Specific DNA aptamer for CD4 positive cell and chimera of specific DNA aptamer

A nucleic acid aptamer and positive cell technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, drug combination, etc., can solve the problem of high cost

Active Publication Date: 2018-09-21
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, one of the hot spots in drug delivery system research is nanomaterials. Polymer nanomaterials can passively or actively target tumor tissue to deliver drugs through physical and chemical properties. Compared with proteins, they are easier to be absorbed through physiological barriers and have relatively few side effects. There are also studies on nanomaterials with controlled release, which have safer and more controllable drug delivery effects. However, there are still stability factors and biosafety factors in the research of nanomaterials, and the cost is relatively high.

Method used

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  • Specific DNA aptamer for CD4 positive cell and chimera of specific DNA aptamer
  • Specific DNA aptamer for CD4 positive cell and chimera of specific DNA aptamer
  • Specific DNA aptamer for CD4 positive cell and chimera of specific DNA aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Construction and verification of CD4 nucleic acid aptamer aptamer and CCL18 siRNA-connected chimera chimera

[0068] 1.1 CD4 DNA aptamer, aptamer-linked siRNA sense strand intermediate and siRNA antisense strand were synthesized and provided by the company (TAKARA, Gemma). Mix the same concentration of intermediates and siRNA antisense strands, add annealing buffer, slowly anneal to 25°C in a 90°C water bath, and store in minus 80°C.

[0069] 1.2 Add the same concentration of CD4 nucleic acid aptamer, intermediate, siRNA antisense strand, and chimera to the loading buffer, and electrophoresis at 150V for 10 minutes in 8% non-denaturing PAGE gel, and observe the position of the chimera constructed in the electrophoresis.

[0070] The secondary structure of the full-length aptamer is predicted in the secondary structure prediction software, and it can be found that the CD4 aptamer has two stem-loop structures such as figure 1 (Top), the morphology of the chime...

Embodiment 2

[0071] Example 2. Verification of uptake rate of chimeras

[0072]2.1 In the TAMs, add the CD4 nucleic acid aptamer labeled with the cy3 fluorescent group at a final concentration of 10 nM. Treat TAMs with an equal amount of PSMA nucleic acid aptamer (also labeled as cy3) as control 1, treat MDA-MB-231 cells with CD4 nucleic acid aptamer as control 2, and liposome transfect fluorescently labeled siRNA double-strand transfected TAMs served as control III.

[0073] 2.2 Collect the treated cells after 24 hours of treatment together, wash off the excess treatment reagent with PBS once, centrifuge at 300g for 5 minutes, discard the supernatant, add 200 microliters of PBS to resuspend, and analyze the uptake of aptamer or siRNA by the cells with a flow cytometer Case.

[0074] 2.3 Immunofluorescence detection: Before the above treatment, plant TAMs or MDA-MB-231 cells on a small glass slide, and perform the above treatment after the cells adhere to the wall. After 24 hours, the s...

Embodiment 3

[0084] Example 3. The constructed chimera inhibits the synthesis and secretion of ccl18

[0085] 3.1 In TAMs, add chimeras at a final concentration of 20 nM, CD4 aptamer as a blank control, chimeras linked to GFP protein siRNA as a negative control, and liposome transfection as a positive control. After 24-36 hours of culture, add 1 ml of trizol to each well to lyse the cells and collect mRNA; after 48-60 hours of culture, collect cell protein and cell supernatant.

[0086] 3.2 RT-QPCR

[0087] 3.2.1 Add 200 microliters of chloroform to the cell mixture containing trizol, shake it vigorously and let it stand for 10 minutes, then centrifuge at 12,000 rpm at 4°C for 15 minutes; after centrifugation, absorb about 400 microliters of the upper transparent liquid phase and add 1 milliliter of isopropyl Alcohol, mix gently and let stand for 5 minutes, centrifuge at 12,000rpm at 4°C for 10 minutes; discard the supernatant after centrifugation, keep the bottom precipitate, add 1 ml of...

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Abstract

The invention discloses a specific DNA aptamer for a CD4 positive cell and a chimera of the specific DNA aptamer. The specific DNA aptamer is characterized in that an aptamer technology with a cell orienting function is combined with an RNAi technology to construct the specific DNA aptamer for the CD4 positive cell, so as to carry anticancer siRNA to be selectively imported into a CD4 positive tumor-associated macrophage. The function of the specific DNA aptamer for the CD4 positive cell as a vector oriented tool for delivering the anticancer siRNA and an antitumor effect are detected in an invitro tumor-associated macrophage and breast cancer cell culture system. The specific DNA aptamer and the chimera thereof disclosed by the invention are based on a novel gene anticancer drug for oriented RNAi and provide a novel idea for RNAi targeting import into target cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA nucleic acid aptamer. Background technique [0002] The incidence of breast cancer in my country is on the rise, especially the cases of advanced metastatic breast cancer are increasing. At present, the treatment of breast cancer is still a comprehensive treatment based on surgery. Since the concept of "breast cancer is a systemic disease" was put forward, more and more attention has been paid to systemic treatment. However, breast cancer that has metastasized throughout the body is still an intractable disease. These cases are often insensitive to multiple chemotherapy and radiotherapy regimens, and various treatment methods have not made breakthroughs except for slightly prolonging life. Molecular targeted therapy came into being. Molecular targeted therapy designs drugs for specific tumor germinal sites, which can directly find tumor cells to exert anti-tumor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115A61K48/00A61K31/7088A61P35/00
CPCA61P35/00A61K31/7088C12N15/115C12N2310/16
Inventor 姚燕丹宋尔卫张明霞李铨黄松音
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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