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Method for inducing stem cell to liver cell directional diferentiation and use of liver cell

A technology of hepatocytes and hepatocyte growth factor, which is applied in the field of inducing stem cells to differentiate into hepatocytes in vitro, can solve the problems of time-consuming, high cost and poor activity of HGF, and achieve great social and economic benefits, as well as the effect of good application prospects.

Active Publication Date: 2007-03-07
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a cytokine with important biological functions, HGF has been widely used in the treatment of clinical diseases (such as acute liver injury, progressive liver fibrosis, etc.), and its source is mainly extracted and purified from human plasma and placenta. This method prepares HGF Time-consuming, labor-intensive, poorly active and expensive

Method used

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  • Method for inducing stem cell to liver cell directional diferentiation and use of liver cell
  • Method for inducing stem cell to liver cell directional diferentiation and use of liver cell
  • Method for inducing stem cell to liver cell directional diferentiation and use of liver cell

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1, the separation of umbilical cord blood mononuclear cells

[0049] Under the condition of aseptic and ACD anticoagulant anticoagulant, blood was collected through umbilical vein puncture, and the umbilical cord blood of healthy full-term neonates was collected, mixed with 0.5% methylcellulose (USA, Sigma company product) at a ratio of 4:1, and settled Erythrocytes: Aspirate the supernatant and place it in a centrifuge tube for centrifugation. After discarding the supernatant, resuspend the cells in PBS, add Ficoll (product of Sigma, USA, relative density 1.077g / ml) and collect human cord blood mononuclear cells after density gradient centrifugation , washed and resuspended in PBS.

Embodiment 2

[0050] Example 2, c-Met derived from human umbilical cord blood + beta 2 m - Isolation of cells (hCBDCC)

[0051] Human umbilical cord blood mononuclear cells resuspended in PBS were mixed with rabbit anti-human β 2 M antibody (USA, product of Santa Cruz Company) and immunomagnetic bead-labeled goat anti-rabbit IgG (Germany, product of Miltenyi Company) were incubated, and separated and purified by MACS high-gradient magnetic field (Germany, product of Miltenyi Company) to obtain β 2 m - Cell group; then the group of cells was incubated with rabbit anti-human c-Met antibody (USA, product of Santa Cruz Company) and immunomagnetic bead-labeled goat anti-rabbit IgG (Germany, product of Miltenyi Company), and then subjected to MACS high-gradient magnetic field Separation and purification to finally obtain c-Met + beta 2 m - cells (hCBDCC).

Embodiment 3

[0052] Embodiment 3, rat bone marrow Thy-1 + beta 2 m - Cell Separation

[0053] Femurs were taken out from male F344 rats after abdominal dissection, bone marrow cells were washed out repeatedly with complete culture medium, filtered through a 200-mesh sieve, resuspended in PBS, and obtained by density gradient centrifugation with Ficoll (ρ=1.083g / ml) Rat bone marrow mononuclear cells were incubated successively with rabbit anti-rat β2M antibody (product of Santa Cruz, USA) and goat anti-rabbit IgG (product of Miltenyi, Germany) labeled with immunomagnetic beads. , Miltenyi company product) separation and purification to obtain β2M - cells; then the collected β2M - Cells were incubated with rabbit anti-rat Thy-1 antibody (USA, product of Santa Cruz Company) and immunomagnetic bead-labeled goat anti-rabbit IgG (Germany, product of Miltenyi Company), and passed through MACS high-gradient magnetic field (Germany, product of Miltenyi Company) Sorting to finally obtain Thy-1 ...

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Abstract

The present invention relates to biological medicine field, concretely relates to a method for inducing stem cell to liver cell directional differentiation and use of liver cell. The invention combines the genetic engineering with the stem cell engineering, uses the latent energy of the stem cell multiway differentiation, chooses the liver stroma cell for stably expressing human liver cell growth factor, induces the stem cell to liver cell directional differentiation, and induces the fore-and-after stem cell to execute comparative identification. The build of the external induce system not only provides proof for the theory of 'adult stem cell plasticity ', but also can work as seed cell for preparing the stem cell transplant, biological artificial liver and microencapsulation stem cell preparations; the induced and differentiated stem cell can be as external pharmacokinetic model for cell drug screen selecting. The method will has excellent application foreground in regeneration medicine based on genetic engineering, cell engineering and organizing engineering, and will generate great social benefit and economic benefit.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for inducing directional differentiation of stem cells to hepatocytes in vitro and its application. Background technique [0002] Orthotopic liver transplantation is currently the only effective treatment for end-stage liver failure caused by various reasons (including viral infection, poison, drug poisoning, tumor, genetic metabolic disease, postoperative liver cancer, etc.). However, due to the extreme shortage of liver donors, the high mortality rate associated with surgery and transplantation itself, the life-long administration of immunosuppressants and the serious or even fatal complications, the widespread clinical application of liver transplantation is greatly limited. With the deepening of stem cell biology research, cell therapy using stem cells as seed cells and gene therapy using stem cells as transport carriers will bring new hope to patients with end-stage liver...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08
Inventor 裴雪涛王韫芳南雪岳文白慈贤闫舫
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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