CN109481682A

CN109481682A - Application of the CFTR inhibitor in the reagent that preparation adjusts Hedgehog signal path

Info

Publication number: CN109481682A
Application number: CN201811337213.8A
Authority: CN
Inventors: 刘凯胜; 王建红; 王飞; 王一飞; 邹畅; 戴勇
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2018-11-12
Filing date: 2018-11-12
Publication date: 2019-03-19
Anticipated expiration: 2038-11-12
Also published as: CN109481682B

Abstract

The present invention provides application of the CFTR inhibitor in the reagent that preparation adjusts Hedgehog signal path, it is related to technical field of bioengineering, several CFTR inhibitor provided can effectively inhibit the expression of key protein Ihh, SMO and Gli albumen of Hedgehog signal path, and the expression of β-catenin, to inhibit Hedgehog signal path, the molecular regulation mechanism that CFTR inhibitor adjusts Hedgehog signal path is disclosed, provides a kind of new approach for the research of the physiological function of CFTR, CF related disease and intestinal tract.

Description

Application of the CFTR inhibitor in the reagent that preparation adjusts Hedgehog signal path

Technical field

The present invention relates to technical field of bioengineering, and in particular to CFTR inhibitor adjusts Hedgehog signal in preparation and leads to Application in the reagent on road.

Background technique

Cystic fibrosis transmembrane conductance regulator (cystic fibrosis transmembrane conductance Regulator, CFTR) encoding gene be referred to as cftr gene, the product of coding is one residual containing 1480 amino acid The protein of base is the chloride channel positioned at apical cell plasma membrane, and is ABC (ATP-blndingeasoette) Unique ionophorous protein in protein family.Containing there are two hydrophobic structural domain, the two structures all have complicated transferring film Part, two ATP binding structural domains and an intracellular domain.CFTR is Cystic fibrosis transmembrane conductance regulator, it It is adjustable the transhipment of epithelial cell membrane electrolyte ion.It is because finding the mutation meeting of the gene that people, which initially recognize cftr gene, Lead to cystic fibrosis (cystic fibrosis, CF).

Cystic fibrosis (CF) is a kind of autosomal recessive hereditary diseases, is mostly occurred in the crowd of Caucasia, is by CFTR Caused by gene mutation, the anion that cftr gene encodes cAMP adjusting on the top film of the epithelial cell of Different Organs is logical Road.It has been determined currently, having more than 2000 mutation in cystic fibrosis, the mutation type of wherein most is phenylalanine Missing in 508 positions (Δ F508) causes CFTR protein folding defect and endoplasmic reticulum to be degenerated, and generation is being more than 80% CF In patient.Cftr gene mutation influences the mucous membrane physiology of breathing, digestion and reproductive system, leads to extensive clinical manifestation, including Pancreatic insufficiency, focal biliary cirrhosis, infertility and chronic airway obstruction etc..

The inhibitor of CFTR mainly includes that the inhibitor and natural products obtained is screened from small molecule combinatorial chemical libraries Inhibitor, the CFTR inhibitor found earliest are aromatic radical aminobenzoic acid esters compound DPC and NPPB.Currently, CFTR Inhibitor is main 2 classes: thiazolidone compound and pyrimidine (sulphur) ketone compounds.Currently, Chinese patent is announced CN101668732 discloses a kind of CFTR inhibitor compound and application thereof, which is CFTR inhibitor Containing hydrazone compound is mainly used for treating diarrhea.Chinese patent bulletin CN104398509B discloses a kind of CFTR inhibitor Application of the CFTRinh-172 in the drug for the disease that preparation prevention and treatment leukaemia cell mediates, CFTRinh-172 pass through inhibition High expression of the CFTR in mononuclearcell effectively to inhibit the proliferation of leukaemia cell.CFTR inhibitor is clinically applied more In secretory diarrhea and cystic fibrosis diseases.

