CN104689341B - Application of the PP2A antagonists in treatment acute liver damage medicine is prepared - Google Patents
Application of the PP2A antagonists in treatment acute liver damage medicine is prepared Download PDFInfo
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- CN104689341B CN104689341B CN201310667422.XA CN201310667422A CN104689341B CN 104689341 B CN104689341 B CN 104689341B CN 201310667422 A CN201310667422 A CN 201310667422A CN 104689341 B CN104689341 B CN 104689341B
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Abstract
The invention discloses applications of the PP2A in acute liver damage drug therapy, application of the PP2A antagonists in treatment acute liver damage medicine is prepared is particularly provided.It can be used for the medicine for preparing treatment acute liver damage present invention firstly discloses the antagonist of PP2A genes and albumen, there is potential, good application prospect in liver protecting drug field.
Description
Technical field
It is prepared by the application the present invention relates to PP2A in acute liver damage drug therapy, more particularly to PP2A antagonists
Treat the purposes in acute liver damage medicine.
Background technology
Liver is an organ based on metabolic function in body, plays removing toxic substances, and deoxidation stores liver glycogen, secretion
Property protein the effect such as synthesis.The blood for having general 70% in vivo flows through liver, therefore liver is oral class medicine thing, alcohol
Deng the exogenous material through intestinal absorption must through place so that liver turn into the device extremely sensitive to chemical lesion
Official.
Diseases range is very wide caused by drug induced hepatic injury, including hepatotoxicity, liver cell lesion, acute fatty disease
Become, hepatitis, cholestasia, liver property granuloma, fat hepatitis, liver cancer etc..The degree of drug induced hepatic injury also differ and
Foot.Due to the aggravation of environmental pollution, the factor such as drinking too much, in China, chemical damage number of patients is the most extensive.And liver
If damage is allowed to deteriorate development, eventually it is changed into hepatic sclerosis, or even it is liver cancer to deteriorate, and can not finally be saved.Therefore, keep away as far as possible
Chemically poisonous substance, iatrogenic medicine in contact-free life and it is excessive drink, carry out associated treatment as early as possible, for taking precautions against and treatment
Hepatic injury relevant disease is significant.
Liver is when tackling acute chemical liver injury, and the liver regeneration of performance, extracellular matrix accumulation, inflammatory cell are invaded
Profit etc. is to participate in regulation and control jointly by many cells.After acute liver damage, HSCs is upset activation after the factor
For myofibroblast like cell, HGF is secreted（HGF）Promote the regeneration of hepatic parenchymal cells, while producing a large amount of
Extracellular matrix act not only as hepatic parenchymal cells growth support, after damage obtains control, the cell of unnecessary accumulation
Epimatrix can be absorbed, and the HSCs activated can then come back to quiescent stage or apoptosis is removed, and otherwise can cause liver
Fibrosis, if uncontrolled may eventually form hepatic sclerosis, or even liver cancer.The liver cell of resting stage is upset to be increased by compensatory
Raw mode carries out one makes liver return to original size to duplication twice, and this process is also referred to as liver regeneration.And in molecular water
On flat, the mark of hepatic stellate cell activator is mainly α smooth muscle actins（α-SMA）With collagen（Collagen1）Deng gene
Up-regulation and expression.
.PP2A it is a kind of highly conserved serine/threonine phosphatase in evolution, is adjusted as expressed by PP2A genes
Control.In cell, the dynamic phosphorylation of protein is exactly based on dephosphorization with the fundamental mechanism that dephosphorylation is signal transduction, PP2A
The function of acidifying makes substrate protein inactivate or activate, so as to cause a series of changes in cell.PP2A holoenzymes are by 65-kDa
Structural subunits（A subunits）, 36-kDa catalytic subunit（C subunits）With multifarious regulation subunit（B subunits）Constitute.
The catalytic subunit among PP2A holoenzymes is knocked out（C subunits）C α subunits whole PP2A can be caused to lose it and remove phosphoric acid
The enzyme activity effect of change.Mainly and cell cycle regulating, the biochemical process such as Apoptosis is related, but studies it also through inventor by PP2A
Regulating and controlling effect can be produced to acute liver damage process.
The content of the invention
In view of this, it is an object of the invention to by building Ade-PP2Ac α/siRNA recombined adhenovirus, investigate PP2A
Expression of the gene in acute liver damage cell model, structure PP2A Gene Knock-Out Animal Models model and clinical hepatic injury patient's liver
Situation and functional activity, confirm the drug target that can PP2A treat as acute liver damage.And it is thin by acute liver damage
Born of the same parents' model and PP2A Gene Knock-Out Animal Models model detect whether its antagonist is effective medicine, and proposition utilizes Ade-
PP2Ac α/siRNA recombined adhenovirus carries out the treatment of acute liver damage, so as to improve the therapeutic effect of acute liver damage patient
And quality of life.
It is that, up to above-mentioned purpose, inventor has investigated PP2A and moved in CCL4 damages hepatocyte model, acute chemical liver injury
Thing model is expressed with patients with liver deficiency tissue detection to be changed, and liver is damaged while having investigated Ade-PP2Ac α/siRNA recombined adhenovirus
The influence of wound and on hepatocyte growth influence.As a result find PP2A in chemical liver injury and liver cirrhosis patient tissue
Middle expression quantity has notable up-regulation.Disturb PP2Ac α（PP2A catalytic subunit, determines PP2A function）Afterwards, in hepatic parenchymal cells
The cyclin related to propagation（cyclin D）Expression quantity is dramatically increased, this reparation that represent damage and degree of injury
Mitigation.Simultaneously in acute property liver damage animal model, it is found that knocking out PP2A helps to subtract reduction Collagen1 expression,
This represents the mitigation of degree of injury.Result above points out us, and Ade-PP2Ac α/siRNA recombined adhenovirus can be for
Carry out the treatment of acute liver damage.
The medicament administration mode of present invention treatment acute liver damage can be oral, particular organization's organ is applied, whole body is applied
With（For example, through skin, nasal inhalation or using suppository）Or parenteral administration（For example, intramuscular, intravenously or subcutaneously）.
Medicine of the invention is formed into injection form, such as with physiological saline or contains glucose and other assistant agents
The aqueous solution prepared by conventional method.The medicine of such as tablet and capsule etc, can be prepared by conventional method.
Injection, solution, tablet and capsule are preferably aseptically manufactured.
In the present invention, described siRNA is that corresponding target sequence is SEQ ID NO in sequence table:1 or 2
The siRNA of DNA target sequence（SEQ ID NO:3 or 4）, control group sequence is SEQ ID NO in sequence table:11 RNA sequences
Row.
.PP2Ac the Ade-PP2Ac α that α siRNA gene recombinant vectors such as the application is built/siRNA restructuring glands
Virus can combine individually or with other functional active gene recombinant expression carriers, then be aided with pharmaceutically acceptable carrier,
Hypersitization medicine to prepare treatment acute liver damage.
It is further, any in transcription and/or translation skill according to the record of the present invention and the common knowledge of this area,
The expression of PP2Ac α genes can be suppressed, the connection such as polypeptide, albumen, fusion protein that can be active individually or with other
Close, then be aided with pharmaceutically acceptable carrier, be used to prepare the medicine for the treatment of acute liver damage.
The beneficial effects of the present invention are：It can be used for present invention firstly discloses the antagonist of PP2A genes and albumen
Treatment acute liver damage is prepared, acute liver damage recovery process is favorably improved, the propagation of liver cell after enhancing is impaired improves anxious
The quality of life of property patients with liver deficiency, has potential, good application prospect in acute liver damage therapy field.
Brief description of the drawings
Fig. 1 is CCl4Damage being successfully established for hepatocyte model.CCl4After stimulation liver cell is damaged.Liver cell number
Amount is reduced.
Fig. 2 is CCl4Damage being successfully established for hepatocyte model.CCl4After stimulation liver cell quantity is reduced.
Fig. 3 shows the potency checking of Ade-PP2Ac α/siRNA recombined adhenovirus.
Fig. 4 is shown in PP2Ac alpha expression amounts in the hepatic injury cell model that CCL4 is caused and significantly risen.
Fig. 5 shows facilitation of the Ade-PP2Ac α/siRNA recombined adhenovirus to hepatocyte growth, and liver cell passes through
Compared after Ade-PP2Ac α/siRNA recombinant adenovirus toxic actions with the cyclin D expression quantity of negative control group, significantly rise.
Fig. 6 shows the expression quantity of PP2Ac α in patient with liver cirrhosis tissue apparently higher than normal structure.
Fig. 7 shows that the liver weight of PP2Ac α hepatic parenchymal cells specific knockdown mouse, liver weight weight ratio compare control group,
In the case of non-damaging（Olive groups）Two groups of liver weight weight ratios do not have difference, in CCL4Increase more after induced liver injury, reflect
More preferable restoring force.
Fig. 8 shows that PP2Ac α hepatic parenchymal cells specific knockdown mouse Collagen1 albumen and gene expression amount are notable
Less than control group.
Fig. 9 shows in HE sections that the necrosis area of PP2Ac α hepatic parenchymal cells specific knockdown mouse is substantially less than control
Group.
Embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.
Following examples are merely to illustrate the present invention rather than the limitation scope of the invention.It is unreceipted specific in embodiment
The experimental method of condition, generally according to normal condition, such as Sambrook et al. writes, Molecular Cloning:A Laboratory guide（New York：
Cold Spring Harbor Laboratory Press, 1989）Described in condition, or according to proposed by manufacturer
Condition.
Materials and methods
Material
293A cell lines are purchased from Wuhan University's cyropreservation center；LO-2 cell lines teach favour by Nanjing Normal University Liu Chang
Give；CCL4It is pure for commercially available analysis,；PP2Ac α are purchased from U.S. company BD with Collagen1 antibody；Reverse transcription reagent box
Traverse-Ace and PCR kit for fluorescence quantitative are purchased from TOYOBO companies；PP2Acα、Collagen1、cyclin D1、
18s primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd（See SEQ ID NO:5-10）；Wild type and PP2Ac
α hepatic parenchymal cells specific knockdown mouse are given by model animal research institute of Nanjing University；Tissue of patient sample is roused by Nanjing
Building hospital provides.
Cell culture
LO-2 is incubated at containing in 10% hyclone, 100U/ml penicillin, the DMEM culture mediums of 100mg/L streptomysins, in
37 DEG C, cultivate in the cell culture incubator under 5%CO2 concentration conditions.
Hepatocellular injury model is prepared
After LO-2 hepatocyte cultures 12h, supernatant is inhaled and abandoned, change nutrient solution and add CCL42mM（It is molten with DMSO in advance
Solution, DMSO final concentrations 1%）, control group, which is only added, collects culture supernatant detection GOT activity in 24 orifice plates after DMSO, effect 6h, together
Degree of injury is evaluated in Shi Jinhang cell counts.
Animal liver injury model is prepared
To 8~12 weeks big wild mouse, CCL4 was injected intraperitoneally in row respectively with PP2Ac α hepatic parenchymal cells specific knockdowns mouse
（1:5 are dissolved in olive oil, 1ul/g）, control group injection same dose olive oil, the mouse after injection raises according to SPF grades of conditions
After supporting 72 hours, put to death, completely take liver to weigh, take hepatic tissue to carry out correlation molecule Biological Detection.
Cell RNA is extracted
By for extracting RNA reagent, pipette tips and container are anticipated with 0.1% DEPC water, and high pressure is damp and hot
Sterilizing.Specific RNA extraction steps are as follows：
1）Exhaust culture medium in six orifice plates, on ice with PBS twice, add 1mL Trizol（Invitrogen）,
5min is stood on ice, and scraping cells are sucked in the EP pipes without RNase with pipette tips
2）Plus 1/5 volume chloroform（200μL）, mix, be stored at room temperature after 3-5 minutes, 12000rpm, centrifuge 15 points
Clock；
3）Upper strata aqueous phase is transferred in a new 1.5mL centrifuge tubes, isometric isopropanol is added, mixed, room temperature 10
~15 minutes, 12000rpm was centrifuged 10 minutes；
4）Abandon supernatant, plus 1mL precoolings 75% ethanol, precipitation is suspended, 7500g is centrifuged 5 minutes；
5）Draw supernatant, drying at room temperature 5~10 minutes, plus 20 μ L DEPC（Baycovin）Solution dissolves RNA, uses
Ultraviolet specrophotometer carries out that RNA is quantitative and analysis of purity.
Tissue specimen RNA is extracted
1mg tissue specimens are taken, 1ml Trizol (Invitrogen) are suspended in, are homogenized 3-5 minutes with tissue homogenizer, plus
1/5 volume phenol chloroform albumen, 12000rpm is centrifuged 10 minutes, extracts supernatant, plus isometric isopropanol, -20 degree, 20
Minute precipitation RNA, 12000rpm, are centrifuged 10 minutes, abandon supernatant, and precipitation is dissolved in 20ul DEPC (baycovin) solution, surveys
Determine concentration.
Immunoblotting assay
Cell culture sucks culture medium to when can carry out Protein Extraction, and ice-cold PBS is washed twice, adds 300ul
Lysis buffer (50.0mmol/L Tris, 150.0mmol/L NaCl, 0.1%SDS, 1.0%NP-40,1.0mmol/L
Na3VO4,2.0mmol/L NaF,100.0μg/ml PMSF,1.0μg/ml leupeptin,and1.0μg/ml
Aprotinin), ice bath 30min, was during which mixed once, scraping cells every 5 minutes, and 12000g is centrifuged 10 minutes, in collection
Clearly.（Or animal tissue is derived from, extract albumen）Protein concentration is determined using Bradford methods.50 μ g total protein is taken to carry out
SDS-PAGE gel electrophoresis are separated, and are transferred to according to a conventional method on pvdf membrane, 4 DEG C of overnight incubations of primary antibody,（antiPP2Acα1:
1000）（anti-GAPDH1:1000) secondary antibody that, is marked with AP and NBT and BCIP colour developings use BM
Chemiluminescence Western Blotting Kit (being purchased from Roche companies) testing result.
Paraffin section makes
Fixed and stayed overnight with 4% paraformaldehyde immediately after liver is in vitro.
（1）Dehydration
（2）It is transparent
Dimethylbenzene 20min × 2.
（3）Waxdip >=3 hour；
After embedding overnight, 5 μm of sections are made.
.H＆E dye
Resinene mounting；
Olympus Optical microscope photographing.
SABC
（1）Dewax aquation
（2）Remove endogenous peroxydase
Slide is placed in 3% hydrogen peroxide（Deionized water is configured）, after room temperature 10min, deionization washing 5min × 3.
（3）Antigen retrieval
1）Section is placed in sodium citrate buffer solution, Microwave method：Middle height fire 2.5min, in low fiery 1min, low fiery 7min；
2）Naturally cool to room temperature；
3）PBS washes 5min × 3.
（4）Closing
30 μ L3% BSA is added dropwise in each sample（PBS is configured）, room temperature 1h.
（5）Primary antibody is added dropwise
Confining liquid is directly drawn, primary antibody is added dropwise（anti-collagen11:100,1%BSA dilutions）, 4 DEG C overnight, and PBST is washed
5min×4。
（6）Secondary antibody is added dropwise
The IgG of horseradish peroxidase（Horse anti-rabbit 1:200,1%BSA dilutions）, PBST washes 5min × 6
（7）DAB develops the color
Nitrite ion needs Fresh, and each nitrite ion A, B, C mono- that be added dropwise is dripped in every 1mL nitrite ions, and concussion is mixed, need to during colour developing
Lucifuge is wanted, depending on developing time needs are according to the time and depth that yellow is shown under light microscope.
Reverse transcription PCR
By Tranverse-Ace reverse transcription reagent box (TOYOBO), reverse transcription is carried out using oligo-dT.Take 800ng
The synthesizing single-stranded cDNA of total serum IgE reverse transcription, flow is as follows：800ng total serum IgEs, add 1ul mol/L OligodT primer 1ul, mend
DEPC water is to 10ul.65 DEG C, react after 5min, be immediately placed on ice, add reaction buffer 4ul, 10mmol/L
DNTP2ul, 20U/ul RNase inhibitor 1ul.After 37 DEG C are reacted 5min, 1ul10U/ul reverse transcriptases are added, 42 DEG C anti-
Answer after 1h in 90 DEG C of processing 10min.
Real-time fluorescence quantitative PCR
Using real-time fluorescence quantitative PCR kit（TOYOBO）, specific system is as follows：
20ul systems, SYBR (2*):10ul, primer (up) (10*):2ul, primer (down) (10*):2ul, cDNA
Template：2ul, plus distilled water is to 20ul.
PP2Ac α real-time quantitative PCR primer sequences：
PP2Acα(Mus)forward:5’-GAACAATAGCCAGTTATTCAGG-3’
PP2Acα(Mus)reverse:5’-TAATGAGCAATGGTAAGGAGC-3’
Other primer sequences refer to sequence table
Amplification condition：
95 DEG C of pre-degenerations 5 minutes, 95 DEG C are denatured 15 seconds, and 60 DEG C are annealed 15 seconds, 72 DEG C of extensions, collect fluorescence signal 45 seconds,
Totally 40 circulations, data analysis.
Glutamic-oxalacetic transaminease（GOT）Expression is detected
Scientific ＆ technical corporation's GOT activity detection kits are built up using Nanjing, concrete operations are as follows：
Respectively in the matrix liquid 25ul for determining hole with 37 DEG C of preheatings being added in control wells, determining in hole also needs addition to treat test sample
This 5ul (control group add the normal incubation medium 5ul containing DMSO), 37 DEG C of water-baths 30 minutes, respectively determine hole with control wells
Also need to add sample to be tested 5ul (normal cultures of the control group addition containing DMSO in addition 2,4-dinitrophenylhydrazine liquid, control wells
Base 5ul), 37 DEG C of water-baths 20 minutes are determining hole with adding 0.4mol/L caustic lye of soda 250ul in control wells, gently respectively
Level is shaken 96 orifice plates and mixed, and room temperature is placed 15 minutes, 510nm wavelength, and ELIASA surveys each hole OD values, with（Absolute OD=measure hole
OD subtracts control wells OD）, standard curve is looked into, corresponding AST/GOT unit of activity is tried to achieve.
.PP2A siRNA is built：
The rule designed according to siRNA designs siRNA, and selected target sequence is as follows：
5'GCAGACAGATCACACAAGTTT-3'/5'-ACTTGTGTGATCTGTCTGCTT-3'(sense), design
PP2A sequences of small interfering RNAs is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
.Ade-PP2Ac the structure of α/siRNA recombined adhenovirus
Recombinant plasmid pATC-PP2Ac α/siRNA is after Pme I linearization for enzyme restriction, with electroporation by itself and plasmid
PAdEasy-1 cotransformation competence bacteria BJ5183 (conditions：Electric pulse 25 μ F, voltage 2.5kV, the Ω of resistance 200), carry out homologous
Restructuring.The plasmid of homologous recombination is identified with Pac I digestions, recon is transformed into Escherichia coli DH10 α with electroporation, greatly
Measure the standby transfections of Prepare restructuring adenoviral plasmid DNA.
The packaging and harvest of recombined adhenovirus
Recombinant plasmid is transfected into HEK-293A cells with calcium phosphate method.7-10 days after transfection, there is cytopathic effect
(cytopathiceffects, CPE), show as attached cell be rounded, core concentration, come off, there is virus plaque.Collect cell,
Multigelation 3 times, centrifugation is collected supernatant and saved backup in -70 DEG C.
The measure of Viral particle concentration
In 24 hole Tissue Culture Dish, when the cell growth of 4 hole 293 cultivated to 90% is converged, nutrient solution is sucked, is added
0.5mL serum-free DMEM nutrient solutions, are added dropwise to 5,10,30 and 40ul viral dilution liquid respectively（About 500 times of dilution）, it is careful mixed
It is even, it is placed in incubator containing 5%CO2 and cultivates.After 1h, the DMEM nutrient solutions of 0.5mL calf serums containing 100mL/L are carefully added into,
Mix, cultivate, observe CPE；After 3d, when CPE phenomenons occurs completely in the hole of 5ul viral dilutions liquid to be added, count cell and count
Calculate the concentration of virion.The concentration of virion=（The volume of the viral dilution liquid of cell number × 20/）× viral dilution multiple.
Cell count
1st, by tally and cover plate wiped clean, and cover plate is covered in tally.2. cell suspension is suctioned out to a little, dropwise addition
At cover plate edge, suspension is full of between cover plate and tally, stand 3min, note there should not be bubble under cover plate, can not allow
Suspension is flowed into the groove of side.3. the big lattice TCS of computing board four, line ball cell only counts left side and top.Then based on formula
Calculate：Big lattice TCS/4 × 10 of cell number/mL=tetra-4Individual/
Embodiment 1CCl4Damage the foundation of hepatocyte model
Using the experimental method of parts such as [0027-0029], [0039] and [0044], CCl has been successfully established4Damage liver
Cell model.As depicted in figs. 1 and 2, CCl4After stimulation liver cell is damaged.Liver cell quantity is reduced.As shown in figure 1,
2mmolCCl is added in LO-2 cell culture mediums4（It is previously dissolved in DMSO solution）6h is handled, it is molten that control group adds equivalent DMSO
Liquid, takes culture medium to carry out GOT detections, it was observed that CCl4 treatment group LO-2 cell damages, glutamic-oxalacetic transaminease respectively（GOT）Vigor liter
It is high；*, with control group than p ﹤ 0.05；*, with control group than p ﹤ 0.01, n >=5.As shown in Figure 2, add in LO-2 cell culture mediums
Enter 2mmolCCl4（It is previously dissolved in DMSO solution）Cell count is carried out respectively at 0h2h6h10h after processing, each time point repeats
Three times, find to add 2mmolCCl4LO-2 cell numbers 0 are substantially reduced to 6h afterwards, are slightly recovered after 10h.It these results suggest that,
It has been successfully established CCl4 damage hepatocyte models.
The potency checking of embodiment 2Ade-PP2Ac α/siRNA recombined adhenovirus
Using the experimental method of the parts such as [0027-0029] [0037-0038] and [0040-0043], Ade- is demonstrated
The potency of PP2Ac α/siRNA recombined adhenovirus.As shown in figure 3, adding Ade-PP2Ac α/siRNA in LO-2 cell culture mediums
After recombined adhenovirus (control group imposes unloaded virus), 48h, LO-2 cell RNAs, detection PP2Ac α expression quantity are extracted, it is seen that
Ade-PP2Ac α/siRNA recombined adhenovirus of synthesis is higher to PP2Ac α efficiency；*, with control group than p ﹤ 0.05；* is and right
According to group than p ﹤ 0.01, n >=5.It these results suggest that, the potency of Ade-PP2Ac α/siRNA recombined adhenovirus is high, can significantly press down
PP2Ac α processed mrna expression amount.
PP2Ac alpha expression amounts significantly rise in the hepatic injury cell model that embodiment 3CCL4 is caused
Using the experimental method of the part such as [0027-0029] and [0033], CCL have detected4The hepatic injury cell caused
PP2Ac α expression in model.As shown in figure 4, adding 2mmol CCl in LO-2 cell culture mediums4（It is previously dissolved in
DMSO solution）Respectively at 0h after processing（ctr）2h6h10h extracts cell protein, and PP2A expression quantity is detected using westernblot
Change with lesion progress, find to add CCl4LO-2 expresses PP2Ac α albumen and dramatically increased afterwards.It these results suggest that, CCL4
PP2Ac alpha expression amounts significantly rise in the hepatic injury cell model caused, and PP2Ac alpha expressions amount has positive correlation pass with hepatic injury
System, PP2Ac α are a potential target spots for treating hepatic injury.
Embodiment 4Ade-PP2Ac α/siRNA recombined adhenovirus has facilitation to hepatocyte growth
Using [0027-0029] [0037-0038] and [0040-0043] experimental method, as shown in figure 5, in LO-2
Added in cell culture medium after Ade-PP2Ac α/siRNA recombined adhenovirus (control group imposes unloaded virus), 48h, extract LO-2
Cell RNA, detects CyclinD1 expression quantity, it is found that knockout group cyclinD1 expression quantity is higher, point out more hepatic parenchymal cellses
In vegetative state, liver cell is being repaired central.*, with control group than p ﹤ 0.05；*, with control group than p ﹤ 0.01, n >=5.
Liver cell is compared after passing through Ade-PP2Ac α/siRNA recombinant adenovirus toxic actions with the cyclin D expression quantity of negative control group,
Significantly rise.It these results suggest that, suppress that after PP2A the increment of normal liver cell can be promoted, promote the reparation of liver cell,
Illustrate again and confirm that PP2A is the target spot for treating hepatic injury.
PP2Ac α expression quantity is apparently higher than normal structure in the patient with liver cirrhosis tissue of embodiment 5
Using the experimental method of parts such as [0030-0032] [0037-0038], it have detected in patient with liver cirrhosis tissue
PP2Ac α expression quantity.As shown in fig. 6, using Q-PCR detect patient with liver cirrhosis sample tissue in CTGF, Collagen1,
PP2Ac α expression quantity, finds the expression quantity of liver fibrosis marker molecule CTGF and Collagen1 in liver cirrhosis
Raise, while PP2Ac α expression quantity also has rise.*, with control group than p ﹤ 0.05；*, with control group than p ﹤ 0.01, n >=5.
Result above confirms that PP2A is the target spot for treating hepatic injury.
Embodiment 6CCL4After induced liver injury, the liver weight of PP2Ac α hepatic parenchymal cells specific knockdown mouse, liver weight body weight
Dramatically increase
Using the experimental method of parts such as [0030], CCL have detected4After induced liver injury, PP2Ac α hepatic parenchymal cellses are special
Liver weight, the liver weight body weights of different in nature knock-out mice.As shown in fig. 7, the liver of PP2Ac α hepatic parenchymal cells specific knockdown mouse
Weight, liver weight weight ratio compare control group, in the case of non-damaging（Olive groups）Two groups of liver weight weight ratios do not have difference, in CCL4
Increase more after induced liver injury, reflect more preferable restoring force.To 8~12 weeks big wild mouse and PP2Ac α hepatic parenchymal cellses
CCL is injected intraperitoneally in row to specific knockdown mouse respectively4（1:5 are dissolved in olive oil, 1ul/g）, control group injection same dose olive
Olive oil, after the mouse after injection is raised 72 hours according to SPF grades of conditions, weighs, puts to death, and takes out liver and weighs, and calculates liver weight body
Compare again, it is found that knockout group liver weight and liver weight weight ratio do not have significant difference in group of buying oil, in CCL4Show after induced damage
Write and be higher than control group, and legend 8 does not show that conspicuousness oedema changes, with reference to legend 5 and legend 9, illustrates liver weight weight ratio increase
React more hepatic parenchymal cellses and participate in regeneration.*, with CCl4Control group is than p ﹤ 0.05；*, with control group CCL4Than p ﹤ 0.01,
n≥5;##, compares p with Olive control groups<0.01.Result above confirms that PP2A is the target spot for treating hepatic injury, also indicates that suppression
PP2A is the effective way for treating hepatic injury.
Embodiment 7PP2Ac α hepatic parenchymal cells specific knockdown mouse Collagen1 albumen and gene expression amount are notable
Less than control group
Using the experimental method of parts such as [0030] [0034-0035], it have detected PP2Ac α hepatic parenchymal cells specificity and strike
Except mouse Collagen1 albumen and gene expression amount.As shown in figure 8, being dyed using H.E, detection knockout group and control group mice
Necrosis area size, is counted after downright bad large area using imageJ, it can be found that knocking out necrosis area after PP2Ac α is less than control
Group.Meanwhile, do not find that knockout group has with control group liver cell size and significantly sexually revise.*, with CCl4Control group is than p ﹤ 0.05；*,
With control group CCL4 than p ﹤ 0.01, n >=5.Result above confirms that PP2A is the target spot for treating hepatic injury, also indicates that suppression PP2A
It is the effective way for treating hepatic injury.
The necrosis area of embodiment 8PP2Ac α hepatic parenchymal cells specific knockdown mouse is substantially less than control group
Using the experimental method of parts such as [0030] [0034-0035], it have detected PP2Ac α hepatic parenchymal cells specificity and strike
Except the necrosis area of mouse.As shown in figure 9, detecting that knockout group expresses feelings with control group mice Collagen1 using SABC
Condition（See that A schemes）, after imageJ statistics collagen1 expression large area, it can be found that knocking out Collagen1 tables after PP2Ac α
To be less than control group up to situation（See that B schemes）, collagen1 genes are detected using Q-PCR, knockout group is after CCL4 stimulations
Collagen1 expression quantity is significantly lower than control group（See that C schemes）.*, with CCl4Control group is than p ﹤ 0.05；*, with control group CCL4
Than p ﹤ 0.01, n >=5.Result above confirms that PP2A is the target spot for treating hepatic injury, and it is treatment hepatic injury to also indicate that suppression PP2A
Effective way.
Claims (3)
- Application of the 1.PP2A antagonists in treatment acute liver damage medicine is prepared；Wherein described PP2A antagonists suppress PP2A genetic transcriptions and/or translation；It is characterized in that:Wherein described PP2A antagonists are the small interference for PP2A RNA, the coded sequence of described siRNA is：5'-GCAGACAGAUCACACAAGUTT-3' （SEQ ID NO:3）Or5'-ACUUGUGUGAUCUGUCUGCTT-3' （SEQ ID NO:4）.
- 2. the application as described in claim 1, wherein described siRNA targets following sequence：5'-GCAGACAGATCACACAAGTTT-3'（SEQ ID NO:1）Or5'-ACTTGTGTGATCTGTCTGCTT-3'（SEQ ID NO:2）.
- 3. application as claimed in claim 2, wherein eukaryotic expression of the PP2A antagonists for the expression siRNA Carrier.
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| CN106478798B (en) * | 2016-10-18 | 2019-07-16 | 南通大学 | The application of PP1 γ truncate |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300824A (en) * | 1999-12-21 | 2001-06-27 | 复旦大学 | Polypeptide-serine/threonine dehybase 37 and polynucleotide for coding this polypeptide |
| CN1364785A (en) * | 2001-01-10 | 2002-08-21 | 上海博德基因开发有限公司 | New polypeptide-phosphate 2A catalytic subunit alpha 14.63 and polynucleotide for encoding such polypeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300824A (en) * | 1999-12-21 | 2001-06-27 | 复旦大学 | Polypeptide-serine/threonine dehybase 37 and polynucleotide for coding this polypeptide |
| CN1364785A (en) * | 2001-01-10 | 2002-08-21 | 上海博德基因开发有限公司 | New polypeptide-phosphate 2A catalytic subunit alpha 14.63 and polynucleotide for encoding such polypeptide |
Non-Patent Citations (3)
| Title |
|---|
| Expression of the ErbB4 receptor causes reversal;Noriko Yumoto等;《Molecular and Cellular Biochemistry 》;20060430;第285卷(第1期);全文 * |
| PP2A在小鼠肝再生终止的研究进展;来珊珊;《中国博士学位论文全文数据库医药卫生科技辑》;20130415(第04期);第3页第三段，第52页倒数第一段，第64页倒数第1段，第67页第2段，第104页倒数第一段 * |
| RNA 干扰 PP2A-Aα 基因表达对结直肠;白雪;《中华普外科手术学杂志》;20120531;第6卷(第2期);全文 * |
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