Pharmaceutical composition containing gene-edited adipose stem cells
A composition and drug technology, applied in the field of pharmaceutical composition of adipose stem cells, can solve the problems of less differentiation research and achieve the effect of lowering blood sugar
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Embodiment 1
[0030] Example 1 Preparation of adipose-derived mesenchymal stem cells
[0031] 15ml of human fat particles, centrifuged at 1000r / min for 5min. The upper layer of adipose tissue and the lower layer of cells were taken. Trypsin and collagenase were added to the adipose tissue at a final concentration of 0.1%. Digest for 30 min in a water bath shaker at 37 °C. Centrifuge at 700g / min for 5min. Leave the lower layer of cells, mix the two cells, add PBS buffer to resuspend the cells, and centrifuge again at 700 g / min for 5 min. The cells were resuspended in α-MEM medium and then inoculated into culture flasks, and cultured in a 37°C, 5% carbon dioxide incubator. Denoted as P0 generation. Observe cell growth. Half of the medium was changed on the third day for the first time, and the medium was changed every 3 days thereafter. When the cells reached 90% confluence, the cells were subcultured, digested with 0.05% trypsin, and subcultured at a ratio of 1:5. Passed to the 3rd ge...
Embodiment 2
[0032] Example 2 Preparation of γ-secretase knockout adipose-derived mesenchymal stem cells
[0033] The sequences in the present invention are all 5'-3'.
[0034] According to the sequence of γ-secretase APH1B, dozens of targets were obtained through target sequence optimization. After identification, it was found that target 1 had the best effect. Specifically, targets 1 and 2 are used as examples.
[0035] Target 1: acctgggcattcttagctgcggg
[0036] sgRNA1 sequence:
[0037] accugggcauucuuagcugcGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGGUGCUUUU (SEQ ID NO: 1)
[0038] Target 2: tagtcagtgtctctgggttttgg
[0039] sgRNA2 sequence:
[0040] uagucagugucucuggguuuGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGGUGCUUUU
[0041] Preparation of AsCpf1 protein:
[0042] Vector construction plasmid pTrcHis2B (purchased from Invitrogen company), plasmid map such as figure 1 As shown, the 3' end of the AsCpf1 gene sequence plus...
Embodiment 3
[0046] Example 3 Preparation of γ-secretase monoclonal antibody
[0047] (1) Protein immunization and antibody preparation
[0048] Screening to obtain high immunogenic epitope peptide sequences, that is, epitope peptides of γ-secretase
[0049] VELGQVALRTSLELWMHTDPVSQKNESVRNQVEDLLATLEK, the polypeptide was emulsified and mixed with Freund's complete adjuvant, and BALB / c mice were subcutaneously immunized. 3d before fusion, booster immunization with antigen without adjuvant by intraperitoneal injection. The fusion method of cells is prepared by conventional methods in the art. The positive cells secreting antibodies were screened by indirect ELISA, and subcloned by limiting dilution method. After three clones, a stable hybridoma cell line was obtained, which was expanded and cultured and cryopreserved. One week later, the hybridoma cell lines were intraperitoneally injected into mice. An anti-γ-secretase antibody 5C6 was successfully prepared.
[0050] (2) Monoclonal anti...
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