Wnt/ β-catenin and Hedgehog (Hh) signal path formed with tumour it is in close relations, in the generation of tumour Unconventionality expression, and there is reciprocation.Hedgehog signal path mainly includes 3 parts: Hh ligandin, including Shh, Ihh And Dhh；Membrane receptor protein Patched (PTCH) and Smoothened (SMO)；Downstream transcription factor Gli (Gli1, Gli2, Gli3).Wherein, Gli1 is the transcription factor in Hedgehog signal path, and it is logical that expression reflects Hedgehog signal The activation degree on road.When lacking Hh signal, PTCH inhibits SMO, to inhibit the cracking of Gli.As ligand binding PTCH, The inhibition of SMO is released from, and for Gli by cutting and transporte to cells core, leading to Hh target gene includes PTCH and Gli itself and proliferation Related gene includes the transcription of cyclin D1.Although in recent years to various risk of cancer in CF patient and CFTR carriers of mutation Concern increase, hyperplasia can be observed in the various tissues and organ of cystic fibrosis (CF), most CF patients are digesting Disease incidence in road cancer increases, such as colorectal cancer, but application of the CFTR inhibitor in terms of colorectal cancer also has not been reported, Inherent mechanism is still unclear.

The application is directed to the vacancy of the prior art, has studied CFTR inhibitor and makees to the adjusting of Hedgehog signal path With, it was found that CFTR inhibitor to the inhibiting effect of Hedgehog signal path, and then proposes CFTR inhibitor and adjusted in preparation Save the application theme of the application in the reagent of Hedgehog signal path.Since Hedgehog signal path and tumour form relationship Closely, the application theme of application of the CFTR inhibitor in the drug that preparation treats and prevents tumour is further provided.

Summary of the invention

The purpose of the present invention is to provide CFTR inhibitor answering in the reagent that preparation adjusts Hedgehog signal path With for the physiological function of CFTR, the research and treatment or diagnosis of CF related disease and intestinal tract provide a kind of new approach.

The present invention provides application of the CFTR inhibitor in the reagent that preparation adjusts Hedgehog signal path.

Specifically, the application is CFTR inhibitor answering in the reagent that preparation inhibits Hedgehog signal path With.

Specifically, the application is CFTR inhibitor in the reagent that preparation lowers β-catenin protein expression Using.

Specifically, the application is CFTR inhibitor in the reagent that preparation lowers Ihh, SMO, Gli1 protein expression Application.

Preferably, the CFTR inhibitor is selected from siRNA, shRNA or Anti-CFTR antibody for CFTR.

It is further preferred that the sequence of the siRNA are as follows:

SiRNA-1:5 '-GUUAAGAAUCCCACCUGCUUUCAGCUU-3 '；

SiRNA-2:5 '-GUGCAAAUUCAGAGCUUUGUGGAACAG-3 '；

The sequence of the shRNA are as follows:

ShRNA-1:5 '-GCTTCTCTGGGACTTGTTACA-3 '；

ShRNA-2:5 '-GCTGCAAAGATCAATGAAAGA-3 '；

ShRNA-3:5 '-GGATCTACTGGAGCAGGAAAG-3 '.

Preferably, the CFTR inhibitor is CFTRinh-172 and CFTR Inhibitor II (GlyH-101)；

The chemical structural formula of the CFTRinh-172 are as follows:

The chemical structural formula of the CFTR Inhibitor II (GlyH-101) are as follows:

Preferably, the CFTR inhibitor is resveratrol oligomer, such as resveratrol dimer (trans- ε- Viniferin, TV), Vaticaffinol (r-2-viniferin, RV)；Licorice root extract, such as isoliquiritigenin (Isoliquiritigenin)；Stevioside glycosides compound, such as steviol (steviol)；Glucosidase procyanidins compound, such as Crofelemer(SP-303)；Rabdosia rubescens extract, such as Oridonin (oridonin) or catechin compounds, such as catechu Element (Catechin, C), epicatechin (Epicatechin, EC), Epigallo-catechin gallate (EGCG) (Epigallocatechin gallate, EGCG)；

It is further preferred that the resveratrol oligomer be resveratrol dimer (trans- ε-viniferin, ) or Vaticaffinol (r-2-viniferin, RV) TV；

Resveratrol dimer:

Vaticaffinol:

It is further preferred that the catechin compounds are that catechin, epicatechin or epigallocatechin do not have Infanticide acid esters；

Catechin:

Epicatechin；

Epigallo-catechin gallate (EGCG):

The technical effect that the present invention invents is as follows:

The present invention reports the molecular regulation mechanism that CFTR inhibitor adjusts Hedgehog signal path for the first time, provided Several CFTR inhibitor can effectively inhibit the expression of key protein Ihh, SMO and Gli albumen of Hedgehog signal path, And the expression of β-catenin, to inhibit Hedgehog signal path, the CFTR inhibitor provided is adjusted in preparation Application in the reagent of Hedgehog signal path, is the physiological function of CFTR, and the research of CF related disease and intestinal tract mentions A kind of new approach is supplied.

