CN106443024B - LGR4 genes/proteins are used to screen the drug for the treatment of hypercholesterolemia - Google Patents
LGR4 genes/proteins are used to screen the drug for the treatment of hypercholesterolemia Download PDFInfo
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- CN106443024B CN106443024B CN201610835255.9A CN201610835255A CN106443024B CN 106443024 B CN106443024 B CN 106443024B CN 201610835255 A CN201610835255 A CN 201610835255A CN 106443024 B CN106443024 B CN 106443024B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses the drugs that LGR4 genes/proteins are used to screen treatment hypercholesterolemia；Using LGR4 genes as molecular target or drug target, the drug for screening or preparing treatment hypercholesterolemia and low density lipoprotein cholesterol.The present invention provides the new applications of glycoprotein hormone receptor LGR4 genes in g protein coupled receptor, a novel targets are provided for the treatment of the treatment of hypercholesterolemia and high low density lipoprotein cholesterol (LDL c) indication, there is good application prospect.
Description
Technical field
The present invention relates to biotechnologys and field of medicaments, specially apply LGR4 genes/proteins as drug target, screening
Treat the drug of hypercholesterolemia.
Background technology
The survey data of the newest announcements of WHO shows that death caused by global ischemic heart disease and apoplexy accounts for dead sum
A quarter, be the most important cause of death of current majority state.Hypercholesterolemia is the independence of ischemic cardiovascular disease
Risk factor, when TC >=5.72mmol/L, acute coronary initiation potential increases by 74%, in ischemic cardiovascular disease event,
5.9% is attributable to hypercholesterolemia.Therefore, the research of the drug to reducing blood cholesterol levels is particularly important.
LGR4 systems g protein coupled receptor (GPCR) family member, is located at chromosome 11p14 sections, and overall length about 106Kb contains
18 exons encode 951 amino acid, wherein extracellular 548 amino acid residues, are rich in leucine, there is 5 glycosylation positions
Point, and belong to glycoprotein hormone receptor.Glycoprotein hormone receptor is divided into three classes by research in recent years：The first kind is classical glucoprotein hormone
Plain receptor, including FSHR (LGR1), LHR (LGR2), TSHR (LGR3)；Second class includes LGR4, LGR5 and LGR6, at present to this
The function of receptoroid is known little；Third class includes LGR7 (relaxin/insulin-like family peptide
Receptor 1, RXFP1) and LGR8 (RXFP2).Early-stage study thinks that the major function of glycoprotein hormone receptor class participates in body
Development and reproductive function, but recent years with stem cell and the continuous progress of related field, to the understanding of LGR4-6 also day
Benefit is goed deep into comprehensive.Studies have shown that LGR4 can regulate and control downstream gene Atf4 expression to participate in bon e formation by cAMP/PKA accesses
With reconstruction；In addition, LGR4 lowers the expression of target gene Pitx2 after knocking out, lead to mouse anterior chamber of eye depauperation；LGR4 can also lead to
Activation Gs subunits are crossed, intracellular cAMP levels are increased, activate PKA signal paths, to regulate and control estrogen receptor (ER) expression,
And then participate in the development of epididymis and convoluted seminiferous tubule.Above-mentioned correlative study all elaborates shadows of the LGR4 to body development process emphatically
It rings, then some researches show that it in recent years being associated with energetic supersession, LGR4 whole body knock-out mice weight is substantially reduced, sugar tolerance
Improve, and the obesity and heredity obesity of high fat diet induction can be resisted, discloses regulating and controlling effects of the LGR4 to adipose tissue, and
Specific effects of the LGR4 to the related organ of other metabolism is not probed into deeply.
In recent years, clinical research has proven to low density lipoprotein cholesterol (LDL-C) there are closely related with coronary heart disease
Property, most LDL-c is to rely on the ldl receptor of liver surface to remove in blood, therefore generation of blood cholesterol levels and liver
It thanks that function is closely related, and discloses influences of the LGR4 to liver there has been no research at present, and its sent out in hepatic cholesterol metabolism
The effect waved.
LGR4 liver specificity knock-out mice models are constructed in this research for the first time, it is solid in the courage of liver for probing into LGR4
The effect played in alcohol metabolic function is of great significance.
Invention content
The purpose of the present invention is to provide the new applications of LGR4 genes, specially provide a kind of LGR4 genes/proteins and are used for
Purposes in the drug of screening treatment hypercholesterolemia.
The purpose of the present invention is achieved through the following technical solutions：
In a first aspect, the use the present invention relates to LGR4 genes and its albumen in the drug of screening treatment hypercholesterolemia
On the way.
Second aspect, the present invention relates to the use of LGR4 genes and its albumen in the drug for preparing treatment hypercholesterolemia
On the way.
