CN105031619B - Applications of the secretion factor GREM2 in diabetes B medicine is prepared - Google Patents

Abstract

The invention belongs to field of medicine preparing technology, more particularly to applications of the secretion factor GREM2 in diabetes B medicine is prepared.This research proves that GREM2 transgenic mice sugar tolerances are obviously improved, and insulin sensitivity substantially increases.Further experiment shows that transgenic mice hepatic insulin downstream P AKT signal paths significantly increase, and prompts hepatic insulin sensitivity increase.Found using ob diabetic mouse models, after ob mouse give adenovirus overexpression GREM2 albumen, blood glucose is obviously improved, fatty liver is obviously improved, insulin sensitivity dramatically increases.So GREM2 can be applied in the medicine for preparing hypoglycemic medicine, improvement diabetes B, new drug target is provided for clinical treatment diabetes B.

Description

Applications of the secretion factor GREM2 in diabetes B medicine is prepared

Technical field

Field of medicine preparing technology of the present invention, more particularly to secretion factor GREM2 is in diabetes B medicine is prepared Application.

Background technology

Diabetes are a kind of clinical syndromes using hyperglycaemia as main feature.Diabetes B accounts for the total crowd of diabetes More than 95%.Diabetes become global health problem, and to current, the whole world has diabetic more than 1.71 hundred million, According to statistics, 3.7 hundred million will be broken through to the year two thousand thirty, global diabetic.Number of patients of the diabetes in China is in sharp increase Trend, how effectively to treat diabetes is researchers' hot spot that urgently generation solves the problems, such as and studies.

The seriousness of diabetes is various acute and chronic complication caused by chronic hyperglycemia.Complication mainly includes The diabetes heart, brain, peripheral blood vessel lesion, diabetic nephropathy, diabetic retinopathy and ocular complications, diabetic neuropathy Become etc..Complication is very harmful, and the death rate is high, seriously affects patients ' life quality.Diabetes have become seriously endangers mankind's life Deposit the important diseases with quality of life, be to endanger one of three big killers of human health 21 century, be only second to angiocardiopathy and Tumour.Blood glucose how is controlled, reduces complication, improves the main target that long-term prognosis is treating diabetes.

Diabetes B is that the aspect of h and E two is coefficient as a result, and pathophysiological basis is pancreas islet Element resistance and B cell hyposecretion.The natural history of diabetes B is since insulin resistance, and insulin secretion is by mistake It is compensatory, compensatory, gradually switch to defect of insulin secretion i.e. decompensation.Diabetes B starts and the major reason of progress is surrounding The insulin resistance of tissue particularly skeletal muscle, fat, the liver of insulin sensitivity.Currently for the medicine of diabetes B treatment Thing treatment mainly has five major classes, i.e.,：The rush of insulin secretes agent, biguanides, the inhibitor of alpha-glucosidase, thiazolidinediones With secretin (Incretin).Sulphur urine class clinically uses for a long time as Insulin secretagogues, and it is concurrent to prevent capilary Disease, but be not ideal selection since β cell functions are lost there are secondary failure in it.In addition, as insulin Sensitizer thiazolidinediones, Rosiglitazone in such medicine, because its cardiovascular risk causes U.S.'s food and medicine supervision The concern of management board.Traditional hypoglycemic medicine not can effectively stop the progress of the deterioration of islet function volume and diabetes B, it is difficult to Continuously and effectively control blood glucose for a long time.Since current medicine still has larger limitation, how sugar is efficiently controlled Sick hyperglycaemia is urinated to reduce the emphasis challenge that the complication risk of diabetes is still modern medicine.For this reason, people are always In the continuous new way for exploring treatment diabetes.

The intervention for being found to be diabetes of new secretion factor provides new potential target spot.Research shows autocrine/side point The blood glucose of diabetic mice can be improved by improving insulin sensitivity by secreting factor FGF1.In recent years, go deep into research, point Important function of factor B MP (Bone Morphogenetic Protein) family in energetic supersession is secreted also to be revealed.BMP families exist natural in vivo Antagonist, wherein the antagonist GREM2 for being similarly secretion factor causes our concern.So far, research report is had no Effects of the road GREM2 in blood glucose-control.GREM2 is a kind of glycosylation albumen of secretion, belongs to CAN in bmp antagonist family (cerberus and dan) subfamily, its C-terminal contain 8 cystine rings.The family includes GREM1, GREM22 member, its Middle GREM2 is far above its hetero-organization in the expression of metabolism linked groups.GREM2 genes are located at No. 1 chromosome 1q43 section of people, comprising 2 extrons, total length 4,170bps, encodes 168 amino acid.GREM2 can be combined with corresponding BMP, final to suppress BMP letters The activation of number path, previously report is related to bon e formation.Know little at present for GREM2 biological functions, especially in blood glucose It there is no and be related in terms of adjusting.

The content of the invention

The present invention provides a kind of applications of secretion factor GREM2 in diabetes B medicine is prepared.Provide improvement The new therapy approach of diabetes.

The present invention relates to applications of the secretion factor GREM2 in diabetes B medicine is prepared.The secretion factor GREM2 improves blood glucose by improving hepatic insulin sensitivity.

