DNA silver nano-clusters and in-situ synthesis method and application of DNA silver nano-clusters in cells

A technology for in situ synthesis of silver nanoclusters, applied in the field of DNA silver nanoclusters, can solve the problems of inability to monitor telomerase activity in real time for a long time and limit the residence time of probes.

Active Publication Date: 2017-11-10
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] However, although DNA-silver nanoclusters synthesized by chemical methods have a wide range of uses and good biocompatibility, they are still easily excreted by the immune system through the kidneys, which limits the residence time of the probe in the organism and cannot monitor telomeres in real time for a long time. Activity of other functional molecules such as enzymes

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  • DNA silver nano-clusters and in-situ synthesis method and application of DNA silver nano-clusters in cells
  • DNA silver nano-clusters and in-situ synthesis method and application of DNA silver nano-clusters in cells
  • DNA silver nano-clusters and in-situ synthesis method and application of DNA silver nano-clusters in cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 In situ synthesis of endogenous DNA silver nanoclusters in cells

[0090] (1) The embodiment up-regulates the intracellular GSH level:

[0091] Cells were cultured in a 25mL cell culture flask, and when the cell coverage reached about 80%, 10 μL of 5 μM α-lipoic acid (α-lipoic acid) was added and co-incubated for 24 hours at 37 degrees Celsius and 5% CO2, and then slowly poured out the culture solution and replaced with fresh medium.

[0092] The above cells were washed once with PBS, collected by centrifugation, and the supernatant was aspirated. Add a protein removal reagent 3 times the volume of the cell pellet, and then use liquid nitrogen and a 37°C water bath to freeze and thaw the sample twice to break up the cells. After 5 minutes at 4°C or in an ice bath, centrifuge at 10,000 g for 10 minutes at 4°C. The supernatant was taken to determine the content of glutathione with DTNB kit.

[0093] (2) Transfection DNA:

[0094] Take the above-mentioned ce...

Embodiment 2 Embodiment 1

[0098] Example 2 Example 1 The DNA silver nanocluster probe synthesized in situ is used for long-term monitoring of stem cell proliferation

[0099] Changes in telomerase activity during differentiation.

[0100] (1) In situ monitoring of telomerase activity changes in human umbilical cord mesenchymal stem cells during proliferation:

[0101] The first-generation human umbilical cord mesenchymal stem cells treated in Example 1 were cultured in a cell culture box. At each passaging, take part of the plate. The corresponding samples were observed with a fluorescent confocal microscope, excited at a wavelength of 488 nm, and co-fluorescent imaging was observed with an objective lens multiple of 63X. And use the software to calculate the average fluorescence intensity in each cell, and compare the relative activity of telomerase in each generation of mesenchymal stem cells.

[0102] The method for calculating the relative activity of telomerase is to use the average fluorescenc...

Embodiment 3

[0106] Embodiment 3 in vitro verification experiment

[0107] In order to evaluate the accuracy of the DNA silver nanocluster probe synthesized in situ in Example 1 of the present invention for long-term monitoring of changes in telomerase activity in the process of stem cell proliferation and differentiation, each generation of mesenchymal stem cells was lysed and telomeres were extracted. Enzymes The telomerase activity of each generation of cells was detected using an ELISA kit. In addition, neural stem cells differentiated from mesenchymal stem cells of each generation were lysed and telomerase was extracted, and the telomerase activity of cells of each generation was detected using an ELISA kit.

[0108] Figure 9 a is a series of cells used in the verification experiment of Example 3. Figure 9 b is the result of lysing and extracting telomerase from each generation of mesenchymal stem cells, and detecting it with an ELISA kit. It can be seen from the figure that the ...

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Abstract

The invention discloses an in-situ synthesis method of DNA silver nano-clusters Cells are subjected to DNA transfection by raising the level of glutathione (GSH) in cells, and therefore the cells are induced to synthesize the DNA silver nano-clusters in situ. The DNA silver nano-clusters synthesized in situ by the method have high biocompatibility, and the retention time of probes in the organisms is prolonged, so that the DNA silver nano-clusters can used for long-term monitoring over the activity of telomerase during the proliferation and differentiation processes of cells. In addition, by replacing sliver nano-clusers modified by oligonucleotide with different sequences as multifunctional fluorescent probes, the method can be used for detecting other functional molecules such as enzymes, glucose, protein, DNA and the like in a high-sensitivity mode and widely applied to the fields of bio-marking, disease monitoring and the like.

Description

technical field [0001] The invention relates to a long-term monitoring of telomerase in the process of proliferation and differentiation of stem cells by using DNA silver nano-clusters prepared by in-situ synthesis of cells, and belongs to the technical fields of material synthesis, biological imaging and sensing. Background technique [0002] Telomerase (Telomerase) is an enzyme responsible for the extension of telomeres. Cells with shortened telomeres have limited replication ability. Telomerase can use its own RNA as a template to extend the length of telomeres, thereby maintaining chromosome stability and cell activity. Plays an important role in cell proliferation and differentiation. Mesenchymal stem cells are a kind of stem cells with multi-directional differentiation potential. They are often used in the clinical treatment of central nervous system damage. Compared with bone marrow-derived and adipose-derived stem cells, human umbilical cord-derived mesenchymal stem ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 田阳董方圆郑婷婷
Owner EAST CHINA NORMAL UNIV
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