mounting, take pictures.

4) liver organization oil red O stain

1. frozen liver tissues section being air-dried 30 minutes in fume hood, 4% paraformaldehyde fixes 10 minutes.It is placed in double Steam in water and slightly wash 10 minutes, to remove the paraformaldehyde that tissue shows.

2. process 1 minute with 60% isopropanol.

3. with oil red O(company sigma, article No. O0625, concentration 0.5 gram/100ml100% isopropanol) dye 30 minutes.

4. with 60% isopropyl alcohol 1 minute × 3 times after, until clean background.

5. with Mayer ' s haematoxylin dye liquor (5) light dye nucleus.

6. water rinsing, promotees indigo plant, fully washes, be washed to nucleus oil blackeite in dilute lithium carbonate aqueous solution.

7. use glycerin gelatine mounting, take pictures.

3. murine liver tissue lipid detection

1. take out liver tissue sample from-80 DEG C of refrigerators, weigh 50mg tissue, use mill to be ground to form by liver organization Powder, is dissolved in 1 mLPBS, is total to overnight incubation with 1 mL chloroform/methanol (2:1) again after mixing.

2. after with 12000g high speed centrifugation 15min, lipid layer material at the bottom of collecting pipe, air-dry, to remove moisture removal.

3. isolated lipid layer material is dissolved in the 200 μ L PBS solution containing 1% Triton X-100, careful pressure-vaccum Mixing.

4. open computer labman software, printer, be then turned on Biochemical Analyzer；

5. select and clean detection probe, cuvette etc., it is ensured that probe is unobstructed, the attachment of cuvette free from admixture, and light absorption value exists The term of reference set；

6. on labman software, check that required Testing index reagent is enough, sets Testing index and detection ordering Deng.

7. by machine testing on prepared mixing liquid, analyze.

Liver general appearance, liver organization lipid components and lipid-metabolism measurement result as shown in Figure 3, by substantially drawing materials Take pictures, it is observed that the liver volume the RECS1 KO mouse of HFD group is slightly big and front compared with the liver of the WT mouse of HFD group Person's liver color and luster yellowing, grease more (such as Fig. 3 A).RECS1 KO mouse no matter liver weight or liver weight in HFD group The WT mouse of body weight ratio the most relatively HFD group with mouse own is high, illustrates that RECS1 KO mouse adipose liver is substantially (such as Fig. 3 B).We Further by histotomy, carrying out HE and oil red O stain, basis of microscopic observation respectively organizes the pathological change of mouse liver tissue. Being dyeed by liver HE, we can observe that under HFD rearing conditions, WT mouse and RECS1 KO mouse liver tissue all have More fat deposition, it can be seen that liver cell generation steatosis, vacuolation and fusion are linked to be sheet, and liver cell form is Destroy the most completely, especially notable RECS1 KO mouse, and more complete (such as Fig. 4 A in NC group mouse equal liver cell form On).Detecting lipid in hepatic tissue by liver oil red O stain, we are it appeared that the liver of RECS1 KO mouse in HFD group In large stretch of red around portal vein, prompting has substantial amounts of lipidosis, around the vena portae hepatica of the WT mouse of HFD group then Having less lipidosis, two groups of mouse lipidosis at NC diet are minimum (under Fig. 4 A).

We have detected again the triglycerides in the liver organization after WT and RECS1 KO mouse high fat diet (triglyceride, TG), T-CHOL (total cholesterol, TC) and free fatty (non-esterIfied Fatty acid, NEFA) content.Quantitative analysis results finds RECS1 KO mouse liver organization TG under the conditions of HFD, TC Compareing WT group with the total amount of NEFA much larger than it, this shows what RECS1 knock out mice removing lipid and AFL were formed Ability is decreased obviously (such as Fig. 4 B).

In this part is studied, it is observed that there is carbohydrate metabolism disturbance, pancreas under the induction of HFD in RECS1 KO mouse Insulin resistance, sugar tolerance is decreased obviously, and the mouse adipose liver pathological changes of RECS1 gene knockout is serious, and accumulation of lipid increases notable.This A little results show, RECS1, to maintaining glycometabolism stable state to have powerful ability of regulation and control, has powerful liver lipids to remove and lipotropism The ability that fat liver is formed.This shows that RECS1 gene has important protective effect in fatty liver, diabetes conditions model.

Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment Limit, the change made under other any Spirit Essence without departing from the present invention and principle, modify, substitute, combine, simplify, All should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (1)

1. centrifugal force and shear stress response gene 1 RECS1 are in preparation prevents, alleviates and/or treat the medicine of diabetes conditions Application.