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Method of differentiating human bone marrow matrix stem cell to form insulin cell

A stem cell differentiation and islet cell technology, applied in artificial cell constructs, bone/connective tissue cells, biochemical equipment and methods, etc., can solve the problems of low number of stem cells, transplant rejection, low differentiation efficiency, etc., and achieve rich sources. , Good repeatability, short time effect

Inactive Publication Date: 2005-04-27
陈立波 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the islet cells formed by the differentiation of these stem cells have significant defects: (1) These stem cells are not from the diabetic patient himself, and if transplanted into the patient's body, it will inevitably cause transplant rejection
(2) The number of these stem cells is small, the differentiation efficiency is low, and the formed islet cells may not meet the needs of clinical islet cell transplantation for treating diabetes

Method used

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  • Method of differentiating human bone marrow matrix stem cell to form insulin cell
  • Method of differentiating human bone marrow matrix stem cell to form insulin cell
  • Method of differentiating human bone marrow matrix stem cell to form insulin cell

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Take 0.1-4 ml of human fresh bone marrow under aseptic conditions, blow and beat it into a single-cell suspension with medium L-DMEM or RPMI1640 or HAM or Fischer containing 0.5-20% FSC, filter it with a 200-mesh sieve, Adjust the cell density according to 1×10 9 / L density inoculation, 37°C, 5% CO 2 After 48 hours of culture in a cell culture box, the culture medium was replaced, and non-adherent cells were discarded, and the medium was changed every 3-4 days thereafter. After about 10 days, when the wall is close to the bottom of the bottle, digest with 0.25-0.5% trypsin, pipette into a single-cell suspension, centrifuge at 1000r / min for 10 minutes, discard the supernatant, and replace with medium containing 0.5-20% FSC L-DMEM or RPMI1640 or HAM or Fischer suspended sediment, according to the ratio of 1:3 ~ 8 passage.

Embodiment 2

[0024] Example 2: Take 0.1-4 ml of human fresh bone marrow under aseptic conditions, blow and beat it into a single-cell suspension with medium L-DMEM or RPMI1640 or HAM or Fischer containing 0.5-20% FSC, filter it with a 200-mesh sieve, Adjust the cell density according to 1×10 9 / L density inoculation, 37°C, 5% CO 2 After 48 hours of culture in a cell culture box, replace the culture medium, discard non-adherent cells, add LIF to the cell culture medium, and change the medium every 3-4 days. After about 10 days, when the wall is close to the bottom of the bottle, digest with 0.25-0.5% trypsin, pipette into a single-cell suspension, centrifuge at 1000r / min for 10 minutes, discard the supernatant, and replace with medium containing 0.5-20% FSC L-DMEM or RPMI1640 or HAM or Fischer suspended sediment, according to the ratio of 1:3 ~ 8 passage.

Embodiment 3

[0025] Example 3: Take 0.1-4 ml of human fresh bone marrow under aseptic conditions, blow it into a single-cell suspension with medium L-DMEM or RPMI1640 or HAM or Fischer containing 0.5-20% FSC, inoculate and culture it for 48 hours, then discard it Non-adherent cells, adherent cells were digested and cultured in serial dilutions to form single cell clones, in situ hybridization, RT-PCR or immunocytochemistry, and flow cytometry were used to detect Nanog expression, and Nanog+ cell clones were selected for culture and passage for 3~ In the 10th generation, retinoic acid (Retinoic Acid) was added to inhibit the expression of Nanog.

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Abstract

The method of differentiating human bone marrow matrix stem cell to form insulin cell includes culturing and screening fresh human bone marrow to obtain human bone marrow matrix stem cell; amplifying human bone marrow matrix stem cell; inducing the amplified human bone marrow matrix stem cell with inducing agent beta-mercaptoethanol and / or niacinamide to obtain insulin cell formed by differentiated human bone marrow matrix stem cell. The bone marrow matrix stem cell may taken from the patient himself, and this can avoid graft rejection and corresponding cell function failure. The present invention has high repeatability and rich material source.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for forming islet cells from human bone marrow stromal stem cells. Background technique [0002] At present, the treatment of diabetes mainly includes diet, drug therapy and organ transplantation. The effect of drug treatment is not durable, does not conform to physiology, and cannot prevent the progression of diabetes and the occurrence of complications such as the heart, blood vessels, and retina. Pancreas transplantation, including islet transplantation, is the most valuable treatment for diabetes. However, the lack of sources of pancreas and islet cells and the rejection of organ transplantation hinder the development of pancreas transplantation. So far, pancreas transplantation in China has not exceeded 50 cases, which is far from meeting the needs of diabetic patients. [0003] In recent years, studies on the differentiation of stem cells into islet cell...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0775
Inventor 陈立波姜晓兵杨炼
Owner 陈立波
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