A kind of spleen-regulated B lymphocyte and its preparation method and application

A technology of B lymphocytes and spleen cells, applied in the field of cell biology, can solve problems such as glomerulonephritis and interstitial fibrosis that cannot be universally applied, and achieve good therapeutic potential, reduced thinning, and tubulointerstitial fibrosis lessening effect

Active Publication Date: 2022-07-29
THE FIRST MEDICAL CENT CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Given the complex phenotypes of B lymphocyte subsets, B lymphocyte depletion therapy targeting a single surface antigen may not be universally applicable to all types of glomerulonephritis and its induced interstitial fibrosis

Method used

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  • A kind of spleen-regulated B lymphocyte and its preparation method and application
  • A kind of spleen-regulated B lymphocyte and its preparation method and application
  • A kind of spleen-regulated B lymphocyte and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This example provides a method for sorting the regulatory B lymphocytes positive for CD1d, CD5 and CD21 / 35 antibodies in mouse spleen.

[0038] Step 1: Harvest mouse spleen cells. Using freshly obtained mouse spleen, on ice, use pre-cooled sharp ophthalmic scissors to cut the spleen tissue into 1mm 3 Fragments were filtered through a 100 μm nylon mesh and cells were resuspended in RPMI1640 cell culture medium.

[0039] Step 2: Surface antibody-labeled mouse spleen cells. The cells obtained in step 1 were subjected to flow cytometry surface antibody staining in the Staining Buffer (eBioscience) system; staining markers: CD1d, CD5 and CD21 / 35; staining incubation conditions: room temperature, dark, 20 minutes.

[0040] Step 3: FACS sorting target cells. The cells stained in step 2 were immediately subjected to FACS cell sorting on the MoFlo XDP ultra-fast flow cytometry sorting system (Beckman Coulter) to sort CD1d, CD5 and CD21 / 35 antibody-positive regulatory B lympho...

Embodiment 2

[0043] This example provides the use of regulatory B lymphocytes in adoptive transfer to the kidney. This application involves the adoptive transfer of regulatory B lymphocytes and the tracking and observation of their distribution in the kidney.

[0044] Step 1: Obtain spontaneous red fluorescent mT / mG mouse-derived spleen CR1 + Breg cells. The sorting method of the cells is the same as that in Example 1.

[0045] Step 2: Spleen CR1 from mT / mG mice with auto red fluorescence + Adoptive transfer of Breg cells. Spleen CR1 from mT / mG mice with auto red fluorescence + The Breg cells were adoptively transferred by the left renal vein injection method.

[0046] Step 3: Detect the fluorescent signal after adoptive transfer. At 15 minutes after the injection procedure in step 2, the bilateral kidneys were exposed from the abdomen, the renal capsule was removed with microtweezers, and CR1 was observed with a two-photon microscope while keeping the animal alive. + Distribution ...

Embodiment 3

[0049] This example provides the application of regulatory B lymphocytes in kidney protection. Specifically, it relates to the application of regulatory B lymphocytes in adoptive transfer therapy for experimental renal tubulointerstitial fibrosis.

[0050] Step 1: Obtain spleen CR1 + Breg cells. The sorting method of the cells is the same as that in Example 1.

[0051] Step 2: Construction of UUO model mice. UUO model mice were obtained by unilateral ureteral ligation.

[0052] Step 3: Spleen CR1 + Adoptive transfer of Breg cells. Immediately after the ureteral ligation procedure in step 2, 5 x 10 5 CR1 + Breg cells were injected into the obstructed kidney of UUO model mice through renal vein, and gauze was used to stop bleeding for 3 minutes after injection.

[0053] Step 4: Staining observation. Step 3: On the 7th day after treatment, the obstructed side kidney of the mouse was taken, and the renal tissue paraffin pathological section was prepared, with a thickne...

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Abstract

The invention provides a spleen-regulated B lymphocyte and a preparation method and application thereof. One aspect of the present invention provides a spleen regulatory B lymphocyte, in which the proportion of CD1d, CD5 and CD21 / 35 antibody positive regulatory B lymphocytes is more than 94.6%. The second aspect of the present invention provides a method for preparing spleen-regulated B lymphocytes. The method for preparing spleen-regulated B lymphocytes includes the following steps: taking a spleen cell suspension and labeling with surface antibodies CD1d, CD5 and CD21 / 35; and sorting the labeled cells by fluorescence activated cell sorting. The third aspect of the present invention provides the application of the above-mentioned spleen-regulated B lymphocytes in the preparation of a preparation targeted for colonization in bilateral kidneys. The fourth aspect of the present invention provides the application of the above-mentioned spleen-regulated B lymphocytes in the preparation of a product for renal protection.

Description

technical field [0001] The invention relates to a spleen-regulated B lymphocyte and a preparation method and application thereof, in particular to a fluorescence-activated cell sorting technique, which belongs to the field of cell biology. Background technique [0002] In recent years, the role of B lymphocytes in fibrosis has gradually attracted attention. B lymphocyte depletion therapy has a potential regulatory effect on the composition of immune cell subsets and the expression of fibroblast phenotype, which may be one of the ways of its anti-fibrotic effect. [0003] Considering the complexity of B lymphocyte subset phenotypes, B lymphocyte depletion therapy targeting a single surface antigen may not be universally applicable to all types of glomerulonephritis and its induced interstitial fibrosis. The reason why B lymphocyte depletion therapy targeting a single surface antigen, especially a single broad-spectrum surface antigen, is not applicable in some diseases and p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0781G01N33/569A61K35/17A61P13/12
CPCC12N5/0635G01N33/56966A61K35/17A61P13/12C12N2509/00G01N2469/10
Inventor 朱凤阁陈香美蔡广研傅博张欢平陈欠欠
Owner THE FIRST MEDICAL CENT CHINESE PLA GENERAL HOSPITAL
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