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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.

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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

干细胞 > 成体干细胞 > 细胞移植
作者: Prakash Baligar
Prakash BaligarAffiliation: Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity University, Noida, UP, India
For correspondence: pbaligar@amity.edu
Bio-protocol author page: a3620
Sebanta Pokhrel
Sebanta PokhrelAffiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
Bio-protocol author page: a3621
 and Asok Mukhopadhyay
Asok MukhopadhyayAffiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
For correspondence: ashok@nii.ac.in
Bio-protocol author page: a3622
Vol 6, Iss 20, 10/20/2016, 937 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1972

[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), Intrasplenic Transplantation(脾内移植), Liver Fibrosis(肝纤维化), Liver regeneneration(肝regeneneration), Hepatocytes(肝细胞)

[Abstract]

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: 00 4353 )
  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: 0 29608 )
  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

  1. Experimental liver fibrosis model
    1. Development of fibrosis mouse model
      1. A liver fibrosis model was established in C57Bl6/J mice by repeated injection of CCl4 (0.8 ml/kg body weight) in mineral oil, twice a week, for a period of 8 weeks (16 doses) via intraperitoneal injection as shown in the Figure 1.
      2. CD45+ cells were isolated from eGFP transgenic mice and transplanted through intrasplenic route.
      3. After 3 days of transplantation, CCl4 dose was reduced to half and continued till the end of experiment to avoid spontaneous regression of the fibrosis.
      4. After 4 weeks of transplantation, mice were sacrificed in excess CO2 gas, serum samples were stored for liver function tests, and liver tissue was fixed in 10% formalin saline for 48-72 h for histological analyses.


        Figure 1. A schematic diagram with timeline and doses for fibrosis model development. B wt. - Body weight; Tx - Transplantation.

    2. Tissue processing for paraffin sectioning
      1. Take fixed tissues, put in the tissue processing cassettes and label properly as per experiment with the help of a pencil.
      2. Dehydrate them with series of isopropyl alcohol gradations (30%, 50%, 70%, 90% and 100% for 30 min intervals in each step).
      3. Place the tissues in 1:1 isopropyl alcohol and xylene for 30 min.
      4. Place in pure xylene for 1 h.
      5. All tissue cassettes dipped in 1:1 xylene and paraffin wax solution for 30 min in 60 °C oven.
      6. Place the cassettes in pure paraffin wax container 1, then 2 and 3 for 1 h at each step in 60 °C oven.
      7. Then embed in the steel or plastic molds with the help of embedding machine. These blocks can be used forever if stored properly in room temperature.
      8. Then cut 5 µm sections into the floater bath (45-50 °C) and take on to the poly-lysine coated slides. Air dry completely in room temperature and use for staining (cut sections can be stored for 2-3 months if stored properly, otherwise loss of antigenicity occurs and is not good for immunohistochemistry).
    3. Measurement of fibrosis
      1. Fibrosis can be measured by Picrosirus red staining. Cut sections are rehydrated, processed for Picrosirus staining as per the protocol provided by the kit manufacturer.
      2. After staining with picrosirus red, fibrosis is determined by using Ishak et al. (1995) scoring system or METAVIR (Group FMCS, 1994) scoring system (Table 1). The representative images of liver sections stained with Picrosirus stain are shown in Figure 2.
        Note: Fibrosis can be measured by many ways by different staining techniques such as Masson’s trichrome staining, Picrosirus red staining, collagen staining with antibodies etc. In addition, percentage of collagen proportionate area in the Picrosirus stained sections using NIH ImageJ software or quantitative estimation of hydroxyproline in liver tissue by calorimetric methods can be followed to measure the extent of fibrosis.

        Table1. The Ishak scoring and the METAVIR scoring system



        Figure 2. Pathological changes of liver in the presence of CCl4. A. No CCl4 dose; B. CCl4 (0.8 ml/kg body weight) in mineral oil, twice a week, for a period of 4 weeks and harvested 3 days after last dose; C. CCl4 (0.8 ml/kg body weight) in mineral oil, twice a week, for a period of 8 weeks and half of CCl4 dose (0.4 ml/Kg body weight, twice a week) was continued till the end of experiment (further 4 weeks); D. CCl4 (0.8 ml/kg body weight) in mineral oil, twice a week, for a period of 8 weeks and dose was stopped for further 4 weeks then harvested (score 1). Scale bars A, C, D = 400 µm, B = 200 µm (100x). E. Ishak and METAVIR scoring system of the representative images A, B, C and D.

  2. Bone marrow CD45+ cells isolation
    1. Six to eight weeks old eGFP transgenic mice are sacrificed by inhaling excess CO2 and then dipped in 70% isopropyl alcohol for 1 min to avoid contamination from the skin.
    2. The mouse is taken to laminar hood and placed on the dissecting board.
    3. Cut the skin at the middle of the trunk towards and along the two sides of the tail with the help of a pair of scissors so that it will be easy to pull the skin towards the claws and claws dissected out at the ankle joints along with the peeled skin. Then hind limbs are dissected out at the connection between hind limb and trunk.
    4. Muscles, ligaments and tendons are carefully disassociated from the tibias and femurs using sterile surgical scalpel and also scrubbed to remove the residual tissue parts.
    5. Tibias and femurs are separated by bending in opposite direction and transferred to a 30 mm Petri plate containing DMEM with 3% FCS.
    6. Repeat the steps B4 and B5 for other hind limb.
    7. The bone is held with a sterile forceps and tip of two ends excised using a micro-dissecting scissors.
    8. BD tuberculin syringe with needle containing DMEM is inserted into the cut tip of the bone and slowly flushed through bone cavity into a 50 ml Falcon tube which is already containing 5 ml of DMEM with 3% FCS. Repeat twice from both ends of the bone or until the bone becomes white.
    9. Repeat steps B7 and B8 for all pieces of bones. The entire procedure for isolation of cells is depicted in Figure 3.
      Note: Bone marrow cells are isolated in two stage process. In the first stage, cells are removed from tibia and femurs by flushing with medium. The firmly adhered cells (mostly stromal cells and primitive stem cells) of the endosteal region are recovered in the second stage by digesting the crushed bone with collagenase type IV (0.03%) and dispase enzyme (2 U/ml).By performing these two stage yield a higher number of cells (Baligar et al., 2016).
    10. Centrifuge at 259 x g for 5 min and supernatant is discarded.
    11. Erythrocytes are lysed by treatment with Gey’s solution as follows, 5 ml of Gey’s solution is added per mouse bone marrow cells pellet and gently suspend with the help of a pipet tip for 90 sec, and then PBS is added up to 50 ml to dilute the Gey’s solution.
      Note: Gey’s treatment is used to lyse RBCs. The time of treatment is important because inadequate treatment period made lead to improper lysis of RBC. If treatment period is exceeded above permitted, it may kill the target cells. Use 5 ml of reagent per mouse bone marrow. After bone marrow flushing, wash with media and add 5 ml (In case of more number of cells are harvested from 2 or 3 mice, accordingly Falcon tube number is increased) on the cells pellet, mix gently with the pipettes for exactly 90 sec. Immediately dilute with 45 ml of PBS and spin down (259 x g for 5 min) then wash with media.
    12. Centrifuge at 259 x g for 5 min and supernatant is discarded and pellet is washed in medium.
    13. Filter cell suspension through a 40 µm cell strainer.
    14. Then count the cells by using hemocytometer and used for anti-CD45 antibody staining.
    15. CD45+ cells are sorted by using FACS AriaIII (70 mm nozzal) technique. Here, we used FACS AriaIII for the CD45 positive cells sorting by labeling with anti-CD45 APC antibody surface antigen (Baligar et al., 2016).


      Figure 3. Isolation of BM stem cells from femur and tibia. A. The sacrificed mouse is dipped in 70% (v/v) ethanol for 1-2 min. B. Mouse is taken in side a biosafety cabinet hood and placed on a dissecting board. C. Carefully skin is removed. D. Claws dissected out at the ankle joints. E. Skin removed legs kept in 100 mm sterile Petri plate. F. Muscles, ligaments, and tendons are removed. G. Tibias and femurs are dissected and kept in complete media. H. BD tuberculin syringe needle is introduced into bone cavity and slowly flush the marrow out into the 50 ml Falcon tube containing 5-10 ml of complete media. The bone cavities are washed twice from both ends again until the bones became whitish.

  3. Cell transplantation
    1. Before transplantation, all surgical materials, suture tread, suture needle, cotton should be autoclaved and arranged in the laminar hood. Pre-arrange a separate suture thread of approximately 10 cm long for the ligation of spleen.
    2. Anesthetize the animals with ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) by intraperitonial injection.
    3. Turn the animal left side and shave an area of 2-3 cm2 underneath the caudal rib (Figure 4A).
    4. Disinfect the shaved areas with 70% isopropyl alcohol.
    5. Incise the skin below the most caudal rib for 0.5 cm at the dorsoventral side using scissors (Video 1).
    6. Lift the spleen carefully with the help of small blunt end forceps (Video 1).
    7. Place the pre-arranged ligation over the spleen and loosely ligate at the tip around the spleen without holding the adipose tissue (do not pull the spleen) as shown in the Figure 4B (Video 1).
    8. Insert the syringe loaded with cells at the tip of spleen before the knot and after passing the needle tighten the knot to avoid the back flow (Avoid the perforation of the spleen and subsequent leakage. Figure 4D, Video 1).
    9. Slowly infuse cell suspension (50-80 μl) over 1-2 min until it is completely injected (cells are infused gently to avoid choking of the vain as shown in Video 1).
    10. Don’t remove the needle immediately after infusion; wait for 2-3 min to avoid back flow (Video 1).
    11. Then remove the needle and set the ligation tight for 2-3 min (Video 1).
    12. Remove the thread and relocate the spleen in the cavity.
      Note: Steps C9-C12 are the critical steps, as cells should be infused very slowly to avoid choking of the vein. After passing all cell suspension, don’t remove the needle suddenly, if you remove suddenly there will be chance getting back flow of cells as well as bleeding. Therefore, at least keep needle inside the spleen at least 2-4 min so that pressure in the spleen is released and clotting will occur at the site of puncture. The remove the needle slowly followed by slight tightening of the knot for at least 1-2 min. it wills also aid back flow and clotting at puncture site. Then slowly remove knot make sure that there should not be a back flow of any fluid.
    13. Close the abdominal peritoneum and then skin with the degradable sterile suture separately.
    14. Rehydrate the animal with subcutaneous injection of 1 ml of sterile saline solution (5 ml/100 g body weight Figure 4E).


      Figure 4. Intrasplenic transplantation of the stem cells. A. Turn the animal left side and shave an area underneath the caudal rib. B. and C. Exposure of the spleen and ligation with the thread. D. Insertion of the needle into the spleen then tightens the thread and infuse cells into spleen. E. Close the peritoneum and skin with suture. F. Rehydration by subcutaneous injection of saline after surgery.

      Video 1. Showing intrasplenic transplantation of cells (steps C5-C11)

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    15. Avoid postoperative analgesia to minimize the potential fulminate liver failure.
    16. Keep the animals into a pre-warmed cage immediately after surgery and allow free access to water and food. Then shift cages in the animal rack for follow-up.

  4. Detection of transplanted cells by Immunohistochemical technique (IHC)
    1. After 30 days of transplantation, sacrifice mice by inhaling excess CO2.
    2. Remove the liver using a scissors.
    3. Dissect the liver lobe into pieces and fix a part of the lobe in 4% paraformaldehyde (cryosections) and a part in 10% formalin saline solution (Paraffin sections).
      Note: Generally, cryosections are good for Immunohistochemistry (IHC) when compared to paraffin sections. However, cryosections may not give clear morphology than paraffin sections. In paraffin sections for IHC, it requires additional step of antigen retrieval at high degree temperature (85-95 °C). It is also possible that some of the antigens may lose during paraffin processing. Considering all these, this protocol used cryosections for the IHC and paraffin sections for picrosirus red staining.
    4. Prepare the cryosections after fixing for 2-3 days. The fixed tissue washed in PBS and keep in 30% sucrose solution for overnight (6-7 h) to get integrity of the sections.
    5. Then tissue is embedded in the tissue freezing medium for solidifying in cryochamber (cryo bar temp approx. -35 °C to -40 °C).
    6. Cut the section at 5 µm thickness and mount on the pre coated slides.
    7. Wash the slides and make border around the each section with the help of nail polish so that antibody should not move out of the sections.
    8. Permeabilize the sections with 0.1% of Triton X-100 for 15 min (3 changes at 5 min intervals).
    9. Block the sections with the serum or with general blocking solution 3% BSA for 1 h.
    10. Place primary antibody against eGFP (mouse monoclonal dilution 1:200) and incubate for 2 h. And also keep another section for isotype control for the primary antibody so that the testing antibody will not show any non-specific binding.
    11. Wash with PBST (PBS containing 0.01% Triton X-100) 3 times.
    12. Put Secondary Alexa Fluor-conjugated anti-mouse 488 (1:400 dilutions - depending on the source) and incubate for 1 h.
    13. Then wash as step D11.
    14. Add DAPI 1:1,000 dilution for 10 min and wash one with PBS.
    15. Mount with Antifade and seal with nail polish. Then observe in fluorescent microscope.
    16. First, adjust the IgG-isotype control, increase the exposure time till no background colour is seen even in high exposure.
    17. Then adjust test slide and set the exposure time, capture the Images (Figure 5).
    Note: Identification of transplanted cell can be done either based on antibody based detection method or in combination with a molecular biology tools. In case of donor cells expressing eGFP or RFP and YFP protein, direct antibody based technique can be followed. This above protocol uses eGFP cells as a donor cells, so Immunohistochemical analysis of tissue sections can be performed using eGFP antibody. In alternative, Western blot method can be adopted.


    Figure 5. Donor cells engraftment after 30 days of transplantation. Upper row shows the immunohistochemistry of transplanted liver tissue section stained with DAPI and GFP antibody indicating the presence of donor cells (See merged photo of DAPI and GFP at the upper right). Lower row is the Isotype control for the same tissue sections showing the absence of non-specific binding of the GFP antibody (See the merged photo of DAPI and GFP at the lower right). Scale bars = 200 µm (200x).

  5. Detection of the fate of donor cells
    1. Follow ‘D’ till step D9, mix appropriately diluted primary antibodies against eGFP (raised in mouse monoclonal) and albumin (raided in goat polyclonal). Don’t forget to make IgG-isotype control.
    2. Follow step D11.
    3. Add secondary antibody AF anti-mouse 488 (1:400 dilutions - depending up on the source) and AF anti-goat 594 (1:400 dilutions - depending up on the source for 1 h)
    4. Then wash as step D11, and follow steps D14-D17. Take separate images of blue (DAPI), green (eGFP) and red (albumin) channel images, then merge them by using Adobe Photoshop or similar program (Figure 6).
    Note: The CD45+ cells are isolated from eGFP mice. This will make easy to identify or track the cells in the host tissue by IHC. The fate of the transplanted cells can be studied by combination staining with donors and potential target cells. For instance, one can perform simultaneously double or triple colour staining of three different antigens and evaluate the fate of the cells. Keeping eGFP antigen as common one can stain various combination with other antigens, for example, albumin (here the target cells are hepatocytes) or vWF/CD31 (here the target cells are endothelial) or F4/80 (here the target cells are macrophages) or CK19 (here the target cells are biliary epithelial cells. For double or triple colour staining, one should use the primary antibodies that have not been raised in same animals. In this study we use eGFP which is raised in mouse and with albumin-specific antibodies which is raised in the goat (both has reactivity in mouse) to detect the engrafted cells that would have differentiated into hepatocytes.


    Figure 6. Donor cells (eGFP) differentiated into hepatocytes, expressing albumin. Upper row shows the Immunohistochemistry of transplanted liver tissue section double stained with GFP and albumin antibodies indicating the presence of donor cells that expressing albumin (See merged photo of DAPI, GFP and albumin at the upper right). Lower row is the Isotype control for the same tissue sections showing the absence of non-specific binding of the GFP and albumin antibodies (See the merged photo of DAPI and GFP at the lower right). Scale bars = 200 µm (200x).

Data analysis

Data analysis has been reported as mean ± SEM. Student’s t-test was carried out to calculate the significance. The number of mice used for different experiments for creation of fibrosis model and intraspleenic transplantation is presented in the original publication (Baligar et al., 2016).

Notes

  1. During creation of the fibrotic model, strictly follow regular intervals of CCl4 treatment, that is twice a week till the end of the experiment.
  2. Continue CCl4 dose (half) even after the cell transplantation till the end of the experiment, otherwise recovery of live due to cell transplantation will be confused with spontaneous regression of fibrosis after discontinuation of CCl4 injection.
  3. Intrasplenic transplantation needs one more person to help during spleen knot and cells infusion.
  4. Avoid more pulling of the spleen, as it is connected with fat and blood vessels.
  5. Don’t remove the needle suddenly after cells infusion is completed. It helps to prevent back flow of cells.
  6. Don’t remove the knot suddenly after removing the needle. It helps to avoid back flow.

Recipes

  1. CCl4 dose preparation
    1 ml CCl4
    5 ml mineral oil
    Notes:
    1. Take CCl4 oil with the help of glass pipette (use pipette man) and add to the 5 ml mineral oil which was taken in the glass vial. Then mix gently.
    2. Don’t use plastic pipettes or vials.
  2. Gey’s solution
    1. Solution A
      3.5 g NH4Cl
      0.185 g KCl
      0.15 g Na2HPO4
      0.012 g KH2PO4
      0.5 g glucose
      2.5 g gelatin
      0.005% phenol red
      100 ml Milli Q water
      Note: Autoclave and store at 4 °C.
    2. Solution B
      0.42 g MgCl2·6H2O
      0.14 g MgSO4·7H2O
      0.34 g CaCl2
      100 ml Milli Q water
      Note: Autoclave and store at 4 °C.
    3. Solution C
      2.25 g NaHCO3
      100 ml Milli Q water
      Note: Do not autoclave, syringe filter and store at 4 °C.
      Autoclave solution A and B, then mix 20 ml of A, 5 ml of B and 5 ml of C (syringe filtered).

Acknowledgments

We are thankful to Department of Biotechnology (DBT), Government of India for generous support in the Center for Molecular Medicine programme.

References

  1. Baligar, P., Mukherjee, S., Kochat, V., Rastogi, A. and Mukhopadhyay, A. (2016). Molecular and cellular functions distinguish superior therapeutic efficiency of bone marrow CD45 cells over mesenchymal stem cells in liver cirrhosis. Stem Cells 34(1): 135-147.
  2. Chang, M. L., Yeh, C. T., Chang, P. Y. and Chen, J. C. (2005). Comparison of murine cirrhosis models induced by hepatotoxin administration and common bile duct ligation. World J Gastroenterol 11(27): 4167-4172.
  3. Group FMCS. (1994). Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Cooperative Study Group. Hepatology 20(1 Pt 1): 15-20.
  4. Ishak, K., Baptista, A., Bianchi, L., Callea, F., De Groote, J., Gudat, F., Denk, H., Desmet, V., Korb, G., MacSween, R. N., Phillips, M.J., Portmann, B.G., Poulsen, H., Scheuer, P.J., Scheuer, M. and Thaler, H. (1995). Histological grading and staging of chronic hepatitis. J Hepatol 22(6): 696-699.

材料和试剂

  1. 猎鹰管
    15ml(Corning,Falcon ,目录号:352097) 50ml(Corning,Falcon ,目录号:352070)
  2. 培养皿(Corning,目录号:430167)
  3. 移液器吸头(Eppendorf,目录号:022491954)
  4. 手术玻片(厚度:1.45mm; 75×25mm& 76×26mm)(Polar Industrial,目录号:Blue Star PIC 2)
  5. BD结核菌素注射器1ml(BD,目录号:309623)
  6. 细胞过滤器(40μm)(Corning,Falcon ,目录号:352340)
  7. 培养皿(30mm,100mm)
  8. 六至八周龄雄性C57BL/6J(THE JACKSON LABORATORY,目录号:000664-C57BL/6J)
  9. 增强的绿色荧光蛋白(eGFP)转基因小鼠[C57BL/6-Tg(UBC-GFP) Scha/J](THE JACKSON LABORATORY,目录号:004353)
  10. 四氯化碳(CCl 4)(Sigma-Aldrich,目录号:319961)
  11. 矿物油(Sigma-Aldrich,目录号:M8410)
  12. CO <2>气体
  13. 福尔马林(Thermo Fisher Scientific,目录号:SF98-4)
  14. 异丙醇(Thermo Fisher Scintific,目录号:A416S-4)
  15. 二甲苯(Thermo Fisher Scintific,目录号:X3S)
  16. 石蜡(EMD Millipore,目录号:107151)
  17. Picrosirus Red染色试剂盒(Polysciences,目录号:24901-250)
  18. Dulbecco's修饰的Eagle's培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:12100-046)
  19. FCS
  20. 磷酸盐缓冲盐水(PBS)(HiMedia Laboratories,目录号:TS1099)
  21. 抗CD45 APC( affymetrix ,eBioscience,目录号:17-0451)
  22. 70%乙醇
  23. Ketamin(nirlife healthcare,目录号:Ketamin)
  24. 赛拉嗪(INDIAN IMMUNOLOGICALS,目录号:21)
  25. 10%福尔马林盐水溶液
  26. PBS
  27. 蔗糖(Thermo Fisher Scintific,目录号:S5)
  28. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P4707)
  29. 指甲油
  30. 氯胺酮
  31. 赛拉嗪
  32. Triton X-100(HiMedia Laboratories,目录号:9002-93-1)
  33. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  34. 胎牛血清(FBS)(Biological Industries,目录号:04121-1A)
  35. DAPI
  36. 同种型小鼠IgG(Jackson ImmunoResearch,目录号:015-000-003)
  37. 同种型山羊IgG(Jackson ImmunoResearch,目录号:005-000-003)
  38. 抗GFP抗体(Takara Bio,目录号:632381)
  39. 抗白蛋白抗体(Bethyl Laboratories,目录号:A90-134B)
  40. Alexa Fluor驴抗小鼠488(Thermo Fisher Scientific,Invitrogen,目录号:A-21202)
  41. Alexa Fluor驴抗山羊594(Thermo Fisher Scientific,Invitrogen,目录号:11058)
  42. Antifade(Thermo Fisher Scientific,Molecular probes TM ,目录号:P36961)
  43. 氯化铵(NH 4 Cl)(HiMedia Laboratories,目录号:TC092)
  44. 氯化钾(KCl)(Thermo Fisher Scintific,目录号:13305)
  45. 磷酸二氢钾(KH 2 PO 4)(Central Drug House,目录号:029608)
  46. 磷酸氢二钠(Na 2 HPO 4)(Thermo Fisher Scintific,目录号:S379)
  47. Glocose(Thermo Fisher Scintific,目录号:D16)
  48. 明胶(Sigma-Aldrich,目录号:G9664)
  49. 酚红(Thermo Fisher Scintific,目录号:P7410)
  50. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-aldrich,目录号:M2670)
  51. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-aldrich,目录号:230391)
  52. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  53. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  54. 组织冷冻培养基(Leica Biosystems Nussloch,目录号:14020108926)
  55. CCl 4剂量准备(参见配方)
  56. Gey的解决方案(参见配方)

设备

  1. 组织处理盒
  2. 烤箱(科学系统公司)
  3. 纯石蜡罐
  4. 嵌入机器
  5. 层流(Esco Micro Pte,型号:AC2-2E9)
  6. 剪刀(2)
  7. 钝镊子(2)
  8. 手术刀的外科刀片
  9. 止血钳
  10. 缝合线
  11. 缝合针
  12. 解剖板
  13. FACS Aria TM III/III(BD,型号:BD FACSARIA III)
  14. 孵育器(SHEL LAB,型号:SCO5A)
  15. 邮递(Eppendorf)
  16. 发光(SAFEMAX)
  17. 离心机(Eppendorf,型号:5810R)
  18. Cryotome(Thermo Fisher Scientific,型号:CryoStar NX50 Cryostat)
  19. 超薄半自动切片机(Histo Line Laboratories,型号:MRS 3500)

软件

  1. Adobe Photoshop

程序

  1. 实验肝纤维化模型
    1. 纤维化小鼠模型的发展
      1. 通过在矿物油中重复注射CCl 4(0.8ml/kg体重),每周两次,持续8周(16个剂量),在C57B16/J小鼠中建立肝纤维化模型通过腹膜内注射,如图1所示。
      2. 从eGFP转基因小鼠分离CD45 +细胞,并通过脾内途径移植。
      3. 移植3天后,CCl 4剂量减少至一半,并持续到实验结束以避免纤维化的自发消退。
      4. 移植4周后,将小鼠在过量的CO 2气体中处死,储存血清样品用于肝功能测试,并将肝组织在10%福尔马林盐水中固定48-72小时用于组织学分析。


        图1.具有纤维化模型发展的时间线和剂量的示意图。 B wt。 - 体重; Tx - 移植
    2. 用于石蜡切片的组织加工
      1. 取固定的组织,放入组织处理盒中,并用铅笔的帮助,根据实验正确标记
      2. 用系列的异丙醇级配(30%,50%,70%,90%和100%,每个步骤30分钟间隔)使它们脱水。
      3. 将组织置于1:1异丙醇和二甲苯中30分钟。
      4. 置于纯二甲苯中1小时。
      5. 所有组织盒在60℃烘箱中浸渍在1:1二甲苯和石蜡溶液中30分钟。
      6. 将盒子放入纯石蜡容器1,然后2和3在60℃烘箱中的每个步骤1小时。
      7. 然后在嵌入机的??帮助下嵌入钢或塑料模具中。如果在室温下正确储存,这些块可以永久使用。
      8. 然后将5微米切片切成浮动槽(45-50℃),并取到聚赖氨酸包被的载玻片上。在室温下完全风干,并用于染色(如果储存适当,切片可以保存2-3个月,否则会发生抗原性丧失,不利于免疫组织化学)。
    3. 纤维化测量
      1. 纤维化可以通过微红霉素红染色测量。切片切片进行再水化,按照试剂盒制造商提供的方案进行微生物染色处理
      2. 在用微红斑病毒红染色后,通过使用Ishak等确定纤维化。 (1995)评分系统或METAVIR(FMCS,1994)评分系统(表1)。用Picrosirus染色的肝切片的代表性图像显示在图2中 注意:纤维化可以通过许多方法通过不同的染色技术例如Masson三色染色,Picrosirus红色染色,用抗体的胶原染色等来测量。此外,使用NIH ImageJ软件在Picrosirus染色切片中胶原比例区域的百分比,可以通过量热法定量估计肝组织中的羟脯氨酸,以测量纤维化的程度
        Table1。 Ishak评分和METAVIR评分系统



        图2.在CCl 4存在下肝脏的病理学变化。 A.没有CCl 4剂量; B.CCl 4(0.8ml/kg体重)在矿物油中的溶液,每周两次,持续4周,并在最后一次给药后3天收获; (0.8ml/kg体重)在矿物油中的溶液,每周两次,持续8周,并且一半CCl 4剂量(0.4ml/kg体重) Kg体重,每周两次)持续到实验结束(进一步4周); (0.8ml/kg体重)在矿物油中的溶液,每周两次,持续8周,并且停止给药另外4周,然后收获(评分1)。比例尺A,C,D =400μm,B =200μm(100x)。 E.代表图像A,B,C和D的Ishak和METAVIR评分系统
  2. 骨髓CD45 + 细胞分离
    1. 通过吸入过量的CO 2将6至8周龄的eGFP转基因小鼠处死,然后浸入70%异丙醇中1分钟以避免来自皮肤的污染。
    2. 将鼠标带到层流罩并放置在解剖板上。
    3. 在一条剪刀的帮助下,在树干的中部朝向和沿着尾部的两侧切开皮肤,使得将容易将皮肤拉向在爪踝关节处解剖出的爪和爪,以及去皮皮肤。然后在后肢和躯干之间的连接处解剖出后肢。
    4. 使用无菌手术刀轻轻地将肌肉,韧带和肌腱与胫骨和股骨分离,并且还擦洗以去除残留的组织部分。
    5. 通过在相反方向上弯曲分离胫骨和股骨,并转移到含有含有3%FCS的DMEM的30mm培养皿中。
    6. 对其他后肢重复步骤B4和B5。
    7. 用无菌镊子夹住骨,用微解剖剪刀切除两端的尖端
    8. 将具有含有DMEM的针的BD结核菌素注射器插入骨的切割尖端,并且缓慢地通过骨腔冲洗到已经包含5ml具有3%FCS的DMEM的50ml Falcon管中。从骨的两端重复两次或直到骨变白。
    9. 对所有骨头重复步骤B7和B8。图3描述了细胞分离的整个过程 注意:骨髓细胞在两个阶段过程中分离。在第一阶段,通过用培养基冲洗从胫骨和股骨去除细胞。在第二阶段通过用IV型胶原酶(0.03%)和分散酶(2U/ml)消化碎骨来回收骨内膜区域的牢固粘附的细胞(主要是基质细胞和原始干细胞)。通过执行这两个阶段产生更高数量的细胞(Baligar等,2016)。
    10. 在259×g离心5分钟,弃去上清液。
    11. 通过用Gey's溶液处理如下裂解红细胞,每小鼠骨髓细胞沉淀加入5ml Gey's溶液,并用移液管尖端轻轻悬浮90秒,然后加入PBS至50ml以稀释Gey的解决方案。
      注意:Gey的治疗用于溶解RBC。治疗的时间是重要的,因为不充分的治疗期导致RBC的不适当裂解。如果超过允许的治疗时间,它可能杀死靶细胞。每只小鼠骨髓使用5毫升试剂。在骨髓冲洗后,用培养基洗涤并加入5ml(在从2或3只小鼠收获更多数量的细胞的情况下,因此Falcon管数目??增加),在细胞沉淀上,用移液管轻轻混合正好90秒。立即用45ml PBS稀释并离心(259×g,5分钟),然后用培养基洗涤。
    12. 在259×g离心5分钟,弃去上清液,在培养基中洗涤沉淀。
    13. 通过40μm细胞过滤器过滤细胞悬浮液。
    14. 然后使用血细胞计数器计数细胞,并用于抗CD45抗体染色
    15. CD45 +细胞通过使用FACS AriaIII(70mm喷嘴)技术分选。在这里,我们使用FACS AriaIII通过用抗CD45 APC抗体表面抗原标记来分选CD45阳性细胞(Baligar等人,2016)。


      图3.从股骨和胫骨分离BM干细胞 A.将处死的小鼠浸入70%(v/v)乙醇中1-2分钟。 B.将鼠标放在生物安全柜的侧面,放在解剖板上。仔细去除皮肤。 D.在踝关节解剖出的爪子。 E.将皮肤移除的腿保存在100mm无菌培养皿中。 F.去除肌肉,韧带和腱。 G.将胫骨和股骨切开并保存在完全培养基中。 H.将BD结核菌素注射器针引入骨腔,并缓慢地将骨髓排出到含有5-10ml完全培养基的50ml Falcon管中。骨腔从两端再次洗涤两次,直到骨头变白。

  3. 细胞移植
    1. 移植前,所有手术材料,缝合胎面,缝合针,棉花应高压灭菌并布置在层流罩中。预先安排约10厘米长的单独的缝合线,用于结扎脾脏。
    2. 通过腹膜内注射用氯胺酮(100mg/kg体重)和甲苯噻嗪(10mg/kg体重)麻醉动物。
    3. 转动动物左侧,并在尾肋下方刮下2-3厘米2的区域(图4A)。
    4. 用70%异丙醇消毒剃须区域。
    5. 使用剪刀(视频1)将背侧肋骨下方的皮肤切开深侧0.5 cm处(视频1)。
    6. 用小钝端钳(视频1)小心地提起脾脏。
    7. 将预先布好的结扎物放在脾脏上,松开地结扎在脾脏周围的尖端,而不握住脂肪组织(不要拉脾脏),如图4B(视频1)所示。
    8. 将装有细胞的注射器插入结扎前的脾尖处,并在通过针头后拧紧结以避免回流(避免脾脏穿孔,然后渗漏)(图4D,视频1)。
    9. 在1-2分钟内缓慢注入细胞悬液(50-80μl),直到完全注射(如视频1所示,轻轻地注入细胞以避免窒息)。
    10. 输液后不要立即取下针头;等待2-3分钟以避免回流(视频1)。
    11. 然后取出针头并将结扎紧紧2-3分钟(视频1)。
    12. 取出螺纹并将脾脏重新放置在腔体中。
      注意:步骤C9-C12是关键步骤,因为细胞应该缓慢注入以避免静脉阻塞。通过所有细胞悬浮液后,不要突然取出针头,如果你突然去除,将有机会回流细胞和出血。因此,至少保持针在脾脏内至少2-4分钟,使得脾脏中的压力被释放,并且在穿刺部位将发生凝结。慢慢地除去针,然后轻微收紧结至少1-2分钟。它还将有助于穿刺部位的回流和凝血。然后慢慢地去除结,确保不应有任何流体的回流。
    13. 关闭腹部腹膜,然后单独用可降解的无菌缝合皮肤
    14. 通过皮下注射1ml无菌盐水溶液(5ml/100g体重,图4E)使动物再水化。


      图4.干细胞的脾内移植 A.转动动物的左侧,并刮除尾部肋骨下的一个区域。 B.和C.脾脏的暴露和与线的连接。 D.将针插入脾中,然后收紧线并将细胞注入脾脏。 E.缝合闭合腹膜和皮肤。 F.手术后皮下注射生理盐水进行再水合
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      视频1.显示细胞的脾内移植(步骤C5-C11)
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    15. 避免术后镇痛,以尽量减少潜在的爆发性肝功能衰竭
    16. 在手术后立即将动物保持在预热笼中,并允许自由获得水和食物。然后在动物架中移动笼子进行随访。

  4. 通过免疫组织化学技术(IHC)检测移植的细胞
    1. 移植30天后,通过吸入过量的CO 2处死小鼠。
    2. 用剪刀去除肝脏。
    3. 解剖肝叶成碎片,并固定在4%多聚甲醛(冷冻切片)和一部分在10%福尔马林盐水溶液(石蜡切片)叶的一部分。
      注意:通常,与石蜡切片相比,冷冻切片对于免疫组织化学(IHC)是有益的。然而,冷冻切片可能不能给出清晰的形态比石蜡切片。在用于IHC的石蜡切片中,其需要在高温度(85-95℃)下进行额外的抗原修复步骤。还可能的是,一些抗原在石蜡加工过程中可能损失。考虑到所有这些,本协议使用IHC的冷冻切片和石蜡切片用于微红斑病毒红染色。
    4. 固定后准备冷冻切片2-3天。固定的组织在PBS中洗涤并保持在30%蔗糖溶液中过夜(6-7小时)以获得切片的完整性。
    5. 然后将组织嵌入组织冷冻介质中,用于在冷冻箱(冷冻棒温度约-35℃至-40℃)中固化。
    6. 切割厚度为5μm的部分,并安装在预涂的载玻片上。
    7. 洗涤幻灯片,并在指甲油的帮助下,使每个部分的边框,使抗体不应移出部分。
    8. 使用0.1%的Triton X-100进行15分钟的渗透处理(3次间隔5分钟)。
    9. 用血清或一般封闭溶液3%BSA封闭切片1小时
    10. 放置针对eGFP的一抗(小鼠单克隆稀释1:200),孵育2小时。并且保留另一部分用于初级抗体的同种型对照,以使测试抗体不显示任何非特异性结合。
    11. 用PBST(含0.01%Triton X-100的PBS)洗涤3次
    12. 将次要的Alexa Fluor缀合的抗小鼠488(1:400稀释 - 取决于来源)并孵育1小时。
    13. 然后按步骤D11清洗。
    14. 加入DAPI 1:1,000稀释10分钟,并用PBS洗涤一次
    15. 装有防褪色和密封与指甲油。然后在荧光显微镜下观察
    16. 首先,调整IgG同种型对照,增加曝光时间,直到没有背景颜色,即使在高曝光。
    17. 然后调整测试幻灯片并设置曝光时间,捕获图像(图5)。
    注意:移植细胞的鉴定可以基于基于抗体的检测方法或与分子生物学工具组合进行。在供体细胞表达eGFP或RFP和YFP蛋白的情况下,可以遵循基于直接抗体的技术。上述方案使用eGFP细胞作为供体细胞,因此可以使用eGFP抗体进行组织切片的免疫组织化学分析。或者,可以采用Western印迹方法。


    图5.移植30天后供体细胞移植。上行显示用DAPI和GFP抗体染色的移植的肝组织切片的免疫组织化学,表明存在供体细胞(参见DAPI和GFP的合并照片右上)。下行是相同组织切片的同种型对照,显示不存在GFP抗体的非特异性结合(参见右下侧的DAPI和GFP的合并照片)。比例尺=200μm(200x)。

