Universal human-derived stem cell suitable for allograft and construction method thereof
A stem cell and cell technology, applied in the medical field, can solve problems such as immune rejection, achieve low immunogenicity, and avoid the effect of allogeneic immune response
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Embodiment 1
[0093] Example 1: IFN-γ Up-regulates HLA-I Gene Expression of MSCs
[0094] This example proves through specific experiments that IFN-γ can up-regulate the expression of the HLA-I gene of MSCs.
[0095] 1.1 Cell culture and treatment
[0096] MSC culture conditions: L-DMEM medium (containing 10% fetal bovine serum), 5% CO2, 37°C constant temperature culture. 24 hours before treatment, MSCs were treated with 5 × 10 5 Each well was seeded into a 6-well plate for culture, and the cell confluency reached 60%-70% the next day.
[0097] The cultured MSCs were divided into three groups, the first group was blank control, the second group added IFN-γ to the medium at a concentration of 100ng / ml, and continued to culture for 24 hours; the third group, IFN-γ was added at a concentration of 100ng / ml -γ was added to the culture medium and continued to culture for 48 hours; the cells were collected for subsequent processing such as RNA extraction.
[0098] 1.2 MSC RNA extraction and fl...
Embodiment 2
[0122] Example 2: Experiments involving the participation of enhancers in the process of IFN-γ stimulating HLA-I expression
[0123] In this example, JQ1 (purchased from: abcam; product number: ab146612; batch number: APN15092-1-1; an enhancer inhibitor) with three concentrations of 125, 250 and 500nmol / ml were used to pretreat MSCs for 2 hours, and then Add 100ng / ml IFN-γ to treat MSC, continue to culture for 48 hours, collect cells and extract RNA for qPCR to detect the expression of HLA-I related genes, and set the corresponding MSC blank control, and MSC blank control plus IFN-γ treatment, A total of five groups of MSCs were compared and detected by q-PCR and flow cytometry respectively. The results are shown in Figure 3. For the specific process and sequence of q-PCR, refer to the process and conditions of Example 1. For the q-PCR results, see Figure 3A , is the expression level of HLA-A, HLA-B, HLA-C and B2M of MSC in the five groups; the results of flow cytometry are s...
Embodiment 3
[0136] Example 3, IFN-γ has a promoting effect on the opening and activation of the enhancer near the B2M gene
[0137] In order to further understand whether there are enhancers involved in the vicinity of HLA-I related genes after IFN-γ stimulation, and whether it is directly related to the expression of that gene, we performed H3K27ac ChIP-Seq analysis and bioinformatics analysis.
[0138] 1. The process of H3K27ac ChIP-Seq analysis is as follows:
[0139] 1 x 10 at room temperature 7 The non-IFN-γ-stimulated MSCs and IFN-γ-stimulated MSCs were respectively fixed with 1% formaldehyde for 10 minutes to obtain DNA-protein crosslinks. After sonicating for 1.5 hours to obtain 200-300bp chromatin fragments, mix with H3K27ac antibody (H3K27ac is a recognized active enhancer marker protein, and use H3K27ac antibody to capture all enhancer DNA bound to H3K27ac protein) at 4°C After overnight incubation, antibody-chromatin complexes were obtained by incubating with Dynabeads Prote...
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