The present invention utilizes GREM2 transgenic mice animal models, the study find that GREM2 transgenic mice sugar tolerances are obvious Improve, insulin sensitivity substantially increases.Further experiment shows that transgenic mice hepatic insulin downstream P-AKT signals lead to Road significantly increases, and prompts hepatic insulin sensitivity increase.Using ob mouse (diabetic mouse model), the study find that ob is small After mouse gives adenovirus overexpression GREM2 albumen, blood glucose is obviously improved, fatty liver is obviously improved, insulin sensitivity significantly increases Add.The present invention provides secretion factor GREM2 and is used to treat evidence of the diabetes B on animal model first, prompts secretion Factor GREM2 clinically may have some improvement diabetes tool.So GREM2, which can be applied to, prepares hypoglycemic Medicine, improve diabetes B medicine in, new drug target is provided for clinical treatment diabetes B.

Beneficial effect：The present invention is that have studied the treatment relation of secretion factor GREM2 and diabetes B, passes through animal mould Type research shows that secretion factor GREM2 can be used for treating diabetes B, and diabetes are controlled for clinically secretion factor GREM2 Treatment provides theoretical foundation and experimental data.

Brief description of the drawings

Fig. 1 is overexpressed efficiency qualification figure for transgenic mice.

Comparison diagrams of the Fig. 2 for GREM2 in the case of normal diet to mouse weight, blood glucose and insulin tolerance.

Comparison diagrams of the Fig. 3 for GREM2 in the case of high fat diet to mouse weight, blood glucose and insulin tolerance.

Fig. 4 is that the influence of the liver gluconeogenesis, liver, liver and adipose tissue insulin of GREM2 transgenic mices contrasts Figure.

Diabetic mice weight, blood glucose, sugar tolerance and insulin sensitivity become after Fig. 5 adenovirus is overexpressed GREM2 albumen Change figure.

Fig. 6 adenovirus is overexpressed diabetic mice fatty liver variation diagram after GREM2 albumen.

Embodiment

With reference to embodiment, the invention will be further described：

Embodiment

1st, GREM2 transgenic mices blood glucose is obviously improved

1) whole body is overexpressed GREM2 Establishment of mouse model.

We undergo vector construction, DNA linearisations and purification, linearize the micro- notes of DNA using C57BL/6J mouse as background 200 embryonated eggs are mapped to, these embryonated eggs are transmitted back to the 3-4 medium experiment flow of replace-conceive female rat fallopian tubal, finally reflect through PCR again Surely 10 positive head of genotype results identification are obtained and build mouse (Founder), remove three obsolete founder：GA After founder has given birth to F1 generation, no longer give birth to；GE founder do not bear positive mice；GJ founder fertility positive rates are low, still There are not enough mouse to be used to identify, the overexpression efficiency of remaining 7 founder by expression efficiency as shown in Figure 1, reflect Fixed, GF mouse overexpression efficiency is higher, for subsequent experimental.

2) whole body is overexpressed phenotypic analysis under GREM2 mouse normal diet and high fat fed conditions.

It is to detect GREM2 to the adjustment effect of blood glucose, we improve normal diet and high fat on the basis of previous work The metabolic phenotype analysis of GREM2 transgenic mices under eating condition.Using SPF grades of C57BL/6 mouse, pressed in standard mice cage The following conditions divide cage to feed:Normal diet is normal business mouse feed (4.5% fat, 4% cellulose, 21% protein), High fat diet feed is purchased from Research Diet (60%Fat).Freely ingest and drink water, the stable artificial lighting cycle (6:00 To 18:00 illumination, 18:00 to 6:00 is dark).

It is as follows to predominantly detect index：

A. under normal diet and high smectic state WT mouse and GREM2TG (Transgenic mice) mouse weight increase it is bent Line records；

B. the measure of sugar tolerance and insulin tolerance：Tried by dextrose tolerance test (IPGTT) and insulin stimulating Test (ITT), tolerance, insulin sensitivity of the analysis GREM2 transgenic mices to glucose.Sugar tolerance experimentation is such as Under：After when mouse starvation 16 is small, the glucose through intraperitoneal injection, the dosage of glucose is 1g/kg, then respectively after injection 15min, 30min, 60min, 120min record blood sugar level.Insulin tolerance tests (ITT) process is as follows：Mouse starvation 6 is small After mouse, through rh-insulin 0.75U/kg (normal diet mouse) or 1.5u/kg (high fat diet mouse) is injected intraperitoneally, respectively 15min, 30min, 60min, 120min record blood sugar level after injection of insulin.

Look first at influences of the GREM2 to blood glucose in the case of normal diet.We select the control mice 10 of 8 week old altogether Only, GREM2 transgenic mices 9, after full diet is fed 16 weeks, influences of the observation GREM2 to mouse weight and blood glucose.As a result It has been shown that, compared with control mice, without substantially changeing (Fig. 2 a), sugar tolerance is obviously improved (Fig. 2 b) transgenic mice weight.Pancreas islet Plain tolerance is the results show that transgenic mice insulin sensitivity is remarkably reinforced (Fig. 2 c).

