CN106138050B - The purposes of Shh signal pathway inhibitor - Google Patents
The purposes of Shh signal pathway inhibitor Download PDFInfo
- Publication number
- CN106138050B CN106138050B CN201510138093.9A CN201510138093A CN106138050B CN 106138050 B CN106138050 B CN 106138050B CN 201510138093 A CN201510138093 A CN 201510138093A CN 106138050 B CN106138050 B CN 106138050B
- Authority
- CN
- China
- Prior art keywords
- mouse
- pkd2
- cre
- vil
- kidney
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses a kind of new applications of Shh signal pathway inhibitor.The new application of Shh signal pathway inhibitor provided by the present invention is specially application of the Shh signal pathway inhibitor in the product of preparation treatment kidney of patients with autosomal dominant polycystic kidney disease (ADPKD)；The Shh signal pathway inhibitor is specially Vismodegib.The present invention is with Vil-Cre:Pkd2f3/f3Mouse is as ADPKD animal model, the results showed that Shh signal pathway inhibitor-Vismodegib can weaken Vil-Cre:Pkd2f3/f3The renal cyst degree of mouse, can reduce Vil-Cre:Pkd2f3/f3The kidney weight ratio of mouse, can reduce Vil-Cre:Pkd2f3/f3The content of creatinine and urea nitrogen in the blood of mouse；Vil-Cre:Pkd2 can be reducedf3/f3The renal fibrosis degree of mouse.Analysis of the present invention for ADPKD pathogenesis all has significance for treating the research and development of drug of ADPKD.
Description
Technical field
The invention belongs to biomedicine fields, are related to the new application of Shh (sonic hedgehog) signal pathway inhibitor,
In particular to application of the Shh signal pathway inhibitor-Vismodegib in treatment kidney of patients with autosomal dominant polycystic kidney disease.
Background technique
Vismodegib is a kind of new oral class drug with selective Hedgehog signal path inhibiting effect,
Ratify via FDA for treating basal-cell carcinoma.
Vismodegib effectively inhibits hedgehog access, is a kind of micromolecular inhibitor of novel and specific synthesis,
IC50 is 3nM.Vismodegib acts on Hedgehog signal path, blocks Hedgehog ligand, i.e. cell surface receptor
The activity of PTCH and SMO, to block Hedgehog signal path.In terms of tissue growth and reparation, Hedgehog signal is logical
Road is significant.The abnormal activation of Hedgehog path signal and uncontrolled cell Proliferation may be coordinated with Hedgehog
The mutation of body, i.e. cell surface receptor PTCH and SMO is related.In vitro, Vismodegib is not inhibiting Stem Cells to be proliferated
Under the premise of prevent the growth of primary pancreas transplantation object, this result has been applied to clinic recently.Vismodegib also inhibits
ABCG2, P- glycoprotein, and with the related important MRP1ABC carrier of MDR.Vismodegib also blocks other a variety of ABC to carry
Body.ABC carrier belongs to a memebrane protein family, its overexpression is related with drug resistance, this is also a weight for the treatment of cancer
Big obstacle.Two kinds of ABC carriers of Vismodegib high inhibition, ABCG2/BCRP and ABCB1/P- glycoprotein are also mild to inhibit
ABCC1/MRP1.In the HEK293 cell of ABCG2 overexpression, fluorescence ABCG2 substrate is can be improved in Vismodegib
The retentivity of BODIPY- prazosin, it is also possible to which mitoxantrone handles these cells again, generates antitumor ABCG2 substrate.It is real
Overexpression P glycoprotein or MRP1 in the kidney cell for making Madin-Darby dog are tested, it is yellowish green that Vismodegib improves calcium
The retentivity of element-AM, then load handles these cells with colchicine.In addition, with mitoxantrone, Hycamtin, SN-38 processing
Overexpression ABCG2 human non-small cell lung cancer cell NCI-H460/par and NCI-H460/MX20, use Vismodegib
It handles again.The IC50 value that Vismodegib acts on ABCG2 and Pgp is respectively 1.4 μM and 3.0 μM.Vismodegib changes
Intracellular Ca2+Balance, and the lung carcinoma cell of anti-cisplatin is acted on, inhibit cell growth.
