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Peer-reviewed

Experimental Liver Fibrosis and Intrasplenic Transplantation of CD45+ Bone Marrow Cells

通过CD45+骨髓细胞的脾内移植来实验性治疗肝纤维化

Prakash Baligar Prakash Baligar
Sebanta Pokhrel Sebanta Pokhrel
AM Asok Mukhopadhyay

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1972 浏览次数: 13837

评审: Anonymous reviewer(s)

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Abstract

Liver fibrosis results from the excessive collagen deposition (collagen scar) by activated hepatic stellate cells (HpSCs), leading to the inhibition of normal liver regeneration and function. Fibrogenesis is a complex mechanism involving both the synthesis and degradation of matrix proteins by different cell types, mainly macrophages in the liver. Carbon tetrachloride-induced fibrosis (CCl4) and cirrhosis is one of the oldest, simplest and probably the most widely used toxin-based experimental model for the induction of fibrosis. Here we have explained experimental animal model of liver fibrosis using CCl4, injecting twice a week for a period of 8 weeks. In these fibrotic mice, bone marrow (BM) derived CD45+ cells were transplanted via intrasplenic route after 8 weeks of CCl4 injection, and half of the CCl4 dose was continued till the end of the experiment to know the effect of transplanted cells on liver fibrosis and regeneration. So far, crude bone marrow (BM) cells or mesenchymal stem cells (MSCs) have been used for the treatment of liver fibrosis. Low survival rate, less fibrolytic and profibrogenic properties of MSCs remain the major concerns for inadequate recovery of liver from fibrosis. This led us to investigate BM cells devoid of mesenchymal lineage that is CD45+ cells for the antifibrotic effect as this population consisting of mononuclear cells which are the precursor of macrophages and may involve in the scar degradation process. Cells transplantation can be followed in different ways like intrasplenic infusion, tail vein injection and ectopic cell transplantation in experimental animal models. The survival of the cells after ectopic transplantation is less when compared to tail vein and intrasplenic infusion. Intrasplenic route of transplantation is effective in engraftment and long term survival of the donor cells especially in case of liver disease models. This protocol describes fibrosis mouse model development, intrasplenic route of cell transplantation and tracking of the donor cells after transplantation.

Keywords: Bone Marrow Cells (骨髓细胞) search Intrasplenic Transplantation (脾内移植) search Liver Fibrosis (肝纤维化) search Liver regeneneration (肝regeneneration) search Hepatocytes (肝细胞) search

