CN104474537B - Applications of NEDL2 protein and encoding gene of NEDL2 protein in treatment of congenital megacolon disease - Google Patents
Applications of NEDL2 protein and encoding gene of NEDL2 protein in treatment of congenital megacolon disease Download PDFInfo
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Abstract
The invention discloses applications of an NEDL2 protein and an encoding gene of the NEDL2 protein in treatment of a congenital megacolon disease. The invention discloses applications of the NEDL2 protein, the encoding gene of the NEDL2 protein or a product for promoting expression of the NEDL2 protein in preparation of products for promoting intestinal tract movement. The NEDL2 protein participates in regulation and control of enteric nervous system development; development of an enteric nervous system can be inhibited through knocking out NEDL2 genes in mice, particularly, multiplication of intestinal neuronal progenitor cells is inhibited, and the intestinal peristalsis ability is obviously reduced; and activation of GDNF/RET signal channels in intestinal neurons can be inhibited by knocking out the NEDL2 genes. The NEDL2 protein has important theoretical value on research of enteric nervous system development, promotion of intestinal neurons multiplication and activation of the GDNF/RET signal channels, and has important application value in treatment of the congenital hirschsprung disease.
Description
Technical field
The present invention relates to a kind of application of NEDL2 albumen and its encoding gene in treatment congenital giant colon disease, category
In biological technical field.
Background technology
Eukaryotic internal protein synthesizes balance the maintaining with important effect for Cell Homeostasis with degraded.Eucaryon
There are two kinds of proteolytic pathway in organism：Lysosomal pathway and Ubiquitin-Proteasome Pathway.Lysosomal pathway mainly drops
Solution enters intracellular foreign protein by endocytosis or pinocytosis, and Ubiquitin-Proteasome Pathway principal degradation intracellular protein.
Ubiquitin-proteasome pathway refers to protein after being identified and being coupled with ubiquitin label, the mistake finally degraded by proteasome
Journey.The ubiquitin for being referred to as " dead label " is tied in ubiquitin kinase (ubiquitin-activating enzyme, E1), ubiquitin
The enzymatic of synthase (ubiquitin-conjugating enzyme, E2) and ubiquitin ligase (ubiquitin ligase, E3)
Protein is marked in cascade reaction and it is recognized by proteasome, is finally completed degradation process, wherein, E3 is determined
The specificity of substrate.E3 found in human genome about 1000, however, clearly having found its substrate at present
E3 be less than the 1/3 of total amount.Substantial amounts of research work finds that Ubiquitin-proteasome systerm participates in numerous cell biology mistakes
Journey, including the regulation and control of the process such as cell cycle, cell differentiation and growth, Apoptosis.The exception of Ubiquitin-Proteasome Pathway
The generation of various diseases, such as cancer, nerve degenerative diseases and autoimmune disease etc. can be caused.At present, for ubiquitin-egg
White enzyme body approach devises kinds cancer medicine, such as proteasome inhibitor Bortezomib (Velcade, PS-
341) generation of various myeloma, can effectively be suppressed in clinical practice, however, due to the medicine of proteasome inhibitor class
Selective poor, the restricted application of such medicine.Therefore, design there is high selectivity to cancer cell and cytotoxicity compared with
Little medicine is the important directions of current drug research.Specificity is selected because E3 has to substrate, so having been for part at present
E3 has carried out the clinic that the inhibitor of RING types E3 such as the drug design for the treatment of correlative diseases, HDM2, IAP has been enter into oncotherapy
Front research.
