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
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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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