Materials and Reagents

  1. Falcon tubes
    15 ml (Corning, Falcon®, catalog number: 352097 )
    50 ml (Corning, Falcon®, catalog number: 352070 )
  2. Culture discs (Corning, catalog number: 430167 )
  3. Pipettes tips (Eppendorf, catalog number: 022491954 )
  4. Surgical glass slides (Thickness: 1.45 mm; 75 x 25 mm & 76 x 26 mm) (Polar Industrial, catalog number: Blue Star PIC 2 )
  5. BD tuberculin syringe 1 ml (BD, catalog number: 309623 )
  6. Cell strainer (40 μm) (Corning, Falcon®, catalog number: 352340 )
  7. Petri plates (30 mm, 100 mm)
  8. Six- to eight-week-old male C57BL/6J (THE JACKSON LABORATORY, catalog number: 000664-C57BL/6J )
  9. Enhanced green fluorescence protein (eGFP) transgenic mice [C57BL/6-Tg(UBC-GFP)30Scha/J] (THE JACKSON LABORATORY, catalog number: 004353 )
  10. Carbon tetrachloride (CCl4) (Sigma-Aldrich, catalog number: 319961 )
  11. Mineral oil (Sigma-Aldrich, catalog number: M8410 )
  12. CO2 gas
  13. Formalin (Thermo Fisher Scientific, catalog number: SF98-4 )
  14. Iso-propyl alcohol (Thermo Fisher Scintific, catalog number: A416S-4 )
  15. Xylene (Thermo Fisher Scintific, catalog number: X3S )
  16. Paraffin wax (EMD Millipore, catalog number: 107151 )
  17. Picrosirus Red Staining Kit (Polysciences, catalog number: 24901-250 )
  18. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 12100-046 )
  19. FCS
  20. Phosphate buffer saline (PBS) (HiMedia Laboratories, catalog number: TS1099 )
  21. Anti-CD45 APC (affymetrix, eBioscience, catalog number: 17-0451 )
  22. 70% ethanol
  23. Ketamin (nirlife healthcare, catalog number: Ketamin )
  24. Xylazine (INDIAN IMMUNOLOGICALS, catalog number: 21 )
  25. 10% formalin saline solution
  26. PBS
  27. Sucrose (Thermo Fisher Scintific, catalog number: S5 )
  28. Poly-L-lysine (Sigma-Aldrich, catalog number: P4707 )
  29. Nail polish
  30. Ketamine
  31. Xylazine
  32. Triton X-100 (HiMedia Laboratories, catalog number: 9002-93-1 )
  33. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  34. Fetal bovine serum (FBS) (Biological Industries, catalog number: 04121-1A)
  35. DAPI
  36. Isotype mouse IgG (Jackson ImmunoResearch, catalog number: 015-000-003 )
  37. Isotype goat IgG (Jackson ImmunoResearch, catalog number: 005-000-003 )
  38. Anti-GFP antibody (Takara Bio, catalog number: 632381 )
  39. Anti-Albumin antibody (Bethyl Laboratories, catalog number: A90-134B )
  40. Alexa Fluor donkey anti-mouse 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21202 )
  41. Alexa Fluor donkey anti-goat 594 (Thermo Fisher Scientific, Invitrogen, catalog number: 11058 )
  42. Antifade (Thermo Fisher Scientific, Molecular probesTM,catalog number: P36961 )
  43. Ammonium chloride (NH4Cl) (HiMedia Laboratories, catalog number: TC092 )
  44. Potassium chloride (KCl) (Thermo Fisher Scintific, catalog number: 13305 )
  45. Potassium dihydrogen ortho phosphate (KH2PO4) (Central Drug House, catalog number: 029608 )
  46. Disodium hydrogen phosphate (Na2HPO4) (Thermo Fisher Scintific, catalog number: S379 )
  47. Glocose (Thermo Fisher Scintific, catalog number: D16 )
  48. Gelatin (Sigma-Aldrich, catalog number: G9664 )
  49. Phenol red (Thermo Fisher Scintific, catalog number: P7410 )
  50. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-aldrich, catalog number: M2670 )
  51. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-aldrich, catalog number: 230391 )
  52. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  53. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  54. Tissue freezing media (Leica Biosystems Nussloch, catalog number: 14020108926 )
  55. CCl4 dose preparation (see Recipes)
  56. Gey’s solution (see Recipes)

Equipment

  1. Tissue processing cassettes
  2. Oven (Scientific Systems Company)
  3. Pure paraffin wax container
  4. Embedding machine
  5. Laminar flow (Esco Micro Pte, model: AC2-2E9 )
  6. Scissors (2)
  7. Blunt forceps (2)
  8. Surgical blades with scalpel
  9. Hemostatic forceps
  10. Suture threads
  11. Suture needle
  12. Dissecting board
  13. FACS AriaTM III (BD, model: BD FACSARIA III )
  14. Incubator (SHEL LAB, model: SCO5A )
  15. Pipets (Eppendorf)
  16. Glows (SAFEMAX)
  17. Centrifuge (Eppendorf, model: 5810R )
  18. Cryotome (Thermo Fisher Scientific, model: CryoStar NX50 Cryostat )
  19. Ultra-Thin semiautomatic microtome (Histo Line Laboratories, model: MRS 3500 )

Software

  1. Adobe Photoshop

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Baligar, P., Pokhrel, S. and Mukhopadhyay, A. (2016). Experimental Liver Fibrosis and Intrasplenic Transplantation of CD45+ Bone Marrow Cells. Bio-protocol 6(20): e1972. DOI: 10.21769/BioProtoc.1972.

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分类

干细胞 > 成体干细胞 > 细胞移植

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