According to design feature and the difference of the mode of action, E3 can be divided to for two big class：RING(really interesting
New gene) Zinc finger domain class and HECT (homologous to E6AP C-terminus) domain class.Wherein, human body
Middle RING classes E3 account for about the 95% of E3 total amounts, and wherein HECT classes E3 are studied more to be clear that Nedd4 (neural
Precursor cells-expressed developmentally down-regulated 4) family, in human genome,
Nedd4 families by 9 member compositions, including Smurf1, Smurf2, Nedd4-1, Nedd4-2, NEDL1, NEDL2, WWP1,
WWP2、AIP4/Itch.Have been reported announcement the family member it is related to numerous biological processes, such as regulation protein transport, across
Bud life, cell autophagy, the congenital immunity escape of participation C. Elegans Automatic Screening of the signal transduction, virus of membrane receptor in infection cell
Deng, meanwhile, it is also closely related with the pathological process such as cancer, osteoporosis, Liddle ' s syndromes.
Congenital giant colon disease (Hirschsprung disease, HSCR) is because of enteric nervous system (Enteric
Nervous System, ENS) developmental defect cause disease, its incidence of disease be 1/5000, wherein M-F be 4:1.Normally
In the case of, baby can just exclude tire external in 24-48h after birth.However, HSCR sick babies then can not be excluded normally
Tire just, normally behaves as serious constipation and the serious flatulence of enteron aisle, and ultimately results in death.Developing the medicine of effectively treatment HSCR is
One problem demanding prompt solution.
The content of the invention
It is an object of the invention to provide a kind of NEDL2 albumen and its encoding gene are in treatment congenital giant colon disease
Using.
The present invention provides NEDL2 albumen, the encoding gene of NEDL2 albumen or promotes the product of NEDL2 protein expressions in system
Application in the standby product for promoting intestines peristalsis or the product for suppressing intestines peristalsis ability to decline；
Or,
Application of the product of NEDL2 protein expressions in the product for reducing intestines peristalsis ability is prepared is suppressed to fall within this
Bright protection domain；
The gastric emptying ability that the promotion intestines peristalsis is embodied as is improved and/or the shortening of stomach and intestine conversion time；
The intestines peristalsis ability decline is embodied as gastric emptying ability and declines and/or the prolongation of stomach and intestine conversion time.
NEDL2 albumen, the encoding gene of NEDL2 albumen promote the product of NEDL2 protein expressions preparing promotion nerve
Application in the product that first cell propagation product or inhibitory neuron ability of cell proliferation decline falls within the protection model of the present invention
Enclose；
Or,
Application of the product of NEDL2 protein expressions in the product for reducing neuronal cell multiplication capacity is prepared is suppressed also to belong to
In protection scope of the present invention.
In above-mentioned application, the neuronal cell is enteric nervous unit cell；
Enteric nervous unit cell is specially enteric nervous unit CFU-GM.
In above-mentioned application, the neuronal cell is neuron progenitor cell；
The neuron progenitor cell is specially enteric nervous unit CFU-GM.
NEDL2 albumen, the encoding gene of NEDL2 albumen promote the product of NEDL2 protein expressions preparing activation GDNF/
Application in the product of RET signal paths falls within protection scope of the present invention；
Or,
Suppress application of the product of NEDL2 protein expressions in the product for suppressing the activation of GDNF/RET signal paths is prepared
Belong to protection scope of the present invention；
The activation of the GDNF/RET signal paths is embodied in the level of pAkt and improves；
The pAkt is Phosphorylated Protein Kinase B.
NEDL2 albumen, the encoding gene of NEDL2 albumen or promote NEDL2 protein expressions product prepare prevention and/or
Application in the product for the treatment of congenital giant colon disease falls within protection scope of the present invention.
In any of the above-described described application, the amino acid sequence of the NEDL2 albumen is as shown in SEQ ID No.1；
The encoding gene of the NEDL2 albumen is as shown in SEQ ID No.2.