  5. 检测供体细胞的命运
    1. 按照"D"直到步骤D9,混合适当稀释的一抗针对eGFP(在小鼠单克隆中产生)和白蛋白(在山羊多克隆中进行)。不要忘记进行IgG同种型控制。
    2. 按照步骤D11。
    3. 加入第二抗体AF抗小鼠488(1:400稀释 - 取决于来源)和AF抗山羊594(1:400稀释 - 取决于来源1小时)。
    4. 然后作为步骤D11进行洗涤,并且遵循步骤D14-D17。分别拍摄蓝色(DAPI),绿色(eGFP)和红色(白蛋白)通道图像,然后使用Adobe Photoshop或类似程序合并(图6)。
    注意:CD45 + 细胞是从eGFP小鼠中分离的。这将容易通过IHC鉴定或追踪宿主组织中的细胞。移植细胞的命运可以通过与供体和潜在靶细胞的组合染色来研究。例如,可以同时对三种不同抗原进行双色或三色染色,并评估细胞的命运。保持eGFP抗原是常见的可以用其他抗原,例如白蛋白(这里靶细胞是肝细胞)或vWF/CD31(这里靶细胞是内皮的)或F4/80(这里靶细胞是巨噬细胞)染色各种组合,或CK19(这里的靶细胞是胆管上皮细胞)对于双重或三重染色,应该使用未在同一动物中产生的一抗。在本研究中,我们使用在小鼠中产生的eGFP,在山羊中产生的抗体(在小鼠中都具有反应性)来检测将分化成肝细胞的植入细胞。


    图6.分化为肝细胞,表达白蛋白的供体细胞(eGFP)。上行显示用GFP和白蛋白抗体双重染色的移植的肝组织切片的免疫组织化学,表明存在表达白蛋白的供体细胞。合并照片的右上角的DAPI,GFP和白蛋白)。下行是相同组织切片的同种型对照,显示不存在GFP和白蛋白抗体的非特异性结合(参见右下侧的DAPI和GFP的合并照片)。比例尺=200μm(200x)。

数据分析

数据分析报告为平均值±SEM。进行Student's t 测试以计算显着性。用于产生纤维化模型和脾内移植的不同实验的小鼠的数目在原始出版物中呈现(Baligar等人,2016)。

笔记

  1. 在创建纤维化模型期间,严格遵循CCl 4处理的定期间隔,即每周两次,直到实验结束。
  2. 即使在细胞移植后直到实验结束,仍然继续CCl 4剂量(一半),否则由于细胞移植导致的活的恢复将与CCl 4停止后纤维化的自发消退混淆注射
  3. 脾肿瘤移植需要一个人在脾结和细胞输注期间帮助
  4. 避免更多的拉脾,因为它与脂肪和血管相连
  5. 细胞输注完成后,不要突然取下针头。它有助于防止细胞的回流。
  6. 取下针后不要突然打结。它有助于避免回流。

食谱

  1. CCl 4剂量准备
    1ml CCl 4
    5ml矿物油
    注意:
    1. 借助于玻璃移液管(使用移液管人)取CCl 4油,并加入放入玻璃小瓶中的5ml矿物油。然后轻轻混合。
    2. 不要使用塑料移液管或小瓶。
  2. Gey的解决方案
    1. 解决方案A
      3.5g NH 4 Cl
      0.185克KCl
      0.15g Na 2 HPO 4
      0.012g KH 2 PO 4 sub/
      0.5克葡萄糖 2.5克明胶
      0.005%酚红
      100ml Milli Q水
      注意:高压灭菌并在4°C保存。
    2. 解决方案B
      0.42g MgCl 2·6H 2 O·dm / 0.14g MgSO 4·7H 2 O·h/v 0.34g CaCl 2
      ml Milli Q水
      注意:高压灭菌并在4°C保存。
    3. 解决方案C
      2.25g NaHCO 3水溶液 100ml Milli Q水
      注意:不要用高压灭菌器,注射器过滤器,并在4℃保存。
      高压灭菌溶液A和B,然后混合20ml的A,5ml的B和5ml的C(注射器过滤)。

致谢

我们感谢印度政府的生物技术部(DBT)对分子医学中心计划的慷ous支持。

参考文献

  1. Baligar,P.,Mukherjee,S.,Kochat,V.,Rastogi,A.和Mukhopadhyay,A.(2016)。  分子和细胞功能区分了骨髓CD45细胞在肝硬化中比间充质干细胞优越的治疗效力。干细胞 34(1):135-147
  2. Chang,ML,Yeh,CT,Chang,PY and Chen,JC(2005)。  通过施用肝毒素和胆总管结扎诱导的鼠肝硬化模型的比较。世界胃肠病学杂志11(27):4167-4172。
  3. 组FMCS。 (1994)。  肝活检解释中的观察者和观察者之间的差异在慢性丙型肝炎患者中。法国METAVIR合作研究组。肝实质 20(1 Pt 1):15-20。
  4. Ishak,K.,Baptista,A.,Bianchi,L.,Callea,F.,De Groote,J.,Gudat,F.,Denk,H.,Desmet,V.,Korb,G.,MacSween,RN, Phillips,MJ,Portmann,BG,Poulsen,H.,Scheuer,PJ,Scheuer,M.and Thaler,H。(1995)。  慢性肝炎的组织学分级和分期。 J Hepatol 22(6):696-699。
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How to cite this protocol: 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; Full Text



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