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Preparation method and application of regulatory T cell

A regulatory and cellular technology, applied in the field of molecular biology and biomedicine, can solve problems such as poor effect, and achieve the effect of prolonging receptor survival and alleviating acute AMR.

Inactive Publication Date: 2017-12-29
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current clinical treatment of AMR in transplanted kidneys is generally not effective. Once acute AMR occurs, 15-20% of recipients will experience graft failure within 1 year; regardless of whether it can be reversed by current anti-rejection therapy, more than 40 % of patients with acute AMR will continue to develop into chronic AMR, and once chronic AMR is diagnosed, the 5-year transplant kidney survival rate is often lower than 50% (A.Djamali, D.B.Kaufman, T.M.Ellis et al.Diagnosis and management of antibody-mediated rejection: current status and novel approaches. Am JTransplant, 2014, 14(2): 255-271; Qiquan Sun, Yang Yang. Late and Chronic Antibody-Mediated Rejection: Main Barrier to Long Term Graft Survival. Clinical and Developmental Immunology, 2013, 2013: 1 -7; M.H.Levine, P.L.Abt. Treatment options and strategies for antibody mediated rejection after renal transplantation. Semin Immunol, 2012, 24(2): 136-142)

Method used

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  • Preparation method and application of regulatory T cell
  • Preparation method and application of regulatory T cell
  • Preparation method and application of regulatory T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Initial CD4 + Sorting of cells and induction of iTreg (taking the spleen of a 5-8w BALB / c mouse as an example)

[0088] 1. Cell preparation

[0089] 1) The mouse spleen was dissected and placed in a 60mm dish filled with a 70um filter and 2mL containing 10% FBS 1640.

[0090] 2) Grind the spleen with a 1 mL syringe, rinse the remaining cells on the syringe and filter membrane with 1640 containing 10% FBS, and pipette the cell suspension into a 15 mL centrifuge tube.

[0091] 3) Centrifuge at 300g at 4°C for 5min, discard the supernatant, add 1mL of erythrocyte lysate, resuspend the cells, let stand at room temperature for 20-30s, add about 5mL of 1640 containing 10% FBS, mix well to stop the lysis.

[0092] 4) Resuspend with 2 mL of 1640 containing 10% FBS and count (usually 5w B6 get 140 million SPcells), put on ice.

[0093] 2. One-step negative selection and sorting method

[0094] 1) Resuspend the cells to 500ul with 1640 containing 10% NCS (or FBS), a...

Embodiment 2

[0108] Example 2 Establishment and Intervention of Mouse Antibody-Mediated Acute Renal Allograft Rejection Model

[0109] Anesthesia was induced and maintained with isoflurane. The operation was performed in an SPF-grade small animal operating room, and the donor and the recipient selected C3H (H-2 k ) and BALB / c (H-2 d ) mice. For skin transplantation, the skin of the tail of the donor is transplanted on the back of the recipient, with a size of about 1x1cm. Kidney transplantation was performed 4 days after skin transplantation. The method of kidney transplantation was based on international reports and our improved method. The artery was sutured with 12-0 non-damaging nylon monofilament suture (Ningbo Lingqiao, Ningbo Medical Suture Needle Co., Ltd.) with 6 intermittent stitches, and a small segment of the donor renal artery was anastomosed end-to-side with the recipient abdominal aorta. The vein was anastomosed continuously with 12-0 nylon suture, and the donor renal ve...

Embodiment 3

[0110] Example 3 Pre-sensitization of skin transplantation to establish antibody-mediated mouse model of acute renal allograft rejection

[0111] In this model, we used C3H mice as donors and BALB / c mice as recipients. Kidney transplantation was performed on the 4th day after skin transplantation presensitization, as the presensitized group (presensitized); no presensitized skin transplantation Sensitized as a non-sensitized group (nonpresensitized). The survival of transplanted kidneys was observed, and the level of DSA in serum and histological manifestations at specific time points were detected. ) The survival curves of the pre-sensitized group and the non-sensitized group are as follows figure 1 Shown (comparison of the two survival curves, ** p figure 2 Shown (the difference in IgG level between the pre-sensitization group and the non-sensitization group was statistically significant ( * p0.05)); the transplanted kidney specimens were obtained on the 5th day after ren...

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Abstract

The invention relates to a preparation method and an application of a regulatory T cell and belongs to the field of molecular biology and biological medicine. The preparation method of the regulatory T cell comprises the following step of preparing the regulatory T cell via TGF (transforming growth factor)-beta in-vitro induction. The invention further provides the application of the regulatory T cell generated by the TGF-beta in-vitro induction in preparing (a) a medicine of preventing and treating antibody mediated transplanted kidney rejection and injuries, or (b) a medicine of improving survival of the transplanted kidney. The use of the regulatory T cell generated by the TGF-beta in-vitro induction for a mouse antibody mediated acute transplanted kidney rejection model proves that the regulatory T cell can prevent and treat the antibody mediated transplanted kidney rejection and injuries, improve the survival of the transplanted kidney and reduce inflammatory cell infiltration.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biomedicine, and specifically relates to a preparation method and application of regulatory T cells. Background technique [0002] Kidney transplantation is currently the preferred method for the treatment of end-stage renal disease (R.A.Wolfe, V.B.Ashby, E.L.Milford et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med, 1999, 341(23):1725-1730). The emergence of new immunosuppressants has significantly reduced T cell-mediated renal transplant rejection, but the incidence of antibody-mediated rejection (AMR) remains high and has been proven to be the primary cause of renal transplant failure (A.Djamali, D.B.Kaufman , T.M.Elliset al.Diagnosis and management of antibody-mediated rejection: current status and novel approaches. Am J Transplant, 2014, 14(2): 255-271; C.Gosset, C.Lefau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61P13/12A61P37/06C12N5/0783
Inventor 孙启全郑颂国廖涛薛有求赵大强李思雯
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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