A kind of construction method of the mouse of NEDL2 gene knockouts falls within protection scope of the present invention, comprises the steps：
The targeting vector of NEDL2 genes is injected into the embryonic stem cell of mouse, then is transplanted to dams intrauterine, obtained on genome
Offspring rat containing target practice sequence, is denoted by F1；F1 is mated with EIIa-Cre mouse, using Cre/LoxP systems, Cre enzymes
Sequence between the loxP sites of target practice sequence described in F1 genomes is deleted, the mouse of NEDL2 genetic heterozygosis is obtained, by it
It is denoted as NEDL2+/-Mouse；By NEDL2+/-Mouse carries out selfing, obtains the mouse that NEDL2 genes are not knocked out, and is denoted by NEDL2+/+Mouse, or the mouse of NEDL2 gene knockouts is obtained, it is denoted by NEDL2-/-Mouse, NEDL2-/-Mouse is purpose mouse；
The mouse of the NEDL2 genetic heterozygosis is NEDL2 gene knockouts on item chromosome, on another item chromosome
The mouse that NEDL2 genes are not knocked out；
The sequence of the targeting vector of the NEDL2 genes is concrete as shown in SEQ ID No.3；
The dams are specially C57BL/6 mouse；
The mouse of the NEDL2 gene knockouts is specially congenital giant colon disease model mouse；
The sequence of the NEDL2 genes is as shown in SEQ ID No.9.
The mouse of the NEDL2 gene knockouts prepared by said method is in exploitation or screens following arbitrary described product
In application fall within protection scope of the present invention：
(1) product for promoting intestines peristalsis or the product for suppressing the decline of intestines peristalsis ability；
The promotion animal intestinal is wriggled and is embodied as the raising of gastric emptying ability and/or the shortening of stomach and intestine conversion time；
The intestines peristalsis ability decline is embodied as gastric emptying ability and declines and/or the prolongation of stomach and intestine conversion time.
(2) product or inhibitory neuron ability of cell proliferation for promoting neuronal cell propagation declines product；
The neuronal cell is enteric nervous unit's cell and/or neuron progenitor cell, specially enteric nervous unit CFU-GM；
(3) product of GDNF/RET signal paths is activated；
The activation of the GDNF/RET signal paths is embodied in the level of pAkt and improves；
The pAkt is Phosphorylated Protein Kinase B；
(4) prevent and/or treat the product of congenital giant colon disease.
Present invention demonstrates that NEDL2 albumen participates in the regulation and control of enteric nervous system development, by knocking out NEDL2 bases in mouse body
Development because suppressing enteric nervous system, the propagation for being embodied in enteric nervous unit CFU-GM is suppressed and intestines peristalsis
Ability is substantially reduced；The knockout of NEDL2 genes can suppress the activation of GDNF/RET signal paths in enteric nervous unit.The present invention is right
In the research of enteric nervous system development, promote the propagation of enteric nervous unit and the activation of GDNF/RET signal paths that there is great reason
Value, for treatment congenital giant colon disease has great using value.
Description of the drawings
Fig. 1 is the structure of model mice.
Fig. 2 is NEDL2+/+And NEDL2-/-Mouse intestinal wriggling situation.
Fig. 3 is NEDL2+/+And NEDL2-/-The proliferative conditions of mouse enteric nervous unit CFU-GM.
Fig. 4 is NEDL2+/+And NEDL2-/-The activation situation of GDNF/RET in mouse enteric nervous unit.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
Quantitative test in following examples, is respectively provided with three repetitions and tests, results averaged.
C57BL/6 mouse are model animal research institute of Nanjing University product.
EIIa-Cre mouse are Jackson Lab Products, and the public can be from Academy of Military Medicine, PLA
Institute of Radiation Medicine obtains.
PAkt (Ser473) antibody, pErk1/2 (Thr202/Tyr204) antibody and BrdU antibody are
CellSignaling Technology Products, catalog number is respectively 4060,5683 and 5292.
PGP9.5 antibody, NEDL2 antibody and Neurofilament antibody are Abcam Products, catalog number point
Wei not ab10404, ab92711 and ab50284.
GDNF (neurotrophic factor in glial cell line source) is Sigma Products, and catalog number is
SRP3200。
The structure of embodiment 1, model mice
NEDL2+/-In the structure schematic diagram such as Fig. 1 of mouse shown in A and B.
