Separation and purification method of human umbilical cord mesenchymal stem cell exosome and application of human umbilical cord mesenchymal stem cell exosome

A technology for the separation and purification of stromal stem cells, which is applied in the application field of human umbilical cord mesenchymal stem cell exosomes, can solve the problems of no substantial improvement in the etiology, increased glucose utilization, and uncontrollable dose safety, so as to reduce immune reactivity, Easy operation and controllable dose effect

Active Publication Date: 2018-06-01
JIANGSU UNIV
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AI Technical Summary

Problems solved by technology

[0003] Many studies at home and abroad have shown that mesenchymal stem cells (MSCs) play an important role in blood sugar regulation and islet cell function repair. MSCs can improve the sensitivity of peripheral tissues to insulin and increase the use of glucose in blood by tissues. , has shown certain effects in the intervention of diabetes, but the application standards of

Method used

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  • Separation and purification method of human umbilical cord mesenchymal stem cell exosome and application of human umbilical cord mesenchymal stem cell exosome
  • Separation and purification method of human umbilical cord mesenchymal stem cell exosome and application of human umbilical cord mesenchymal stem cell exosome
  • Separation and purification method of human umbilical cord mesenchymal stem cell exosome and application of human umbilical cord mesenchymal stem cell exosome

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Embodiment 1

[0061] The main materials and sources are as follows:

[0062] MSC culture reagents: α-MEM, fetal bovine serum (Gibco company product), trypsin (Sigma company product), carbon dioxide incubator (Forma company), serum-free medium (Shanghai Yikesai company; inverted microscope, fluorescence microscope, Biological microscope, electron microscope, ultra-clean bench, desktop centrifuge;

[0063] Exosome extraction reagent: heavy water (D 2 O, Shanghai Chuangsai Company), analytically pure sucrose (Guangzhou Chemical Reagent Factory), rabbit anti-human CD9 antibody (U.S. BioworldTechnology Company), rabbit anti-human CD63 antibody (U.S. Epitomics Company), BCA protein quantification kit, horseradish peroxidation HRP-labeled goat anti-rabbit IgG secondary antibody (Beijing Kangwei Century Company), HRP chemiluminescent substrate, 100-kDa MWCO ultrafiltration centrifuge tube, 0.22 μm sterile filter membrane (Millipore, USA); transmission electron Microscope (FEITecnai 12, Philips Co...

Embodiment 2

[0070] Construction of type 2 diabetic rat model and the therapeutic effect of MSC-ex on type 2 diabetic rats

[0071] The main materials and sources used in this embodiment are as follows:

[0072] Materials required for the animal model: 8-week-old male SD rats, common feed (Shuke Beta Company), 45% high-fat feed (Jiangsu Medison Company), streptozotocin (Sigma Company), blood glucose meter and test strips (Roche Jinrui), glycogen staining kit (Beijing Suo Lai Bao G1280), related antibodies: rabbit anti-mouse AKT antibody (SAB, USA), rabbit anti-mouse p-AKT antibody (SAB, USA), rabbit anti-mouse GSK Antibody (SAB, U.S.), rabbit anti-mouse β-GSK antibody (SAB, U.S.), rabbit anti-mouse Actin antibody (U.S. Bioworld Technology Company), rabbit anti-mouse IRS1 antibody (SAB, U.S.), rabbit anti-mouse p-IRS1 ( SAB, USA), rabbit anti-mouse Glycogen synthase antibody (abclonal, USA). Other equipment: disposable insulin injection syringe, insulin (Sigma company), glucose (edible), ...

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Abstract

The invention provides a separation and purification method of a human umbilical cord mesenchymal stem cell exosome and an application of the human umbilical cord mesenchymal stem cell exosome, and belongs to the technical field of medicines. The human umbilical cord mesenchymal stem cell exosome provided by the invention is prepared by cultivating human umbilical cord mesenchymal stem cells via aserum-free medium, collecting supernatant liquid, implementing centrifuging as well as ultra-filtration and concentration, transferring a concentrated solution onto a 30% sucrose-heavy water densitypad and implementing further purification through sucrose density centrifuging, so that the human umbilical cord mesenchymal stem cell exosome is obtained. According to the separation and purificationmethod that the human umbilical cord mesenchymal stem cell exosome is obtained through separation and purification, immunoreactivity is effectively reduced, and a controllable dosage when the human umbilical cord mesenchymal stem cell exosome is used is guaranteed. The human umbilical cord mesenchymal stem cell exosome, by improving a degree of activating an insulin signaling pathway of a type IIdiabetes animal model, can inhibit composition and decomposition of hepatic glycogen, so that glucose metabolism homeostasis can be achieved; and meanwhile, the sensitivity of the type II diabetes animal model to insulin and an insulin secretion function of pancreatic [beta] cells can be improved and a blood glucose concentration can be reduced, so that the application of the human umbilical cordmesenchymal stem cell exosome to the preparation of medicines for treating type II diabetes can be achieved.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for separating and purifying human umbilical cord mesenchymal stem cell exosomes and the application of human umbilical cord mesenchymal stem cell exosomes. Background technique [0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia due to defects in insulin secretion or insulin action. In the past 30 years, the incidence of diabetes in China has increased from less than 1% to more than 10%. The number of diabetic patients has exceeded 100 million, ranking first in the world, and they are getting younger. Diabetes has become a serious public health problem in my country. 90% to 95% of diabetic patients are type 2 diabetes mellitus (T2DM). Patients with type 2 diabetes mellitus regulate their blood sugar through diet control, exercise and oral hypoglycemic drugs in the early stage. With the development of the disease, most patients ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P3/10
Inventor 许文荣孙瑶湘钱晖张斌史惠纪成孙丰田严永敏张徐毛飞
Owner JIANGSU UNIV
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