Vismodegib has been used to the medulloblastoma for the treatment of animal model.Vismodegib inhibits primary pancreatic transplanting
Tumor growth, but do not inhibit cell Proliferation.Vismodegib presses 25mg/kg or more dosage medulloblastoma Ptch
(+/-) Xenograft Model, causes cellular degeneration, handles the colorectal cancer that two kinds of ligands rely on by 92mg/kg dosage
Model D5123 and 1040830, daily processing twice, inhibit tumour growth.Analysis Hh pathway activity and PK/PD model are shown
Vismodegib inhibits Gli1, and IC50 value and Vismodegib act on the IC50 value difference of medulloblastoma and D5123 model not
More (respectively 0.165 μM ± 11.5% and 0.267 μM ± 4.83%).
Kidney of patients with autosomal dominant polycystic kidney disease (Autosomal Dominant Polycystic Kidney
Disease, ADPKD) disease is one of most common monogenic inheritance nephrosis.Its main clinic symptoms is bilateral renal shape
At the spherical fluidity tumour largely to differ in size, and progressive increases, and destroys the structure and function of kidney, eventually leads to renal function
Failure.ADPKD usually falls ill in adult, disease incidence 1:400-1000, is the third of current terminal kidney failure in the world
The big cause of disease.In addition to nephrosis phenotype, the non-renal tract lesion phenotype of ADPKD patient is also fairly obvious: 83% ADPKD patient
There is hepatic cyst, cerebral aneurysm occurs in 16% patient.Other Pathologies include aortic root, and Thoracic arteries are abnormal, two points
Valve prolapsus and stomach wall hernia.Terminal kidney failure will occur after 60 years old in about 50% patient.The urea nitrogen (BUN) of normal person
Term of reference is 2.14-7.14mmol/L, and serum creatinine (CRE) term of reference is 44-133 μm of ol/L, when serum creatinine is more than 133 μ
Mol/L is the inflammation damnification phase, and it is renal failure stage more than 451umol/L that more than 186 μm ol/L, which are the renal impairment phase,.
Summary of the invention
The object of the present invention is to provide the new applications of Shh signal pathway inhibitor.
The new application of Shh signal pathway inhibitor provided by the present invention can be controlled for Shh signal pathway inhibitor in preparation
Treat the application in the product of kidney of patients with autosomal dominant polycystic kidney disease (ADPKD).
Further, Shh signal pathway inhibitor also belongs to protection of the invention in the application prepared in following any product
Range:
(1) weaken the product of sickened body renal cyst degree；
(2) product of sickened body kidney weight ratio is reduced；
(3) product of creatinine and/or urea nitrogen content in sickened body blood is reduced
(4) product of sickened body renal fibrosis degree is reduced.
In the present invention, the Shh signal pathway inhibitor is specially Vismodegib.
The sickened body is the body with kidney of patients with autosomal dominant polycystic kidney disease (ADPKD) clinical symptoms, as usual
Autosomal dominant heredity polycystic kindey (ADPKD) patient.In the present invention, with Vil-Cre:Pkd2f3/f3Mouse is dynamic as ADPKD
Object model, as with the body of kidney of patients with autosomal dominant polycystic kidney disease (ADPKD) clinical symptoms, above-mentioned (1) then specific table
It is now decrease Vil-Cre:Pkd2f3/f3The renal cyst degree of mouse；Above-mentioned (2), which are then embodied in, reduces Vil-Cre:Pkd2f3 /f3The kidney weight ratio of mouse；Above-mentioned (3), which are then embodied in, reduces Vil-Cre:Pkd2f3/f3Creatinine in mouse blood and/
Or urea nitrogen content；Above-mentioned (4), which are then embodied in, reduces Vil-Cre:Pkd2f3/f3The renal fibrosis degree of mouse.
It is also another object of the present invention to provide a kind of products.
The active constituent of product provided by the present invention is Shh signal pathway inhibitor, which has following on the way
At least one:
(a) kidney of patients with autosomal dominant polycystic kidney disease is treated；
(b) weaken sickened body renal cyst degree；
(c) sickened body kidney weight ratio is reduced；
(d) creatinine and/or urea nitrogen content in sickened body blood are reduced；
(e) sickened body renal fibrosis degree is reduced.