First, first with Cre/LoxP systems, 5,6,7 three extrons of NEDL2 genes are chosen as target practice sequence,
Its two ends adds respectively LoxP sites, meanwhile, insert other sites on targeting vector, such as Neo (resistant gene, screening is used),
IRES LacZ (expression LacZ albumen, NEDL2 expression spike use) and can be used for the FRT sites of set up the condition type knock-out mice,
The sequence of targeting vector is as shown in SEQ ID No.3.
2nd, targeting vector is expelled in the embryonic stem cell of C57BL/6 mouse, and it is further transplanted to C57BL/
The intrauterine of 6 mouse, obtains being integrated with the filial generation positive mice of target practice sequence on genome, is denoted by F1.
3rd, F1 is mated with EIIa-Cre mouse, using Cre/LoxP systems, Cre enzymes are by sequence of practicing shooting in F1 genomes
LoxP sites between sequence deleted such that it is able to produce NEDL2LacZ/+(i.e. NEDL2+/-) generation mice, NEDL2+/-Mouse
NEDL2 is obtained Jing after selfing-/-Mouse and NEDL2+/+Mouse.
Wherein, NEDL2+/-Mouse is (NEDL2 gene knockouts, another dyeing on item chromosome of NEDL2 genetic heterozygosis
NEDL2 genes are not knocked out on body) mouse；NEDL2+/+Mouse is the mouse that NEDL2 genes are not knocked out；NEDL2-/-Mouse is
The mouse of NEDL2 gene knockouts.
The amino acid sequence of the NEDL2 albumen being knocked in above-mentioned mouse as shown in SEQ ID No.8, its encoding gene sequence
Row are as shown in SEQ ID No.9.
4th, NEDL2+/-Mouse, NEDL2-/-Mouse and NEDL2+/+The identification of mouse
(1) NEDL2 is extracted+/-The genomic DNA of the self progeny of mouse, respectively the genomic DNA with each mouse is as mould
Plate, to identify the primer P1 of NEDL2 wild types (WT) sequence：5'-GATTGTGTTGATGGTATTTGAGAGTT-3'(SEQID
) and P2 No.4：5'-GTATGAAAGAGGAAGAGGGCGTTATT-3'(SEQ ID No.5), or to identify 5,6, the 7 of NEDL2
Three extrons are knocked the primer P3 of (KO)：5'-GTTGGTCTGGTGTCAAAAAT-3'(SEQ ID No.6) and P4：5'-
ATCAGGATAAGATGAGGGTG-3'(SEQ ID No.7) it is primer, enter performing PCR amplification, as a result as shown in C in Fig. 1.
In Fig. 1 C, 1 is NEDL2-/-Mouse, 2 is NEDL2+/-Mouse, 3 is NEDL2+/+Mouse.
Fig. 1 C show, NEDL2-/-The purpose band that mouse is only obtained with KO primers as primer amplification；NEDL2+/-Mouse
Both there is the purpose band obtained for primer amplification as primer with WT or had there is the purpose bar obtained as primer amplification with KO primers
Band；NEDL2+/+The purpose band that mouse is only obtained with WT primers as primer amplification, as a result shows, each mouse genotypes build
Success.
(2) NEDL2 is extracted-/-Mouse and NEDL2+/+The albumen of mouse, with NEDL2 antibody western blot inspections are carried out
Survey, while with Hsp90 as internal reference, as a result as shown in figure ip.
Fig. 1 D show, further prove NEDL2-/-Without the expression of NEDL2 albumen in mouse.
Embodiment 2, NEDL2+/+And NEDL2-/-Mouse intestinal wriggling situation analysis
The NEDL2 that embodiment 1 builds-/-Mouse interior all death in 2 weeks after birth, and NEDL2-/-Mouse intestinal occurs tight
Weight flatulence, similar to congenital giant colon disease symptom, points out NEDL2-/-Mouse intestinal is short of power.