Equally, in the case of we observe high fat diet again, influences of the GREM2 to mouse blood sugar and insulin sensitivity.I Same choose 8 week old mouse, control mice 15, GREM2 transgenic mices 14.16 Zhou Houguan of mouse High-fat diet Examine influences of the GREM2 to mouse weight and blood glucose.The results show (Fig. 3), compared with control mice, GREM2 transgenic mices Weight is without substantially changeing, through abdominal cavity glucose tolerance test the results show that transgenic mice sugar tolerance is obviously improved.Starvation is fed again Experimental result again shows that GREM2 transgenic mice sugar tolerances significantly improve.Likewise, transgenic mice insulin sensitivity It is remarkably reinforced.

2nd, GREM2 transgenic mices hepatic insulin sensitivity is remarkably reinforced

From test result indicates that, GREM2 transgenic mice fasting blood-glucoses substantially reduce, and sugar tolerance is obviously improved, insulin Sensitiveness is remarkably reinforced.Fasting blood-glucose is adjusted by liver gluconeogenesis, and transgenic mice fasting blood-glucose improves, if is gluconeogenesis energy Caused by power declinesNext, we have detected first key gene FBP1, G6Pase of liver gluconeogenesis, Pepck, The expression quantity of Pgc-1 α, the results showed that transgenic mice liver gluconeogenesis is without substantially changeing (Fig. 4 a).In order to further detect Influences of the GREM2 to liver, the method that we are dyed by HE detect liver morphology.Normal diet mouse liver tissue is with 4% Paraformaldehyde fix, after specimens paraffin embedding slices, contaminated through roasting piece-dewaxing-aquation-hematoxylin dyeing-dehydration-Yihong Color-dehydration-transparent and etc., micro- Microscopic observation after resinene mounting.Mouse liver HE coloration results show (Fig. 4 b), There are more small fat drips in control mice liver, and the fat drips in transgenic mice liver are less, prompts transgenic mice liver Fatty infiltration is few.

Skeletal muscle, liver, fat are the important target organs of insulin.Next, we further detect mouse liver and The sensitiveness of adipose tissue insulin.Mouse is divided into two groups：It is small after the mouse of (basal) and insulin stimulating under base state Mouse.Insulin treatment group：Mouse peritoneal insulin injection 1.5U/kg, 15min clocks are rapidly separated mouse adipose after insulin processing Tissue and liver.Further extract liver and visceral adipose tissue albumen, Western blot methods detection insulin downstream letter The change of number path p-AKT.The results show (Fig. 4 c), under base state (basal), transgenic mice hepatic insulin downstream letter Number path p-AKT is remarkably reinforced, and close to the level after insulin stimulating, prompts GREM2 transgenic mices hepatic insulin sensitive Property is apparently higher than control mice.And transgenic mice visceral adipose tissue insulin sensitivity and control group no significant difference (figure 4d)。

3rd, blood glucose is obviously improved after ob mouse adenoviruses are overexpressed GREM2 albumen

The homozygote mouse (Lepob, usually writes ob or ob/ob) of fat spontaneous mutation, is leptin gene mutations Mouse.Ob can see phenotype during mouse about four weeks.Homozygous mutation mouse weight increases sharply, up to wild type control mouse Three times of normal type.In addition to obesity, mutation mouse also shows hyperphagia, and diabetes sample hyperglycaemia syndrome is sugared resistance to Measure bad, plasma insulin rise.Ob mouse are common diabetes B mouse models.

GREM2 is a kind of secretory protein.Next, we are injected GREM2 adenovirus using the method for tail vein injection In ob Mice Bodies.This research uses the ob mouse of 8 week old, control group virus treated 4, GREM2 adenovirus treatment group 4, gland The dosage of virus is 10⁹IU/ is only.After mouse injection adenovirus, the expression of general 3~4 days destination proteins peaks, after a week It is gradually reduced, the continuous expression time was at two weeks or so.Continuous monitoring blood glucose and weight after adenovirus processing.Give GREM2 adenopathies After poison processing, we are respectively the 5th day after virus treated, the 7th day, the 9th day detection mouse random blood sugar, the results showed that, ob is small After mouse gives adenovirus overexpression GREM2 albumen, mouse random blood sugar is obviously improved (Fig. 5 b), and sugar tolerance improves (Fig. 5 c), pancreas Island element sensitiveness is remarkably reinforced (Fig. 5 d).

Equally, we dye the change of detection liver morphology by HE.The results show that there are serious fat for ob mouse Liver, and the mouse fatty liver of GREM2 adenovirus processing is obviously improved (Fig. 6).

Research passes through two kinds of animal models of transgenic mice and diabetic mice (ob mouse) above, discloses secretion factor GREM2 improves blood glucose, improves the new function of insulin sensitivity, and new drug target is provided for clinical treatment diabetes B.

The above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within Enclose.

Claims (1)

1. applications of the secretion factor GREM2 in diabetes B medicine is prepared, the secretion factor GREM2 passes through raising Hepatic insulin sensitivity improves blood glucose.