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Adult rat hepatic oval cell (rHOC) line and application thereof

A cell line and liver technology, applied in the field of medical biology, can solve the problem of low cell purity and achieve the effect of uniform composition and stable characteristics

Active Publication Date: 2012-11-28
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Liver oval cells were first treated with ethionine and 2-acetylaminofluorene (2-AAF) by Farber (Cancer Res, 1956, 16(2): 142-148.) in 1956. Found and named in rat liver tissue, proved to be adult by Thorgeirsson et al. Rat liver precursor cells, previously (Cancer Res, 1988, 48(2): 368-378. Hepatology, 1997, 25(2): 329-334.) usually based on the principle that different types of cell densities have differences, using Density gradient medium is used to obtain the cells by density gradient centrifugation, but it is inevitable to mix a small amount of cells with similar cell density, resulting in the problem of low cell purity

Method used

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  • Adult rat hepatic oval cell (rHOC) line and application thereof
  • Adult rat hepatic oval cell (rHOC) line and application thereof
  • Adult rat hepatic oval cell (rHOC) line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Isolation and primary culture of adult rat liver oval cells

[0040] 1. Experimental materials and reagents

[0041] 1. Experimental animals: 150 g SD rats; experimental animal feed: choline-deficient rat feed containing 0.1% ethionine (Sigma-Aldrich) was prepared by Beijing Experimental Animal Center.

[0042] 2. Perfusion buffer: D-Hank's balanced salt solution containing 20mM HEPES.

[0043] 3. Circulating buffer: Hank's balanced salt solution containing 0.1% collagenase (Sigma-Adlrich).

[0044]4. Enzyme buffer: containing 0.1% collagenase (Sigma-Adlrich), 0.1% protease E (Sigma-Adlrich), 0.025% trypsin (Sigma-Adlrich), 0.02% EDTA, 0.004% DNase I (Sigma-Adlrich) of PBS solution.

[0045] 5. DMEM / F12 liquid medium and fetal bovine serum are all products of Gibco

[0046] 6.Percoll is a product of Pharmacia

[0047] 7. Complete medium: containing 10% fetal bovine serum, 0.5U / ml insulin (Sigma-Adlrich), 1ng / ml epidermal growth factor (Epithelial growth ...

Embodiment 2

[0063] Example 2: Subculture and cryopreservation of adult rat liver oval cells

[0064] 1. Reagents

[0065] 1. Trypsin solution: PBS containing 0.25% trypsin (Sigma-Adlrich), 0.02% EDTA.

[0066] 2. Freezing medium: DMEM / F12 medium containing 10% DMSO and 40% fetal bovine serum.

[0067] 2. Experimental method:

[0068] 1. When the cells grow to 90% confluence, discard the old medium, add 2ml PBS and gently swirl the culture flask to wash the cells.

[0069] 2. Discard the PBS, then add 2ml of PBS to wash the cells once.

[0070] 3. Discard PBS, add trypsin solution so that it just covers the cells, observe the digestion situation under an inverted phase-contrast microscope, and quickly discard the digestion solution when the cell processes retract.

[0071] 4. Add 2ml of complete medium to stop the action of trypsin, draw the medium in the bottle and gently blow the cells repeatedly.

[0072] 5. Collect the blown cells and place them in a 15ml centrifuge tube, mix well...

Embodiment 3

[0077] Example 3: Identification of adult rat liver oval cells

[0078] 1. Reagents

[0079] 1. Paraformaldehyde fixative: PBS containing 4% paraformaldehyde

[0080] 2. Saponin membrane breaker: PBS containing 0.1% saponin

[0081] 3. Triton X-100 membrane breaker: PBS containing 0.3% Triton X-100

[0082] 4. Mouse anti-rat oval cell marker (hepatic oval cell marker, OV-6) antibody is a product of Santa Cruz, USA

[0083] 5. Rabbit anti-human Drosophila Notch ligand Delta-like protein (delta drosophila homolog-like 1, Dlk) antibody is a product of Abcam, USA

[0084] 6. Mouse anti-human alpha-fetoprotein (α-fetoprotein, AFP) antibody is a product of American R&D Company

[0085] 7. Rabbit anti-rat albumin (albumin, ALB) antibody is a product of Bethyl company in the United States

[0086] 8. Mouse anti-human cytokeratin 19 (cytokeratin19, CK19) antibody is a product of American R&D Company

[0087] 9. Alexa Fluro488-labeled chicken anti-rabbit IgG and Alexa Fluro594-lab...

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Abstract

The invention discloses an adult rat hepatic oval cell (rHOC) line. The cell line is collected in the China General Microbiological Culture Collection Center (CGMCC) on July 23rd, 2010 with the collection registration number of CGMCC No.4057. The cell has the biological characteristics that: the cell is in a typical epithelial cell form, and has larger multiplication capacity; contact inhibition and density inhibition phenomena occur in the growing process; expressed hepatic progenitor cell markers include a hepatic oval cell marker (OV-6), a Notch ligand Delta drosophila homolog-like (Dlk), alpha-fetoprotein (AFP), albumin (ALB) and cytokeratin 19 (CK19); after the cell is continuously subcultured for 48 months to a hundredth generation, the form characteristic, growth and multiplicationproperties and immunophenotype are still kept; and the cell has stable property and uniform components, and is a good hepatic progenitor cell research model.

Description

technical field [0001] The invention relates to an adult rat liver oval cell line and belongs to the field of medical biotechnology. Background technique [0002] Although organ transplantation is currently a relatively safe and effective method for the clinical treatment of end-stage liver disease, the shortage of donor livers has led to the death of many patients with severe liver failure while waiting for liver sources. The most anticipated option for end-stage liver failure. Because mature hepatocytes are difficult to expand and culture in vitro and their liver-specific functions are gradually lost during the culture process, the application of mature hepatocyte transplantation in the treatment of liver diseases is severely limited. Since hepatic stem / progenitor cells not only have strong proliferative ability, but also have the ability to differentiate into functional mature hepatocytes, hepatic stem / progenitor cells should be used instead of liver organ transplantatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12R1/91
Inventor 王萍贾继东尤红马红丛敏刘天会唐淑珍
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV