Extracellular matrix biomaterial for resisting cell premature senility and preparation method and application thereof
A biomaterial and extracellular matrix technology, applied in the field of extracellular matrix biomaterials against premature cell aging and their preparation, can solve problems such as premature cell aging
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Embodiment 1
[0037] Example 1 Preparation of extracellular matrix biomaterial (ECM) against premature cell aging
[0038] Human UC-MSCs umbilical cord-derived mesenchymal stem cells, UC-MSCs) at 37℃, 5%CO 2 Cultivated at 175cm in the incubator 2 In the culture bottle of complete medium (the volume ratio of each component of the medium is: 89% α-MEM, 10% fetal bovine serum, 1% penicillin, 1% streptomycin), change the medium every three days . When the cell density is close to 90%, digest the cells with 0.25% trypsin-EDTA at 5000 / cm 2 Inoculate in a well plate and cultivate until reaching 90% confluency, add 100 μM ascorbic acid to continue culturing for 8 days, and change the medium once every three days. After 8 days, in order to obtain free ECM, the cultured cells were added to the decellularization mixture (prepared from pH 7.4 phosphate buffer solution, containing 0.5% Triton X-100 and 20mM ammonia water) and incubated at 37°C for 5 minutes. Then the decellularized ECM was washed 3...
Embodiment 2
[0039] Example 2 ECM to various concentrations of H 2 o 2 Effects of SA-β-Gal staining positive rate, intracellular ROS and cell cycle distribution in stem cells after treatment
[0040] Human UC-MSCs were stored at 37°C, 5% CO 2 Cultivated at 175cm in the incubator 2 In the culture bottle (the volume ratio of each component of the medium is 89% α-MEM, 10% fetal bovine serum, 1% penicillin, streptomycin), the medium is changed every three days. When the cell density is close to 90%, digest the cells with 0.25% trypsin-EDTA at 5000 / cm 2 Inoculate in well-plates of various specifications (12 wells) pretreated (the method is to incubate the TCPS plate with 0.2% gelatin at 37°C for 1 hour, then incubate with 1% glutaraldehyde and 1M ethanolamine at room temperature for 30 minutes) Plate for SA-β-Gal staining, 6-well plate for ROS content, cell cycle, RT-PCR determination, 75cm 2 Flasks for p16 INK4α protein analysis) culture until reaching 90% confluency, adding 100 μM asco...
Embodiment 3
[0051] Example 3 ECM to various concentrations of H 2 o 2 p16 in stem cells after treatment INK4α effects on gene and protein expression.
[0052] A. ECM for various concentrations of H 2 o 2 p16 in stem cells after treatment INK4α The impact of gene level changes ECM construction and cell culture methods are the same as in Example 1. The adherent cells are lysed with TRIzol reagent and then total RNA is extracted. According to the operation steps of the cDNA reverse transcription kit (Thermo Fisher Company, USA), 1 μg of total RNA is reversed. recorded as cDNA. To quantify mRNA expression, cDNA equivalent to 50 ng of RNA was used with the Universal Fluorescent Dye Supermix Kit (iTap TM UniversalSYBR Green Supermix kit) in CFX96 TM The polymerase chain reaction was performed on the real-time PCR reaction machine according to the operation steps provided by the manufacturer. We detected the senescence-associated gene p16 INK4α , GAPDH was used as an internal reference...
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