Multi-modal tracing umbilical cord mesenchymal stem cell as well as preparation method and application thereof
A stem cell, multi-modal technology, applied in the field of biomedicine, can solve the problems of high requirements for detection objects, MRI comparison, and the difficulty of single-modal tracer to meet the application requirements, and achieve the effect of convenient efficacy evaluation.
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Embodiment 2
[0101] Example 2 Construction of multimodal lentiviral expression vector
[0102] 1) Amplify the rFTH1-T2A gene fragment:
[0103] Using the plasmid containing the rFTH1-T2A gene as a template, use the following primer pair to amplify the rFTH1-T2A gene fragment by PCR, and introduce XbaI and AgeI restriction sites at both ends of the rFTH1-T2A gene fragment respectively;
[0104] PRTE-F:
[0105] 5'-CGGTGAATTCCTCGAGACTAGTTCTAGAGCCACCATGACCACCGCGTCT-3';
[0106] PRTE-R:
[0107] 5'-CTCCTCGCCCTTGCTCACCATGATACCGGTAGGGCCGGGATTCTCCTCCA-3';
[0108] 2) Use the pLVX-EGFP plasmid as a vector, digest it with XbaI and BamHI and use it for later use;
[0109] 3) Homologous recombination connection:
[0110] The rFTH1-T2A gene fragment obtained in step 1) is connected with the vector obtained in step 2) under the action of homologous recombinase to obtain the vector pLVX-rFTH1-T2A-EGFP;
[0111] 4) Synthesize the gene P2A-SR39RK fragment (synthesized by Jinweizhi Biotechnology Co.,...
Embodiment 3
[0114] Example 3 Lentiviral Packaging
[0115] 1) Spread 293T cells in a 10 cm culture dish one day before virus packaging, and carry out lentivirus packaging when the cell fusion degree is about 80%;
[0116] 2) Configure plasmid transfection systems A and B respectively:
[0117] System A includes 2mL OPTI and 50 μL PEI, system B includes 2mL OPTI and mixed plasmids, the mixed plasmids include 6.5 μg PMDL, 3.5 μg REV, 2.5 μg VSVG and 10 μg pLVX-rFTH1-P2A-SR39TK-T2A obtained in step 2) -EGFP;
[0118] 3-3) After the system A and system B are prepared, let them stand for 5 minutes respectively, then add system B dropwise to system A at a uniform speed, blow and suck several times, and incubate for 10 minutes to obtain the AB mixture;
[0119] 3-4) Discard the medium above the 293T cells, then slowly add the AB mixture along the side wall of the culture dish, and cultivate;
[0120] 3-5) Discard the AB mixture above the 293T cells after 8 hours, then slowly add complete medi...
Embodiment 4
[0126] Example 4 Establishment of multimodal tracer mesenchymal stem cell lines
[0127] 1) Recombinant lentivirus transfection: Plate the umbilical cord mesenchymal stem cells obtained in Example 1 18-24 hours before transfection, then add the recombinant lentivirus solution obtained in Example 3, incubate in a constant temperature incubator at 37°C, and transfect When the cell density is controlled at 70% to 90%;
[0128] 2) Pick a single clone: After 48 hours, culture the umbilical cord mesenchymal stem cells infected with the recombinant lentivirus with 2ml of 0.25% Trypsin in a 10cm culture dish, and digest them in a 37°C incubator for 1-2min. The specific time depends on to the disappearance of cell synapses as a reference;
[0129] 3) Aspirate the Trypsin, add 2ml medium to stop the digestion and pipette the cells evenly into a cell suspension;
[0130] 4) Transfer the cell suspension to a 10ml centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernata...
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