Application of artificially constructed antisense nucleotide fragment Ri111 in Th1 cell polarization

An antisense nucleotide and cell differentiation technology, applied in the application field of antisense nucleotide fragment Ri111 in Th1 cell polarization, can solve infection and other problems, and achieve the effect of simple operation, efficient transfection and cell culture

Active Publication Date: 2021-09-03
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the clinical application for the treatment of autoimmune system diseases caused by immune disorders often relies on hormone drugs or immunosuppressants, but it is often accompanied by problems such as drug resistance and infection caused by long-term immunosuppression

Method used

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  • Application of artificially constructed antisense nucleotide fragment Ri111 in Th1 cell polarization
  • Application of artificially constructed antisense nucleotide fragment Ri111 in Th1 cell polarization
  • Application of artificially constructed antisense nucleotide fragment Ri111 in Th1 cell polarization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 mouse Isolation of CD4+ T lymphocytes

[0046] The method of isolating and extracting mouse spleen lymphocytes is as follows: take the spleen of a six-week-old C57BL / 6 mouse under aseptic conditions, place it in a 1640 medium, cut the spleen with ophthalmic scissors, and transfer it to a 200-mesh sterile screen , grind with a glass rod, drop 1640 medium while grinding, until all the splenocytes that are ground and dispersed flow out through the filter, collect the splenocytes and centrifuge at 450g for 10min, remove the supernatant and resuspend, add an equal volume of lymphocyte separation medium, and centrifuge at 450g for 30min , absorb the lymphocyte layer, add PBS to resuspend, centrifuge at 400g for 10min, add red blood cell lysate to stand for 5min after washing with PBS, centrifuge at 1500rpm for 10min, collect cells for counting, and use Miltenyi CD4 + T cell isolation kit, isolated from splenic lymphocytes CD4 + T lymphocytes.

Embodiment 2

[0047] Example 2 Induced differentiation of mouse Th1 cells

[0048] CD4 + After T cell sorting, counting, according to 1 × 10 6 / mL seeded in a 6-well plate, placed in 1640 complete medium containing 10% FBS, adding mixed factors to induce Th1 cell differentiation, the concentration of each factor is: IL12: 10ng / mL, IL2: 5ng / mL, anti CD28: 0.5 μg / mL, anti IL4: 1 μg / mL, anti CD3ε: 1 μg / ml, add NEAA and 55 μM β-mercaptoethanol, and culture for 72h to 96h. When the cells were cultured for 36 h, the medium was changed in half, 1 mL of the original medium was removed, and 1 mL of 1640 complete medium containing 10% FBS was mixed with 2 times the concentration of factor, NEAA and 55 μM β-mercaptoethanol.

Embodiment 3

[0049] Example 3 Design, preparation and transfection of the artificially constructed antisense nucleotide fragment Ri111

[0050] 1. Preparation of the artificially constructed antisense nucleotide fragment Ri111

[0051] In order to reduce the RNAi cascade expansion reaction caused by mRNA degradation, the inventors set the RNAi interference target at the 3'UTR region of the target gene, and searched for duplications in the 3'UTR region of IFN-γ, STAT4, Tbx21, IL12Rβ1 and IL12Rβ2 Match the fragment to ensure that the fragment can highly specifically target and bind the target 3'UTR, and simply inhibit mRNA transcription; at the same time, the sense strand or antisense strand of the fragment is designed to match the target 3'UTR to a certain extent, and tested by mouse whole Genome-wide blast comparison found no potential interference with the expression of other genes. It can specifically target the expression of Th1 polarization-related genes IFN-γ, STAT4, Tbx21, IL12Rβ1 an...

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Abstract

The invention belongs to the technical field of cellular immune regulation, and particularly discloses application of an artificially constructed antisense nucleotide fragment Ri111 in Th1 cell polarization. The invention provides the artificially constructed antisense nucleotide fragment Ri111. The nucleotide sequence of the Ri111 is as shown in SEQ ID NO: 1 and/or SEQ ID NO: 2. According to the application, the regulation and control effect of Ri111 on the differentiation capability of mouse CD4 + T lymphocytes to Th1 cells is utilized, and after Ri111 is introduced, the differentiation capability of mouse young CD4 + T lymphocytes to Th1 cells is obviously reduced; and therefore, a biological preparation for inhibiting differentiation of the mouse type 1 helper T lymphocytes can be applied to regulating differentiation of mouse CD4 + T lymphocytes to Th1 cells and controlling immune response.

Description

technical field [0001] The invention belongs to the technical field of cellular immune regulation, and in particular relates to the application of artificially constructed antisense nucleotide fragment Ri111 in Th1 cell polarization. Background technique [0002] The immune system is an important physiological defense line of the organism. By activating and producing immune cells and related cytokines, it recognizes and eliminates foreign pathogens and self-produced damaged cells or tumor cells, etc., and plays the role of immune surveillance, defense, regulation, etc., and maintains the stability of the internal environment of the body. , to protect the health of the body. [0003] An uncontrolled or dysfunctional immune response originates from an abnormally activated immune system, and abnormal immune attack is also the main cause of many autoimmune diseases (such as Crohn's disease, type 1 diabetes, graft-versus-host disease, etc.). Maintaining the homeostasis of the im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N5/10A61K31/7088A61P37/02A61P37/08A61P37/06A61P25/00
CPCC12N15/1136C12N5/0636A61K31/7088A61P37/02A61P37/08A61P37/06A61P25/00C12N2310/11C12N2320/30C12N2500/40C12N2501/2302C12N2501/2312C12N2501/2304C12N2501/515C12N2501/51C12N2510/00
Inventor 张毅李雪张晗刘元林刘伟江王洋
Owner ACADEMY OF MILITARY MEDICAL SCI
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