CN102719428A

CN102719428A - Adrenergic alpha1 acceptor subtype fluorescence probe combination and application thereof

Info

Publication number: CN102719428A
Application number: CN2012100982059A
Authority: CN
Inventors: 陈鹏慧; 阮怀珍
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2012-04-06
Filing date: 2012-04-06
Publication date: 2012-10-10
Anticipated expiration: 2032-04-06
Also published as: CN102719428B

Abstract

The invention discloses an adrenergic alpha1 acceptor subtype fluorescence probe combination, comprising an adrenergic alpha1A acceptor (alpha1A-AR) fluorescence probe and an adrenergic alpha1B acceptor (alpha1B-AR) fluorescence probe. Nucleotide sequences of the alpha1A-AR fluorescence probe and the alpha1B-AR fluorescence probe are shown in SEQIDNo.1 and SEQIDNo.2 respectively. With application of the above fluorescence probe combination and a fluorescence in-situ hybridization technology, in the brain of a transgenic mouse where oligodendrocyte precursor cells (OPC) are marked by the fluorescence, the OPC expresses different kinds of the alpha1-AR in different brain regions, which shows that the alpha1A-AR and the alpha1B-AR can be used as marks for OPC heterogeneity. The fluorescence probe combination of the invention can be used as an OPC reagent to discriminate different brain regions, and can be used for OPC heterogeneity research.

Description

The fluorescent probe combination and the application thereof of suprarenin alpha1 receptor subtype

Technical field

The invention belongs to the nucleic acid detection technique field, relate to a kind of nucleic probe combination and application thereof.

Background technology

Neurogliocyte accounts for 90% of central nervous system cell sum, and its importance in neural system comes into one's own day by day, becomes one of focus of Neuroscience Research gradually.Previously have the neurobiology investigator in cns, to find a kind of have typical bipolar projection, the little and irregular cell of cell space, its form is difficult to be included into known neurogliocyte category, and similar jejune spongiocyte.Can breed and can be divided into the characteristic of oligodendrocyte according to it, the investigator with this cell called after oligodendrocyte precursor cells (oligodendrocyte precursor cell, OPC).Chondroitin sulfate proteoglycan (NG2) is specific immunity affinity tag and the standard of perfection of OPC.Previous research thinks that OPC is that mammal embryo is grown early stage oligodendrocyte precursor cells, and it is differentiation and maturation after birth, becomes into the oligodendrocyte of myelin.But research shows recently; Also express a large amount of NG2 immunity positive reaction cells in the sophisticated brain; These cells are all distinguished with astrocyte, oligodendrocyte, microglia etc. on form to some extent, and cell space is big slightly, and projection is short and abundant; Have than multi-branched; Think one type of sophisticated spongiocyte in the brain from morphological feature tendency, and the extensive distribution of these cells in grey matter, white matter and hippocampus indicates that also it possibly not strictly belong to the precursor cell of oligodendrocyte system, but in cns with oligodendrocyte, the different a kind of new spongiocyte type of astrocyte.Because the white matter infringements due to research demonstration OPC and nerve degenerative diseases, toxicant effect, Nutrition and Metabolism obstacle and the Hypoxia and ischemia in recent years etc. are all closely related, thereby become the focus of Neuroscience Research recently.White matter has different with grey matter OPC form in the mammal brain.Whether OPC is similar with astrocyte, also has heterogeneity in the different cerebral district, significant in the body function for illustrating it.

Summary of the invention

In view of this, the objective of the invention is to heterogeneity research, design and make up a kind of fluorescent probe to OPC.

For achieving the above object, the present invention provides following technical scheme:

1, the fluorescent probe of suprarenin alpha1 acceptor (alpha1-AR) hypotype combination; Form by suprarenin alpha1A acceptor (alpha1A-AR) fluorescent probe and suprarenin alpha1B acceptor (alpha1B-AR) fluorescent probe; The nucleotide sequence of said alpha1A-AR fluorescent probe is shown in SEQ ID No.1, and the nucleotide sequence of alpha1B-AR fluorescent probe is shown in SEQ ID No.2; Said alpha1A-AR fluorescent probe and alpha1B-AR fluorescent probe carry out mark with the fluorescence molecule of fluorescence spectrum non-overlapping copies respectively.