Detailed description of the invention

Fig. 1 is that the small intestinl channel H.E. of mouse in embodiment 1 dyes observation figure under simple microscope；

Fig. 2 is the expression figure of small intestine PCNA albumen under fluorescence microscope of mouse in embodiment 1；

Fig. 3 is the polyacrylamide gel electrophoresis figure and expression quantity histogram of CFTR and PCNA albumen in embodiment 2；

Fig. 4 is the polyacrylamide gel electrophoresis figure and expression quantity column of PTCH, Ihh, SMO and Gli albumen in embodiment 3 Figure；

Fig. 5 is the polyacrylamide gel electrophoresis figure and expression quantity histogram of Gli albumen in embodiment 4；

Fig. 6 is expression figure of the small intestine in fluorescence microscopy microscopic observation PCNA albumen of mouse in embodiment 4；

Fig. 7 be in embodiment 5 after siRNA and shRNA processing CFTR, β-catenin, Ihh, SMO in IEC-18 cell With the expression quantity histogram of Gli1 albumen；

Fig. 8 be in embodiment 5 after the processing of Anti-CFTR antibody CFTR, β-catenin, Ihh, SMO in IEC-18 cell With the expression quantity histogram of Gli1 albumen；

Fig. 9 be in embodiment 5 after the processing of chemical classes CFTR inhibitor CFTR, β-catenin in IEC-18 cell, Ihh, The expression quantity histogram of SMO and Gli1 albumen.

Specific embodiment

The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article It encloses.Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material Material, preferred method and material are enumerated by place herein.

Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention The normally understood identical meaning of those of ordinary skill.

In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art (such as with reference to J. Pehanorm Brooker etc. write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or Person carries out according to product description.

Mouse used is female reproductive tract Δ F508-CFTR (F508) mouse in embodiment, has the cftr gene of mutation, 3-bp missing, eliminates phenylalanine, this is corresponding with the most common CFTR mutation of the mankind.

Embodiment 1

F508 mouse intestinal epithelial cell hyper-proliferative and crypts are deepened

Take respectively the small intestine of wild type (WT) and F508 mouse carry out paraffin tissue sections, by H.E. (hematoxylin-she It is red) dyeing, it is observed under simple microscope, the result is shown in Figure 1.

The small intestine of wild type and F508 mouse is taken to be sliced, dyed respectively, in fluorescence microscopy microscopic observation proliferative cell The expression of nuclear antigen (Proliferating Cell Nuclear Antigen, PCNA), is as a result shown in Fig. 2.

The results show that the crypts of small intestine of F508 mouse is compared with wild type type, depth is dramatically increased, and PCNA positive cell is bright It is aobvious to increase.The increase for showing Crypt depth is as caused by the proliferation increase of cell.

Embodiment 2

F508 mouse intestinal epithelial cell hyper-proliferative and CFTR protein expression

CFTR and PCNA albumen in wild-type mice and F508 mouse intestinal is analyzed using Western blot.

Specific step is as follows:

Each 100mg of small intestine for weighing 7 wild-type mices and 6 F508 mouse respectively, is put in EP pipe, and every pipe is added RIPA fine melt liquid (RIPA+1%PMSF) 1ml shreds tissue, Ultrasonic Pulverization 3 times, each 15s, it is abundant to stand 30min on ice Cracking；12000rpm is centrifuged 25min under the conditions of 4 DEG C；Taking supernatant is protein sample, measures protein content with BCA method, according to BCA quantitative result takes 20 μ g/ porin loadings, carries out polyacrylamide gel electrophoresis (SDS-PAGE), and β-actin is used as internal reference It compares, after transferring film, room temperature closes 1h, is washed film 3 times with containing 0.05%Tween-20TBST, and primary antibody is (dilute accordingly after addition dilution Degree of releasing 1:3000), it is placed on shaking table and stays overnight for 4 DEG C, washed film 3 times with containing 0.05%Tween-20TBST, is added after dilution accordingly Secondary antibody (dilution 1:3000) is incubated at room temperature 1h, is washed film 3 times with containing 0.05%Tween-20TBST, enhances chemistry hair with standard The detection of light (ECL) method.As a result see Fig. 3.

