CN101280306A - Novel use of gene DCF1 related to neural stem cell differentiation - Google Patents
Novel use of gene DCF1 related to neural stem cell differentiation Download PDFInfo
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- CN101280306A CN101280306A CNA2008100376989A CN200810037698A CN101280306A CN 101280306 A CN101280306 A CN 101280306A CN A2008100376989 A CNA2008100376989 A CN A2008100376989A CN 200810037698 A CN200810037698 A CN 200810037698A CN 101280306 A CN101280306 A CN 101280306A
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Abstract
The invention relates to a new usage of a neural stem cell differentiating related gene DCF1. The invention sieves protein ATP1B1 interacted with DCF1 through utilizing a yeast-two hybrid technology and is positioned altogether by using cell fluorescence, and the interaction is confirmed through immunity and precipitation. According to a literature report, the interaction between ATP1B1 and BACE1 exists, but BACE1 is an essential gene of the development of AD (Alzheimer disease). After DCF1 is muted through a RNAi method, the expression of ATP1B1 and BACE1 also changes remarkably the results prove that the expression of BACE1 can be adjusted and controlled through certain signal passages of DCF1, certain relation exists in the occurrence and the development of DCF1 and AD, which is possible to be a new drug effect target spot of AD treatment, the interaction of DCF1 and ATP1B1 is found, and the invention has a vital significance to a signal conducting mechanism during the understanding process of the development and the progression of AD.
Description
Technical field
The present invention relates to a kind of and the gene DCF 1 related new purposes of cell differentiation of nerve cord.
Background of invention
(neural stem cells, but NSCs) self-replacation and have multidirectional differentiation potential have important use and are worth neural stem cell in the treatment of central nervous system injury and degenerative disease.Neural stem cells transplantation to damaged part, can be divided into the specific neurocyte of body, finish specific neural function.Yet there are many technical difficult problems in neural stem cells transplantation, and the ability of wherein transplanting the back cell differentiation of nerve cord and participating in neural network reconstruction becomes the focus that people pay close attention to.The differentiation mechanism of illustrating neural stem cell is for having overcome the above problems most important theories meaning and practical value.
In neural stem cell, the dendritic cell factor (dentritic cell factor 1, DCF1) do not appear in the newspapers by the function of gene.Discover the DCF1 gene in undifferentiated neural stem cell high expression level and in the neurocyte of eventually end differentiation very low (the Tieqiao Wen of expression amount, Ping Gu and Fuxue Chen (2002) Discovery of twonovel functional genes from differentiation of neural stem cells in thestriatum of the fetal rat.Neuroscience Letters 329:101-105), infer that thus this gene may be relevant with the differentiation of neural stem cell; With rear clone and in the mouse neural stem cell this gene of overexpression, find to compare with control group, the cell axon growth of overexpression DCF1 gene is suppressed, the DCF1 gene can be kept the state of neural stem cell.And the expression of inhibition DCF1, then neural stem cell can be broken up.In order further to study the function of DCF1 gene in neural system and the molecular mechanism of effect thereof, the present invention utilizes yeast-two hybrid technique screening and the interactional albumen of DCF1, and locatees altogether with cell fluorescence, and co-immunoprecipitation confirms its interaction.And after the means discussion DCF1 silence with RNAi, AD (A Zihaimo disease) Expression of Related Genes changes.
Summary of the invention
One of the objective of the invention is to be to provide that a kind of cell differentiation of nerve cord is gene DCF 1 related is regulating Na
+, K
+Application during-ATP enzyme β 1 protein subunit is expressed.
Two of purpose of the present invention is to provide a kind of cell differentiation of nerve cord gene DCF 1 related application in the expression of regulating the gene BACE1 sick relevant with A Zihaimo.
The cDNA of DCF1 entire reading frame is cloned in the present invention from mouse brain cDNA with PCR, and it is recombined into the cloning vector pGBKT7 of yeast two-hybrid system, be built into pGBKT7-DCF1, the bait plasmid pGBKT7-DCF1 that makes up does not have toxic action to host bacterium AH109, does not have the ability of self activation reporter gene expression yet.With bait plasmid pGBKT7-DCF1 and mouse brain cDNA library plasmid cotransformation yeast AH109, screening positive clone on the auxotroph substratum, and send order-checking.The significative results that order-checking obtains is backcrossed with bait plasmid once more, gets rid of false positive, obtains ATPase, Na+/K+transporting, and beta 1 polypeptide (being abbreviated as ATP1B1) is positive.DCF1 and ATP1B1 are building up to fluorescence respectively locate carrier altogether, observe with laser confocal microscope behind the transfection C17.2 cell.DCF1 and ATP1B1 are building up to the co-immunoprecipitation carrier respectively, carry out co-immunoprecipitation.Cell fluorescence is located the interaction that has all proved DCF1 and ATP1B1 with co-immunoprecipitation altogether.According to the literature, there are interaction in ATP1B1 and BACE1, and BACE1 is the key gene of AD (A Zihaimo disease), so DCF1 may also have certain relation with AD.With the reticent DCF1 of RNAi technology, find that the expression of ATP1B1 is significantly reduced, the expression of ATP1B1 related gene B ACE1 is significantly downward modulation also.More than these results prove that DCF1 passes through the expression that certain signal path can be regulated and control BACE1, the generation development of DCF1 and AD exists certain and gets in touch, it might become the new drug target of AD treatment, and the signal transduction mechanism that the interaction partners of discovery DCF1 and ATP1B1 is understood in the AD generation evolution has great importance.
Description of drawings
(1-does not insert segmental pGBKT7 expression vector to Fig. 1 for pGBKT7 expression vector EcoRI and SalI double digestion electrophorogram; 2-inserts segmental pGBKT7 expression vector; The 3-DCF1 fragment), illustrate that the DCF1 gene has inserted in the pGBKT7 carrier
Fig. 2 xeroxs for the betagalactosidase activity filter membrane and analyzes, and wherein 1-3 classifies Yeast pGBKT7-DCF1 as, and the 4th classifies negative control as, and the 5th classifies positive control as, illustrates that AH109 (pGBKT7-DCF1) does not have betagalactosidase activity.
Fig. 3 is for detecting the expression of beta-galactosidase gene
Fig. 4 illustrates that ATP1B1 has inserted in the pDSRED2 carrier for pDSRED2 carrier EcoRI and BamHI double digestion electrophorogram (1:marker 2:pDSRED2-ATP1B1 recombinant plasmid 3:pDSRED2-C1 empty plasmid)
Fig. 5 illustrates that for fluorescence is total to location map (upper left: upper right under the green exciting light: lower-left under the red exciting light: the bright field bottom right: the picture left above and top right plot MERGE) DCF1 and ATP1B1 can present completely location altogether in tenuigenin
Fig. 6 illustrates that for pCMV-HA and pCMV-MYC carrier EcoRI and BglII double digestion electrophorogram (1:pCMV-HA-DCF1 recombinant plasmid 2:pCMV-HA empty plasmid 3:pCMV-MYC-ATP1B1 recombinant plasmid 4:pCMV-MYC empty plasmid 5:marker) DCF1 and ATP1B1 have inserted in pCMV-HA and the pCMV-MYC carrier
Fig. 7 co-immunoprecipitation figure (1.HA-DCF1 and the proteic lysis supernatant liquor of MYC-ATP1B1 protein immunization co-precipitation sample 2. blank 3.C17.2 cell expressing MYC-ATP1B1, as positive control 4.HA and MYC-ATP1B1 protein immunization co-precipitation sample, as negative control) illustrate that co-immunoprecipitation can take place for DCF1 and ATP1B1
Embodiment
Embodiment one: the structure of bait plasmid pGBKT7-DCF1 and evaluation
Utilize the upstream and downstream primer of Premier 5.0 software design DCF1, introduce the restriction enzyme site of EcoRI and SalI respectively at its upstream with in the downstream primer.It is synthetic that this gives birth to the worker to primer by Shanghai.With mouse brain cDNA is template, with synthetic upstream and downstream primer amplification DCF1 full-length cDNA, EcoR I and SalI double digestion, be connected to same in the pGBKT7 carrier of EcoR I and SalI double digestion.Enzyme is cut, and PCR identifies that order-checking is correct, sees Fig. 1.AH109 (pGBKT7-DCF1) can not grow at SD/-Trp/-His, xeroxs in the analysis at the betagalactosidase activity filter membrane, and bacterium colony keeps white in 8 hours, becomes blue, sees Fig. 2.Prompting pGBKT7-DCF1 plasmid does not have self to activate the expression of reporter gene.Can be used for follow-up experiment.
Embodiment two: mouse brain cDNA library screening
Mouse brain cDNA library plasmid behind the purifying and pGBKT7-DCF1 bait plasmid cotransformation yeast cell AH109 obtain 5.2X10 altogether from 100 SD/-Trp/-Leu (100mm)
7Individual transformant.Collect yeast and titration from flat board, its titre is 0.59X10
8Get 1.57X10
8Individual yeast is coated the SD/-Trp-Leu-His flat board, grows 1820 clones altogether; Use toothpick to choose flat board these clones, obtain 105 mono-clonals to SD/-Trp-Leu-His-Ade.Detected the expression (indigo plant is screened in vain) of these 105 mono-clonal beta-galactosidase genes with fliter-assay, the result has 20 clones and is blue (promptly expressing beta-galactosidase gene), positive clone as shown in Figure 3.From above-mentioned 20 positive colonies that obtain, extract the plasmid contain the pACT2 carrier, with its respectively electricity be transformed in the bacillus coli DH 5 alpha and increase, extract plasmid.The above-mentioned pairing clone of non-repetition segment who obtains is delivered the living worker in Shanghai to check order.Homology analysis: the result who carries out blast at NCBI show some sequences respectively with ATPsynthase F0 subunit 6, DTX3, Creatine Kinase, the high homology (99%) that has ATP1B1 etc. will contain above these segmental pACT2 carriers and bait carrier cotransformation yeast AH109 once more, backcross, the result shows that ATP1B1 still can interact with DCF1
Embodiment three: fluorescence is located the interaction of checking DCF1 and ATP1B1 altogether
Utilize Premier 5.0 software design ATPase, Na+/K+transporting, the upstream and downstream primer of beta 1polypeptide is introduced the restriction enzyme site of EcoRI and BamHI respectively at its upstream with in the downstream primer.It is synthetic that this gives birth to the worker to primer by Shanghai.With mouse brain cDNA is template, with synthetic upstream and downstream primer amplification ATPase, Na+/K+transporting, beta 1 polypeptide full-length cDNA, EcoRI and BamHI double digestion, be connected to equally in the pDSRED2-C1 carrier of EcoRI and BamHI double digestion, enzyme is cut, PCR identifies correctly, sees Fig. 4.PDSRED2-ATP1B1 and pEGFP-DCF1 (its construction process, referring to Lei Wang, Jiao Wang, YiliuWu, Jie Wu, Shuya Pang, Rong Pan, Tieqiao Wen (2008) A Novel Function of dcflDuring the Differentiation of Neural Stem Cells In vitro.Cell Mol Neurobiol).Cotransfection c17.2 neural stem cell system was observed under laser confocal microscope after 24 hours, saw Fig. 5.Behind pDSRED2-ATP1B1 and the pEGFP-DCF1 cotransfection cell, both can be positioned tenuigenin altogether, have further proved their interaction
Embodiment four: the interaction of co-immunoprecipitation checking DCF1 and ATP1B1
Utilize Premier 5.0 softwares to design DCF1 and ATPase respectively, Na+/K+transporting, the upstream and downstream primer of beta 1polypeptide is introduced the restriction enzyme site of EcoRI and BglII respectively at its upstream with in the downstream primer.With mouse brain cDNA is template, with synthetic upstream and downstream primer increase respectively DCF1 and ATPase, Na+/K+transporting, beta 1polypeptide full-length cDNA, cutting the back purifying with EcoRI and BglII enzyme reclaims, adding is through the pCMV-HA and the pCMV-MYC of identical double digestion, and 16 ℃ of connections are spent the night, and connects product transformed into escherichia coli DH5 α.After growing bacterium colony, extract plasmid according to a small amount of plasmid extraction test kit operation instructions of the vast Imtech in Beijing, EcoR I and BglII double digestion, PCR identify correct, see Fig. 6.PCMV-HA-DCF1 and pCMV-MYC-ATP1B1 cotransfection c17.2 neural stem cell system, extracting cell protein after 72 hours.Adding anti-HA antibody and proteinA carry out co-immunoprecipitation in the lysis supernatant liquor, with sample through the SDS-PAGE electrophoresis, change film, it is shown in Figure 7 to carry out western blotting. result with anti-MYC antibody again, when by anti-HA antibody deposit D CF1, ATP1B1 is simultaneously by coprecipitation, and the pCMV-HA empty carrier then can not make the ATP1B1 co-precipitation in the negative control group, and this further shows the interaction of DCF1 and ATP1B1.
Embodiment five: the variation of the reticent DCF1 research of RNAi AD genes involved
There is document to show that ATP1B1 can interact with AD related gene B ACE1, the expression of BACE1 in neural system can cause the generation of AD, conjecture DCF1 and BACE1 exist certain and get in touch, so with BACE1 after the means research DCF1 silence of RNAi, ATP1B1 change of Expression.PsiRNA-1, psiRNA-3 (upsetting sequence), psiRNA (empty carrier) (more than be this laboratory and make up in earlier stage, document [2] sees reference) spend intracellular toxin test kit purifying after, distinguish transfection c17.2 cell, transfection was distinguished the RNA of the above 3 kinds of cells of extracting after 72 hours, reverse transcription, realtimePCR detects, and detects beta actin, dcf1 respectively, ATP1B1, these 4 kinds of expression of gene of BACE1 are calculated as benchmark with beta actin, the psiRNA-1 group, the psiRNA-3 group is with respect to the gene expression amount of psiRNA group, as shown in table 1, above data can judge tentatively that the downward modulation of DCF1 has remarkably influenced for the expression of ATP1B1 and BACE1, DCF1 may be by the expression of certain signal path regulation and control BACE1, and it exists certain with the generation development of AD and gets in touch.
Table 1 calculates as benchmark with beta actin, the psiRNA-1 group, and the psiRNA-3 group is with respect to the genetic expression spirogram of psiRNA group
| The psiRNA-1 group | The psiRNA-3 group | |
| DCF1 | Reduce 32 times | Raise 4 times |
| ATP1B1 | Reduce 512 times | Substantially constant |
| BACE | Reduce 2000 times | Reduce 8 times |
Table 1 is to calculate as benchmark with beta actin, the psiRNA-1 group, and the psiRNA-3 group is with respect to the genetic expression spirogram of psiRNA group, and above data can judge tentatively that the downward modulation of DCF1 has remarkably influenced for the expression of ATP1B1 and BACE1.
Claims (2)
1. cell differentiation of nerve cord is gene DCF 1 related is regulating Na
+, K
+Application during-ATP enzyme β 1 protein subunit is expressed.
2. the gene DCF 1 related application in the expression of regulating the gene BACE1 sick relevant of cell differentiation of nerve cord with A Zihaimo.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653766A (en) * | 2012-05-03 | 2012-09-05 | 上海大学 | Gene with function of controlling bZIP transcription factors as well as cloning method and application of gene |
| CN102766635A (en) * | 2012-05-25 | 2012-11-07 | 上海大学 | Construction method of human dcf1 gene transgenic drosophila melanogaster model |
| CN102776236A (en) * | 2012-06-06 | 2012-11-14 | 上海大学 | Specific interaction of dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes |
| CN102925483A (en) * | 2012-05-25 | 2013-02-13 | 上海大学 | New uses of DCF1 gene |
| CN102940890A (en) * | 2012-10-11 | 2013-02-27 | 上海大学 | Gene application in inhibition and apoptosis of glioma cell |
| CN105903017A (en) * | 2016-05-28 | 2016-08-31 | 上海大学 | Application of inhibiting Dcf1 gene expression on disposing capacity of Alzheimer diseases |
| CN105974126A (en) * | 2016-05-31 | 2016-09-28 | 上海大学 | Application of dcf1 gene to regulation and control over expression of ATP1B1 |
| CN106913875A (en) * | 2016-05-28 | 2017-07-04 | 上海大学 | Suppress application of the expression of Dcf1 genes in treatment antiobesity agents are prepared |
| CN106983864A (en) * | 2016-05-28 | 2017-07-28 | 上海大学 | Knock out DCF1 genes and prepare the application in alleviating alzheimer symptom medicine |
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2008
- 2008-05-20 CN CNA2008100376989A patent/CN101280306A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653766A (en) * | 2012-05-03 | 2012-09-05 | 上海大学 | Gene with function of controlling bZIP transcription factors as well as cloning method and application of gene |
| CN102653766B (en) * | 2012-05-03 | 2013-11-20 | 上海大学 | Gene with function of controlling bZIP transcription factors as well as cloning method and application of gene |
| CN102766635A (en) * | 2012-05-25 | 2012-11-07 | 上海大学 | Construction method of human dcf1 gene transgenic drosophila melanogaster model |
| CN102925483A (en) * | 2012-05-25 | 2013-02-13 | 上海大学 | New uses of DCF1 gene |
| CN102766635B (en) * | 2012-05-25 | 2013-10-16 | 上海大学 | Construction method of human dcf1 gene transgenic drosophila melanogaster model |
| CN102776236A (en) * | 2012-06-06 | 2012-11-14 | 上海大学 | Specific interaction of dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes |
| CN102940890A (en) * | 2012-10-11 | 2013-02-27 | 上海大学 | Gene application in inhibition and apoptosis of glioma cell |
| CN105903017A (en) * | 2016-05-28 | 2016-08-31 | 上海大学 | Application of inhibiting Dcf1 gene expression on disposing capacity of Alzheimer diseases |
| CN106913875A (en) * | 2016-05-28 | 2017-07-04 | 上海大学 | Suppress application of the expression of Dcf1 genes in treatment antiobesity agents are prepared |
| CN106983864A (en) * | 2016-05-28 | 2017-07-28 | 上海大学 | Knock out DCF1 genes and prepare the application in alleviating alzheimer symptom medicine |
| CN106913875B (en) * | 2016-05-28 | 2020-01-17 | 上海大学 | Application of inhibiting expression of Dcf1 gene in preparation of medicine for treating obesity |
| CN105974126A (en) * | 2016-05-31 | 2016-09-28 | 上海大学 | Application of dcf1 gene to regulation and control over expression of ATP1B1 |
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Open date: 20081008 |