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Preparation for treating ischemic diseases caused by peripheral atherosclerosis and preparation method thereof

A technology for atherosclerosis and ischemic diseases, applied in the field of preparations for ischemic diseases, can solve the problems of low survival rate, actual efficiency of surviving cells, restrictions on the promotion and application of stem cell therapy, and low content

Active Publication Date: 2013-12-04
盛泰英诺(嘉兴)医疗科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, a series of deficiencies in technology and application at this stage greatly limit the further promotion and application of stem cell therapy in clinical practice. These limitations include but are not limited to: (1) The extremely low levels of such stem cells in bone marrow and blood content (approximately 0.01%); (2) the extremely large number of cells (1×10 5 -1×10 6 (one / kg body weight); (3) the extremely low survival rate and actual efficiency of surviving cells (less than 10%) after transplantation of cells into the body; Malfunction caused by influence

Method used

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  • Preparation for treating ischemic diseases caused by peripheral atherosclerosis and preparation method thereof
  • Preparation for treating ischemic diseases caused by peripheral atherosclerosis and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Preparation of monocytes.

[0033] 100 mL of blood was added to the density gradient agent Histopaque-1077 (Sigma), and 30 mL of blood was added per 15 mL of Histopaque. Centrifuge the test tube containing the blood and the gradient reagent at 400G for 30 minutes at room temperature. After stratification, use a sterile disposable needle to draw the middle opaque layer, which is the suspension rich in mononuclear cells. Depending on the situation, every 100ml of blood can get 1x10 8 ~1x10 10 monocytes.

Embodiment 2

[0034] Example 2. Acquisition of EPC cells.

[0035] For the acquisition method of type I EPC cells: the mononuclear cell-rich suspension was added with 10% human serum albumin and 1% growth factor additive EGM (purchased by Swiss Lonza Company, which contains vascular endothelial growth factor VEGF- 1. Basic fibroblast growth factor FGF-2, epidermal growth factor EGF, and insulin-like growth factor IGF-1) under the EBM-2 medium of 1 × 10 per square centimeter 6 The density of the cells was cultured for 4 days, and the adherent cells were digested with trypsin and collected at 2×10 per square centimeter. 5 density for 2 days. The obtained type I EPCs are spindle-shaped cells with typical endothelial cell characteristics of endocytosing acetylated-LDL and binding UEA-1 lectin, and expressing a large amount of vascular endothelial cells Cell growth factor receptors KDR, CD31, CD14, CD45, a small amount of expression of CD34, CD133 and VE-cadherin.

[0036] For the acquisition...

Embodiment 3

[0040] Example 3. Acquisition of preparations for treating ischemic diseases caused by peripheral atherosclerosis.

[0041] The type I EPC cells obtained in Example 2 were placed in phosphate buffered saline (PBS) without any growth factors (2×10 per square centimeter). 5 cells), cultured in an environment with an oxygen concentration of 1.5% for 2 days, and supplemented with 1% medical human serum albumin. After the culture was completed, the cell-free medium rich in cell growth factors was collected, and the adherent EPC cells were discarded. The collected cell-free medium was passed through a filter with a pore size of 0.2 microns to remove cell impurities and debris, and then stored in a -80°C freezer for later use. As the case may be, every 1x10 6 Each EPC cell can prepare 2-5 ml of the preparation for treating ischemic diseases caused by peripheral atherosclerosis.

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Abstract

The invention relates to a preparation for treating ischemic diseases caused by peripheral atherosclerosis. The preparation method comprises the following steps of: obtaining endothelial progenitor cells (EPC) by inducing mononuclear cells, culturing the EPC cells under low-serum and low-oxygen conditions, obtaining cell-free culture medium by separating the EPC cells, and performing corresponding treatment on the cell-free culture medium to obtain the preparation of the invention. In vitro and in vivo experiments show that the preparation of the invention can treat ischemic diseases caused by peripheral atherosclerosis.

Description

technical field [0001] The invention relates to a preparation capable of treating ischemic diseases caused by peripheral atherosclerosis and a preparation method thereof. Background technique [0002] With the advent of an aging society, people often encounter the problem of peripheral atherosclerosis (pAD) in their daily work. Especially in intermittent claudication, early limb pain leads to reduced activity and prolonged bed rest, which seriously affects the quality of life of the elderly. Peripheral vascular disease is often of the same origin as cardiovascular and cerebrovascular diseases, and it accompanies and brakes early, causing a large number of vascular smooth muscle cells to atrophy and aggravating muscle atrophy of limbs, resulting in decompensation of vital organs. [0003] Epidemiological surveys show that intermittent claudication is closely related to age, and there is a clear correlation between the incidence and age, and the incidence increases significan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789A61K35/14A61K35/28A61P9/10
Inventor 杨肖泱杨子江顾丽娅
Owner 盛泰英诺(嘉兴)医疗科技有限公司