In the present invention, the Shh signal pathway inhibitor is specially Vismodegib.
The sickened body is the body with kidney of patients with autosomal dominant polycystic kidney disease (ADPKD) clinical symptoms, as usual
Autosomal dominant heredity polycystic kindey (ADPKD) patient.In the present invention, with Vil-Cre:Pkd2f3/f3Mouse is dynamic as ADPKD
Object model, as with the body of kidney of patients with autosomal dominant polycystic kidney disease (ADPKD) clinical symptoms, above-mentioned (b) then specific table
It is now decrease Vil-Cre:Pkd2f3/f3The renal cyst degree of mouse；Above-mentioned (c), which is then embodied in, reduces Vil-Cre:Pkd2f3 /f3The kidney weight ratio of mouse；Above-mentioned (d), which is then embodied in, reduces Vil-Cre:Pkd2f3/f3Creatinine in mouse blood and/
Or urea nitrogen content；Above-mentioned (e), which is then embodied in, reduces Vil-Cre:Pkd2f3/f3The renal fibrosis degree of mouse.
In above-described each application or product, the product concretely drug.
The above all of Vil-Cre:Pkd2f3/f3Mouse constructs to obtain according to the method included the following steps:
(I) by Pkd2f3/f3Mouse (is recorded in " lngyu Kim, Tianbing Ding, Yulong Fu, et al.
Conditional Mutation of Pkd2Causes Cystogenesis and Upregulatesβ-Catenin.J Am
Soc Nephrol, 2009 (20): a 2556-2569 " text) and the transgenic mice of Vil-Cre recombinase is had (purchased from The
Jackson Laboratory, Strain Name:B6.SJL-Tg (Vil-cre) 997Gum/J, Stock Number:
004586, it is detailed in network address: http://jaxmice.jax.org/strain/004586.html) hybridized, obtain F1 generation
(genotype for wherein theoretically having 1/2 offspring is Vil-Cre:Pkd2+/f3；The genotype of 1/2 offspring is Pkd2+/f3)；
(II) F1 generation (Vil-Cre:Pkd2 for obtaining step (I)+/f3) and the Pkd2f3/f3Mouse is returned, and is obtained
To backcross progeny；
(III) Pkd2 gene pure is filtered out from the backcross progeny that step (II) obtains, while expresses Vil-Cre's
Mouse, as Vil-Cre:Pkd2f3/f3Mouse.
Wherein, " Pkd2 gene pure is filtered out from the backcross progeny that step (II) obtains, together described in step (III)
When express Vil-Cre mouse ", can realize as follows: using the mouse genome of the backcross progeny as template, use
For primer (three: 5 '-TCT GAC TTG CAG ACT the GTG GG-3 ', 5 '-AGG TAG GGG AAG of Pkd2 gene
GTC AGG GTT GG-3 ', 5'-TTTACGTCCAGCCAAGCT-3') and for Cre gene primer (two: 5 '-CCA
GGT TAC GGA TAT AGT TCA TG-3 ', 5 '-TGC CAC GAC CAA GTG ACA GC-3 ') PCR expansion is carried out respectively
Increase；PCR amplification is carried out for Pkd2 gene and obtains the single band (Pkd2 gene pure) that size is 650bp, while being directed to Cre
Gene carries out PCR amplification and obtains the mouse for the band (expression Vil-Cre) that size is 650bp to be the Vil-Cre:
Pkd2f3/f3Mouse.
The present invention is with Vil-Cre:Pkd2f3/f3Mouse has carried out the inhibition of Shh signal path as ADPKD animal model
The effect disquisition that agent-Vismodegib treats kidney of patients with autosomal dominant polycystic kidney disease (ADPKD), the results showed that, Shh letter
Number pathway inhibitor-Vismodegib has the effect of certain, specific body for treatment kidney of patients with autosomal dominant polycystic kidney disease
It is present: (1) that Vil-Cre:Pkd2 can be weakenedf3/f3The renal cyst degree of mouse；(2) Vil-Cre:Pkd2 can be reducedf3/f3Mouse
Kidney weight ratio；(3) Vil-Cre:Pkd2 can be reducedf3/f3The content of creatinine and/or urea nitrogen in the blood of mouse；(4) may be used
Reduce Vil-Cre:Pkd2f3/f3The renal fibrosis degree of mouse.The present invention is for kidney of patients with autosomal dominant polycystic kidney disease
(ADPKD) analysis of pathogenesis has for treating the research and development of drug of kidney of patients with autosomal dominant polycystic kidney disease (ADPKD)
It is significant.
