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Method for transferring bone marrow filling dry cell by TPO/FL

A bone marrow mesenchymal and stem cell technology, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., to achieve the effect of improving self-renewal in vitro and reducing costs

Inactive Publication Date: 2006-08-16
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, because some cytokines (such as TPO and FL) are expressed very little in bone marrow stromal cells, it is still necessary to add exogenous cytokines to assist in the expansion culture system.

Method used

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  • Method for transferring bone marrow filling dry cell by TPO/FL
  • Method for transferring bone marrow filling dry cell by TPO/FL
  • Method for transferring bone marrow filling dry cell by TPO/FL

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Embodiment 1

[0027] The technology of the present invention adopts Trizol reagent (Invitrogen, Carlsbad, CA) to isolate and extract human fetal liver total RNA, then adopts cDNA first-strand synthesis kit (Fermentas company) to carry out cDNA first-strand synthesis, then uses cDNA first-strand as PCR amplification The template was added to amplify the TPO and FL gene sequences respectively. TPO gene amplification primers: 5’-GGG CGT TAACAT GGA GCT GAC TGA ATT GC-3’ and 5’-TAT GGG ATC CTT ACC CTT CCT GAG ACA G-3’. The total volume of TPO gene PCR reaction was 25 μl, 30 cycles after 95°C for 5 minutes pre-denaturation, each cycle including 94°C for 60 seconds, 55°C for 45 seconds, and 72°C for 60 seconds. The TPO gene amplification product is about 1100bp. FL gene amplification primers: 5’-GGGCGT TAA CAT GAC AGT GCT GGC GCC AGC CT-3’ and 5’-ATT AGG ATC CTT ACG GGGCTG TCG GGG CT-3’. The total volume of the FL gene PCR reaction was 25 μl, and 30 cycles were performed after 4 minutes of pre-d...

Embodiment 2

[0031] The technology of the present invention adopts LipofectAMINE transduction kit (Life Technologies, Grand Ialand, NY) to prepare recombinant virus vector suspension and transduce human bone marrow mesenchymal stem cells. According to the instructions of the kit, the recombinant viral vector suspension was prepared by using the amphophagic packaging cell line PA317 (ATCC, Rockefeller, MA), and the recombinant viral vector was screened with 1 mg / mL G418 (Merck, Germany) for 4 days. With the assistance of 8 μg / mL 1,5-dimethyl-1,5-diazaundecylidene polymethyl bromide (Polybrene; Sigma, St.Louis, MO), in a 10-cm Petri dish ( Nunc) Medium Pair 2.0×10 5Individual bone marrow mesenchymal stem cells (Qiu Liyan et al., 2004) were transduced with recombinant viral vectors for 8 hours. Cells were then washed with phosphate buffered saline (PBS) and selected with 1 mg / mL of G418 for 48 hours. Cell transduction efficiency was analyzed by enhanced green fluorescent protein (GFP) fluor...

Embodiment 3

[0034] The technology of the present invention adopts immunoblotting method and RT-PCR method to detect the expression levels of TPO and FL genes in transduced human bone marrow mesenchymal stem cells.

[0035] Immunoblot method: Cells were incubated with 50mM Tris-HCl (tris(hydroxymethyl)aminomethane-HCl, pH7.4) (AMRESCO, Shanghai Bioengineering Co., Ltd.), 1% Nonidet P40 (NP40; Sigma), 150mM NaCl (Shanghai Biotechnology Co., Ltd. Engineering Co., Ltd.) and protease inhibitors (Sigma) buffer solution, take 50 μ g of the lysate and electrophoresis on a 12.5% ​​SDS-polyacrylamide gel (Shanghai Bioengineering Co., Ltd.). After electrophoresis, use a semi-dry transfer instrument ( Bio-Rad Laboratories, Hercules, CA) were transferred to nitrocellulose membranes (Millipore Corporation), with anti-TPO antibody (R&D Systems, Minneapolis, MN) and anti-FL antibody (BIODESIGN International, Saco, ME) as primary antibodies , using horseradish peroxidase-conjugated antibody (Amersham Phar...

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Abstract

The invention provided a method of transducing the marrow stem cell by the TPO / FL gene. (1) The mRNA of the TPO and the FL is separated from the human fetus liver, then to synthesis the two cDNA by the RT-PCR and clone it to the pBluescript carrier to construct the FL-IRES-TPO fragment, then to transform the fragment to the reverse transcription virus carrier to construct the pfl30 expression carrier. (2) transducing the marrow stem cell by the pfl30 expression carrier to construct the marrow stem cell with the high expression of the exogenous TPO and the FL. The stem cell is in the hemopoietic stem / ancestor cell in vitro culture system, so it can save the cost and improve the refresh, amplified and differential ability of the hemopoietic stem cell.

Description

technical field [0001] The invention belongs to biological technology and relates to a new biological technology for constructing bone marrow mesenchymal stem cells exogenously expressing thrombopoietin (TPO) and fms-like tyrosine kinase-3 ligand (FL). This technique enables bone marrow mesenchymal stem cells to highly express two cytokines, TPO and FL, thereby improving the ability to support the expansion of hematopoietic stem / progenitor cells in vitro. Background technique [0002] The existing hematopoietic stem / progenitor cell in vitro expansion technology methods can be roughly classified into two categories: the first method is to add different combinations of recombinant cell growth factors in the culture system, the purpose is to use cell growth factors to stimulate hematopoietic stem / progenitor cells. proper regulation of proliferation and differentiation. Such technical methods can increase the expansion factor of hematopoietic stem / group cells to a certain exten...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/09
Inventor 王金福
Owner ZHEJIANG UNIV
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