A neural stem cell marker identification probe, its preparation method and application

Abstract

The present invention provides a probe for identifying neural stem cell markers, a preparation method thereof and an application, comprising the following steps: (1) preparing a compound shown in formula (I); (2) adding rhodamine and the compound shown in formula (I) into a DNA solution, heating to 85-95 °C and reacting for 3-8 minutes to obtain a synthetic solution A; (3) dialyzing the solution A through a dialysis membrane with a molecular weight exceeding 3000 DA in methanol or ethanol, and removing the dialysate to obtain the probe for identifying neural stem cell markers. The probe for identifying neural stem cell markers of the present invention forms a complex with Zn<supgt;2+< / supgt; to generate a fluorescence signal in the range of 530-570 nm. The rhodamine molecular group serves as a pH-responsive molecular group. In an H<supgt;+< / supgt> environment, the above-mentioned probe for identifying neural stem cell markers generates a specific fluorescence signal in the range of 590-630 nm. The signals of Zn<supgt;2+< / supgt> and H<supgt;+< / supgt> do not interfere with each other, and other Ca<supgt;2+< / supgt>, Cu<supgt;2+< / supgt>, Fe<supgt;3+< / supgt> do not interfere with the response signal, enabling the simultaneous detection of neural stem cell markers Zn<supgt;2+< / supgt> and H<supgt;+< / supgt>.

Description

Technical Field

[0001] The present invention relates to the field of stem cell marker detection, and particularly to a probe for identifying neural stem cell markers, a preparation method thereof, and an application thereof. Background Art

[0002] Substances related to the regulation of neural stem cells, Zn in cells 2+ and Cu 2+ are related to learning and memory. In addition, metal ions can directly regulate the activity of neurotrophic factors, possibly by conformational changes, or indirectly regulate the neurogenesis of stem cells by activating their downstream signaling in a neurotrophic factor-independent mode. As an important ion in biological fluids, H+ is one of the key factors determining cell function and viability. In the nervous system, the acidic environment affects neuronal function by triggering proton-activated ion channels (including acid-sensing ion channel ASIC1a). Therefore, neural stem cells can be indirectly detected by detecting substances related to the regulation of neural stem cells, but most of the detection methods in the prior art are directed at a single substance related to the regulation of neural stem cells. Summary of the Invention

[0003] An object of the present invention is to overcome the deficiencies of the prior art and provide a probe for identifying neural stem cell markers, a preparation method thereof, and an application thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is: A preparation method of a probe for identifying neural stem cell markers, comprising the following steps:

[0005] (1) Prepare a compound shown in formula (Ⅰ),

[0006]

[0007] (2) Add rhodamine and the compound shown in formula (Ⅰ) into a DNA solution, heat to 85 - 95 °C and react for 3 - 8 minutes to obtain a synthetic solution A;

[0008] (3) Dialyze the solution A through a dialysis membrane with a molecular weight exceeding 3000 DA in methanol or ethanol, and remove the dialysis solution to obtain a probe for identifying neural stem cell markers.

[0009] The above preparation method of the probe for identifying neural stem cell markers is obtained by reacting the amino group on the DNA strand with the carboxyl group on rhodamine and the compound shown in formula (Ⅰ), coupling the DNA strand, rhodamine, and the compound shown in formula (Ⅰ), and the compound shown in formula (Ⅰ) can react with Zn 2+ to form a complex reaction. The above probe for identifying neural stem cell markers reacts with Zn 2+The formation of the complex produces a fluorescence signal in the range of 530 - 570 nm. The rhodamine molecular group serves as a pH-responsive molecular group. In an H + environment, the above-mentioned neural stem cell marker identification probe produces a specific fluorescence signal in the range of 590 - 630 nm. The signals of Zn 2+ and H + do not interfere with each other, and the signals of other Ca 2+ , Cu 2+ , Fe 3+ do not interfere with the response signal either. It is possible to simultaneously detect the neural stem cell markers Zn 2+ and H + .

[0010] Preferably, in step (2), the weight ratio of the compound shown in formula (I), rhodamine, and DNA is 1:(0.8 - 1.2):(90 - 150).

[0011] Preferably, the DNA contains an amino group.

[0012] Preferably, the 5'-segment of the strand is labeled with a lysosome-targeting molecule (MP).

