Method for preparing BM-MSCs conditioned medium, conditioned medium and application

Abstract

The invention provides a method for preparing a BM-MSCs conditioned culture medium, the conditioned culture medium and application. The method comprises the following steps of (1) constructing PHD2. shRNA on a lentiviral gene transfer vector pGCSIL-GFP; (2) under the condition that the multiplicity of infection(MOI) is 10, transfecting the BM-MSCs by using a lentivirus containing shPHD2-GFP; (3) determining stably transfected BM-MSCs by detecting GFP fluorescence and PHD2 protein expression, carrying out cell sorting and enrichment, and then culturing the BM-MSCs in a new serum-free basic culture medium for 48 hours to obtain a basic conditioned culture medium containing paracrine substances efficiently expressed by BM-MSCs; and (4) performing ultrafiltration concentration on the basic conditioned culture medium by 50 times, controlling the molecular weight cut-off to be 5kDa to obtain a concentrated conditioned culture medium, and then storing the obtained conditioned culture medium at minus 20 DEG C. In the basic conditioned culture medium obtained in the step (3), 1mL of non-concentrated conditioned culture solution is equivalent to the secretion volume of 1.5-2.5 * 10 <5> BM-MSCs. The conditioned culture medium prepared by the invention can enhance the paracrine effect of the BM-MSCs.

Description

[0001] This research was supported by National Natural Science Foundation of China (No.81300279, 81741067), Guangdong Natural Science Foundation of China (No.2016A030313815, 2017A030313464), Guangdong Science and Technology Project (No.2015A020212029), Guangzhou Science and Technology Project (No.201707010419, 201804010050) ), Guangdong Provincial Bureau of Traditional Chinese Medicine scientific research project (No.20201009, 20201010), high-level hospital construction project (Dengfeng Program) (No.DFJH201803, KJ012019099). technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing BM-MSCs conditional medium with high paracrine expression by silencing PHD2, conditional medium and application. Background technique [0003] NEC (Necrotizing enterocolitis) is the most common acute and severe gastrointestinal disease in newborns. It occurs in 7%-10% of premature infants, and the mortality rate is as high as ...

AI Technical Summary

Problems solved by technology

In the prior art, attempts have been made to modify genes such as AKT, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (BFGF) and insulin-like growth factor (IGF-1) to enhance the effect of MSC paracrine, but many Clinical studies also suggest that the expression of a single angiogenic factor may not be sufficient to promote angiogenesis and repair tissue damage

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