Detailed description of the invention
Fig. 1 is the PCR testing result of backcross progeny murine genes type.Wherein, A be for Pkd2 gene PCR testing result,
Pkd2f3/f3For Pkd2 gene pure, Pkd2+/f3For Pkd2 genetic heterozygosis；B is the PCR testing result for Cre gene, Vil-
Cre is expression Vil-Cre；WT is not express Vil-Cre.
Fig. 2 is Vil-Cre:Pkd2f3/f3The disease phenotype of mouse simulation mankind ADPKD.Wherein, A Vil-Cre:
Pkd2f3/f3Mouse kidney form；B is Vil-Cre:Pkd2f3/f3Mouse liver form；C is Vil-Cre:Pkd2f3/f3Mouse pancreas
Gland form；D is Vil-Cre:Pkd2f3/f3The kidney cross section of mouse；E is Pkd2f3/f3The kidney cross section of mouse.
Fig. 3 is the kidney HE coloration result of two groups of mouse.Wherein, A indicates control group；B indicates treatment group.In A and B, 1,
2,3, the 4 kidney HE coloration result for indicating two groups of Different Individual mouse.
Fig. 4 is the kidney weight ratio measurement result of two groups of mouse.
Fig. 5 is the assay result of urea nitrogen (BUN) and creatinine (CRE) in the blood of two groups of mouse.Wherein, A is indicated
Creatinine content；B indicates urea nitrogen content.
Fig. 6 is the renal fibrosis degree measurement result of two groups of mouse.Wherein, A indicates treatment group's mouse kidney Masson
Fibrosis coloration result；The comparison of B expression control group mice renal fibrosis area；C indicates control group and treatment group's Mouse Kidney
The comparison of dirty fibrosis area.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Pkd2f3/f3Mouse: " lngyu Kim, Tianbing Ding, Yulong Fu, et are recorded in
al.Conditional Mutation of Pkd2Causes Cystogenesis and Upregulatesβ-Catenin.J
Am Soc Nephrol, 2009 (20): in a 2556-2569 " text.It is constructed and qualification process is substantially as follows: firstly,
Two sites loxP are inserted into 3 exon two sides of Pkd2 gene, have the site FRT in the two sides of Positive selectable Neo, due to
The structure does not block the expression of normal Pkd2 gene completely, so generated with this carrier by gene targeting
Pkd2nf3Mouse can avoid dying of embryonic period, embryonic phase.Again by Pkd2nf3Mouse and ACTB-Flep mouse hybrid are produced without Neo member
The Pkd2 of partf3/f3Mouse.In view of in Pkd2f3/f3More capsule phenotypes are not observed in mouse, also there is no changing for the expression of PC2
Become, it is believed that this Pkd2f3Allele not will lead to the expression inactivation of Pkd2.
Transgenic mice with Vil-Cre recombinase: The Jackson Laboratory, Strain Name are purchased from:
B6.SJL-Tg (Vil-cre) 997Gum/J, Stock Number:004586, is detailed in network address: http: //
jaxmice.jax.org/strain/004586.html。
Vismodegib (GDC-0449): Chemie Tek company, catalog number ctg0449.Molecular weight: 421.3；Change
Formula: C19H14Cl2N2O3S；No. CAS: 879085-55-9；Dissolubility (25 DEG C): DMSO 84mg/mL, water < 1mg/mL, ethyl alcohol
4mg/mL；Stability: 2 years -20 DEG C of powderies, 2 weeks 4 DEG C be dissolved in DMSO, be dissolved in DMSO -80 DEG C of June.
Embodiment 1, Vil-Cre:Pkd2f3/f3The building and identification of mouse model
Vil-Cre:Pkd2f3/f3Mouse is the mouse mould that Pkd2 can be knocked out with conditionity using the foundation of Cre-loxP system
Type can produce and mankind's kidney of patients with autosomal dominant polycystic kidney disease (Autosomal Dominant Polycystic Kidney
Disease, ADPKD) similar to clinical phenotypes, and early stage dies of kidney failure, can be used as ADPKD animal model.It is constructed and identification
Process is as follows:
One, Vil-Cre:Pkd2f3/f3The building of mouse model
By Pkd2f3/f3Mouse and the transgenic mice (B6.SJL-Tg (Vil-cre) for having Vil-Cre recombinase
997Gum/J) hybridized, obtaining F1 generation, (genotype for theoretically having 1/2 offspring is Vil-Cre:Pkd2+/f3；1/2 offspring
Genotype be Pkd2+/f3)；By Vil-Cre:Pkd2 in F1 generation+/f3Mouse (carries out PCR identification, specific method for Cre gene
It see below, identified to obtain the F1 generation mouse that size is 650bp purpose band) and Pkd2f3/f3Mouse is returned, and is returned
Hand over offspring.
From backcross progeny obtained as above, screening obtains Pkd2 gene pure, while can express the mouse of Vil-Cre, has
Gymnastics is made as follows:
Backcross progeny rat-tail genome is extracted, using it as template, PCR mirror is carried out to Pkd2 gene and Cre gene respectively
It is fixed, wherein the PCR the primer sequence for Pkd2 gene is following three: 5 '-TCT GAC TTG CAG ACT GTG GG-
3 ', 5 '-AGG TAG GGG AAG GTC AGG GTT GG-3 ', 5'-TTTACGTCCAGCCAAGCT-3'；For Cre gene
PCR the primer sequence be following two: 5 '-CCA GGT TAC GGA TAT AGT TCA TG-3 ', 5 '-TGC CAC
GAC CAA GTG ACA GC-3'.When use carries out PCR amplification for three primers of Pkd2 gene in same reaction system
When, if obtaining two bands that size is respectively 650bp and 350bp, corresponding backcross progeny mouse is that Pkd2 gene is miscellaneous
Close (Pkd2+/f3), if obtaining the single band that size is 650bp, corresponding backcross progeny mouse is Pkd2 gene pure
(Pkd2f3/f3)；When carrying out PCR amplification using two primers for Cre gene, if obtaining the purpose that size is 650bp
Band, then corresponding backcross progeny mouse expresses Vil-Cre, right if not obtaining the purpose band that size is 650bp
The backcross progeny mouse answered does not express Vil-Cre.
As a result as shown in Figure 1, Pkd2 in Fig. 1 in Af3/f3For Pkd2 gene pure, the Vil-cre in Fig. 1 in B is can
Express Vil-Cre.By identifying above, the Pkd2 gene pure obtained in the backcross progeny, while can express Vil-Cre's
Mouse, as Vil-Cre:Pkd2f3/f3Mouse.Vil-Cre is the Cre enzyme for being controlled by Villin1 promoter, wherein villin1
Promoter is expressed in the mice embryonic phase the 12.5th day enterocyte, and mouse, can be on a cellular level under the action of Cre enzyme
Generate Pkd2d3/d3, due to Pkd2d3/d33 exons on allele have been cut away, and then generate frameshift mutation, are caused
3 exon downstreams nearby generate terminator, and then Pkd2 gene product PC2 is made to become truncated protein.
Two, Vil-Cre:Pkd2f3/f3The identification of mouse model
The Vil-Cre::Pkd2 that conventinal breeding step 1 obtainsf3/f3Mouse observes its existing state.To four monthly age of mouse,
Its kidney, liver, pancreas are taken, lesion situation is observed；In addition carrying out HE staining analysis to kidney, (concrete operations are referring to embodiment 2
Correlation step).Pkd2 is set simultaneouslyf3/f3Mouse is as control.
As the result is shown: the Vil-Cre:Pkd2 that step 1 obtainsf3/f3Mouse is in 4~May or so death, kidney, liver
There is serious tumour in dirty, pancreas, and the HE coloration result of kidney further demonstrates that kidney without kidney essence, and renal function is serious
It is impaired, determine Vil-Cre:Pkd2f3/f3Mouse dies of kidney failure caused by renal cyst, and brood Pkd2 of the same agef3/f3Mouse
Then phenotype is normal.Concrete outcome is as shown in Figure 2.
