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Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases

A diagnostic kit and liver disease technology, applied in the field of medical bioengineering, can solve the problems of specific separation and purification of liver stem cells, cumbersome methods, and low purity of liver stem cells

Inactive Publication Date: 2016-07-27
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 adhesion (LiWL, SuJ , YaoYC, TaoXR, YanYB, YuHY, WangXM, LiJX, YangYJ, LauJT, HuYP, Isolation and characterization of bipotent liver progenitor cells from adult mouse, StemCells, 24(2), 322-332), this method of isolating and purifying hepatic stem cells is cumbersome and difficult to obtain from normal liver tissue hepatic stem cells
In recent years, hepatic stem cells have been isolated from normal livers or livers with induced biliary responses by using surface markers of hepatic stem cells such as Foxl1, CD133, and Lgr5 combined with flow cytometry or magnetic bead sorting (ShinS, WaltonG, AokiR, BrondellK, SchugJ , FoxA, SmirnovaO, DorrellC, ErkerL, ChuAS, WellsRG, GrompeM, GreenbaumLE, KaestnerKH, Foxl1-Cre-markedadulthepaticprogenitorshaveclonogenicandbilineagedifferentiationpotential, GenesDev. DuncanAW,FinegoldMJ,SanderM,KaestnerKH,GrompeM,Prospectiveisolationofabipotentialclonogenicliverprogenitorcellinadultmice.GenesDev.25(11):1193-1203;HuchM,DorrellC,BojSF,vanEsJH,LiVS,vandeWeteringM,SatoT,HamerK,SasakiN,FinegoldMJ,HaftA,VriesRG,GrompeM,CleversH , InvitroexpansionofsingleLgr5+liverstemcellsinducedbyWnt-drivenregeneration, Nature.494(7436):247-250.), but these markers can not 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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  • Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases
  • Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases
  • Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1. Localization of CD63 hepatic stem cells in the liver

[0093] 1、正常肝脏和DDC诱导胆管反应3周后的肝脏(PreiseggerKH,FactorVM,FuchsbichlerA,StumptnerC,DenkH,ThorgeirssonSS,Atypicalductularproliferationanditsinhibitionbytransforminggrowthfactorbeta1inthe3,5-diethoxycarbonyl-1,4-dihydrocollidinemousemodelforchronicalcoholicliverdisease,LabInvest.79(2):103-109) After embedding with OCT embedding agent (product number: 4583, purchased from SAKURA Company), frozen sections were 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.

[0094] 2. Immunofluorescence staining: After blocking the liver section treated in step 1 with 1% BSA (product number: A1933, purchased from sigma company) at room temperature for 30 minutes, add dropwise the mouse a...

Embodiment 2

[0097] Example 2. Rapid isolation and expansion of CD63-positive hepatic stem cells

[0098] 1. Two-step in situ collagenase perfusion digested liver (WangMJ1, ChenF, LiJX, LiuCC, ZhangHB, XiaY, YuB, YouP, XiangD, LuL, YaoH, BorjiginU, YangGS, WangensteenKJ, HeZY, WangX, HuYP, Reversalofhepatocytesenescenceaftercontinuousinvivocellproliferation, Hepatology .60(1):349-361.), the digestion mixture was further incubated with 0.25% collagenase (product number: 11088858001, purchased from Roche) at 37° C. for 30 minutes.

[0099] 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 DensityGradientMedium (Catalog No.: D1556, s...

Embodiment 3

[0105] Example 3. Differentiation of CD63-positive liver stem cells into mature hepatocytes

[0106] 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.

[0107] 2. After the cells are congested, replace the liver induction medium (DMEM / F12, 10% FBS, 10ng / mlHGF, 10ng / mlEGF, 10-7MDex, 1%DMSO, 10ng / mlOSM, 20% Matrigel) and continue to culture for 6 days , and change the liquid every other day.

[0108] 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 (Alb...

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Abstract

The invention relates to the technical field of medical bioengineering, provides a liver stem cell specific surface molecular marker CD63 and a use thereof and especially relates to a use of CD63 in preparation of a kit for liver disease diagnosis or a drug for preventing or treating liver diseases. A method for fast separating, culturing and amplifying liver stem cells from liver by the molecular marker comprises fast separating liver stem cells from a liver cell suspension through the liver stem cell specific surface molecular marker CD63 through a flow cytometer. The liver stem cell with a CD63 positive result has the characteristics of self-renewal and bidirectional differentiation in vitro and has the effects of treating a damaged mouse liver. The separated liver stem cells can be used as seed cells for liver drug screening, tissular engineering liver and liver disease cell therapy and provide an ideal cell model for liver development and liver stem cell biological characteristic research.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, and provides a specific surface molecular marker CD63 of liver stem cells and its application, especially the application of CD63 in the preparation of liver disease diagnostic kits or the preparation of drugs for the prevention or treatment of liver diseases, and through the A method for the rapid isolation and culture expansion of hepatic stem cells from the liver based on surface markers. 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, w...

Claims

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Application Information

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IPC IPC(8): G01N33/68A61K35/407A61P1/16
Inventor 胡以平何志颖陈费王敏君于兵
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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