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shRNA for inhibiting STAT3 expression and preventing chronic graft-versus-host disease and use thereof

A graft-versus-host, RNA interference technology, applied in the field of shRNA, can solve the problems of uncontrollable cGVHD, fatal infection, secondary tumors, etc., and achieve the effect of broad application prospects and economic value.

Active Publication Date: 2015-10-28
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current studies believe that cGVHD is mainly due to immune rejection caused by the activation of donor self-reactive T cells. Most of the existing immunosuppressive treatments are based on the inhibition of T cell activation, although these measures can reduce transplant rejection to a certain extent. However, there are still relapses such as leukemia, and even complications such as fatal infection and secondary tumors caused by excessive immunosuppression, while insufficient suppression makes cGVHD difficult to control

Method used

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  • shRNA for inhibiting STAT3 expression and preventing chronic graft-versus-host disease and use thereof
  • shRNA for inhibiting STAT3 expression and preventing chronic graft-versus-host disease and use thereof
  • shRNA for inhibiting STAT3 expression and preventing chronic graft-versus-host disease and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] In this example, mice were infected with STAT3-RNAi lentivirus cells, and screen out the best STAT3-RNAi.

[0040] 1. Preparation of RNAi lentiviral vector

[0041] 1. shRNA target design

[0042] Gene name: STAT3 Species: Mouse

[0043] For the STAT3 gene sequence, multiple RNA interference target sequences were designed using the RNA interference sequence design principles provided in the public website, and three targets with the best kinetic parameters were selected to enter the subsequent experimental process.

[0044] The results are shown in the following table (Table 1).

[0045] Table 1 Mouse STAT3-shRNA interference target sequence

[0046]

[0047] At the same time, some recognized sequences in RNA interference experiments, such as the RNAi negative control (Negative Control, NC) Scramble sequence (TTCTCCGAACGTGTCACGT) were used as negative controls.

[0048] Gene information: Scramble sequence

[0049] Core sequence: TTCTCCGAACGTGTCACGT

[0050] T...

Embodiment 2

[0106] In this case, flow cytometry was used to detect the distribution of Th subgroups.

[0107] (1) Infect mice with STAT3-shRNA2(22253-1) lentivirus with the best interference effect cells; same as before.

[0108] (2) Harvest the cells of each group at 96 hours after infection, take 100 μL x 2 tubes for each well after mixing, and one tube is used for post-stimulation labeling; the other tube is centrifuged at 300×g for 5 minutes, the supernatant is completely removed, and the flow staining buffer FBS ( Configuration: PBS + 0.1% NaN3 + 5% fetal bovine serum) wash once, centrifuge at 300×g for 5 minutes, completely remove the supernatant; resuspend in FBS and label flow antibodies in groups;

[0109] (3) Detection of Treg cells:

[0110] 1) Add 1 μL Anti-MouseCD4-PE, 1.5 μL Anti-MouseCD25 Alexa-Fluor700, and incubate at room temperature for 15 minutes in the dark.

[0111] 2) After resuspending the cells by vortexing, add Foxp3 / Transcription Factor Staining Buffer fixat...

Embodiment 3

[0125] This example is to detect the effect of STAT3-shRNA2(22253-1) on mouse bone marrow (c-kit) CD117 + effects on cell proliferation and differentiation.

[0126] 1. Prepare semi-solid medium

[0127] 2.2% methylcellulose solution: Prepare according to literature method. Weigh 1.1 g of methylcellulose powder, wrap it in clean white paper, add it to 25 mL of boiled double-distilled water, and stir with a magnetic stirrer for 2 hours to fully dissolve it. After sterilizing at 120°C, cool down to about 37°C, then add 25ml of 2×RPMI1640 solution, and continue stirring for 4 hours. Put it at 4°C overnight, so that all the foam disappears, and it is in a transparent and shiny liquid state. Store at 4°C after aliquoting. When configuring the culture system, draw directly with a 1mL syringe.

[0128] 2. Configuration and dispensing of cytokines

[0129] SCF storage concentration: 100 μg / mL working solution concentration: add 990 μL 0.1% BSA diluent to 10 μL, aliquot (1 μg / mL)...

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Abstract

The invention relates to a shRNA for inhibiting STAT3 expression and preventing chronic graft-versus-host disease (cGVHD) and provides a use of STAT3-shRNA in influence on mouse spleen CD4<+>CD62L<+>naiveT cell differentiation, action safety on mouse marrow CD117<+> early stem and progenitor cell and treatment on a mouse cGVHD model. The STAT3-shRNA can influence a Th17 / Treg ratio, cGVHD clinical scoring and target organ pathological scoring. The shRNA provides an experiment basis for preventing and treating human cGVHD research in living animals. The shRNA has important meanings for development of a novel anti-cGVHD drug and improvement of cGVHD treatment effects.

Description

technical field [0001] The invention relates to an shRNA, in particular to an shRNA small molecule targeted drug capable of inhibiting the expression of STAT3 and preventing and treating chronic graft-versus-host disease and its application. Background technique [0002] Chronic graft versus host disease (cGVHD) is the main complication of long-term survivors of allogeneic hemapoietic stem cells transplantation (allo-HSCT), which not only seriously affects the quality of life of patients, but also leads to Leading cause of non-relapse death. With the widespread development of allo-HSCT, about 30% to 70% of patients may develop different degrees of cGVHD in a median of 4 to 6 months after transplantation, which severely limits the application and development of allo-HSCT. GVHD is mainly due to the reaction of immunocompetent donor-derived T cells with recipient tissues. Current studies believe that cGVHD is mainly due to immune rejection caused by the activation of donor se...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P37/06
Inventor 翁建宇杜欣黄欣陈晓梅童嘉琦耿素霞罗琼曾令基赖沛龙陆泽生吴穗晶钟立业罗成伟凌伟邓程新晁志
Owner GUANGDONG GENERAL HOSPITAL
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