The above result shows that the Vil-Cre:Pkd2 that step 1 obtainsf3/f3Mouse phenotype is similar to mankind ADPKD patient,
It can be used as ADPKD animal model.
The application of embodiment 2, Vismodegib in treatment kidney of patients with autosomal dominant polycystic kidney disease
The present embodiment is with Vil-Cre:Pkd2f3/f3Mouse researchs and analyses Vismodegib and is controlling as ADPKD animal model
Treat the application in kidney of patients with autosomal dominant polycystic kidney disease.It is specific as follows:
One, experimental method
1, experimental group and processing
Vismodegib solution: Vismodegib is dissolved with DMSO, until its concentration be 80mg/mL, after use physiological saline
It is diluted to 2mg/ml.
Grouping and processing: it is divided into treatment group and control group, every group of each 10 Vil-Cre:Pkd2f3/f3Mouse.Go out from mouse
Start within the tenth day after life, carry out continuous subcutaneous injection in one week, after be changed to be injected intraperitoneally, daily administration stops up to two monthly age of mouse
Medicine, every mouse for the treatment of group are administered by the dosage of 50mg/kg (every kg weight gives 50mg Vismodegib)；It is control group every small
Mouse injects isometric physiological saline containing equivalent DMSO daily.
2, each group mouse treatment condition is analyzed
(1) the renal cyst phenotype of two groups of mouse of HE staining analysis
After two monthly age of mouse is discontinued, every group randomly selects 5 mouse, after execution, extracts bilateral renal.In electronic balance
On be precisely weighed, calculate kidney weight ratio (g/g), i.e. bilateral renal weight (g)/weight (g).Afterwards by bilateral renal along arrow
Shape face is cut, and fixes 24 hours with 10% neutral formalin solution, rear graded ethanol dehydration, then with paraffin wax embedding routine
Embedding, wax stone row slice and HE dyeing after.
A. dehydration embedding
Specific steps: it is 1. dehydrated: 70% ethyl alcohol of ethyl alcohol 1h → 75% ethyl alcohol of 1h → 80% ethyl alcohol 1h → 90% of 1h → 85%
The ethyl alcohol of ethyl alcohol 1h → 95% ethyl alcohol of 1h → 100% 1h → dimethylbenzene 15min × 2；2. embedding: paraffin wax embedding is embedded.
B. it is sliced
Kidney wax stone is made into the continuous renal histotomy of 3 μ m thicks on cycle type slicer, is gently propped up with writing brush,
It being sufficiently spread out in warm water, then picks up nephridial tissue slice with the processed slide of poly-D-lysine, electric air drier is dried,
Then in being put in electric heating constant-temperature blowing drying box, 70 DEG C of constant temperature are toasted 1 hour, so that histotomy and slide close adhesion,
It is placed in spare in box.
C.HE pathological staining
Principle: hematoxylin (hematoxylin) dye liquor is alkalinity, mainly makes endonuclear chromatin and intracytoplasmic core
Sugared body hyacinthine；Yihong (eosin) is acid dyes, mainly makes the ingredient red coloration in cytoplasm and extracellular matrix.
Step:
1. dimethylbenzene dewaxes, washed away afterwards with 100% ethyl alcohol；
2. the ethyl alcohol of various concentration successively aquation；
It is washed 5 minutes 3. phosphate buffer shaking table is slow；
4. hematoxylin extracts 8 times；
5. flowing water rinses 5 minutes；
6. hydrochloride alcohol extracts 8 times；
7. flowing water rinses 2 minutes；
8. eosin stains 10 minutes；
9. the ethyl alcohol of various concentration is successively dehydrated；
10. dimethylbenzene is transparent, neutral gum mounting is used afterwards；Micro- sem observation.
(11) as the result is shown: cytoplasm is red, nucleus bluish violet.
