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Small RNA numerator for differentiation of mesenchyma stem cell into hematopoiesis cell and function target point thereof

A technology of mesenchymal stem cells and hematopoietic stem cells, applied in the field of gene medicine, can solve problems such as inability to transform bone marrow mesenchymal stem cells into hematopoietic stem cells

Inactive Publication Date: 2009-02-18
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been recently reported that bone marrow mesenchymal stem cells can be induced to differentiate into hematopoietic stem cells under appropriate culture conditions (2), most researchers still believe that in vitro culture of bone marrow mesenchymal stem cells cannot be transformed into hematopoietic stem cells. stem cells (4-5)

Method used

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  • Small RNA numerator for differentiation of mesenchyma stem cell into hematopoiesis cell and function target point thereof
  • Small RNA numerator for differentiation of mesenchyma stem cell into hematopoiesis cell and function target point thereof
  • Small RNA numerator for differentiation of mesenchyma stem cell into hematopoiesis cell and function target point thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Cells and Cell Culture

[0024] The bone marrow samples were obtained from 30 healthy volunteers (aged 20-50 years old). The bone marrow was obtained in accordance with the guidelines of the Peking Union Medical College Hospital Management Committee for scientific research on the use of human materials and with the consent of the donors. Isolated from bone marrow with the phenotype Flk1 + CD31 - CD34 -The basic steps of the stem cells are: mononuclear cells are obtained by sucrose gradient density centrifugation (centrifugal force is 1.077g / ml). The hematopoietic stem cells in the obtained monocytes were eliminated by using magnetic beads bound with CD5, GlyA and CD34. In order to obtain cell clones derived from single cells, the bone marrow cells of each patient were serially diluted to 9600 wells of fibronectin (fibronectin, catalog number W1509, purchased from Genitix, UK) and collagen (collagen , Cat. No. W1557, purchased from Genitix, UK) on culture ...

Embodiment 2

[0025] Example 2. Construction and transfection of the expression vector of the short hairpin small nucleic acid pre-shR337

[0026] We discovered a short hairpin small RNA gene named shr-337 from the first intron of the human protein-coding gene SH3PXD2B using bioinformatics. In order to confirm that this small RNA gene can produce corresponding functional small RNA, we detected its expression in hematopoietic stem cells by cloning sequencing technology ( figure 1 A). In order to construct a viral vector capable of expressing this small RNA, short hairpin DNA sequences corresponding to the sequence of pre-shR337 were synthesized by chemical synthesis: 5'-GATCCCGTGTAACACAATGGTGAGTATTTGactagaatataCAAATACTCACCATTGTGTTACATTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAATGTAACACAATGGTGAGTATTTGtatattctagtCAAATACTCACCATTGGT-GTTAC', The two synthesized DNAs were annealed to form double-stranded DNA, and the above-mentioned DNA was cloned into the retroviral vector pMSCV (Cat. No. 35140, Invitro...

Embodiment 3

[0027] Example 3. Overexpression of shR-337 can induce bone marrow mesenchymal stem cells to differentiate into hematopoietic stem cells

[0028] figure 2 B shows the expression level of shR-337 in cells detected by standard quantitative RT-PCR after the viral vector containing shR-337 was transfected into the human bone marrow mesenchymal stem cells cultured in Example 1. Compared with the situation of control and false small RNA transfection, transfection shR-337 virus expression vector (expression vector map schematic diagram sees figure 2 A) Afterwards, the expression level of shR-337 in the cells was significantly increased ( figure 2 B). Moreover, it can be maintained in cells for a long time after transfection with shR337. 2 days after highly expressing shR-337, the target gene EID1 of shR337 can be detected with slight changes in its transcription level by RT-PCR reaction ( figure 2 C), but the protein expression level of EID1 was significantly reduced by weste...

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Abstract

The invention provides small RNA (shR337) capable of differentiating hematopoietic stem cells (MSC) from human nonhematopoietic stem cells (HSC) such as mesenchymal stem cells (MSC). The discovery of the small RNA also provides an action target capable of differentiating the hematopoietic stem cells from the human nonhematopoietic stem cells such as the bone marrow mesenchymal stem cells. The small RNA can restrict the protein translation of EIDI genes after being transfected to human bone marrow mesenchymal stem cells to transform the human bone marrow mesenchymal stem cells into hematopoietic stem cells with CD45<+>surface antigen positive, and the hematopoietic stem cells with CD45<+>surface antigen positive can be further differentiated into myeloid cells and lymphoid cells. Mice in vivo experiments show that the differentiation of the small RNA to the mesenchymal stem cells is directional and undivided.

Description

technical field [0001] The invention belongs to the field of gene medicine, and more specifically relates to a small hairpin RNA with a specific sequence, which can differentiate mesenchymal stem cells into hematopoietic cells by inhibiting the expression of EIA-like differentiation inhibitors. Background technique [0002] Hematopoietic stem cell transplantation is a promising emerging therapeutic technique for the treatment of certain incurable diseases. However, the source of hematopoietic stem cells is still one of the main obstacles preventing the clinical efficacy of hematopoietic stem cell therapy technology. Finding stem cells from other sources and making them differentiate into hematopoietic stem cells is an important topic for scientists to study. Mesenchymal stem cells (MSCs) are a type of stem cells that have been studied by scientists from various countries. Like hematopoietic stem cells (HSCs), both have the ability to self-renew and maintain the hematopoieti...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85C12N5/08A61K31/7088A61K31/713A61P7/00
Inventor 殷勤伟赵春华谷同军边春景张洪杰
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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