Using methylene blue (being Beijing Lei Gen Bioisystech Co., Ltd product, catalog number is ZC00485) to reality
Apply the NEDL2 of the structure of example 1+/+And NEDL2-/-Mouse carries out gavage process, and every mouse stomach volume is 100 μ l, 5min and
Survey the length of the enteron aisle that the absorbance and mouse intestinal Methylene Blue of each Mouse Stomach Methylene Blue are marked after 15min respectively
Degree, analyzes mouse intestinal wriggling situation, including gastric emptying ability and stomach and intestine conversion time, and specific formula for calculation is as follows：
(formula 1)
(formula 2)
In formula 1, A600(5min)And A600(15min)Represent respectively after gavage 5min and 15min, Mouse Stomach Methylene Blue
Solution is in the absorbance that wavelength is at 600nm.
In formula 2, methylene blue mark lengths(15min)Represent the small intestinal length marked by methylene blue after gavage 15min.
As a result it is as shown in Figure 2.
In Fig. 2A, St- stomaches, Si- small intestines, Ce- caecums, Co- colons.
Fig. 2A is that methylene blue marks gastrointestinal peristalsis schematic diagram.
Fig. 2 B show, NEDL2-/-Mouse Stomach emptying ability is compared with NEDL2+/+Mouse is weak.Fig. 2 C show, NEDL2-/-Mouse Stomach
Intestines conversion time is compared with NEDL2+/+Mouse length.The above results difference is notable.
Result above shows, NEDL2-/-Mouse intestinal wriggling ability is substantially reduced.
Embodiment 3, NEDL2+/+And NEDL2-/-The proliferative conditions analysis of mouse enteric nervous unit CFU-GM
According to BrdU:Mouse Weight is 100mg:The ratio of 1kg, by BrdU (Chinese entitled 5- bromodeoxyuridines nucleosides,
Thymidine can be replaced) it is injected into the NEDL2 of the structure of embodiment 1+/-Pregnant mouse (12.5 days pregnant ages) in vivo, puts to death pregnant mouse after 3h, take
(embryonic gene type is respectively NEDL2 to go out mice embryonic-/-And NEDL2+/+, authentication method is with the step of embodiment 1 four) and enteron aisle group
Knit and be fixed (4% formalin), paraffin section, and carried out with the labelled protein PGP9.5 antibody of BrdU antibody and neuron
Immunofluorescence test, Immunofluorescence test figure is as shown in Figure 3A.And calculate the double positive cells of BrdU and PGP9.5 and account for PGP9.5
The ratio of positive cell quantity, as a result as shown in Figure 3 B.
Fig. 3 B show, NEDL2-/-The quantity of the enteric nervous unit CFU-GM of mouse propagation is considerably less than NEDL2+/+Mouse breeds
Enteric nervous unit CFU-GM quantity.
Embodiment 4, NEDL2+/+And NEDL2-/-The activation situation analysis of GDNF/RET in mouse enteric nervous unit
GDNF/RET signal paths are a most important signal paths in regulation and control enteric nervous system development.GDNF/RET believes
The activity of number path is embodied in the activity of two signal paths downstream, i.e. (phosphatidylinositol3 3 kinase/albumen swashs PI3K/Akt
Enzyme B) and Ras/Erk (Mitogen activated protein kinase) signal path.
First, in situ detection NEDL2+/+And NEDL2-/-The activation situation of GDNF/RET in mouse enteric nervous unit
Take the NEDL2 that 9 -day-old of embodiment 1 builds+/+And NEDL2-/-Mouse intestinal is organized, and is repaiied as 5mm length, with
Stimulated with 50ng/ml GDNF afterwards, detected pErk (phosphorilated extracellular signal-regulated kinase) group, GDNF stimulation times
For 5min；Detection pAkt (protein kinase B of phosphorylation) group, GDNF stimulation times are 30min.The tissue that GDNF was processed is entered
Row is fixed, paraffin section, finally carries out immunofluorescence respectively with pAkt (Ser473), pErk1/2 (Thr202/Tyr204) antibody
The level height of the pAkt and pErk of each mouse before and after detection GDNF stimulations.And with Neurofilament antibody labeling neurons.