The measurement of (2) two groups of mouse ADPKD related biochemical indicators
The present inventor goes out the improvement degree for the treatment of group's Mouse Kidney liver function for accurate response, further to two groups
Urea nitrogen (BUN) and the content of creatinine (CRE) are determined in mouse blood, with the variation feelings of this precise reaction renal function
Condition reflects the severity of tumour.Concrete operations are as follows:
At two monthly age of mouse, by experimental group and control group mice etherization, mouse blood is extracted from inferior caval vein, from
It is serum that supernatant is obtained after the heart.Creatinine and urea nitrogen content can be measured by chemically examining serum.
(3) the kidney fibrosis phenotype of two groups of mouse of Masson staining analysis
After two monthly age of mouse is discontinued, every group randomly selects 5 mouse, after execution, extracts bilateral renal.By bilateral renal
It is cut along sagittal plane, fixes 12 hours with BouinShi liquid, rear flowing water rinses an evening, graded ethanol dehydration, then with paraffin packet
It buries machine routinely to embed, wax stone row slice and Masson dyeing after.The Kidney sections of Masson dyeing calculate fibrosis after taking pictures
Area.
A.Masson dyeing
Principle: collagenous fibres are contaminated blue by aniline blue when Masson is dyed, and muscle fibre is contaminated by acid fuchsin in red
Color.
Step:
1. tissue is fixed on BouinShi liquid, flowing water is rinsed an evening, conventional dehydration embedding；
2. slice dewaxes to water；
3. WeigerShi Garapa uniformly dyeing 5-10 minutes；
4. flowing water is slightly washed, the differentiation of 1% hydrochloride alcohol；
5. Ponceaux acid fuchsin liquid contaminates 5-10 minutes；
6. distilled water slightly rinses, 1% phosphomolybdic acid aqueous solution is handled about 5 minutes；
7. not being washed with water, directly redyed 5 minutes with aniline blue liquid；
8. 1% glacial acetic acid is handled 1 minute；
9. 95% dehydration of alcohol is multiple；
10. absolute alcohol is dehydrated, dimethylbenzene is transparent, neutral gum sealing
(11) as the result is shown: collagenous fibres are blue, and muscle fibre takes on a red color.
B. fibrosis area calculates
After the Kidney sections of Masson dyeing are taken pictures, picture runs on GNU Image Manipulation Program.
Picture pixels are adjusted to 800 × 598.By picture adjusted run addition grid program, set each grid as size be 22
× 22 pixels.It calculates fibrosis region number of grid and fibrosis area ratio can be obtained in the total number of grid ratio in kidney region
Example.
Two, experimental result
1, the renal cyst phenotype of two groups of mouse
HE coloration result as shown in figure 3, it can be seen from the figure that treatment group has more kidneys essence compared with control group, and
The renal cyst phenotype for the treatment of group mouse is considerably less than control group.In addition, to the kidney weight ratio (g/ for the treatment of group and control group mice
G) measurement result as shown in figure 4, as the result is shown the kidney weight ratio (g/g) for the treatment of group significantly less than control group (p=0.0099),
With statistical significance.
2, the measurement of two groups of mouse ADPKD related biochemical indicators
The assay result of urea nitrogen (BUN) and creatinine (CRE) in the blood of two groups of mouse are as shown in figure 5, result is aobvious
Showing, urea nitrogen (BUN) has downward trend compared with the content of creatinine (CRE) is compared with control group in the blood for the treatment of group mouse,
There were significant differences for middle urea nitrogen content, p=0.048.
3, the kidney fibrosis phenotype of two groups of mouse
The renal fibrosis degree measurement result of two groups of mouse is as shown in Masson coloration result in Fig. 6.From Fig. 6 A and
B can be seen that treatment group and be substantially reduced compared with control group degree of fibrosis in Fig. 6.C is control group and treatment group's Mouse Kidney in Fig. 6
The comparison of dirty fibrosis area.The kidney fibrosis area that C can be seen that treatment group mouse from Fig. 6 is considerably less than control group (p
=0.021), there is statistical significance.
The experimental result of comprehensive the present embodiment, it is seen that Vismodegib is in treatment kidney of patients with autosomal dominant polycystic kidney disease
(ADPKD) it is had a certain effect in.
Claims (3)
- Application of the 1.Vismodegib in the product of preparation treatment kidney of patients with autosomal dominant polycystic kidney disease.
