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Extraction method of autologous peripheral blood stem cells used for treating type 1 diabetes mellitus

A technology of peripheral blood stem cells and extraction methods, applied in the field of stem cell extraction, can solve the problems of ineffective prevention of microvascular disease and its complications, and inconvenience in treating patients

Inactive Publication Date: 2021-05-18
上海南滨江细胞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although insulin therapy can improve the patient's glucose metabolism disorder to a certain extent, long-term insulin injection not only brings inconvenience to the patient, but also cannot effectively prevent or reverse the microangiopathy caused by diabetes and its complications. disease

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for extracting autologous peripheral blood stem cells for treating type I diabetes, comprising the following steps:

[0025] Step A1. Collect peripheral venous blood, mix it with PBS equilibrium solution in an ultra-clean workbench to obtain plasma, add human lymphocyte separation solution into a centrifuge tube after autoclaving, and slowly add the mixed plasma to the separation solution centrifuge at 25°C and 40kHz for 25min;

[0026] Step A2. After centrifugation, the supernatant is divided into 3 layers, suck out the middle buffy coat layer, add it to a centrifuge tube filled with PBS balance solution, mix well, and centrifuge at 18°C ​​and 40kHz for 5 minutes, discard the supernatant, and add red blood cells to lyse After mixing, stand still for 3 minutes, centrifuge at 1000r / min for 4min, then pour out the erythrocyte lysate in the centrifuge tube, add PBS balance solution and mix well, then centrifuge at 18°C ​​and 40kHz for 5min, retain the precipitate,...

Embodiment 2

[0037] A method for extracting autologous peripheral blood stem cells for treating type I diabetes, comprising the following steps:

[0038] Step A1. Collect peripheral venous blood, mix it with PBS equilibrium solution in an ultra-clean workbench to obtain plasma, add human lymphocyte separation solution into a centrifuge tube after autoclaving, and slowly add the mixed plasma to the separation solution centrifuge at 25°C, 45kHz for 28min;

[0039] Step A2. After centrifugation, the supernatant is divided into 3 layers, suck out the middle buffy coat layer, add it to a centrifuge tube filled with PBS balance solution, mix well, and centrifuge at 18°C ​​and 45kHz for 5 minutes, discard the supernatant, and add red blood cells to lyse After mixing, stand still for 4 minutes, centrifuge at 1000r / min for 4 minutes, then pour out the erythrocyte lysate in the centrifuge tube, add PBS balance solution and mix well, then centrifuge at 18°C ​​and 45kHz for 5 minutes, retain the preci...

Embodiment 3

[0050] A method for extracting autologous peripheral blood stem cells for treating type I diabetes, comprising the following steps:

[0051] Step A1. Collect peripheral venous blood, mix it with PBS equilibrium solution in an ultra-clean workbench to obtain plasma, add human lymphocyte separation solution into a centrifuge tube after autoclaving, and slowly add the mixed plasma to the separation solution centrifuge at 25°C, 50kHz for 30min;

[0052] Step A2. After centrifugation, the supernatant is divided into 3 layers, suck out the middle buffy coat layer, add it to a centrifuge tube filled with PBS balance solution, mix well, and centrifuge at 18°C ​​and 50kHz for 5 minutes, discard the supernatant, and add red blood cells to lyse After mixing, stand still for 5 minutes, centrifuge at 1000r / min for 4 minutes, then pour out the erythrocyte lysate in the centrifuge tube, add PBS balance solution and mix well, then centrifuge at 18°C ​​and 50kHz for 5 minutes, retain the preci...

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PUM

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Abstract

The invention discloses an extraction method of autologous peripheral blood stem cells used for treating type 1 diabetes mellitus. The extraction method includes the following steps: step A1, collecting peripheral venous blood to mix with a PBS balanced solution in a clean bench so as to obtain plasma; step A2, supernatant being divided into three layers after centrifugation, sucking the middle buffy coat, adding the buffy coat in a centrifuge tube holding the PBS balanced solution, adding a PBMC culture medium, and adding the mixture in a 24-well culture plate after uniform mixing, each well being 1 mL; step A3, counting mononuclear cells with counterstar cell counting chambers every day, resuspending cells with the PBMC culture media, and adding 0.5 mL in the 24-well culture plate for each well; and step A4, removing the PBMC culture media after resuspending is finished, and performing continuous rinsing with normal saline for 3 times; and performing induction culture in a 37 DEG C incubator with 5% of CO2 for 48-96 h so as to obtain the autologous peripheral blood stem cells used for treating type 1 diabetes mellitus.

Description

technical field [0001] The invention belongs to the technical field of extracting stem cells, and in particular relates to an extracting method of autologous peripheral blood stem cells for treating type I diabetes. Background technique [0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. Diabetic patients have disorders of glucose metabolism due to insulin production disorders or insulin failure to exert physiological functions, resulting in microvascular disease, causing a series of serious complications such as limb necrosis, blindness and renal failure. Currently there is no cure for diabetes, but diabetes can be controlled through a variety of treatments, and insulin therapy is one of the effective methods. Although insulin therapy can improve the patient's glucose metabolism disorder to a certain extent, long-term insulin injection not only bri...

Claims

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Application Information

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IPC IPC(8): C12N5/0789A61P3/10
CPCC12N5/0647A61P3/10C12N2509/00C12N2509/10C12N2500/38
Inventor 占震锋李庆静
Owner 上海南滨江细胞生物科技有限公司