As a result as shown in Figure 4 A.
Fig. 4 A show, before and after either GDNF stimulates, with NEDL2+/+Compare, NEDL2-/-PAkt in mouse enteric nervous unit
Level it is significant lower, and the level of pErk is without significant change.
2nd, vitro detection NEDL2+/+And NEDL2-/-The activation situation of GDNF/RET in mouse enteric nervous unit
Separate the NEDL2 that 9 -day-old of embodiment 1 builds+/+And NEDL2-/-Mouse enteric nervous unit, is trained with Neurobasal A
Foster base carries out in vitro culture 3 days, subsequently carries out the GDNF of final concentration of 50ng/ml and stimulates, and equally, detects pErk groups, GDNF thorns
The sharp time is 5min；Detection pAkt groups, GDNF stimulation times are 30min.Indirect immunofluorescene assay is subsequently carried out, pAkt is used
(Ser473), pErk1/2 (Thr202/Tyr204) antibody carries out respectively each mouse before and after Immunofluorescence test GDNF stimulations
The level height of pAkt and pErk.
As a result as shown in Figure 4 B.
Fig. 4 B show, consistent with early stage in situ detection result, before and after no matter GNDF stimulates, with NEDL2+/+Compare,
NEDL2-/-The level of pAkt is significant lower in mouse enteric nervous unit, and the level of pErk is without significant change.
Result above shows that the knockout of NEDL2 have impact on the normal development of enteric nervous system, GDNF/RET signal paths
Activation is suppressed.
Claims (4)
1. application of the product of NEDL2 protein expressions in the product for reducing intestines peristalsis ability is prepared is suppressed；The NEDL2 eggs
White amino acid sequence is as shown in SEQ ID No.1.
2. application of the product of NEDL2 protein expressions in the product for reducing neuronal cell multiplication capacity is prepared is suppressed；
The amino acid sequence of the NEDL2 albumen is as shown in SEQ ID No.1；
The neuronal cell is enteric nervous unit cell.
3. a kind of construction method of the mouse of NEDL2 gene knockouts, comprises the steps：The targeting vector of NEDL2 genes is noted
Enter the embryonic stem cell of mouse, then be transplanted to dams intrauterine, obtain being integrated with the offspring rat of target practice sequence on genome,
It is denoted by F1；F1 is mated with EIIa-Cre mouse, using Cre/LoxP systems, Cre enzymes will be practiced shooting described in F1 genomes
Sequence is deleted between the loxP sites of sequence, obtains the mouse of NEDL2 genetic heterozygosis, is denoted by NEDL2+/-Mouse；Will
NEDL2+/-Mouse carries out selfing, obtains the mouse that NEDL2 genes are not knocked out, and is denoted by NEDL2+/+Mouse, or obtain NEDL2
The mouse of gene knockout, is denoted by NEDL2-/-Mouse, NEDL2-/-Mouse is purpose mouse；
The mouse of the NEDL2 genetic heterozygosis is NEDL2 gene knockouts on item chromosome, NEDL2 bases on another item chromosome
Because of the mouse not knocked out；
The sequence of the targeting vector of the NEDL2 genes is as shown in SEQ ID No.3；
The dams are C57BL/6 mouse；
The mouse of the NEDL2 gene knockouts is congenital giant colon disease model mouse.
4. the mouse of the NEDL2 gene knockouts for being prepared by the method described in claim 3 is in exploitation or screens following arbitrary
Application in described product：
(1) product for promoting intestines peristalsis or the product for suppressing the decline of intestines peristalsis ability；
(2) product or inhibitory neuron ability of cell proliferation for promoting neuronal cell propagation declines product；The neuron is thin
Born of the same parents are enteric nervous unit cell；
(3) product of GDNF/RET signal paths is activated；
(4) prevent and/or treat the product of congenital giant colon disease.
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