[0013] Preferably, the sequence of the DNA is

[0014] 5'-MP-TCTAGTCTCTACGTCAAGTAAGAACCTTAGCTGCGCGGATGACTCAACTGCCTGGTGATACGA-3' or 5'-MP-CGGTATTGGACCCTCGCATGACATCCGCGCAGCTAAGGTTCAAAGTTCCTGCCGCTTCACGGA-3'.

[0015] Preferably, the compound shown in formula (I) is prepared and purified by a Schiff base reaction of 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid, and 2,3-diamino-2-butenedinitrile.

[0016] Preferably, 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde is formed by reacting 2,4-dihydroxybenzaldehyde with N-(2-chloroethyl)pyrrolidine hydrochloride under alkaline conditions, and 3-formyl-4-hydroxybenzoic acid is obtained by hydrolysis and purification of methyl 3-formyl-4-hydroxybenzoate.

[0017] Preferably, the molar ratio of 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid, and 2,3-diamino-2-butenedinitrile in the compound shown in formula (I) is (0.9 - 1.1):(0.9 - 1.1):1.

[0018] The present invention also provides an intermediate for preparing a probe for identifying neural stem cell markers, and the intermediate is a compound represented by formula (I).

[0019]

[0020] The present invention also provides a probe for identifying neural stem cell markers. The probe for identifying neural stem cell markers has a DNA strand, a lysosome-targeting molecule is labeled at the 5'-segment of the DNA strand, and rhodamine and a compound represented by formula (I) are bonded to the amino group of the DNA strand.

[0021]

[0022] Preferably, it is a probe for identifying neural stem cell markers prepared by the preparation method of any one of the above-mentioned probes for identifying neural stem cell markers.

[0023] The present invention also provides the application of any one of the above-mentioned probes for identifying neural stem cell markers in simultaneously detecting neural stem cell markers Zn 2+ and H + .

[0024] The beneficial effects of the present invention are as follows: The present invention provides a probe for identifying neural stem cell markers, its preparation method and application. The preparation method of the probe for identifying neural stem cell markers is obtained by reacting the amino group on the DNA strand with the carboxyl group on rhodamine and the compound represented by formula (I), coupling the DNA strand, rhodamine and the compound represented by formula (I). The compound represented by formula (I) can react with Zn 2+ to form a complex and generate a fluorescence signal in the range of 530-570 nm. The rhodamine molecular group is used as a pH-responsive molecular group and generates a specific fluorescence signal in the range of 600-640 nm in an H + environment. The signals of Zn 2+ and H + do not interfere with each other, and other Ca 2+ , Cu 2+ , Fe 3+ will not interfere with the response signal, and it is possible to simultaneously detect neural stem cell markers Zn 2+ and H + . BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a result diagram of the probe for identifying neural stem cell markers in the embodiment of the present invention testing neural stem cell markers.

[0026] Figure 2 It is a result diagram of the probe for identifying neural stem cell markers in the embodiment of the present invention testing neural stem cell markers.

[0027] Figure 3 It is a result graph of a neural stem cell marker identification probe of an embodiment of the present invention for testing neural stem cell markers.

[0028] Figure 4 It is a chemical structure diagram of an intermediate product of a neural stem cell marker identification probe of an embodiment of the present invention. Detailed implementation manners

[0029] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] Example 1

[0031] As a neural stem cell marker identification probe of an embodiment of the present invention, the neural stem cell marker identification probe has a DNA strand, the 5'-segment of the DNA strand is labeled with a lysosome targeting molecule, and rhodamine and a compound shown in formula (I) are bonded to the amino group of the DNA strand.

[0032] The preparation method includes the following steps:

[0033] (1) Synthesis and preparation of the compound shown in formula (I);

[0034] (a) First, react 2,4-dihydroxybenzaldehyde with N-(2-chloroethyl)pyrrolidine hydrochloride under the condition of 0.1 moL / L sodium hydroxide aqueous solution to generate 2-hydroxy-4-(2-pyrrolidyl)ethoxybenzaldehyde.

[0035] (b) After hydrolyzing methyl 3-formyl-4-hydroxybenzoate in 0.1 moL / L sodium hydroxide aqueous solution, add chloroform for extraction, collect the extraction phase, and concentrate to obtain 3-formyl-4-hydroxybenzoic acid.

[0036] (c) 2-Hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid, and 2,3-diamino-2-butenedinitrile undergo a Schiff base reaction in toluene solvent. Since both 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde and 3-formyl-4-hydroxybenzoic acid contain formaldehyde, side reactions will occur, resulting in by-products where 2,3-diamino-2-butenedinitrile is simultaneously linked to 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde and by-products where 2,3-diamino-2-butenedinitrile is simultaneously linked to 3-formyl-4-hydroxybenzoic acid. The reaction liquid is first extracted with cyclohexane, and the cyclohexane phase is collected. Then, the cyclohexane phase is extracted with hexane. In the hexane phase, analyzed by a mass spectrometer, the molecular weight is mainly 473.17. According to the ratio of the response signal intensity of the compound with a molecular weight of 473.17 to the total response signal intensity of all Schiff base substances in the hexane phase, the compound shown in formula (I) accounts for 87.6% of the total amount of all Schiff base by-products and the compound shown in formula (I) in the hexane phase. The hexane phase is concentrated and evaporated to dryness at a temperature below 35 °C to obtain a mixture mainly composed of the compound shown in formula (I).

[0037] (2) Rhodamine and the compound shown in formula (I) are added to the DNA solution and heated to 90 °C for reaction for 5 minutes to obtain synthesis solution A; the weight ratio of rhodamine, the compound shown in formula (I) (calculated according to the actual amount of the compound shown in formula (I) in the mixture mainly composed of the compound shown in formula (I)), and DNA is 1:1:100; the sequence of the DNA is 5’-MP-TCTAGTCTCTACGTCAAGTAAGAACCTTAGCTGCGCGGAT GACTCAACTGCCTGGTGATACGA-3’;

[0038] (3) Solution A is passed through a dialysis membrane with a molecular weight cutoff exceeding 3000 DA in methanol or ethanol, and the dialysis solution is removed and concentrated and evaporated to dryness at a temperature below 35 °C to obtain a neural stem cell marker identification probe.

[0039] Example 2

[0040] As the neural stem cell marker identification probe of this embodiment of the present invention, the only difference between this embodiment and Example 1 is that the sequence of the DNA is

[0041] 5’-MP-CGGTATTGGACCCTCGCATGACATCCGCGCAGCTAAGGTTCAAAGTTCCTGCCGCTTCACGGA-3’.

[0042] Experimental method

[0043] I. Materials

[0044] DNA was purchased from Takara Bio Inc.

[0045] II. Fluorescence Characterization

[0046] 1. Prepare a 50 mg / L identification probe solution for neural stem cell markers in Example 1, using citric acid-sodium citrate buffer solution with pH 7.4 as the solvent.

[0047] Prepare zinc chloride solutions with concentrations of 10 μMol / L, 20 μMol / L, 50 μMol / L, 100 μMol / L, and 200 μMol / L, and the concentration is measured according to Zn 2+ measurement.

[0048] After mixing 200 μL of zinc ion solution with 1.8 mL of the identification probe solution for neural stem cell markers for 5 minutes respectively, detect the fluorescence emission spectrum at an excitation wavelength of 418 nm. The experimental results are as Figure 1 shown. It can be seen from Figure 1 that the identification probe for neural stem cell markers of the present invention forms a complex with Zn 2+ to generate a fluorescence signal in the range of 530 - 570 nm, with the maximum response at 538 nm, and the fluorescence signal intensity increases with the increase of Zn 2+ increase.

[0049] 2. Prepare citric acid-sodium citrate buffer solution with pH 7.4, and add citric acid to prepare citric acid-sodium citrate buffer solutions with pH of 6, 5, 4, 3, and 2 respectively. Prepare 50 mg / L identification probe solutions for neural stem cell markers in Example 1 using citric acid-sodium citrate buffer solutions with pH of 7.4, 6, 5, 4, 3, and 2 respectively;

[0050] After mixing 200 μL of 50 μMol / L zinc ion solution with 1.8 mL of identification probe solutions for neural stem cell markers with different pH values for 5 minutes respectively, detect the fluorescence emission spectrum at an excitation wavelength of 418 nm. The experimental results are as Figure 2 shown. It can be seen from Figure 2 that in the H + environment, the above-mentioned identification probe for neural stem cell markers generates a specific fluorescence signal in the range of 590 - 630 nm, and the response value increases with the increase of the concentration of H + with the increase of acidity. The fluorescence signal generated in the range of 530 - 570 nm is the signal after mixing 5 μMol / L zinc ion with the identification probe solution for neural stem cell markers with pH, and it is basically not affected by the change of pH.