- 2.Vismodegib is preparing the application in following any product:(1) weaken the product of sickened body renal cyst degree；(2) product of sickened body kidney weight ratio is reduced；(3) product of creatinine and/or urea nitrogen content in sickened body blood is reduced；(4) product of sickened body renal fibrosis degree is reduced；The sickened body is the body with kidney of patients with autosomal dominant polycystic kidney disease clinical symptoms.
- 3. application according to claim 1 or 2, it is characterised in that: the product is drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510138093.9A CN106138050B (en) | 2015-03-26 | 2015-03-26 | The purposes of Shh signal pathway inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510138093.9A CN106138050B (en) | 2015-03-26 | 2015-03-26 | The purposes of Shh signal pathway inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106138050A CN106138050A (en) | 2016-11-23 |
| CN106138050B true CN106138050B (en) | 2019-10-11 |
Family
ID=57339489
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510138093.9A Expired - Fee Related CN106138050B (en) | 2015-03-26 | 2015-03-26 | The purposes of Shh signal pathway inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106138050B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903996B (en) * | 2022-06-22 | 2023-10-20 | 武汉大学中南医院 | Application of specific inhibitor of Hedgehog pathway in preparation of medicine for treating intrauterine adhesion |
-
2015
- 2015-03-26 CN CN201510138093.9A patent/CN106138050B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Role of Primary Cilia in the Pathogenesis of Polycystic Kidney Disease;Bradley K. Yoder;《Journal of the American Society of Nephrology》;20071231;第18卷(第5期);第1381-1388页 * |
| Shh信号通路在ADPKD发病机制中的作用;王文仪;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20150115(第1期);第33页 * |
| 王文仪.Shh信号通路在ADPKD发病机制中的作用.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2015,(第1期),第25、27、33页. * |
| 转化生长因子β1在人多囊肾病发病中的作用;汤兵 等;《医学研究生学报》;20050930;第18卷(第9期);第793-799页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106138050A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chang et al. | Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements | |
| Yamaguchi et al. | fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. | |
| Dixon et al. | Increased levels of apoptosis in the prefusion neural folds underlie the craniofacial disorder, Treacher Collins syndrome | |
| Ando et al. | Nitric oxide is proangiogenic in the retina and choroid | |
| Chen et al. | Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity | |
| Hong et al. | Cdon mutation and fetal ethanol exposure synergize to produce midline signaling defects and holoprosencephaly spectrum disorders in mice | |
| Depew et al. | Dlx5 regulates regional development of the branchial arches and sensory capsules | |
| Hendershot et al. | Conditional deletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derived noradrenergic sympathetic ganglion neurons | |
| Handrigan et al. | Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata) | |
| Horne et al. | Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models | |
| Friedel et al. | Plexin-B2 controls the development of cerebellar granule cells | |
| Billmyre et al. | Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation | |
| Gould et al. | The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes | |
| Fuerst et al. | Cell autonomy of DSCAM function in retinal development | |
| Kallop et al. | A death receptor 6-amyloid precursor protein pathway regulates synapse density in the mature CNS but does not contribute to Alzheimer's disease-related pathophysiology in murine models | |
| WO2010058227A2 (en) | NOVEL USES OF VEGFxxxB | |
| Byrd et al. | Loss of Gbx2 results in neural crest cell patterning and pharyngeal arch artery defects in the mouse embryo | |
| Griffin et al. | Interplay between FGF, one-eyed pinhead, and T-box transcription factors during zebrafish posterior development | |
| Clouthier et al. | Cell-autonomous and nonautonomous actions of endothelin-A receptor signaling in craniofacial and cardiovascular development | |
| Zaffran et al. | Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations | |
| Badawi et al. | Risperidone mitigates enhanced excitatory neuronal function and repetitive behavior caused by an ASD-associated mutation of SIK1 | |
| Gau et al. | Disruption of profilin1 function suppresses developmental and pathological retinal neovascularization | |
| Iizuka-Kogo et al. | Requirement of DLG1 for cardiovascular development and tissue elongation during cochlear, enteric, and skeletal development: Possible role in convergent extension | |
| US9945845B2 (en) | Methods of screening using amphibians | |
| CN106138050B (en) | The purposes of Shh signal pathway inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191011 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |