CN108707660A - Application of the rat gene in the reagent for preparing screening drug - Google Patents
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Abstract
The invention discloses application of the rat gene in the reagent for preparing screening drug, the gene includes 36 target genes and/or circadian rhythm passageway related genes and/or serotonin energy nerve synapse passageway related genes, the drug is antidepressant, and the reagent is used to detect the expression of rat and depressive disorder related gene.
Description
Technical field
The present invention relates to animal gene engineering technology fields, are related to radix bupleuri low polarity component in rat CUMS depression models
Application.
Background technology
Depression is also known as depressive disorder, is caused by being participated in jointly by many factors, with it is permanent it is depressed be main
The mood disorder of Clinical symptoms.With the rapid development of society, the daily pressure faced of people is stepped up, incidence of depression
Rise year by year.According to World Health Organization's recent statistics, mood disorder population ratio is suffered from up to 12.8% in the whole world, and expects 2020
Year, depression will become the second largest Disease Spectrum source.In recent years, many scholars are dedicated to studying this disease both at home and abroad, because of it
The features such as pathogenesis is complicated, and western medicine side reaction is larger, more and more research steering tcm fields.It summarizes existing
Number it has been found that with soothing liver-qi stagnation bupleurum Chinese and its compound preparation account for about the 1/3 of Chinese medicine antidepression medication, as Xiaoyao San,
Chai Hu Shu Gan San, Xiao Chaihu Tang etc. are widely used in clinic, and achieve good effect, it is shown that radix bupleuri is antidepressant heavy
The property wanted.
Radix bupleuri original name Asia puecon Hu, first recorded in《Sheng Nong's herbal classic》, it is classified as top grade, the medication for having more than 2000 years in China is gone through
History.《Chinese Pharmacopoeia》2010 editions are recorded radix bupleuri（Also known as：Bupleurum Chinese,Bupleurum chinense DC.）And radix bupleuri scorzoneraefolii（B. scorzonerifolium Willd.）Dry root be bupleurum Chinese（Bupleuri Radix）Plant origin.《Legendary god of farming's sheet
Grass warp》It records：" the main fever and chills of radix bupleuri, the heresy of the few positive diseases caused by external factors of fever and chills person.It calls again and ties gas in its main trusted subordinate's stomach, diet accumulation, really
With the reason of the five-element, wood can dredge soil, and the wood gas of sun is lacked for radix bupleuri Starch 1500, then the gas self energy dredging stomach soil of few sun is strongly fragrant, and it ties gas
Diet gathers self-digestion." this is not only the basic effect of radix bupleuri and the theoretical foundation of radix bupleuri " soothing liver-qi stagnation ".With radix bupleuri
For monarch drug in a prescription Chaihu prescription melancholia treatment in significant effect, this is closely related with the pharmacological action of radix bupleuri.Guo Xiao holds up etc. logical
Behaviouristics and metabolism group are crossed the study found that the Xiaoyao San being made of Bupleurum Chinese and RADIX BUPLEURI SCORZONERAEFOLII all there is significant antidepression to make
With.Results of animal shows that radix bupleuri can significantly reduce prefrontal lobe serotonin in depression model rat cerebral tissue（5-HT）
And dopamine（DA）Content, raise hippocampus brain-derived neurotrophic factor（BDNF）Expression.Radix bupleuri antidepressant effect
Active ingredient is also the emphasis of Recent study.The main component of radix bupleuri is saikoside, is resisted research shows that saikoside has
Depression effect.Saikoside can influence neurotransmitter second by inhibiting the expression of cerebral hippocampus area cholinacetyltranslase albumen
Phatidylcholine（ACH）Metabolism, reduce nerve cell apoptosis and play antidepressant effect.Separately there is the bavin it is experimentally confirmed that suitable dosage
Hu saponin(e can be effectively increased the expression of depression rat hippocampus BDNF and generate antidepressant effect.
It is transcription by the process of templated synthesis mRNA of DNA, specific cell, tissue or organ are in physiology or pathological state
Under, in all ribonucleic acid that specific time can transcribe（RNA）Summation be known as transcript profile.Transcription group is in integral level
Type, structure and function and the transcriptional control of gene whole transcript in a certain cell of the specific space-time of upper research, tissue or organ
The subject of rule.By the research of transcription group, not only it will be seen that the function element of cell, tissue or organ genome, takes off
Show its molecular chaperones, can also recognize, predict biological processes and the pathogenetic mechanism of disease.The country, Zeng Kun establish depression
Society defeat animal model, RNA-seq sequencings are carried out to the hippocampal tissue total serum IgE of mouse, difference table is carried out to the data obtained
Up to genetic analysis, the analysis of GO function classifications and related pathways analysis, filter out EGFc, Pik3r5, Pik3r6, Gprl03,133,
179 and GABAA γ, 3 genes have carried out careful analysis, and inquire into their contacting between depression, are provided for further research
The molecule resource of transcriptional level.Doctor Wang Yu establishes rat model, subordinate act and rat brain using chronic restricted stress (CRS)
Organize different parts（Hippocampus, frontal lobe and hypophysis）Transcription group level visits depression and needle thorn antidepressant effect mechanism
It begs for.Mahajan GJ etc. are to clinical 23 major depressive disorders（MDD）The hippocampus dentation of patient and 23 spiritual normal controls
It returns tissue perforation sample to be collected, using RNA-seq sequencing analysis technologies, as a result shows MDD patient's hippocampal dentate tissue
Related ERK/MAPK signal paths occur with neuroinflamation over-express, show that neuroinflamation is played in MDD to closing weight
The effect wanted.The appearance and rapid development of transcription group technology provide completely new research means for depression and present good
Good research effect.Chronic mild unpredictable stress stimulus depression（CUMS）Model as a kind of effective depression model, by
It is widely used in the basic research and drug screening of depressive disorder.
Invention content
It is an object of the invention to study the change of 36 genes in rat CUMS models to take part in rat Depressive behavior
Pathogenesis；Purposes of the radix bupleuri low polarity component in improving Depressive behavior；Radix bupleuri low polarity component is by adjusting back this 36 targets
Gene can be effectively improved rat Depressive behavior；Radix bupleuri low polarity component is by regulating and controlling circadian rhythm passageway related genes and/or 5-
Hydroxytryptamine energy nerve synapse passageway related genes, can be effectively improved rat Depressive behavior.
The purpose of the present invention can be achieved through the following technical solutions：
It is a kind of to be with the relevant rat gene discovery procedure of depressive disorder：It is analyzed by high-throughput Illumina sequencing technologies empty
White group（ZK）And model group（CM）, caused significant difference gene 291 after CUMS modelings is obtained, is analyzed, is found out by KEGG
20 notable accesses that differential gene participates in；Pass through high-throughput Illumina sequencing technologies analysis model group（CM）With radix bupleuri group
（C6）, obtain and give caused significant difference gene 322 after radix bupleuri low polarity component, analyzed by KEGG, find out difference base
Because of 20 notable accesses of participation；Blank group, model group, radix bupleuri group gene are taken into intersection, obtain differential gene, this differential gene
Take part in the depressed pathogenesis of rat CUMS.
Application of the rat gene in the reagent for preparing screening drug, the rat gene are selected from：Npas2,Prlr,
Kcnn2、Cyp7b1、Avpr1a、Cyp21a1、Nceh1、Cyp26a1、Cdkn1a、Zbtb16、Rprm、Sema3g、Mcm5、
A1bg、Ptk6、Nkx1-2、Plppr1、LOC691960、Acpp、Kif26b、Adamts19、Dtx1、St6galnac3、Hspa8、
In Zfp541, Tvp23a, Por, Crym, Plekhd1, Sh2d4b, Srms, Aif1l, Klhdc8a, Sytl5, Irs3, Slc6a6
A combination of one or more.
Preferably, the rat gene be Npas2, Prlr, Kcnn2, Cyp7b1, Avpr1a, Cyp21a1, Nceh1,
Cyp26a1、Cdkn1a、Zbtb16、Rprm、Sema3g、Mcm5、A1bg、Ptk6、Nkx1-2、Plppr1、LOC691960、
Acpp、Kif26b、Adamts19、Dtx1、St6galnac3、Hspa8、Zfp541、Tvp23a、Por、Crym、Plekhd1、
The genome of Sh2d4b, Srms, Aif1l, Klhdc8a, Sytl5, Irs3, Slc6a6 composition.
Preferably, the rat gene further includes circadian rhythm passageway related genes, the choosing of circadian rhythm passageway related genes
From：One or more of Npas2, Cry1, Bhlhe41, Nr1d1, Arntl, Per1, Per2, Per3, Rora, Rorc
Combination.
It is furthermore preferred that the circadian rhythm passageway related genes be Npas2, Cry1, Bhlhe41, Nr1d1, Arntl,
The genome of Per1, Per2, Per3, Rora, Rorc composition.
Preferably, the rat gene further includes serotonin energy nerve synapse passageway related genes, and serotonin can god
It is selected from through cynapse passageway related genes：The group of one or more of Kcnn2, Kcnd2, Prkacb, Prkaca, Adcy5
It closes.
It is furthermore preferred that the serotonin energy nerve synapse passageway related genes be Kcnn2, Kcnd2, Prkacb,
The genome of Prkaca, Adcy5 composition.
Preferably, the drug be antidepressant or adjust circadian rhythm drug, including chemicals, biologics,
Chinese medicine or Chinese medical extract.
It is furthermore preferred that the antidepressant is selected from：Iricyclic antidepressants, monoamine antioxidase inhibitor, selection
Property serotonin reuptake inhibitor, serotonin and norepinephrine reuptake inhibitors, norepinephrine and specific
Serotonin reuptake inhibitor, selective norepinephrine reuptake inhibitors.
It is furthermore preferred that the adjusting circadian rhythm drug is the drug that gets jet lag.
Most preferably, the drug is bupleurum extract.
In a preferred embodiment of the invention, the drug is the low polar extract of radix bupleuri or is detached by bupleurum extract
The substance arrived, the substance can also be the composition of monomeric compound with monomeric compound.
Preferably, the reagent is used to detect the expression of rat and depressive disorder related gene.
The antidepressant that a kind of expression screening by detecting rat gene obtains, the drug is by adjusting back rat base
Because improving Depressive behavior；Alternatively, the drug is by regulating and controlling circadian rhythm passageway related genes；Alternatively, the drug passes through
Regulating and controlling serotonin energy nerve synapse passageway related genes improves Depressive behavior.
The adjusting circadian rhythm drug that a kind of expression screening by detecting rat gene obtains, the drug are to get jet lag
Drug, the drug adjust circadian rhythm by regulating and controlling circadian rhythm passageway related genes.
A kind of low polar extract of radix bupleuri, is obtained by following preparation method：
（1）Radix bupleuri soaked in solvent；（2）Effective component extracting；（3）It is concentrated into medicinal extract；（4）Extraction；（5）Concentration, it is dry.
Preferably, the step（1）In radix bupleuri be selected from radix bupleuri medicine materical crude slice or Chinese Thorowax Root, it is furthermore preferred that radix bupleuri be selected from bavin
Hu medicine materical crude slice.
Preferably, the step（1）In solvent be selected from：N, N- dimethylformamide, acetic acid, dichloromethane, methanol,
Ethyl alcohol, isopropanol, the tert-butyl alcohol, acetonitrile or acetone；It is furthermore preferred that the solvent is selected from：80%-95% ethanol solutions；Most preferably
, the solution is selected from：95% ethanol solution.
Preferably, the step（1）In soaking time be 9-16 h；It is furthermore preferred that soaking time is 12-15 h；Most
Preferably, soaking time is 12 h.
Preferably, the step（2）In extracting method be selected from：Solvent extraction method, decocting method, circumfluence method, surpasses percolation
Sound extraction method, steam distillation, sublimed method, resin adsorption partition method, gel chromatography separation method；It is furthermore preferred that the extraction
Method is selected from：Circumfluence method.
Preferably, the step（4）In extraction mode be selected from ultrasonic extraction, extractant be selected from petroleum ether.
Preferably, the step（5）In drying mode be selected from：Spray drying, vacuum drying, freeze-drying, near-infrared
Dry or microwave drying；It is furthermore preferred that the drying mode is selected from vacuum drying.
Preferably, the step（5）In drying temperature be 50-80 DEG C；It is furthermore preferred that the drying temperature is 60
℃。
The present invention passes through high-throughput Illumina sequencing technologies combination KEGG database analysis, it was found that participates in rat suppression
20 notable accesses of strongly fragrant pathogenesis；Specify radix bupleuri low polarity component change 20 of depression rat physiological function it is significantly logical
Road；It was found that 36 genes participate in rat depression pathogenesis, while radix bupleuri low polarity component passes through the table of 36 target genes of readjustment
Up to amount, participate in improving rat Depressive behavior；Radix bupleuri low polarity component is by regulating and controlling circadian rhythm passageway related genes and/or 5- hydroxyls
Tryptamines energy nerve synapse passageway related genes, can also be effectively improved rat Depressive behavior.
Description of the drawings
Fig. 1 blank groups are analyzed with model group significant difference gene KEGG annotations and enrichment
Fig. 2 radix bupleuri group is analyzed with model group significant difference gene KEGG annotations and enrichment
Fig. 3 expresses variation diagram with relevant 36 genes of depression in transcript profile, compared with model group,*P<0.05,**P<
0.01,***P<0.001；Compared with blank group,#P<0.05,##P<0.01,###P<0.001。
For Fig. 4 compared with model group, blank group and radix bupleuri group are total to differential expression Vean diagram
Fig. 5 radix bupleuri low polarity components regulate and control the proof diagram of circadian rhythm passageway related genes, compared with model group,*P<0.05,**
P<0.01,***P<0.001；Compared with blank group,#P<0.05,##P<0.01,###P<0.001。
Fig. 6 radix bupleuri low polarity components regulate and control the proof diagram of serotonin energy nerve synapse passageway related genes, with model group
Compare,*P<0.05,**P<0.01；Compared with blank group,#P<0.05。
Specific implementation mode
Embodiment 1 prepares the low polar extract of radix bupleuri
5 kg of radix bupleuri medicine materical crude slice, 95% ethyl alcohol impregnates 12 h, and twice, 2 h, merging filtrate, recycle ethyl alcohol to nothing to refluxing extraction every time
Alcohol taste, adds water-dispersed, is concentrated into medicinal extract, isometric petroleum ether ultrasonic extraction is then added（30 min/ times）, until extract liquor is close
It is colourless, merge petroleum ether extract, recycling design is concentrated into medicinal extract, in vacuum drying chamber（60℃）Middle drying, it is low to obtain radix bupleuri
Polar extract（150g）.
2 CUMS rat depression zooperies of embodiment
Adult male SD（SPF grades）It is limited to be purchased from Beijing dimension tonneau China experimental animal technology for rat 32, weight 200g ± 20g
Company, animal credit number SCXK (capital) 2012-0001.Animal feeding temperature is 23-27 DEG C, humidity 30% ~ 70%, naturally round the clock
Rhythm and pace of moving things illumination is tested after adapting to 1 week.32 male SD rats are randomly divided into blank group, model group, radix bupleuri group, positive drug
Group（VENLAFAXINE HCL）.In addition to blank group, other three groups of rats give modeling stimulation, including prohibit water for 24 hours, 50 DEG C of thermostimulations
5min, ultrasound 3h, folder tail 2min, constraint 3h, electric shock（36v, 10 times）, fasting overturn 12h, the swimming of 4 DEG C of ice water for 24 hours, round the clock
5min.Take the mode being administered in modeling, Therapy lasted 21 days.In addition to blank group rat prohibits water for 24 hours before being tested in syrup,
The remaining time normally raises, and does not award any stimulation；Positive drug group dosage is 0.035g/kg, and radix bupleuri group dosage is 50g/
kg；Blank group, model group give the physiological saline of equivalent.When testing completion at 21 days blood is acquired in such a way that femoral artery takes blood
Liquid, while the heart, liver, spleen, lung, kidney and hippocampal tissue are won, it is wrapped up, is frozen in -80 DEG C of refrigerators with masking foil respectively.Experiment
Each group rat body weight is weighed respectively at 0d, 7d, 14d and 21d in the process, its changes of weight is observed and records, this index can be with
Reflect the basic survival condition of tested rat, the results are shown in Table 1；ASP flooding test is carried out respectively at 0d, 7d, 14d and 21d, it should
Item test is intended to the active degree to animal subject and the curiosity to surrounding environment is probed into, as a result as shown in table 2, table 3；
Syrup preference test is carried out respectively at 0d, 7d, 14d and 21d, this index can reflect the missing shape of the pleasant sensation of tested rat
Condition, the results are shown in Table 4.
Influence of the 1 radix bupleuri low polarity component of table to CUMS rat model weight（g）（X ± SD, n=8）
Compared with blank group（*p<0.05, **P<0.01）；Compared with model group（△P<0.05, △△P<0.01）
The initial value of rat body weight statistical result showed, each group rat body weight is not significantly different, the model after 7d after modeling
Group rat body weight increases slow；At 21 days, each group rat body weight is above model group, and all has significant difference（P<
0.05）.
2 radix bupleuri low polarity component of table wears CUMS rat model levels the influence of lattice number（X ± SD, n=8）
Compared with blank group（*p<0.05, **P<0.01）；Compared with model group（△P<0.05, △△P<0.01）
Tested rat level wears lattice number the results show that when modeling 14 days, and blank group is worn lattice number with model group rats level and shown
Significant difference（P<0.05）, show CUMS rat model modelings success；At 21 days, radix bupleuri group and positive drug group rat level are worn
Lattice number is close to or higher than blank group, and significant difference is all had compared with model group（P<0.05）.
Influence of the 3 radix bupleuri low polarity component of table to the upright number of CUMS rat models（X ± SD, n=8）
Compared with blank group（*p<0.05, **P<0.01）；Compared with model group（△P<0.05, △△P<0.01）
The upright number of rat is the results show that when modeling 14 days, and the upright number of blank group rat is apparently higher than model group, and with system
Meter learns difference（P<0.05）, show CUMS rat model modelings success；When modeling 21 days, the upright number of radix bupleuri group is notable（p<
0.05）Higher than model group.
Influence of the 4 radix bupleuri low polarity component of table to CUMS rat model syrup preference rates（%）（X ± SD, n=8）
Compared with blank group（*p<0.05, **P<0.01）；Compared with model group（△P<0.05, △△P<0.01）
Rat syrup preference rate the results show that when experiment starts each group rat syrup preference rate there are no significant difference, with
The passage of time, blank group rat syrup preference rate is more steady, remaining each group rat then has different degrees of reduction；Modeling 14
It when, blank group syrup preference rate be higher than model group, and have significant difference（P<0.05）, show CUMS rat model modelings
Success；When modeling 21 days, radix bupleuri group rat syrup preference rate has significant difference close to blank group compared with model group
（P<0.05）.
3 rat liver transcript profile of embodiment is sequenced
The upper biotech inc Shanghai's style Sen Nuo of liver tissues of rats transcript profile examining order commission.
4 36 differential gene expression screenings of embodiment
By transcription group model group compared with blank group result, amount to 291 significant difference genes；By transcription group model group with
Radix bupleuri group result is compared, and 322 significant difference genes are amounted to；The two significant difference gene takes intersection, amounts to 36 genes.This 36
The change of a gene takes part in the pathogenesis of rat Depressive behavior；And radix bupleuri low polarity component is by adjusting back 36 target genes
Expression, improve rat Depressive behavior.
Embodiment 5 takes verification of the qPCR technologies to circadian rhythm access
Blank group, model group, radix bupleuri group rat liver total serum IgE are extracted respectively, to participating in the upstream and downstream gene of circadian rhythm access
It takes qPCR technologies to be verified, the Ct values of qPCR is obtained according to standard curve, and be converted into relative quantification data, gene expression
Using 2-ΔΔCTMethod, with mean ± standard deviation（Mean±SD）It indicates.
Embodiment 6 takes verification of the qPCR technologies to serotonin energy nerve synapse access
Blank group, model group, radix bupleuri group rat liver total serum IgE are extracted respectively, to participating in serotonin energy nerve synapse access
Upstream and downstream gene takes qPCR technologies to be verified, and the Ct values of qPCR are obtained according to standard curve, and is converted into relative quantification number
According to gene expression uses 2-ΔΔCTMethod, with mean ± standard deviation（Mean±SD）It indicates.
The above specific implementation mode only schematically illustrates the content of present invention, does not represent the limitation of the content of present invention.Ability
Field technique personnel are it is conceivable that concrete structure can have other versions in the present invention.
Claims (10)
1. application of the rat gene in the reagent for preparing screening drug, the rat gene are selected from：Npas2,Prlr,
Kcnn2、Cyp7b1、Avpr1a、Cyp21a1、Nceh1、Cyp26a1、Cdkn1a、Zbtb16、Rprm、Sema3g、Mcm5、
A1bg、Ptk6、Nkx1-2、Plppr1、LOC691960、Acpp、Kif26b、Adamts19、Dtx1、St6galnac3、Hspa8、
In Zfp541, Tvp23a, Por, Crym, Plekhd1, Sh2d4b, Srms, Aif1l, Klhdc8a, Sytl5, Irs3, Slc6a6
A combination of one or more.
2. application according to claim 1, which is characterized in that the rat gene further includes circadian rhythm access dependency basis
Cause, circadian rhythm passageway related genes are selected from：Cry1,Bhlhe41,Nr1d1,Arntl,Per1,Per2,Per3,Rora,Rorc
One or more of combination.
3. application according to claim 1, which is characterized in that the rat gene further includes serotonin energy nerve synapse
Passageway related genes, serotonin energy nerve synapse passageway related genes are selected from：Kcnn2,Kcnd2,Prkacb,Prkaca,
The combination of one or more of Adcy5.
4. application according to claim 1, which is characterized in that the drug is antidepressant.
5. application according to claim 2, which is characterized in that the drug is to adjust circadian rhythm drug.
6. application according to claim 5, which is characterized in that the adjusting circadian rhythm drug is the drug that gets jet lag.
7. application according to claim 1, which is characterized in that the reagent is used to detect the table of the rat gene
It reaches.
8. a kind of low polar extract of radix bupleuri, is obtained by following preparation method：
（1）Radix bupleuri soaked in solvent；（2）Effective component extracting；（3）It is concentrated into medicinal extract；（4）Extraction；（5）Concentration, it is dry.
9. the antidepressant that a kind of expression screening by detecting rat gene obtains, the drug is by adjusting back claim
Rat gene described in 1 improves Depressive behavior；Alternatively, the drug is by regulating and controlling the circadian rhythm access described in claim 2
Related gene；Alternatively, the drug is by regulating and controlling the serotonin energy nerve synapse passageway related genes described in claim 3
Improve Depressive behavior.
10. the adjusting circadian rhythm drug that a kind of expression screening by detecting rat gene obtains, the drug are to get jet lag
Drug, the drug adjust circadian rhythm by regulating and controlling the circadian rhythm passageway related genes described in claim 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109821009A (en) * | 2019-01-22 | 2019-05-31 | 南京医科大学 | The medical usage of axon guidance cue recombinant protein Semaphorin3G |
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| CN116098123A (en) * | 2022-11-16 | 2023-05-12 | 山东大学 | Application of Per2 gene in preparing diphasic disorder animal model |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109821009A (en) * | 2019-01-22 | 2019-05-31 | 南京医科大学 | The medical usage of axon guidance cue recombinant protein Semaphorin3G |
| CN110250108A (en) * | 2019-05-16 | 2019-09-20 | 苏州大学 | RPRM gene knock-out mice model and its construction method and application |
| CN110250108B (en) * | 2019-05-16 | 2021-10-15 | 苏州大学 | RPRM gene knockout mouse model and construction method and application thereof |
| CN110305956A (en) * | 2019-08-09 | 2019-10-08 | 北京大学 | Influence main effect label and its application of Depressive behavior or antidepression behavior |
| CN116098123A (en) * | 2022-11-16 | 2023-05-12 | 山东大学 | Application of Per2 gene in preparing diphasic disorder animal model |
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