an initial state cd4 + Specific biomarkers for T cells and/or their differentiated regulatory T cells

A regulatory and cellular technology, applied in the fields of biotechnology and medicine, can solve the problems of lack of research on lncRNA and the immune system, etc.

Active Publication Date: 2020-11-10
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, the role of lncRNA in the physiological process of life and disease process has attracted continuous attention, but there are not many studies on the relationship between lncRNA and the immune system, especially the reports on the role of lncRNA in the maintenance of immune homeostasis involving regulatory T cells. blank

Method used

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  • an initial state cd4 <sup>+</sup> Specific biomarkers for T cells and/or their differentiated regulatory T cells
  • an initial state cd4 <sup>+</sup> Specific biomarkers for T cells and/or their differentiated regulatory T cells
  • an initial state cd4 <sup>+</sup> Specific biomarkers for T cells and/or their differentiated regulatory T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1. Initial state CD4 + T cell differentiation into regulatory T cell culture process

[0078] Mouse-derived naïve CD4 in vitro according to literature + T cells were differentiated into regulatory T cells (Roychoudhuri, R. et al. BACH2 represses effector programs to stabilize T(reg)-mediated immune homeostasis. Nature 498, 506-510 (2013).).

[0079] naive CD4 + T cell isolation kit (Stemcell) isolated naive CD4 from mouse spleen cells + T cells. The initial state CD4 obtained by sorting + T cells were cultured in a cell culture incubator at 37°C [24-well plate, 1×10 6 cells / well, 1mL / well RPMI-1640 (PAA) cell culture fluid, which contains 10% (v / v) FCS (PAA), plus anti-CD3 (1μg / ml), anti-CD28 mAbs (1μg / ml ), anti-IFN-γ (10μg / ml), anti-IL-4 (10μg / ml), IL-2 (5ng / ml) and recombinant human TGF-β (10ng / ml)]. On the third day of culture, the initial CD4 + T cells differentiate into mature regulatory T cells.

Embodiment 2

[0080] Example 2: Quantitative real-time PCR (qRT-PCR) detection of lnc-Smad3 and genes near its genome

[0081] Get the initial state CD4 that utilizes kit sorting to obtain in embodiment 1 + T cells ( figure 1 ) or cells at various time points of regulatory T cell differentiation culture ( figure 2 ), using TRIzol (Invitrogen Company) to extract the RNA samples therein. qRT-PCR uses SYBR RT-PCR kit (Takara Company, SYBR Green Realtime PCR Master MixCode: QPK-201), and operates on LightCycler (Roche Company) real-time quantitative PCR instrument.

[0082] The quantitative primers for qRT-PCR detection of lnc-Smad3 are:

[0083] 5'-AGGCCAACGATCCAGGTTTA-3' (upstream, SEQ ID NO: 2);

[0084] 5'-ACATGTCTGGAGGCAATGGA-3' (downstream, SEQ ID NO: 3).

[0085] The quantitative primers used for Iqch qRT-PCR detection of genes near the lnc-Smad3 genome as no change control are:

[0086] 5'-TGGGCATGTTAACAATCGGTG-3' (upstream, SEQ ID NO: 4);

[0087] 5'-TCTTCCTCCTCTTTGCTCTGT-3' (...

Embodiment 3

[0101] Example 3: Quantitative real-time PCR (qRT-PCR) detection of lnc-Smad3

[0102] mouse CD4 + The cDNA of T cells was used as a template, and primers were designed to amplify the mouse lnc-Smad3 sequence by PCR respectively; the amplified fragment was introduced into the pCDH-CMV-MCS-EF1-puroEGFP vector, and the expression lnc-Smad3 was constructed using the Lentivectors expression system (SystemBiosciences) lentivirus (lenti-lnc-Smad3).

[0103] CD4 + During the differentiation and development of T cells into regulatory T cells, cells were infected with lnc-Smad3-expressing lentivirus and its control vector (lenti-CTR) (MOI=100). On the third day, the cells were harvested and RNA samples were extracted using TRIzol (Invitrogen). qRT-PCR uses the SYBR RT-PCR kit (Takara Company, SYBR GreenRealtime PCR Master Mix Code: QPK-201), and operates on a LightCycler (Roche Company) real-time quantitative PCR instrument.

[0104] The quantitative primers for qRT-PCR detection o...

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Abstract

The invention relates to the field of biotechnology, specifically to a specific biomarker of an initial CD4<+>T cell and / or a regulatory T cell differentiated from the initial CD4<+>T cell and / or maturity state and function. The invention provides application of long non-coding RNA (namely lnc-Smad3) as shown in the SEQ ID NO:1 or complementary or homologous RNA sequence as a biomarker for identifying the initial CD<+>T cell and / or a regulatory T cell differentiated from the initial CD4<+>T cell, application of a reagent for detecting RNA sequence level in the preparation of a kit for identifying the an initial CD4<+>T cell and / or a regulatory T cell differentiated from the initial CD4<+>T cell, and a corresponding kit and a method thereof. The invention provides a convenient and effective specific biomarker and a method for clinical and scientific research and identification of an initial CD4<+>T cell and / or a regulatory T cell differentiated from the initial CD4<+>T cell and maturity degree thereof. The specific biomarker has a wide application prospect.

Description

technical field [0001] The present invention relates to the fields of biotechnology and medicine, specifically, a long non-coding RNA——lnc-Smad3 as the initial CD4 + The use and application method of a novel specific biomarker for the identification, maturation state and / or function determination of T cells and / or regulatory T cells differentiated from them. Background technique [0002] Mammalian genomes can transcribe a variety of long noncoding RNAs (long noncoding RNAs, lncRNAs), but the sequences and functions of only a few lncRNAs have been clarified. In recent years, the role of lncRNA in the physiological process of life and disease process has attracted continuous attention, but there are not many studies on the relationship between lncRNA and the immune system, especially the reports on the role of lncRNA in the maintenance of immune homeostasis involving regulatory T cells. blank. [0003] Regulatory T cells are important regulatory cells in immune homeostasis, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q2600/158C12Q2600/178
Inventor 夏梦曹雪涛林虹羽薛逸荃
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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