The result shows that the expression of CFTR albumen is suppressed in the small intestine of F508 mouse, PCNA egg compared with wild-type mice White expression significantly rises.Show the mutation of cftr gene, CFTR, which is suppressed or lowers, can promote intestinal epithelial cell increasing It grows, further illustrates, CFTR is suppressed or lowers the proliferation for participating in crypts of small intestine.

Embodiment 3

Cftr gene inhibition from mutation intestinal epithelial cell Hedgehog signal path

Hedgehog in 7 wild-type mices and 6 F508 mouse intestinals is analyzed using Western blot in embodiment 2 Key protein in signal path, including PTCH, Ihh, SMO and Gli albumen.As a result see Fig. 4.

The result shows that PTCH is activated in the small intestine of F508 mouse compared with wild-type mice, significant up-regulation is expressed, Ihh, SMO and Gli are significantly lowered.Show the mutation of cftr gene, CFTR, which is suppressed or lowers, inhibits Hedgehog signal logical Ihh, SMO and Gli albumen in road.

Embodiment 4

The activation of β-catenin signal inhibits Intestinal epitheliual cell proliferation

Wild-type mice and F508 mouse are handled with LiCl, in mouse vivo activation β-catenin signal, with unused LiCl The wild-type mice of processing and F508 mouse are analyzed as control (control, Con) using Western blot in embodiment 2 The expression of Gli1.As a result see Fig. 5.

To 4 groups of above-mentioned mouse using the method in embodiment 1, its small intestine is taken to be sliced, dyed, in fluorescence microscope The expression of lower observation PCNA albumen, is as a result shown in Fig. 6.

Fig. 5's the result shows that, the downward of the reversible Gli1 of activation of β-catenin；Fig. 6's the result shows that, F508 mouse Increased PCNA positive cell can be by activating β-catenin to reduce in small intestine, these are the result shows that β-catenin signal Activation inhibit Intestinal epitheliual cell proliferation, promote Hedgehog access, that is to say, that the mutation of cftr gene, CFTR are pressed down System is lowered, and the reduction of β-catenin level inhibits Hedgehog access.

Embodiment 5

CFTR inhibitor inhibits Hedgehog signal path

Take rat ileum cell IEC-18 (purchased from Shanghai Bang Jing Industrial Co., Ltd., article No.: BJ-ATCC0007) 89% Cell recovery is carried out in DMEM (high sugar) dual anti-culture medium of+10%FBS+1%, it is good to choose growth conditions for secondary culture IEC-18 cell carries out cell count after pancreatin digestion, with 1 × 10⁶The density in/hole is inoculated in tissue culture plate, works as experiment When group is for siRNA the or shRNA lentiviruses transduction particle of CFTR, control group turns for siRNA-nc or shRNA-nc slow virus Particle is led, then transfects, cultivate；When experimental group is antibody or other chemical classes CFTR inhibitor, equivalent is added in control group DMSO (dimethyl sulfoxide) is put into 37 DEG C containing 5%CO₂Cell incubator in cultivate 12h after, in experimental group and control group Cell is counted, and the results are shown in Table 1 (* indicates P≤0.05)；Albumen is extracted, is analyzed using Western blot in embodiment 2 As a result the expression of CFTR, β-catenin, Ihh, SMO and Gli1 albumen are shown in Fig. 7-Fig. 9.

CFTR inhibitor used is the siRNA lentiviruses transduction particle for CFTR (purchased from Shanghai Ji Ma pharmaceutical technology Co., Ltd, article No.: D- series), shRNA lentiviruses transduction particle (be purchased from Shanghai JiMa pharmacy Technology Co., Ltd, article No.: E-S-1), Anti-CFTR antibody (being purchased from Shanghai Rui Qi Biotechnology Co., Ltd, article No.: Rs-1277R) and chemical classes inhibit Agent；

Chemical classes CFTR inhibitor include: CFTRinh-172 (purchased from Sigma-Aldrich company, article No. CAS: 307510-92-5), (trade name: GlyH-101 is purchased from Sigma-Aldrich company, article No. to CFTR Inhibitor II No. CAS: 328541-79-3), TV and RV utilizes the " discovery and molecule pharmacology of Zhang Yaofang resveratrol oligomer CFTR inhibitor Research [D] Liaoning Normal University is learned, the method in 2014. " is from Chinese wild grape rattan (Vitisamurensis Rupr.) Isolated, isoliquiritigenin (being purchased from Shanghai Wa Lan Biotechnology Co., Ltd, article No.: BIO-0481), steviol are (purchased from upper Hai Guduo biotechnology company, article No.: GD-0029), Crofelemer (be purchased from U.S. Salix company, trade name: Fulyzaq), Oridonin (being purchased from Shanghai Gu Duo biotechnology company, article No.: GD-125-188), C are (purchased from Shanghai Gu piece Biotechnology company, article No.: GD-125-46), EC (be purchased from Shanghai Gu Duo biotechnology company, article No.: GD-125-47) or EGCG (is purchased from Shanghai Gu Duo biotechnology company, article No.: GD-125-50)；