The third aspect, the present invention relates to LGR4 genes and its albumen to reduce low density lipoprotein cholesterol (LDL- in screening
C) the purposes in drug.
Fourth aspect, the present invention relates to LGR4 genes and its albumen to prepare reduction low density lipoprotein cholesterol (LDL-
C) the purposes in drug.
Preferably, the LGR4 genes are the LGR4 genes of people or mouse.
Preferably, the LGR4 genes are as molecular target or drug target.
Preferably, the drug includes the plant extract developed using LGR4 genes as molecular target or drug target
The apparent compound of object, chemical constitution, peptide matters, nucleic acid material, polysaccharose substance, viral vectors, liposome vectors or
Nanoparticle vector.
The present invention establishes the mouse model and Lgr4 of adenovirus mediated liver specificity LGR4 gene knockouts；Leptin
(Lgr4m/m；Ob/ob) dual-gene mutant mice model finds that knock out mice blood cholesterol levels change by Phenotypic Observation
Kind, total cholesterol and low-density lipoprotein cholesterol level significantly reduce.
In the case of taking in same food in different mouse models, LGR4 knock out mice total cholesterol and low
Density lipoprotein-cholesterol level reduces a lot than control group.
Compared with prior art, the present invention has the advantages that：
1) present invention is solid for screening and preparing the high courage for the treatment of using LGR4 genes as molecular target and medicament research and development target spot
The drug of alcoholemia, these drugs can reduce blood cholesterol levels, reduce the onset risk of ischemic cardiovascular disease.
2) present invention is using LGR4 genes as molecular target and medicament research and development target spot, for screening and preparing reduction low-density
The drug of lipoprotein cholesterol (LDL-c), these drugs can significantly reduce low-density lipoprotein cholesterol level.
3) present invention is the treatment of the treatment of hypercholesterolemia and high low density lipoprotein cholesterol (LDL-c) indication
A novel targets are provided, there is good application prospect.
Description of the drawings
Fig. 1 is in db/db mouse and mouse high in fat, liver specificity LGR4 knock out mice (Ad-sh-LGR4) with it is right
Serum total cholesterol according to a group mouse (Ad-sh-CTRL) and the horizontal comparison schematic diagram of serum LDL cholesterol,
In, A is db/db mice serum total cholesterol levels, and B is mice serum total cholesterol level high in fat, and C is db/db mice serums
Low-density lipoprotein cholesterol level, D are mice serum low-density lipoprotein cholesterol level high in fat；
Fig. 2 is Lgr4；Dual-gene mutant mice (the Lgr4 of Leptinm/m；Ob/ob) with the serum of control group mice (ob/ob)
Total cholesterol and the horizontal comparison schematic diagram of serum LDL cholesterol, wherein A is each group mice serum total cholesterol water
Flat comparison, B are that each group mice serum low-density lipoprotein cholesterol level compares.
Specific implementation mode
The following describes the present invention in detail with reference to examples.Following embodiment will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that those skilled in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention
Protect range.
Embodiment 1 establishes adenovirus mediated liver specificity LGR4 knock out mice
20 db/db Mouse feeders are in endocrine research institute of Medical College, Shanghai Communication Univ. animal house, environment temperature 22
DEG C, 12 hours/12 hours light and shade alternatings, db/db mouse adaptability is raised after a week, random to be grouped, and is calibrated by weight, respectively
For two groups of control group (Ad-sh-CTRL) and LGR4 knockouts group (Ad-sh-LGR4).Adenovirus tail vein injection is given after grouping,
Control group gives 2*109pfu shNT adenoviral injections, and LGR4 knockout groups give 2*109pfu shLGR4 adenoviral injections.
24 Mouse feeders high in fat are in endocrine research institute of Medical College, Shanghai Communication Univ. animal house, environment temperature 22
DEG C, 12 hours/12 hours light and shade alternatings are tested, grouping and the same db/db of subsequent processing after being raised 4 months with high lipid food
Mouse.
Embodiment 2 establishes Lgr4；Leptin(Lgr4m/m；Ob/ob) dual-gene mutant mice
Lgr4 chimeric mices obtain the Lgr4 chimeric mices of C57BL/6 backgrounds with C57BL/6 mouse backcross at least 6 generations.This
A little Lgr4 chimeric mices are denoted as F2 with the mouse that ob mouse mate, in the offspring that F2 is obtained for mouse hybrid Lgr4 and
The bis- mutant mices of Leptin are for testing, and ob mouse are for compareing.Three monthly age hero mouse and female mice press 1：1 and 1：2 ratios mate friendship
Match.Three weeks young rats from breast, mark, divide cage, genotype identification.
Embodiment 3
Rearing conditions：For Mouse feeder under the conditions of cleaning grade, stocking density is 5 per cages, in standard mice cage by with
Lower condition divides cage to feed：Normal business mouse feed (4.5% fat, 4.0% cellulose, 21.0% protein, 1.404 kilocalories/
Gram), high fat diet feed is purchased from Research Diet (60.0% fat), and feed is replaced according to every mouse food ration next day,
Ensure the freshness of feed.20-22 degrees Celsius of room temperature, freely ingests and drinks water, the stable artificial lighting period (6:00 to 18:
00 illumination, 18:00 to 6:00 is dark).
Two kinds of adenoviral injection mouse models are drawn materials after injecting adenovirus 10 days, Lgr4；Leptin(Lgr4m/m；
Ob/ob) dual-gene mutant mice model is drawn materials when 4 monthly age, and materials noon before that day removes each group animal at 17 points
Feed makes animal fasting 16 hours, uses yellow Jackets anesthetized mice at 9 points, mouse is put to death after anesthesia, collect whole blood, adopt
Blood about 0.6-0.8ml, is put into EP pipes, after being placed at room temperature for 2-4 hours, with 4 degree Celsius of 3000 revolutions per minute on refrigerated centrifuge
The pelleted by centrifugation of clock 10 minutes.Carry out packing serum with 100 μ l/ pipes, to be placed on -80 degrees Celsius of refrigerators spare；Blood sampling finishes
Afterwards, tissues such as quick clip liver, epididymal adipose tissues, subcutaneous fat, gastrocnemius, and being pre-chilled in liquid nitrogen, be put in later liquid nitrogen or-
80 degrees Celsius of refrigerators are spare.
As a result：
One, the results show that in db/db mouse and mouse high in fat, liver specificity LGR4 knock out mice (Ad-sh-
LGR4 serum total cholesterol level) is significantly lower than control group mice (Ad-sh-CTRL) (such as Figure 1A and B), serum low-density fat
Protein cholesterol level is also substantially less than control group (such as Fig. 1 C and D).
Two, Lgr4；Dual-gene mutant mice (the Lgr4 of Leptinm/m；Ob/ob serum total cholesterol level) is significantly lower than
Control group mice (ob/ob) (such as Fig. 2A), serum LDL cholesterol level are also substantially less than control group (such as Fig. 2 B).
According to the above results, LGR4 genes participate in cholesterol metabolic process, property relevant with hypercholesterolemia.LGR4 bases
Because missing can reduce blood cholesterol levels, a drug target is can be used as, for screening or preparing for treating hypercholesteremia
Disease, the drug for reducing ischemic cardiovascular disease occurrence risk.
Claims (4)
- The purposes of 1.LGR4 genes and its albumen in the drug that screening reduces low density lipoprotein cholesterol, the LGR4 genes As molecular target or drug target.
- The purposes of 2.LGR4 genes and its albumen in preparing the drug for reducing low density lipoprotein cholesterol, the LGR4 genes As molecular target or drug target.
- 3. purposes as claimed in claim 1 or 2, which is characterized in that the LGR4 genes are the LGR4 genes of people or mouse.
- 4. purposes as claimed in claim 1 or 2, which is characterized in that the drug includes using LGR4 genes as molecular target Or compound, viral vectors, liposome vectors or the nanometer that the plant extracts of drug target exploitation, chemical constitution are apparent Particulate vector.
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| CN106443024B true CN106443024B (en) | 2018-08-24 |
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| CN110090299A (en) * | 2019-05-29 | 2019-08-06 | 上海交通大学医学院附属瑞金医院 | A kind of LGR4 is preparing the application in cholesterol-lowering drug |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103357026A (en) * | 2013-07-17 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | LGR4 gene used for screening medicines used for treating obesity |
| CN103638531A (en) * | 2013-07-17 | 2014-03-19 | 上海交通大学医学院附属瑞金医院 | LGR4 gene for screening of medicines promoting browning of white fat |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103357026A (en) * | 2013-07-17 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | LGR4 gene used for screening medicines used for treating obesity |
| CN103638531A (en) * | 2013-07-17 | 2014-03-19 | 上海交通大学医学院附属瑞金医院 | LGR4 gene for screening of medicines promoting browning of white fat |
Non-Patent Citations (4)
| Title |
|---|
| LGR4 and its role in intestinal protection and energy metabolism;Ziru Li et al.;《Frontiers in Endocrinology》;20150825;第6卷;第1-9页 * |
| LGR4 对遗传性肥胖小鼠的体重和能量代谢调节作用;陈茂培 等;《中华医学会第十一次全国内分泌学学术会议论文汇编》;20120829;第345页 * |
| 血清总胆固醇与低密度脂蛋白胆固醇水平的相对关系;王抒 等;《中国动脉硬化杂志》;20031231;第11卷(第2期);摘要 * |
| 血脂异常防治建议;方圻 等;《中华心血管病杂志》;19970630;第25卷(第3期);第169-173页 * |
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