Preferably, any one in said alpha1A-AR fluorescent probe and the alpha1B-AR fluorescent probe carries out mark with fluorescence molecule Cy5, and another kind carries out mark with fluorescence molecule Alexa Fluor488.

2, the combination of the fluorescent probe of alpha1-AR hypotype is as the application of the reagent of distinguishing different cerebral district OPC.

Beneficial effect of the present invention is: the present invention designs and has made up a kind of fluorescent probe combination of alpha1-AR hypotype; Comprise alpha1A-AR fluorescent probe and alpha1B-AR fluorescent probe; Use this fluorescent probe combination and fluorescence in situ hybridization technique; In fluorescent mark OPC Transgenic Mice Brain, find that different cerebral district OPC expresses the alpha1-AR Type-Inconsistencies, alpha1A-AR and alpha1B-AR can be used as the mark of distinguishing different cerebral district OPC; This fluorescent probe combination can be used as the reagent of distinguishing different cerebral district OPC, is used for the heterogeneity research of OPC.Because the antibody to alpha1A-AR and alpha1B-AR in the prior art does not still have the ability that specificity is distinguished alpha1A-AR and alpha1B-AR; And the fluorescent mark technology has simple, highly sensitive (with respect to conventional labeling technique), the high specificity advantages such as (with respect to the acceptor detection techniques) of detection, and fluorescent probe combination of the present invention has a good application prospect.

Description of drawings

Fig. 1 has shown the mass spectrometric detection result of Alpha1AAR-1 oligonucleotide, and single absorption peak is at 16382Da.

Fig. 2 has shown the mass spectrometric detection result of Alpha1BAR-1 oligonucleotide, and single absorption peak is at 14491Da.

Fig. 3 has shown hippocampus CA1 district OPC expression alpha1A-AR (shown in the arrow) but has not expressed alpha1B-AR.

Fig. 4 has shown hippocampus CA2 district OPC expression alpha1A-AR (shown in the arrow) but has not expressed alpha1B-AR.

Fig. 5 has shown that pallium grey matter OPC neither expresses alpha1A-AR and also do not express alpha1B-AR.

Fig. 6 has shown that pallium white matter OPC had both expressed alpha1A-AR (shown in the arrow) and also expressed alpha1B-AR.

Embodiment

In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is carried out detailed description below.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, the condition described in the molecular cloning experiment guide (third edition, J. Sa nurse Brooker etc. work) for example, or the condition of advising according to manufacturer.

One, the preparation of alpha1A-AR fluorescent probe and alpha1B-AR fluorescent probe

Specific oligonucleotide probe sequence according to mRNA full length sequence (accession number NM_007416.3) the design alpha1A-AR of the mRNA full length sequence (accession number NM_013461.3) of the alpha1A-AR that logins among the GenBank and alpha 1B-AR and alpha1B-AR is following:

The oligonucleotide probe Alpha1AAR-1:5 ' of alpha1A-AR-gcatcacgaggaagaacggcagccagcagaggacg-aagcatcccaccacaa-3 ' (SEQ ID No.1);

The oligonucleotide probe Alpha1BAR-1:5 ' of alpha1B-AR-aaggcgcacagctccacgggtggctgcgttccacga-cccaggtat-3 ' (SEQ ID No.2).

The BLAST comparison result of Alpha1AAR-1 probe sequence and alpha1A-AR mRNA full length sequence is seen table 1.The BLAST comparison result of Alpha1BAR-1 probe sequence and alpha 1B-AR mRNA full length sequence is seen table 2.

The BLAST result of table 1 Alpha1AAR-1 and alpha1A-AR mRNA full length sequence

Figure 2012100982059100002DEST_PATH_IMAGE001

The BLAST result of table 2 Alpha1BAR-1 and alpha1B-AR mRNA full length sequence

The fluorescent mark of probe adopts end-labelling, holds all on the mark with a kind of fluorescence molecule at the 5 ' end and 3 ' of every oligonucleotide probe.In the present embodiment with Cy5 mark Alpha1AAR-1 probe, with Alexa Fluor488 mark Alpha1BAR-1 probe.The excitation wavelength of Cy5 is 649nm, and emission wavelength is 670nm; The excitation wavelength of Alexa Fluor488 is 490nm, and emission wavelength is 520nm; The two fluorescence spectrum non-overlapping copies, thus interfering with each other in the follow-up fluoroscopic examination avoided.Certainly, the present invention also can use Alexa Fluor488 mark Alpha1AAR-1 probe, with Cy5 mark Alpha1BAR-1 probe; Also can adopt other fluorescence molecule difference mark Alpha1AAR-1 probe and Alpha1BAR-1 probe of fluorescence spectrum non-overlapping copies.

The composition sequence of Alpha1AAR-1 probe is through DNA mass spectroscopy (Fig. 1), and single absorption peak appears at 16382Da, and 16382Da is consistent with predicted molecular weight.The composition sequence of Alpha1BAR-1 probe is through DNA mass spectroscopy (Fig. 2), and single absorption peak appears at 14491Da, and 14491Da is consistent with predicted molecular weight.

Two, the preparation of fluorescence transgenic mice brain tissue slice

With red fluorescence DsRed mark OPC transgenic mice (available from U.S. Jaxson laboratory; Variety name is STOCK Tg (Cspg4-DsRed.T1) 1Akik/J; Be numbered 008241) be experiment mice, this mouse starts the DsRed.T1 expression of gene under chondroitin sulfate proteoglycan 4 (Cspg4) promoter regulation, the Cspg4 translation specific expressed NG2 in back; Thereby make all OPC cell expressing red fluorescent protein DsRed.T1; The excitation wavelength of DsRed.T1 is 554nm, and emission wavelength is 586nm, with the fluorescence spectrum non-overlapping copies of Cy5 and Alexa Fluor488.With mouse from the left ventricle aorta along the body circumfusion, earlier use the 20mL SPSS, use 20-30mL 4% paraformaldehyde solution again; Peel off full brain then, put in 4% paraformaldehyde solution and fix 24 hours, to sinking to the bottom fully, frozen section (40 μ m) floats in the 0.01M of no RNA lytic enzyme PBS (pH7.4) with 30% aseptic sucrose solution dehydration.

Three, the heterogeneity of hybridization in situ experiment research OPC

The Alpha1AAR-1 probe of Cy5 mark, the Alpha1BAR-1 probe of Alexa Fluor488 mark and the nucleic acid in the red fluorescence DsRed mark OPC transgenic mice brain tissue slice are hybridized the mRNA of alpha1A-AR and alpha1B-AR in the in situ detection brain tissue slice.Because the fluorescent probe that the present invention makes up is a fluorescence speed mark, so adopt direct method in situ hybridization, the formed crossbred of target nucleic acid is directly observed with fluorescent microscope or laser confocal scanning microscope without immunohistochemical assay in probe and the histocyte.Concrete experimental technique is: brain tissue slice is washed 3 times with 0.01M PBS (pH7.4) earlier, each 5 minutes, soaks 4 hours in 37 ℃ with 0.3% Triton X-100 again; Use 2 * SSC to wash again 10 minutes, adding concentration is the probe working fluid of 5ng/ μ L, hybridizes 16 hours for 42 ℃; Application 2 * SSC, 1 * SSC, 0.5 * SSC, 0.2 * SSC respectively wash 3 times successively again; Each 10 minutes, rinsing and paster were redyed nucleus with 1 μ g/mL DAPI solution among the most rearmounted 0.01M PBS (pH7.4); The glycerine mounting is observed, and is contrast with the brain tissue slice (cspg4 (NG2) DsRed) of not hybridizing.The result is shown in Fig. 3 ~ 6; It is thus clear that mouse different cerebral district OPC expresses the Type-Inconsistencies of alpha1-AR; The OPC of hippocampus only expresses alpha1A-AR and does not express alpha1B-AR, and pallium grey matter zone OPC neither expresses alpha1A-AR and also do not express alpha1B-AR, and pallium white matter zone OPC had both expressed alpha1A-AR and also expresses alpha1B-AR; Explain that alpha1A-AR and alpha1B-AR can be used as the mark of distinguishing different cerebral district OPC; The Alpha1BAR-1 probe combinations of the Alpha1AAR-1 probe of Cy5 mark and Alexa Fluor488 mark can be used as the reagent of distinguishing different cerebral district OPC, is used for the heterogeneity research of OPC.

Alpha1-AR is a kind of g protein coupled receptor, activates the back through Gq albumen performance biological action.Wherein, the proteic alpha subunit of Gq produces like InsP3 (IP3) and DG second couriers such as (DAG) through hydrolytic phosphatide acyl inositol, and activator enzyme C (PKC) regulating cell function produces effect; The proteic β γ of Gq subunit can the direct regulation and control cytolemma K _CaPassage influences K in the cell ⁺Balance, mediated cell propagation or apoptosis.Bibliographical information; The alpha1A-AR that is expressed in hippocampus all plays an important role in the differentiation of regulating the newborn mice NSC and regulation and control adult mice spongiocyte propagation; Also participated in attention and memory process, the generation with synaptic plasticity and depression, emotional handicap etc. in the growth of hindbrain all has certain relation.In addition, find in enclosing living phase hypoxic-ischemic brain damage (HIBD) experimental model that injured brain tissue is interior to be that typical catecholamine neurotransmitter release has remarkable increase with sympathin, thereby activates the alpha1-AR of postsynaptic membrane.Before initial phase, clinical symptom occur, possibly produce irreversible damage characteristic, especially to the pathologic damage of postsynaptic neurogliocyte.OPC is the most responsive vulnerability cell in the brain paralysis that HIBD causes, is the critical cellular target of pathologic process such as periventricular leukomalacia.The alpha1-AR function of this and its specifically expressing possibly have certain relation.Its effect possibly be that the HIBD pathologic process is early stage owing to the sympathin that stress discharge, and activates the alpha1-AR of postsynaptic OPC, through the KCa permeability of regulation and control OPC, influences K in the cell ⁺Level changes cell membrane potential and cell excitement property, thereby has quickened OPC in the early stage vulnerability of hypoxic-ischemic brain damage.And the OPC alpha1-AR hypotype expressed in the different cerebral district is inconsistent, has formed the heterogeneity of OPC in the different cerebral district, has provided also that to enclose in the living phase brain paralysis with white matter lesion and the damage of oligodendrocyte system be master's pathology foundation.

Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

< 110>Military Medical Univ No.3, P.L.A

< 120>combination of the fluorescent probe of suprarenin alpha1 receptor subtype and application thereof

<160> 2

<210> 1

<211> 51

<212> DNA

< 213>artificial sequence

<220>

< 223>sequence oligonucleotide probe of suprarenin alpha1A acceptor

<400> 1

gcatcacgag?gaagaacggc?agccagcaga?ggacgaagca?tcccaccaca?a 51

<210> 2

<211> 45

<212> DNA

< 213>artificial sequence

<220>

< 223>sequence oligonucleotide probe of suprarenin alpha1B acceptor

<400> 2

aaggcgcaca?gctccacggg?tggctgcgtt?ccacgaccca?ggtat 45

Claims

1. the fluorescent probe of suprarenin alpha1 receptor subtype combination; It is characterized in that; Form by suprarenin alpha1A acceptor fluorescence probe and suprarenin alpha1B acceptor fluorescence probe; The nucleotide sequence of said suprarenin alpha1A acceptor fluorescence probe is shown in SEQ ID No.1, and the nucleotide sequence of suprarenin alpha1B acceptor fluorescence probe is shown in SEQ ID No.2; Said suprarenin alpha1A acceptor fluorescence probe and suprarenin alpha1B acceptor fluorescence probe carry out mark with the fluorescence molecule of fluorescence spectrum non-overlapping copies respectively.

2. the fluorescent probe combination of suprarenin alpha1 receptor subtype according to claim 1; It is characterized in that; In said suprarenin alpha1A acceptor fluorescence probe and the suprarenin alpha1B acceptor fluorescence probe any one carries out mark with fluorescence molecule Cy5, and another kind carries out mark with fluorescence molecule Alexa Fluor488.

3. the combination of the fluorescent probe of the described suprarenin alpha1 of claim 1 receptor subtype is as the application of the reagent of the oligodendrocyte precursor cells of distinguishing the different cerebral district.