Specific surface molecular marker cd63 of liver stem cells and its application

A technology of hepatic stem cells and CD63, applied in the field of medicine and bioengineering, can solve the problems of the method of specific separation and purification of hepatic stem cells, the low purity of hepatic stem cells, and the inability to effectively distinguish hepatic stem cells from bile duct cells.

Inactive Publication Date: 2018-01-30
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

Li et al. used senecone combined with 2 / 3 hepatectomy to induce bile duct reaction, prepared the liver into a cell suspension, and removed hepatic parenchymal cells by fractional centrifugation, and then purified hepatic stem cells by differential attachment (Li WL, Su J, Yao YC, Tao XR, Yan YB, Yu HY, Wang XM, Li JX, Yang YJ, Lau JT, Hu YP, Isolation and characterization of bipotent liver progenitor cells from adult mouse, StemCells, 24(2), 322- 332), this method for isolating and purifying hepatic stem cells is cumbersome, and it is not easy to isolate hepatic stem cells from normal liver tissue
In recent years, hepatic stem cells have been isolated from normal livers or livers with induced biliary responses by means of Foxl1, Lgr5 and other surface markers of hepatic stem cells combined with flow cytometry or magnetic bead sorting (Shin S, Walton G, Aoki R, Brondell K , Schug J, Fox A, Smirnova O, Dorrell C, Erker L, Chu AS, Wells RG, Grompe M, Greenbaum LE, Kaestner KH, Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential, Genes Dev.25( 11):1185-1192; Dorrell C, Erker L, Schug J, Kopp JL, Canaday PS, Fox AJ, Smirnova O, Duncan AW, Finegold MJ, Sander M, Kaestner KH, Grompe M, Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice. Genes Dev.25(11):1193-1203; Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, SatoT, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H, In vitroexpansion of single Lgr5+liver stem cells induced by Wnt-driven regeneration, Nature.494(7436):247-250.), but these markers cannot effectively distinguish liver stem cells and cholangiocytes
Therefore, the existing methods for isolating hepatic stem cells are complicated to operate or the purity of the isolated hepatic stem cells is low. At present, there is no method that can quickly and specifically isolate and purify hepatic stem cells from liver tissue.

Method used

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  • Specific surface molecular marker cd63 of liver stem cells and its application
  • Specific surface molecular marker cd63 of liver stem cells and its application
  • Specific surface molecular marker cd63 of liver stem cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1. Localization of CD63 hepatic stem cells in the liver

[0084] 1. Normal liver and liver after 3 weeks of DDC-induced biliary reaction (Preisegger KH, Factor VM, Fuchsbichler A, Stumptner C, Denk H, Thorgeirsson SS, Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl- 1,4-dihydrocollidine mouse model for chronic alcoholicliver disease, Lab Invest.79(2):103-109) was embedded with OCT embedding agent (product number: 4583, purchased from SAKURA company), and the frozen section was 7um thick. Fix with 4% paraformaldehyde (PFA) (product number: P-6148, purchased from Sigma) at room temperature for 10 minutes, remove the fixative, and wash three times with PBS (product number: BS7016, purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) , stored at 4°C for later use.

[0085]2. Immunofluorescence staining: After blocking the liver section treated in step 1 with 1% BSA (product number: A1933, purc...

Embodiment 2

[0088] Example 2. Rapid isolation and expansion of CD63-positive hepatic stem cells

[0089] 1. Two-step in situ collagenase perfusion digested liver (Wang MJ1, Chen F, Li JX, Liu CC, Zhang HB, Xia Y, Yu B, You P, Xiang D, Lu L, Yao H, Borjigin U, Yang GS, Wangensteen KJ, He ZY, Wang X, Hu YP, Reversal of hepatocyte senescence after continuous in vivo cellproliferation, Hepatology.60(1):349-361.), the digestion mixture continued to be digested with 0.25% collagenase (product number: 11088858001, purchased from Roche) and incubated at 37°C for 30 minutes.

[0090] 2. Centrifuge the digested cell suspension at 50g centrifugal force twice for 30 minutes at low speed, discard the precipitate (large hepatocytes), collect the supernatant and continue to centrifuge at 300g centrifugal force for 5 minutes, and use serum-free DMEM medium (product number: SH30022.01 , purchased from Hyclone Company) resuspended cells, counted, and diluted the cells to 10 7 / ml, using OptiPrep TM Den...

Embodiment 3

[0096] Example 3. Differentiation of CD63-positive liver stem cells into mature hepatocytes

[0097] 1. Press 5×10 4 cells / cm 2 Sow CD63-positive hepatic stem cells on type 1 collagen-coated 35mm culture dishes, add SCM-A medium, and store at 37°C, 5% CO 2 Cultivate in the incubator until the cells are congested, and change the medium every other day.

[0098] 2. After the cells are overgrown, replace the liver induction medium (DMEM / F12, 10% FBS, 10ng / ml HGF, 10ng / mlEGF, 10-7M Dex, 1% DMSO, 10ng / ml OSM, 20% Matrigel) to continue Cultured for 6 days, the medium was changed every other day.

[0099] 3. Detection of mature hepatocytes formed by induction and differentiation of CD63-positive liver stem cells: the cells formed after induction were collected to extract RNA, and Real-Time detection showed that the expression levels of markers (Abcg2, CK19, Gja1, Ggt1) of stem cells and cholangiocytes Compared with before induction, the expression levels of liver cell markers (Al...

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Abstract

The invention relates to the technical field of medical bioengineering. The invention provides a specific surface molecular marker CD63 of hepatic stem cells, and further provides an application of the specific surface molecular marker of hepatic stem cells. In the present invention, the method for rapidly separating, culturing and expanding hepatic stem cells from the liver by using the molecular marker, specifically, using the surface-specific marker CD63 of the hepatic stem cells, is used to rapidly separate the hepatic stem cells from the prepared liver cell suspension by flow cytometry. stem cell. CD63+ hepatic stem cells have self-renewal and bidirectional differentiation properties in vitro, and have therapeutic effects on injured mouse liver. The hepatic stem cells isolated by the invention can be used as seed cells for liver drug screening, tissue engineering liver and liver disease cell therapy, and also provide an ideal cell model for research on liver development and cell biological characteristics of hepatic stem cells.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, provides a specific surface molecular marker CD63 of hepatic stem cells and its application, and a method for rapidly separating, culturing and expanding hepatic stem cells from the liver through the surface marker. It specifically involves the application of specific markers on the surface of hepatic stem cells, the rapid separation of hepatic stem cells from the liver cell suspension by flow sorting, and the realization of the expansion and two-way differentiation of hepatic stem cells in vitro, as well as in animal models of liver diseases. Replantation within the liver. Background technique [0002] Tissue stem cells refer to undifferentiated cells present in differentiated tissues of an individual, which have the ability to self-renew and specialize to form the cells of the tissue. Tissue stem cells exist in various tissues and organs of the body, and are the structural and f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074C12N5/071G01N33/569G01N33/542A61K35/407A61P1/16
Inventor 胡以平李思光何志颖陈费张坤山王敏君于兵
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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