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Preparation method and application of mesenchymal stem cells

A stem cell and bone marrow technology, applied in the field of life sciences, can solve the problems of difficulty in finding a transplant source, high recurrence rate, and difficulty in carrying out, and achieve the effects of reducing the probability of pollution, promoting proliferation, and improving the success rate.

Pending Publication Date: 2019-09-13
GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bone marrow-derived hematopoietic stem cell transplantation has been carried out at home and abroad for the treatment of SLE, and has achieved certain curative effect, but there are problems such as difficulty in finding the source of transplantation and high cost, making it difficult to carry out clinically.
Moreover, autologous hematopoietic stem cell transplantation (HSCT, hematopoietic stem cell transplant) has the disadvantage of high recurrence rate, and allogeneic HSCT has a high mortality rate due to rejection and other reasons

Method used

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  • Preparation method and application of mesenchymal stem cells
  • Preparation method and application of mesenchymal stem cells
  • Preparation method and application of mesenchymal stem cells

Examples

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Embodiment 1

[0040] A method for preparing mesenchymal stem cells, comprising the following steps:

[0041] (1) Collect aliquots of bone marrow (BM) aspirate from the femur and tibia of GFP transgenic mice, and the bone marrow (BM) aspirate is carried out under sterile conditions;

[0042] (2) Bone marrow mononuclear cells were selected by Ficoll-Paque density gradient centrifugation;

[0043] (3) At 37°C and 5% CO 2 75-cm 2 In the flask, it is cultivated in the modified EAG medium (MEM-α) that is supplemented with a volume fraction of 20% fetal bovine serum (FBS), 100 μl / ml penicillin and 100 μg / ml streptomycin;

[0044] (4) Change the medium containing non-adherent cells every 4-5 days;

[0045] (5) After reaching 90% confluency, the adherent cells were harvested by trypsinization; under this condition, Sca-1 positive cells exceeded 96.67%.

[0046] In the step (2), when carrying out the density gradient centrifugation, the bone marrow aspirate placed in the centrifuge tube is first ...

Embodiment 2

[0055] A method for preparing mesenchymal stem cells, comprising the following steps:

[0056] (1) Collect aliquots of bone marrow (BM) aspirate from the femur and tibia of GFP transgenic mice, and the bone marrow (BM) aspirate is carried out under sterile conditions; Centrifuge at 900g-1200 for 10-15 minutes to pellet cellular material;

[0057] (2) Bone marrow mononuclear cells were selected by Ficoll-Paque density gradient centrifugation; the remaining bone marrow supernatant was collected, frozen and stored at (-25°C) ~ (-30°C) for further use.

[0058] (3) At 37°C and 5% CO 2 75-cm 2 In the flask, it is cultivated in the modified EAG medium (MEM-α) that is supplemented with a volume fraction of 20% fetal bovine serum (FBS), 100 μl / ml penicillin and 100 μg / ml streptomycin;

[0059] (4) Change the medium containing non-adherent cells every 4-5 days;

[0060] (5) After reaching 90% confluency, the adherent cells were harvested by trypsinization; under this condition, Sca...

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Abstract

The invention relates to a preparation method of mesenchymal stem cells. The preparation method comprises the following steps of (1) collecting the same parts of bone marrow (BM) aspiration substancesfrom thigh bones and shin bones of a green fluorescent protein transgenic mouse under the aseptic condition; (2) selecting bone marrow mononuclear cells by a Ficoll-Paque density gradient centrifugation; (3) performing culturing in improved EAG culture mediums (MEM-alpha) to which 20vol% of fetal calf serum (FBS), 100[mu]l / ml of penicillin and 100[mu]g / ml of streptomycin are supplemented, in a flask of 75-cm<2> under the condition of 37 DEG C and 5% of CO2; (4) replacing culture mediums containing non-adherent cells once every 4-5 days; and (5) when 90% confluence is achieved, harvesting adherent cells through trypsin digestion, wherein under the condition, Sca-1 positive cells exceed 96.67%. According to the preparation method disclosed by the invention, the cell proliferation speed canbe substantially increased, ample nutrition is provided for cells, protein synthesis is promoted, and the treatment effects of treating systemic lupus erythematosus through stem cells can be effectively increased and reinforced.

Description

technical field [0001] The invention belongs to the technical field of life sciences, and in particular relates to a method for preparing mesenchymal stem cells for treating lupus erythematosus and its application. Background technique [0002] Systemic lupus erythematosus (SLE for short) is a common immune disease. The etiology is not yet clear, but it is characterized by dysfunction of the entire immune system, abnormal cell apoptosis, and abnormal lymphocyte function. Its serum contains a variety of autoantibodies represented by antinuclear antibodies, causing chronic damage to multiple system organs throughout the body. Traditional hormonal immunosuppressant treatments have severe side effects, and patients need to take the medicine for life. Systemic lupus erythematosus is an autoimmune disease involving multiple systems and organs. The immune system in patients is dysfunctional, and T and B lymphocytes are abnormally activated, producing a variety of autoantibodies an...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P37/02
CPCC12N5/0663A61K35/28A61P37/02C12N2509/00
Inventor 聂瑛洁张湘燕陈辉胡荣贵余福勋罗振华叶贤伟查艳许永杰
Owner GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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