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Specific biomarker for human dendritic cells maturity and functions

A technology of dendritic cells and cells, which is applied in the fields of biotechnology and medicine, can solve the problems that there are not many studies on lncRNA and immune system, and there are no research reports on lncRNA and dendritic cells.

Inactive Publication Date: 2015-11-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some lncRNAs have been reported to play an important role in certain processes and diseases, there are not many studies on the relationship between lncRNAs and the immune system, let alone any research reports on the relationship between lncRNAs and dendritic cells

Method used

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  • Specific biomarker for human dendritic cells maturity and functions
  • Specific biomarker for human dendritic cells maturity and functions
  • Specific biomarker for human dendritic cells maturity and functions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Culture process from monocytes to dendritic cells

[0089] According to literature records, human monocytes were differentiated and matured into DCs in vitro [Liu, S., Yu, Y., Zhang, M., Wang, W. & Cao, X. "The involvement of TNF-alpha-related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN-beta-stimulated human dendritic cells to tumor cells". J Immunol. 2001; 166(9): 5407-5415.].

[0090] Peripheral blood samples from healthy individuals were obtained from the Blood Center of Shanghai Changhai Hospital. Human peripheral blood mononuclear cells (PBMCs) were obtained after density gradient centrifugation with Ficoll-Hypaque (Mediatech Cellgro). ). Monocytes in PBMC were obtained by magnetic bead sorting with anti-CD14 microbeads (Miltenyi Biotech).

[0091] The sorted monocytes were cultured in a cell culture incubator at 37°C (24-well plate, 5×10 5 cells / well, 1mL / well RPMI-1640(PAA) cell culture medium containing 10% (v / v) FCS(PAA), plus ...

Embodiment 2

[0092] Example 2: Quantitative real-time PCR (qRT-PCR) detection of lnc-DC and genes near its genome

[0093] Take the primary human peripheral blood cells or mononuclear cells used for sorting in Example 1 to cells at various time points of dendritic cell culture, and other various immune cells, and use TRIzol (Invitrogen Company) to extract RNA samples therein . The resulting RNA was reverse transcribed, followed by qRT-PCR.

[0094] The various immune cells are as follows:

[0095] Monocyte-derived macrophages were also cultured from monocytes from PBMCs. The culture condition is RPMI-1640 cell culture medium containing 10% (v / v) FCS plus 20ng / mL human M-CSF (R&D Systems), and monocyte-derived macrophages can be obtained after culturing until the fifth day.

[0096] Monocytes, plasmacytoid dendritic cells, conventional dendritic cells, neutrophils, eosinophils, CD4 + T cells, CD8 + Other primary immune cells such as T cells, B cells and natural killer cells were sort...

Embodiment 3

[0118] Example 3: Quantitative real-time PCR (qRT-PCR) detection of lnc-DC and DC of lnc-DC RNAi

[0119] Insert the lnc-DC siRNA sequence RNAi-1, RNAi-2 or irrelevant control sequence shown below into the shRNA expression vector pSIF siRNA expression vector (purchased from System Biosciences), thereby obtaining the expression lnc-DC shRNA vector and its control vector.

[0120] RNAi-1 (SEQ ID NO: 11): 5'-GAG TTA TCT TAA GGA TCA T-3';

[0121] RNAi-2 (SEQ ID NO: 12): 5'-GGA GTT CCT TGA CTA GG-3';

[0122] Irrelevant control RNAi (SEQ ID NO: 13): 5'-TTC TCC GAA CGT GTC ACG T-3'.

[0123] Then, Lentivirus was packaged in HEK293T cells using Lentivector Expression Systems (System Biosciences). During the differentiation of monocytes into DCs as described in Example 1, cells were infected with the packaged lnc-DC RNAi lentivector and its control vector (control RNAi) on day 1 (MOI=100). Cells were harvested in mature DC (day 7). RNA was extracted with TRIzol (Invitrogen). ...

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Abstract

The invention provides a specific biomarker for human dendritic cells maturity and functions. The invention specifically relates to an application of a sequence (namely 1nc-DC) as shown in SEQ ID NO:1 or SEQ ID NO:2 or its hybridization or homologous sequence or expression product used as a biomarker for identifying conventional dendritic cells and / or their maturity and / or functions, an application of a reagent for detecting the level of the sequence or expression product in the preparation of a kit for identifying the conventional dendritic cells and / or their maturity and / or functions and corresponding kit and method. The invention provides a convenient and effective specific biomarker and a method for clinic and scientific identification of the conventional dendritic cells and / or their maturity and / or functions. The specific biomarker has a wide application prospect.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine. Specifically, the present invention relates to the use and application method of a long non-coding RNA—lnc-DC as a novel specific biomarker for identification, maturation status and / or function determination of human dendritic cells. Background technique [0002] Mammalian genomes can transcribe a variety of long noncoding RNAs (long noncoding RNAs, lncRNAs), but only a few lncRNAs have been identified for their functions. Although some lncRNAs have been reported to play an important role in certain processes and diseases, there are not many studies on the relationship between lncRNAs and the immune system, let alone any research reports on the relationship between lncRNAs and dendritic cells. [0003] Dendritic cells (DC) are important antigen-presenting cells in the immune system, which play an important role in the body's anti-tumor, anti-infection, transplant rejection and preventi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 曹雪涛王品
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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