The sequence of siRNA are as follows:

SiRNA-1:5 '-GUUAAGAAUCCCACCUGCUUUCAGCUU-3 '；

SiRNA-2:5 '-GUGCAAAUUCAGAGCUUUGUGGAACAG-3 '；

SiRNA-cn:5 '-CUUCCUCUCUUUCUCUCCCUUGUGA-3 '.

The sequence of shRNA are as follows:

ShRNA-1:5 '-GCTTCTCTGGGACTTGTTACA-3 '；

ShRNA-2:5 '-GCTGCAAAGATCAATGAAAGA-3 '；

ShRNA-3:5 '-GGATCTACTGGAGCAGGAAAG-3 '；

ShRNA-cn:5 '-CGAAGAGACCCTGAACAATGT-3 '.

Table 1: the cell quantity in experimental group and control group

As can be seen from Table 1, the cell quantity in experimental group is dramatically increased compared with control group, likewise, chemical classes CFTR presses down Preparation TV, RV, isoliquiritigenin, steviol, Crofelemer, Oridonin, C, EC, EGCG also have the effect of similar, say These bright CFTR inhibitor promote rat ileum cell Proliferation.

Fig. 7-Fig. 9's the results show that these CFTR inhibitor make CFTR, β-catenin, Ihh, SMO and Gli1 albumen Expression is significant to lower, and illustrates that these CFTR inhibitor inhibit the expression of CFTR, it is suppressed that β-catenin, Ihh, SMO and The expression of Gli1 albumen, CFTR inhibitor is by inhibiting the key protein in β-catenin expression and Hedgehog signal path Ihh, SMO and Gli1 albumen inhibit Hedgehog access.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims

Application of the 1.CFTR inhibitor in the reagent that preparation adjusts Hedgehog signal path.
2. application according to claim 1, it is characterised in that: CFTR inhibitor inhibits Hedgehog signal path in preparation Reagent in application.
3. application according to claim 2, it is characterised in that: CFTR inhibitor makes β-catenin protein expression in preparation Application in the reagent of downward.
4. application according to claim 2, it is characterised in that: CFTR inhibitor makes Ihh, SMO, Gli1 albumen table in preparation Application up in the reagent of downward.
5. application according to claim 1, it is characterised in that: the CFTR inhibitor be selected from for CFTR siRNA, ShRNA or Anti-CFTR antibody.
6. application according to claim 5, it is characterised in that:

The sequence of the siRNA are as follows:

SiRNA-1:5 '-GUUAAGAAUCCCACCUGCUUUCAGCUU-3 '；

SiRNA-2:5 '-GUGCAAAUUCAGAGCUUUGUGGAACAG-3 '；

The sequence of the shRNA are as follows:

ShRNA-1:5 '-GCTTCTCTGGGACTTGTTACA-3 '；

ShRNA-2:5 '-GCTGCAAAGATCAATGAAAGA-3 '；

ShRNA-3:5 '-GGATCTACTGGAGCAGGAAAG-3 '.
7. application according to claim 1, it is characterised in that: the CFTR inhibitor is CFTRinh-172 and CFTR Inhibitor II；

The chemical structural formula of the CFTRinh-172 are as follows:

The chemical structural formula of the CFTR Inhibitor II are as follows:
8. application according to claim 1, it is characterised in that: the CFTR inhibitor is resveratrol oligomer, sweet Grass roots extract, stevioside glycosides compound, glucosidase procyanidins compound, Rabdosia rubescens extract or catechin compounds.
9. application according to claim 8, it is characterised in that: the resveratrol oligomer is resveratrol dimer Or Vaticaffinol；

The resveratrol dimer are as follows:

The Vaticaffinol are as follows:
10. application according to claim 8, it is characterised in that: the catechin compounds are catechin, table catechu Element or Epigallo-catechin gallate (EGCG)；

The catechin are as follows:

The epicatechin are as follows:

The Epigallo-catechin gallate (EGCG) are as follows: