CN103667284B - The siRNA of a kind of special suppression COTL1 genetic expression and application thereof - Google Patents
The siRNA of a kind of special suppression COTL1 genetic expression and application thereof Download PDFInfo
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Abstract
The invention discloses siRNA and the application thereof of a kind of special suppression COTL1 genetic expression.A kind of composition suppressing COTL1 genetic expression provided by the invention, is made up of siRNA254 and siRNA395; The double-stranded RNA of described siRNA395 for being made up of the RNA shown in sequence 2 in the RNA shown in sequence in sequence table 1 and sequence table; The double-stranded RNA of described siRNA254 for being made up of the RNA shown in sequence 4 in the RNA shown in sequence in sequence table 3 and sequence table.Experiment of the present invention proves, the invention provides a kind ofly to suppress the composition of COTL1 genetic expression by differential high efficient, and it is two kinds of siRNA mixtures.Said composition can be applied on the medicine of non-alcohol fatty liver study of incident mechanism and treatment non-alcohol fatty liver.
Description
Technical field
The present invention relates to biological technical field, particularly relate to siRNA and the application thereof of a kind of special suppression COTL1 genetic expression.
Background technology
RNA disturbs (RNAinterference, RNAi) be a kind of upper conservative defense mechanism resisting transgenosis or adventitious viruses infringement of evolving, refer to endogenous or the exogenous double-stranded RNA (double-strandedRNA that there is homologous complementary sequence with the transcription product mRNA of target gene, dsRNA) this mRNA of degrade specifically in cell, thus cause the process that specific gene is effectively closed, it is a kind of sequence-specific PTGS (post-transcriptiona1genesilencing, PTGS).RNAi technology has been successfully applied to the research of multiple biological gene function.Build siRNA for goal gene, utilize RNAi technology to close the expression of this gene, can the function of analyzing gene according to the change of phenotype.Gene Knockout needs the long period permanently to close the expression of certain gene, and RNAi technology then in 1 week, even can controllably close 10 genes in 2 days, and therefore RNAi can must be used for modeling and follow-up Mechanism Study more flexible, fast.Take this, RNAi technology has been widely used in the research of disease incidence mechanism.
The siRNA of naked siRNA(unmodified) easily to degrade, its transformation period is short, and be difficult to picked-up, the shortage of targeting brings out Mammals innate immune reaction, and severe patient can cause cell and experimental animal death, therefore range of application is wideless.And the siRNA of chemically modified can make it have good thermodynamic stability, cell-penetrating power, target gene silencing activity and drug metabolism feature, there is incomparable advantage.
In recent years, along with the change of people's living habit and dietary structure, the morbidity of non-alcohol fatty liver (nonalcoholicfattyliverdisease, NAFLD) increases day by day, and China reaches 15.35%, and in constantly raising trend; In western countries especially up to 20-25%, become first hepatopathy; Closely related with metabolic diseases such as obesity, diabetes, hyperlipidaemias.According to pathological change and clinical manifestation, the natural history of NAFLD can be divided into pure NAFLD and nonalcoholic fatty liver disease (nonalcoholicsteatohepatitis, NASH).Generally speaking, pure NAFLD is a kind of benign disease, but NASH can be in progress gradually as the End-stage liver disease such as liver cirrhosis, liver cancer.Retrospective study finds, in 13 years, patient NASH of about 41% can develop into hepatic fibrosis, and patient's NASH progress of 5.4% is End-stage liver disease even liver cancer.Current NASH has become the most important inducement of cryptogenic cirrhosis.In addition, increase because fat becomes the susceptibility of liver to a lot of medicine and poisonous substance, add clinical application risk; Moreover NAFLD promotes the development of diabetes, coronary heart disease etc. by aggravation organism metabolic disorder, cause the occurred frequently of metabolism syndrome related neoplasms and cardiovascular event; If fat becomes liver as donor, liver Primary gastrointrestinal lymphoma after transplanting can be caused and cause graft failure.Therefore strengthen NAFLD study of incident mechanism and find that new means of prevention is extremely urgent.
The remarkable homology of COTL1 and coactosin, so gain the name.Coactosin is a kind of F-actin associated proteins in dictyostelium discoideum source.Similarly, COTL1 and F-actin stoichiometrically combines than the mode for 1:2, but closes not effect to the aggregation disaggregation of actin.COTL1 can go up mediation with 1:1 in conjunction with ALOX5, COTL1 and modify ALOX5 activity under non-calcium relies on, and stablizes ALOX5 molecule.ALOX5 is the rate-limiting enzyme that conversion of arachidonic acid (Arachidonicacid) generates leukotriene (Leukotriene, LT) class biologically active substance, plays an important role in cell LTs synthesizes.LTs is relevant with inflammation and allergic disorder.Now more existing research report COTL1 is relevant to some diseases associated with inflammation.And inflammation can tolerate thus cause the generation of NAFLD by mediate insulin.Have been reported forage feed ob/ob mouse containing ALOX5 inhibitor after 2 weeks, its liver fat range degree compared with normal diet ob/ob mouse alleviates.But there is not yet the research of COTL1 and NAFLD relation.
Summary of the invention
An object of the present invention is to provide a kind of composition suppressing COTL1 genetic expression.
Composition provided by the invention, is made up of siRNA254 and siRNA395;
The double-stranded RNA of described siRNA395 for being made up of the RNA shown in sequence 2 in the RNA shown in sequence in sequence table 1 and sequence table;
The double-stranded RNA of described siRNA254 for being made up of the RNA shown in sequence 4 in the RNA shown in sequence in sequence table 3 and sequence table.
Above-mentioned siRNA395 is specially and the RNA annealing in the RNA shown in sequence in sequence table 1 and sequence table shown in sequence 2 is formed; Above-mentioned siRNA254 is specially and the RNA annealing in the RNA shown in sequence in sequence table 3 and sequence table shown in sequence 4 is formed.
In above-mentioned composition, in described siRNA254 and described siRNA395, in each RNA, 2 ' hydroxyl of the ribose of pyrimidine nucleotide is methoxyl group 2 ' hydroxyl.
RNA after the modification obtained after the present invention not only protects siRNA also to protect to be modified by siRNA.Chemically modified is carried out to the siRNA of synthesis, the stability of siRNA can be increased, effectively suppress the expression of goal gene simultaneously.The chemically modified of siRNA mainly contains three classes: phosphate backbones is modified, ribose is modified and base modification.Wherein, ribose is modified is the most important mode of siRNA chemically modified.Resisting the ability of nuclease hydrolysis for improving siRNA in vivo, in the present invention, 2 '-O-methyl (2 '-OMe) being carried out to the nucleotide sequence of siRNA and modifying.This modification mode of 2 '-O-methyl can strengthen its stability in serum, reduces immunostimulation response intensity.Specific as follows: the 2 ' hydroxyl modifying the ribose of the pyrimidine nucleotide in siRNA with methoxyl group, RNA after the modification obtained.
In above-mentioned composition, the mass ratio of described siRNA254 and described siRNA395 is 1:1.
In above-mentioned composition, described siRNA254 and described siRNA395 is independent packaging.
In above-mentioned composition, the nucleotides sequence of described COTL1 gene is classified as the sequence 7 in sequence table.Above-mentioned COTL1 gene source is in mouse.
In above-mentioned composition, the mode that described composition uses is: 3 packaged amounts, described every packaged amount is siRNA395 described in siRNA254 described in 40 μ g and 40 μ g.
The use object of above-mentioned Packing Unit is mouse, and use-pattern is the composition of the packaged amount of every 5d Hydrodynamic injection 1, injects 3 times altogether.
Another object of the present invention is to provide one and prevents and/or treats non-alcohol fatty liver product.
Product provided by the invention, its activeconstituents is following 1) or 2):
1) above-mentioned composition;
2) be made up of the solution 1 containing described siRNA254 and the solution 2 containing described siRNA395;
The described solution 1 containing described siRNA254 is that described siRNA254 is dissolved in 5%(mass percentage) solution 1 that obtains in D/W, the concentration of described siRNA254 in described solution 1 is 20 μ g/ml;
The described solution 2 containing described siRNA395 is that described siRNA395 is dissolved in 5%(mass percentage) solution 2 that obtains in D/W, the concentration of described siRNA395 in described solution 2 is 20 μ g/ml.
COTL1 gene or albumen are also the scope of protection of the invention in design, exploitation or the screening application prevented and/or treated in non-alcohol fatty liver medicine.
The material of COTL1 genetic expression is suppressed also to be the scope of protection of the invention in the preparation application prevented and/or treated in non-alcohol fatty liver product.
The composition of above-mentioned suppression COTL1 genetic expression or above-mentioned product are also the scope of protection of the invention in suppression COTL1 genetic expression or the preparation application prevented and/or treated in non-alcohol fatty liver product.
In above-mentioned application, the nucleotides sequence of described COTL1 gene is classified as the sequence 7 in sequence table; The described non-alcohol fatty liver that prevents and/or treats realizes by suppressing COTL1 genetic expression.
In above-mentioned application, described non-alcohol fatty liver is mouse non-alcohol fatty liver; Described mouse non-alcohol fatty liver is specially choline-methionine deficiency feed (MCD feed) induction to be caused;
In above-mentioned application, described in prevent and/or treat non-alcohol fatty liver and be embodied in following 1)-4) and at least one: 1) improve body weight, liver weight and/or liver anharmonic ratio; 2) TG, ALT and/or AST content in serum is reduced; 3) reduce fat in liver and drip content; 4) TG content in liver is reduced.
Experiment of the present invention proves, the invention provides a kind ofly to suppress the composition of COTL1 genetic expression by differential high efficient, and it is two kinds of siRNA mixtures.Be expelled in Mice Body, results from vivo experiments shows, COTL1siRNA can suppress COTL1 genetic expression in differential high efficient ground, reduces the deposition of the Models of Nonalcoholic Fatty Liver Disease liver tg of MCD induction, alleviates liver injury.Therefore, said composition can be applied on the medicine of non-alcohol fatty liver study of incident mechanism and preventing/treating non-alcohol fatty liver.
Accompanying drawing explanation
Fig. 1 is that Westernblotting detects normal diet and MCD diet 2 weeks C57BL/6J mouse NAFLD model liver COTL1 expression levels.
**P<0.01
Fig. 2 is that immunohistochemical methods detects normal diet and MCD diet 2 weeks C57BL/6J mouse NAFLD model liver COTL1 expression levels.
Fig. 3 is that Westernblotting detects normal diet and MCD diet 2 weeks interior Hydrodynamic injections COTL1-s3 rear C57BL/6J mouse liver COTL1 protein expression level.
**P<0.01
Fig. 4 strikes low liver COTL1 to express that body weight on MCD diet and normal diet mouse, liver are heavy, the impact of liver anharmonic ratio.Compared with MCDCOTL1-neg group,
#p<0.05, compared with chowCOTL1-neg group,
§ §p<0.01,
*p<0.05,
*p<0.01
Fig. 5 strikes the impact that low liver COTL1 expresses blood sugar, blood fat and liver function on MCD diet and normal diet mouse.
*P<0.05，
**P<0.01
Fig. 6 strikes low liver COTL1 to express impact on MCD diet and normal diet mouse liver lipid content.
**P<0.01
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Detection by quantitative in following embodiment is all tested in triplicate, results averaged.
Statistical method in following embodiment: adopt the analysis of SPSS11.5 statistical analysis software, the comparison of each sample average adopts Studentttest to analyze.
Male C 57 BL/6 J mouse in following embodiment, in 8 week age, body weight 20g ~ 22g, purchased from Beijing dimension concerted effort China Experimental Animal Center.Normal diet is purchased from Beijing dimension concerted effort China Experimental Animal Center; MCD feed purchased from MPBiomedicals company, article No. 960439.COTL1 antibody (10781-1-AP), GAPDH antibody (10494-1-AP) are purchased from ProtienTech company; Anti-rabbit IgG bis-anti-(2B-2301), Hematorylin (ZLI-9610) are purchased from Beijing company of Zhong Shan Golden Bridge; FineFIX tissue fixative solution is purchased from Milestone; Oil red O powder is purchased from Amresco(0684); Triglyceride determination test kit (E1003), RIPA lysate (C1053) are purchased from Puli's Lay company; BCA method protein determination kit (23227), chemical luminous substrate SupersignalWestPico(34078) purchased from Thermo; Serum GLU(F006), TG(F001-1), TC(F002-1), ALT(C009), AST(C010) measure test kit and build up Bioengineering Research Institute purchased from Nanjing; 5% glucose injection (500ml) is purchased from Shijiazhuang Siyao Co., Ltd.
The acquisition of the siRNA composition of embodiment 1, special suppression COTL1 genetic expression
By Shanghai JiMa pharmacy Technology Co., Ltd (factory site: No. 1,011 602, Pudong Zhangjiang Halley road, postcode: 201203) carry out designing and chemosynthesis.Specific as follows: the sequence (NM028071.3) being obtained mouse COTL1mRNA by US National Biotechnology Information center (NCBI) database, follow according to RNAi principle, design and synthesis is for the siRNA of 2 sections of 21nt of mouse COTL1 gene.Carry out BLAST comparison inspection to ensure not having homology with other genes.
No. 395 COTL1-siRNA(are called for short COTL1-s395) double stranded RNA sequences be:
5 '-GAUCCAAGUUUGCCCUCAUtt-3 ' (positive-sense strand, sequence 1);
5 '-AUGAGGGCAAACUUGGAUCtt-3 ' (antisense strand, sequence 2).
No. 254 COTL1-siRNA(are called for short COTL1-s254) double stranded RNA sequences be:
5 '-GGGUGACUUUCAGAUACGAtt-3 ' (positive-sense strand, sequence 3);
5 '-UCGUAUCUGAAAGUCACCCtt-3 ' (antisense strand, sequence 4).
Negative control COTL1-negsiRNA(is called for short COTL1-neg) double stranded RNA sequences be:
5 '-UUCUCCGAACGUGUCACGUtt-3 ' (positive-sense strand, sequence 5);
5 '-ACGUGACACGUUCGGAGAAtt-3 ' (antisense strand, sequence 6).
With ribonucleoside triphosphote (NTP) for raw material, utilize ABI3900 nucleic acid synthesizer chemosynthesis single stranded RNA respectively, finally under the condition of annealing buffer, each single stranded RNA annealing forms double-strand siRNA.
For improving siRNA stability in vivo, before each chain chemosynthesis, the methoxyl group utilizing chemical reaction the ribose of the pyrimidine nucleotide (A U) in each chain synthesis material to be carried out 2 ' hydroxyl substitutes to be modified.
The application of embodiment 2, COTL1-siRNA
One, the structure of NAFLD mouse model C57BL/6J and the detection of expression of COTL1 thereof
1, MCD diet builds NAFLD mouse model
1) NAFLD mouse model is built
12 male C 57 BL/6 J mouses are divided into 2 groups at random by body weight randomized blocks, often organize each 6:
Chow group: raise 2 weeks with normal diet; MCD group: MCD diet 2 weeks.
Experiment mice is raised in Beijing Proteome Research Center's Animal House.Feeding environment condition: temperature 20-22 DEG C, humidity 50-55%, each 12 hours of light and shade.Experiment mice can ad lib and drinking-water, and 3 cages are raised.
All mouse are tested after raising one week by normal diet adaptability.
2) COTL1 expression in NAFLD mouse model liver
(1) sample collecting
At each time point, experiment mice is carried out to fasting overnight, can't help water, pluck eyeball next day and get blood and obtain cervical dislocation after whole blood sample and put to death mouse.By blood sample 4 DEG C of hold over night, then use the centrifugal 10min of whizzer 3000rpm, take out supernatant and be serum.Part serum is used for GLU, TG, TC, ALT, AST and measures, and remainder is in-80 DEG C of freezen protective.Cervical dislocation dissects mouse after putting to death mouse immediately, and take out liver, at liver maximum blde pitch edge, 5mm place cuts small pieces hepatic tissue, fixes, in order to the use of pathology detection with FineFIX.Residue tissue is placed in liquid nitrogen and saves backup after rinsing by 4 DEG C of 1 × PBS solution.
(2) liver organization total protein extraction
Freezing liver is put into the mortar grind into powder of Liquid nitrogen precooler, add 0.8mL protein lysate (30mMTris, 7Murea according to 0.1g tissue, 2Mthiourea, 4%CHAPS, adds 1%DTT and 20 μ Lcocktail temporarily) ratio carries out the extraction of liver organization whole protein, abundant vortex mixing.With the condition ice-bath ultrasonic 60s of " pulseon0.2s; pulseoff1.0s; amplitude 21% ", mixing 30min is rotated at normal temperatures with rotary mixer, normal temperature 40000g centrifugal 60min reject lower floor's insoluble substance and upper strata lipid, middle layer clarified liq is liver organization protein solution, takes a morsel and preserves on ice, adopt Bradford method protein quantification test kit to carry out determining the protein quantity, all the other be placed in-80 DEG C frozen.
(3) Westernblotting detection liver COTL1 represents level
Add LB in each tissue sample and be made into the identical 1 × LB protein solution of protein concentration, get 30 μ g loadings.Before loading after 95 DEG C of heat denatured 5min the centrifugal 5min of 12000g, get supernatant loading, the SDS-PAGE electrophoresis of row 12%, after protein is fully separated, transfer on NC film, transferring film condition is 100mA4h or 100V80min, 1h is closed by the confining liquid room temperature containing 5% skim-milk TBST, take out film TBST and clean one time, film is cut off at suitable molecular weight place, dilute with COTL1(1:2505% skim-milk TBST respectively) and GAPDH(1:30005% skim-milk TBST dilute) corresponding primary antibodie room temperature (25 DEG C) hatches 2h or room temperature 2h and adds 4 DEG C and spend the night and add room temperature 1h, TBST washes film 3min × 5 time, add the two anti-incubated at room 1h that corresponding HRP marks again, TBST washes film 3min × 5 time, film and chemical illuminating reagent are hatched 5min and carries out color reaction, in darkroom, press X film to expose, development, fixing, scan film.Semi-quantitative analysis is carried out with QuantityOne software.
As shown in Figure 1, A is the Westernblotting figure of COTL1 expression level in different treatment group liver to result; B is that Westernblotting detects COTL1 expression level sxemiquantitative histogram; The above results can be found out, compared with normal diet control mouse (Chow), and in the NAFLD mouse model liver organization of MCD diet induced, COTL1 expresses and significantly raises (P<0.05).
(4) immunohistochemical methods detection liver COTL1 represents level
After liver fixes nearly 24 hours, liver is carried out routine dehydration, paraffin embedding, paraffin section (thick 4 μm), 60 DEG C of oven for baking 5h, cooling 10min, puts into dimethylbenzene (I) 15min, dimethylbenzene (II) 15min dewaxes; 100% ethanol (I) 10min, 100% ethanol (II) 10min, 90% ethanol (I) 10min, 90% ethanol (II) 10min, 80% ethanol 10min, washing more than 10min gradient aquation; 3%H
2o
2methanol solution room temperature lucifuge hatch 20min to eliminate endogenous peroxidase activity; 1 × PBS will cut into slices as 0.01mol/L citrate buffer (pH6.0) after embathing 5min × 3 time, microwave heating to 98 DEG C, repair 30min, cool in room temperature; 1 × PBS embathes 5min × 3 time, and drip the PBST solution of 3%BSA, room temperature closes 20min; Get rid of serum deprivation, drip COTL1 primary antibodie (the PBST dilution of 1:1003%BSA), 4 DEG C are spent the night, and hatch as negative control using the PBST solution of 3%BSA; 1 × PBS embathes 5min × 3 time, drips goat anti-rabbit IgG antibody-HRP polymer, incubated at room 30min; 1 × PBS embathes 5min × 3 time, and control colour developing under DAB mirror, with specificity, background is substantially not painted is as the criterion, and immediately water termination reaction is put in section after colour developing; Hematorylin redyes core, and tap water embathes and returns indigo plant; Put into 80% ethanol 1min, 100% ethanol 2min gradient alcohol dehydration, the transparent 5min of dimethylbenzene, neutral gum sealing.
Amplify 400 times of observationss as shown in Figure 2, brown particle calmness place is COTL1 positive cell, has brown particle calm in Hepatic nonparenchymal cell.Compared with normal diet control mouse (Chow), the middle brown particle of NAFLD mouse model liver organization (MCD) of MCD diet induced is more and dyeing is darker.
Above-mentioned Westernblotting and ImmunohistochemistryResults Results all show compared with normal diet control mouse, illustrate that MCD diet can build NAFLD mouse model in 2 weeks, and COTL1 up-regulated in the NAFLD mouse model liver organization of MCD diet induced.
Two, the NAFLD Establishment of mouse model that low liver COTL1 expresses is struck in COTL1-siRNA body
24 male C 57 BL/6 J mouses, are divided into 4 groups at random by body weight randomized blocks, often organize each 6:
Normal group (ChowCOTL1-neg): raise 2 weeks with normal diet, Hydrodynamic injection 2.0mL contains 5% glucose of 80 μ g negative control COTL1-neg simultaneously;
Normal experimental group (ChowCOTL1-s): raise 2 weeks with normal diet, Hydrodynamic injection 2.0mL contains 5% glucose of 40 μ gCOTL1-s254 and 40 μ gCOTL1-s395 simultaneously;
MCD control group (MCDCOTL1-neg): raise 2 weeks with MCD, Hydrodynamic injection 2.0mL contains 5% glucose of 80 μ g negative control COTL1-neg simultaneously;
MCD experimental group (MCDCOTL1-s): raise 2 weeks with MCD, Hydrodynamic injection 2.0mL contains 5% glucose of 40 μ gCOTL1-s254 and 40 μ gCOTL1-s395 simultaneously.
Above-mentioned Hydrodynamic injection is every 5d once, totally 3 times, within the 4th day after the 3rd injection, puts to death mouse, carries out sample collecting.
Before experiment starts, all mouse normal diet adaptability is raised one week.
Hydrodynamic injection requires solution to carry out short period of time large bolus injection from mouse tail vein, and time controling is at 5-8s, and solution dosage is close to 8% of Mouse Weight.
Three, the NAFLD mouse model that low liver COTL1 expresses is struck in detection bodies
The each group of mouse of above-mentioned two is detected as follows:
1, Westernblotting detects COTL1 expression level
(1) sample collecting: with above-mentioned one 1 2) in (1) identical;
(2) liver organization total protein extraction: with above-mentioned one 1 2) in (2) identical;
(3) Westernblotting detects liver COTL1 and represents level: with above-mentioned one 1 2) in (3) identical;
As shown in Figure 3, A is the Westernblotting figure of COTL1 expression level in different treatment group liver to result; B is that Westernblotting detects COTL1 expression level sxemiquantitative histogram.NC is the mouse liver protein sample that Hydrodynamic injection contains 5% glucose solution of negative control COTL1-neg80 μ g; S is the mouse liver protein sample that Hydrodynamic injection contains 5% glucose solution of COTL1-s25440 μ g and COTL1-s39540 μ g.Spleen represents that Normal group (ChowCOTL1-neg) expresses the positive control of COTL1.GAPDH is internal reference, experiment repetition 3 times.
**P<0.01。Can find out, the normal diet and the MCD diet mouse liver COTL1 expression level that accept Hydrodynamic injection COTL1-s are all remarkable in Hydrodynamic injection COTL1-neg(P<0.01) mouse; MCD diet mouse accepts COTL1 expression level similar to the normal diet mouse of Hydrodynamic injection COTL1-neg (P>0.05) after Hydrodynamic injection COTL1-s.
2, the NAFLD mouse model Phenotypic examination that low liver COTL1 expresses is struck
1) sample collecting
At each time point, each group of experiment mice is carried out to fasting overnight, can't help water, pluck eyeball next day and get blood and obtain cervical dislocation after whole blood sample and put to death mouse.By blood sample 4 DEG C of hold over night, then use the centrifugal 10min of whizzer 3000rpm, take out supernatant and be serum.Part serum is used for GLU, TG, TC, ALT, AST and measures, and remainder is in-80 DEG C of freezen protective.Cervical dislocation dissects mouse after putting to death mouse immediately, and take out liver, at liver maximum blde pitch edge, 5mm place cuts small pieces hepatic tissue, fixes, in order to the use of pathology detection with FineFIX.It is quantitative that partial homogenate carries out liver TG, and partial row OCT embeds, and in order to cooking frozen section, carries out histology oil red O stain as early as possible.Residue tissue is placed in liquid nitrogen and saves backup after rinsing by 4 DEG C of 1 × PBS solution.
2) generalized case record
Record weekly the body weight of mouse, food-intake, observe mouse fur, active situation, the mental status etc., record mouse liver and calculating liver anharmonic ratio.
As shown in Figure 4 A, each treatment group Mouse Weight changes broken line graph to the body weight result of mouse, compared with MCDCOTL1-neg group, and #P<0.05, compared with ChowCOTL1-neg group, § § P<0.01;
Normal group (ChowCOTL1-neg) is respectively 23.3 ± 1.1g, 23.4 ± 1.2g, 24.4 ± 1.6g in the normal diet body weight of the 0th, 1,2 week of feeding;
Normal experimental group (ChowCOTL1-s) is respectively 23.1 ± 0.7g, 22.8 ± 0.7g, 23.8 ± 0.8g in the normal diet body weight of the 0th, 1,2 week of feeding;
MCD control group (MCDCOTL1-neg) is respectively 23.6 ± 1.4g, 20.4 ± 1.3g, 18.4 ± 0.9g in the MCD feed body weight of the 0th, 1,2 week of feeding;
MCD experimental group (MCDCOTL1-s) is respectively 23.1 ± 1.1g, 21.3 ± 2.0g, 20.4 ± 1.3g in the MCD feed body weight of the 0th, 1,2 week of feeding;
After 2 weeks, as shown in Figure 4 B, B is each treatment group Mouse Liver weight to the liver weight result of each group mouse;
*p<0.05,
*p<0.01.
Normal group (ChowCOTL1-neg) liver weight of normal diet after 2 weeks of feeding is 0.92 ± 0.06g;
Normal experimental group (ChowCOTL1-s) liver weight of normal diet after 2 weeks of feeding is 0.90 ± 0.04g;
MCD control group (MCDCOTL1-neg) liver weight of MCD feed after 2 weeks of feeding is 0.62 ± 0.05g;
MCD experimental group (MCDCOTL1-s) liver weight of MCD feed after 2 weeks of feeding is 0.70 ± 0.04g;
After 2 weeks, as shown in Figure 4 C, C is each treatment group Mouse Liver anharmonic ratio histogram to liver anharmonic ratio (liver weight/body weight) result of each group mouse;
*p<0.05,
*p<0.01.
Normal group (ChowCOTL1-neg) the liver anharmonic ratio of normal diet after 2 weeks of feeding is 0.039 ± 0.004;
Normal experimental group (ChowCOTL1-s) the liver anharmonic ratio of normal diet after 2 weeks of feeding is 0.039 ± 0.002;
MCD control group (MCDCOTL1-neg) the liver anharmonic ratio of MCD feed after 2 weeks of feeding is 0.036 ± 0.004;
MCD experimental group (MCDCOTL1-s) the liver anharmonic ratio of MCD feed after 2 weeks of feeding is 0.040 ± 0.003;
As can be seen from the above results, in modeling process, MCD diet COTL1 interference group mouse dies unexpectedly one (MCDCOTL1-s5 mouse) when second time Hydrodynamic injection, be MCD diet equally, Hydrodynamic injection COTL1-s is better than the injection COTL1-neg mouse mental status, hair is glossy, food ration increases, activity increases.After normal diet mouse tail vein high-pressure injection, continued weight increases, and after MCD diet mouse tail vein high-pressure injection, continued weight alleviates, and significantly lower than the normal diet mouse same period (P<0.01); The MCD diet 2 weeks Mouse Weights continuing the liver COTL1 striking low abnormal up-regulated expression comparatively do not strike low MCD diet mouse the same period significantly to be increased (P<0.05), but not yet returns to normal diet Hydrodynamic injection COTL1-neg mouse level (P<0.01) (Fig. 4 A).
After Hydrodynamic injection COTL1-neg, MCD diet Mouse Liver heavy compared with normal diet mouse significantly alleviates (P<0.01); The MCD diet 2 weeks Mouse Liver weights continuing the liver COTL1 striking low abnormal up-regulated expression comparatively do not strike low MCD diet mouse the same period significantly to be increased (P<0.05), but not yet returns to normal diet Hydrodynamic injection COTL1-neg mouse level (P<0.01) (Fig. 4 B).
After Hydrodynamic injection COTL1-neg, MCD diet Mouse Liver anharmonic ratio compared with normal diet mouse reduces, but does not have significant difference; The MCD diet 2 weeks Mouse Liver anharmonic ratioes continuing the liver COTL1 striking low abnormal up-regulated expression comparatively do not strike low MCD diet mouse the same period significantly to be increased (P<0.05), and with normal diet Hydrodynamic injection COTL1-neg mouse level close (P>0.05) (Fig. 4 C).
3) Serologic detection
At different time points serum GLU(blood sugar), TC(total cholesterol), TG(triglyceride level), ALT(gpt), AST(glutamic-oxal(o)acetic transaminase) measure test kit and detect different diet mice serum GLU, TC, TG, ALT, AST content (completing detection at 307 hospitals' biochemical room 7600-110 full automatic biochemical apparatus).
Result is as shown in 5A-5E, and A-E is respectively the horizontal histogram of each treatment group mice serum GLU, TC, TG, ALT, AST.
*p<0.05,
*p<0.01 detected result;
Do not inject normal diet group (Chow), do not inject MCD feed group (MCD), the GLU content of Normal group (ChowCOTL1-neg), normal experimental group (ChowCOTL1-s), MCD control group (MCDCOTL1-neg), MCD experimental group (MCDCOTL1-s) is respectively 7.64 ± 1.27nmol/L, 4.42 ± 1.25nmol/L, 8.39 ± 1.69nmol/L, 9.00 ± 1.31nmol/L, 4.14 ± 0.92nmol/L, 4.80 ± 0.15nmol/L;
Do not inject normal diet group (Chow), do not inject MCD feed group (MCD), the TC content of Normal group (ChowCOTL1-neg), normal experimental group (ChowCOTL1-s), MCD control group (MCDCOTL1-neg), MCD experimental group (MCDCOTL1-s) is respectively 2.65 ± 0.28nmol/L, 1.42 ± 0.11nmol/L, 2.18 ± 0.38nmol/L, 2.24 ± 0.06nmol/L, 1.30 ± 0.10nmol/L, 1.33 ± 0.08nmol/L;
Do not inject normal diet group (Chow), do not inject MCD feed group (MCD), the TG content of Normal group (ChowCOTL1-neg), normal experimental group (ChowCOTL1-s), MCD control group (MCDCOTL1-neg), MCD experimental group (MCDCOTL1-s) is respectively 0.99 ± 0.17nmol/L, 0.88 ± 0.08nmol/L, 0.74 ± 0.06nmol/L, 0.90 ± 0.09nmol/L, 0.84 ± 0.07nmol/L, 0.68 ± 0.05nmol/L;
Do not inject normal diet group (Chow), do not inject MCD feed group (MCD), the ALT content of Normal group (ChowCOTL1-neg), normal experimental group (ChowCOTL1-s), MCD control group (MCDCOTL1-neg), MCD experimental group (MCDCOTL1-s) is respectively 29 ± 5U/L, 294 ± 83U/L, 32 ± 7U/L, 48 ± 10U/L, 370 ± 139U/L, 149 ± 58U/L;
Do not inject normal diet group (Chow), do not inject MCD feed group (MCD), the AST content of Normal group (ChowCOTL1-neg), normal experimental group (ChowCOTL1-s), MCD control group (MCDCOTL1-neg), MCD experimental group (MCDCOTL1-s) is respectively 107 ± 8U/L, 266 ± 50U/L, 138 ± 37U/L, 178 ± 85U/L, 288 ± 68U/L, 175 ± 47U/L;
Result shows, MCD diet mice serum GLU and TC content reduce to the nearly half (P<0.01) of normal diet, the MCD diet mouse accepting Hydrodynamic injection COTL1-neg compared with the same period normal diet mouse change similarly, strike low liver COTL1 and express the serum GLU level of MCD diet and TC level without impact (P>0.05) (Fig. 5 A, B).Similar to not accepting Hydrodynamic injection mouse, serum TG and normal diet mouse indifference (P>0.05) after MCD diet 2 weeks mouse tail vein high-pressure injection COTL1-neg, MCD diet mouse continue to strike low liver COTL1 express 2 weeks afterwards serum TG do not strike low mouse and significantly reduce (P<0.05), and with normal diet Hydrodynamic injection COTL1-neg mouse level close (P>0.05) (Fig. 5 C).MCD diet mice serum ALT and AST level are elevated to about 10 times of normal diet and 2 times (P<0.01) respectively, the MCD diet accepting Hydrodynamic injection COTL1-neg compared with the same period normal diet mouse change similarly, MCD diet mouse continues to strike low liver COTL1 and expresses 2 weeks Serum ALT and AST level are not struck low mouse and be significantly dropped by nearly half (P<0.05) afterwards, ALT level not yet returns to normal diet Hydrodynamic injection COTL1-neg mouse level (P<0.05), and AST level returns to normal diet Hydrodynamic injection COTL1-neg mouse similar level (P>0.05) (Fig. 5 D, E).
4) mouse liver lipid content is evaluated
(1) liver HE dyes
After each group of liver fixes nearly 24 hours, liver is carried out routine dehydration, paraffin embedding, paraffin section (thick 4 μm), 60 DEG C of oven for baking 5h, cooling 10min, can long-term 4 DEG C of preservations.Paraffin section is put into dimethylbenzene (I) 15min, dimethylbenzene (II) 15min dewaxes; 100% ethanol (I) 10min, 100% ethanol (II) 10min, 90% ethanol (I) 10min, 90% ethanol (II) 10min, 80% ethanol 10min, washing more than 10min gradient aquation; Hematorylin 5min contaminates core, and clear water embathes 5s, and acidic alcohol differentiation 2-3s, washes 3 times, and it is anti-blue that clear water soaks more than 15min, and Yihong 2-3min contaminates endochylema, washing 2-3 time, 80% ethanol 1min, 100% ethanol 2min, dimethylbenzene 5min, neutral gum mounting.Experiment repetition 3 times.
**P<0.01。
Amplify 400 times of observationss as shown in Figure 6A, the normal diet 2 weeks mouse (ChowCOTL1-neg) accepting Hydrodynamic injection COTL1-neg are with not accept normal diet 2 weeks mouse (Chow) pathology that venous hypertension injects similar, liver lobule clear in structure, cell rope marshalling, liver cell is without obvious pathology, and nuclear structures is clear.The MCD diet 2 weeks mouse (MCDCOTL1-neg) accepting Hydrodynamic injection COTL1-neg are with not accept MCD diet that venous hypertension injects 2 weeks mouse (MCD) similar, there is obvious steatosis in hepatic parenchymal cells, even visible inflammatory cell, without Hepatocellular ballooning.MCD diet mouse continue to strike low liver COTL1 express 2 weeks (MCDCOTL1-s) afterwards in hepatic parenchymal cells steatosis obviously reduce, to such an extent as to HE dyeing is difficult to find out fat vacuole.
(2) liver oil red O stain
Detect by sensitiveer oil red O stain method and accept MCD diet 2 weeks mouse (MCDCOTL1-neg) of Hydrodynamic injection COTL1-neg and accept MCD diet 2 weeks mouse (MCDCOTL1-s) of Hydrodynamic injection COTL1-s, specific as follows: the liver taking out OCT embedding carries out frozen section, slice thickness is 10 μm, frozen section 70% ethanol is fixed 1min, oil red O diluent dye 8min, it is clear to break up to interstitial under 60% ethanol mirror, after washing, MayerShi Hematorylin redyes core 30s, tap water returns blue 30min, glycerine mounting.To the section photomicrography after dyeing.Experiment repetition 3 times.
**P<0.01。
Amplify 400 times of observationss as shown in Figure 6B, the MCD diet accepting Hydrodynamic injection COTL1-neg has oil red O painted in mouse (MCDCOTL1-neg) hepatic parenchymal cells endochylema in 2 weeks, in circle not of uniform size, COTL1 strikes fat in low mouse (MCDCOTL1-s) each hepatic parenchymal cells, and to drip number less, form is less, and the hepatic parenchymal cells not having oil red O painted is more.
(3) liver TG assay
Measure test kit specification sheets according to tissue T G to operate.Specific as follows: every 50mg liver organization adds 1mL test kit lysate in proportion, with manual glass homogenizer Assisted Cleavage tissue, getting half lysate adopts BCA method protein quantification test kit to carry out determining the protein quantity, all the other lysates transfer to 600 μ L centrifuge tubes, 70 DEG C of heating in water bath 30min, the centrifugal 5min of room temperature 2000rpm, supernatant liquid is used for enzymatic determination.In 96 hole enzyme plates, volume shown in list in standard substance and testing sample and working fluid by specification is mixed, detect under 490nm wavelength by microplate reader after 37 DEG C of reaction 10min, also calculate protein concentration and TG concentration respectively with Excel drawing standard curve, correct TG content with every mg protein concentration.Experiment repetition 3 times.
**P<0.01。
As shown in Figure 6 C, in the liver of Normal group (ChowCOTL1-neg), normal experimental group (ChowCOTL1-s), MCD control group (MCDCOTL1-neg), MCD experimental group (MCDCOTL1-s), TG content is respectively 138 ± 26nmol/mg albumen, 250 ± 62nmol/mg albumen, 2189 ± 149nmol/mg albumen, 1396 ± 23nmol/mg albumen to result;
The above results shows, Hydrodynamic injection COTL1-negMCD diet mouse liver compared with normal diet TG content increases nearly ten times (P<0.01), continue to strike low liver COTL1 to express 2 weeks MCD diet mouse liver TG content and do not strike low mouse and reduce by about 1/3rd (P<0.01), but not yet return to Hydrodynamic injection COTL1-neg normal diet mouse liver TG level (P<0.01).
Liver HE dyes, oil red O stain and liver tg measure all promptings continue to strike low liver COTL1 express 2 weeks MCD diet mouse liver lipid contents comparatively COTL1 do not strike low person and reduce.
Claims (8)
1. prevent and/or treat a non-alcohol fatty liver product, its activeconstituents is following 1) or 2):
1) suppress the composition of COTL1 genetic expression, described composition is made up of siRNA254 and siRNA395;
The double-stranded RNA of described siRNA395 for being made up of the RNA shown in sequence 2 in the RNA shown in sequence in sequence table 1 and sequence table; The double-stranded RNA of described siRNA254 for being made up of the RNA shown in sequence 4 in the RNA shown in sequence in sequence table 3 and sequence table;
2) be made up of the solution 1 containing described siRNA254 and the solution 2 containing described siRNA395;
The described solution 1 containing described siRNA254 is described siRNA254 is dissolved in the solution 1 obtained in 5% (mass percentage) D/W, and the concentration of described siRNA254 in described solution 1 is 20 μ g/ml;
The described solution 2 containing described siRNA395 is described siRNA395 is dissolved in the solution 2 obtained in 5% (mass percentage) D/W, and the concentration of described siRNA395 in described solution 2 is 20 μ g/ml.
2. product according to claim 1, is characterized in that: in described siRNA254 and described siRNA395, in each RNA, 2 ' hydroxyl of the ribose of pyrimidine nucleotide is methoxyl group 2 ' hydroxyl.
3. product according to claim 1 and 2, is characterized in that: the mass ratio of described siRNA254 and described siRNA395 is 1:1.
4. product according to claim 1 and 2, is characterized in that: described siRNA254 and described siRNA395 is independent packaging.
5. suppress the material of COTL1 genetic expression preparing the application prevented and/or treated in non-alcohol fatty liver product;
The material of described suppression COTL1 genetic expression is the composition suppressing COTL1 genetic expression, and described composition is made up of siRNA254 and siRNA395;
The double-stranded RNA of described siRNA395 for being made up of the RNA shown in sequence 2 in the RNA shown in sequence in sequence table 1 and sequence table; The double-stranded RNA of described siRNA254 for being made up of the RNA shown in sequence 4 in the RNA shown in sequence in sequence table 3 and sequence table.
6. the arbitrary described product of claim 1-4 is preparing the application prevented and/or treated in non-alcohol fatty liver product.
7. application according to claim 6, is characterized in that: described in prevent and/or treat non-alcohol fatty liver and realize by suppressing COTL1 genetic expression.
8. the application according to claim 5,6 or 7, is characterized in that:
Described non-alcohol fatty liver is mouse non-alcohol fatty liver; Described mouse non-alcohol fatty liver is specially choline-methionine deficiency feed induction to be caused;
The described non-alcohol fatty liver that prevents and/or treats is embodied in following 1)-4) middle at least one: 1) improve body weight, liver weight and/or liver anharmonic ratio; 2) TG, ALT and/or AST content in serum is reduced; 3) reduce fat in liver and drip content; 4) TG content in liver is reduced.
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