[0051] 3. Mix 200 μL of 50 μMol / L zinc ion solution with 1.8 mL of 50 mg / L identification probe solution for neural stem cell markers in Example 1 (pH 7.4), and mix it with 20 μL of 500 μMol / L Ca 2+ ion solution (calcium chloride), 20 μL of 500 μMol / L Fe3+ Ionic solution (ferric chloride), 20 μL of 500 μMol / L Cu 2+ After mixing the ionic solution (copper chloride) solution for 5 minutes, Ca was detected respectively 2+ ions, Fe 3+ ions, Cu 2+ Under the ionic environment, at an excitation wavelength of 418 nm, the fluorescence emission spectrum was detected. The experimental results are as Figure 3 shown. The fluorescence emission spectra of the three samples coincide, and are basically the same as the spectrum of mixing 200 μL of 50 μMol / L zinc ion solution with 1.8 mL of the neural stem cell marker identification probe solution (pH 7.4) of Example 1, indicating that the neural stem cell marker identification probe of the present invention forms a complex with Zn 2+ to produce a fluorescence signal in the range of 530 - 570 nm. The rhodamine molecular group serves as a pH-responsive molecular group. In an H + environment, the above-mentioned neural stem cell marker identification probe produces a specific fluorescence signal in the range of 590 - 630 nm. The signals of Zn 2+ and H + do not interfere with each other, and other Ca 2+ , Cu 2+ , Fe 3+ will not interfere with the response signal, and it is possible to simultaneously detect the neural stem cell markers Zn 2+ and H + .

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a neural stem cell marker identification probe, characterized in that: The following steps are involved: (1) preparing a compound represented by formula (I), Formula (I); (2) adding rhodamine and the compound represented by formula (I) to a DNA solution and heating the solution to 85-95°C for 3-8 minutes to obtain a synthetic solution A; the DNA contains an amino group, and the sequence of the DNA is 5'-MP-TCTAGTCTCTACGTCAAGTAAGAACCTTAGCTGCGCGGATGACTCAACTGCCTGGTGATACGA-3' or 5'-MP-CGGTATTGGACCCTCGCATGACATCCGCGCAGCTAAGGTTCAAAGTTCCTGCCGCTTCACGGA-3'; (3) Solution A is passed through a dialysis membrane with a molecular weight greater than 3000DA in methanol or ethanol, and the dialysate is removed to obtain a neural stem cell marker identification probe.

2. The method for preparing the neural stem cell marker identification probe according to claim 1, characterized in that: In step (2), the weight ratio of the compound represented by formula (I), rhodamine and DNA is 1: (0.8-1.2): (90-150).

3. The method for preparing the neural stem cell marker identification probe according to claim 1, characterized in that: The compound shown in formula (I) is prepared and purified by Schiff base reaction of 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid and 2,3-diamino-2-butenedinitrile.

4. The method for preparing the neural stem cell marker identification probe according to claim 3, characterized in that: 2-Hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde is prepared by the reaction of 2,4-dihydroxybenzaldehyde and N-(2-chloroethyl)pyrrolidine hydrochloride under alkaline conditions, and 3-formyl-4-hydroxybenzoic acid is obtained by hydrolysis and purification of 3-formyl-4-hydroxybenzoic acid methyl ester.

5. The method for preparing the neural stem cell marker identification probe according to claim 3, characterized in that: The molar ratio of the 2-hydroxy-4-(2-pyrrolidinyl)ethoxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid, and 2,3-diamino-2-butenedinitrile is (0.9-1.1):(0.9-1.1):

1.

6. An intermediate for preparing a neural stem cell marker identification probe, characterized in that: The intermediate is a compound as shown in formula (I), Formula (I).

7. A neural stem cell marker identification probe, characterized in that: The neural stem cell marker identification probe comprises a DNA chain, wherein the 5' segment of the DNA chain marks a lysosome targeting molecule, and the sequence of the DNA is 5'-MP-TCTAGTCTCTACGTCAAGTAAGAACCTTAGCTGCGCGGATGACTCAACTGCCTGGTGATACGA-3' or 5'-MP-CGGTATTGGACCCTCGCATGACATCCGCGCAGCTAAGGTTCAAAGTTCCTGCCGCTTCACGGA-3'; and the amino group of the DNA chain is bonded with rhodamine or a compound as shown in formula (I), Formula (I).

8. The neural stem cell marker identification probe according to claim 7, characterized in that: A neural stem cell marker identification probe prepared according to the method for preparing a neural stem cell marker identification probe as described in any one of claims 1 to 5.

9. The neural stem cell marker identification probe as claimed in claim 7 or 8 is used to simultaneously detect the neural stem cell marker Zn when the purpose is not to diagnose and treat a disease. 2+ and H + Application in.

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