Method for reprogramming immortalized lymphocyte line into induced pluripotent stem cells
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A technology of lymphocyte lines and lymphocytes, applied in the field of reprogramming immortalized lymphocyte lines into induced pluripotent stem cells, to achieve the effect of reducing the risk of cell tumorigenesis and expanding the value of clinical research
Active Publication Date: 2021-06-15
ZHEJIANG UNIV
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[0004]
Although a lot of research on iPSC-related technologies has been carried out in various countries, there is no mature technical system for inducing human immortalized lymphocyte lines into iPSCs.
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Embodiment 1
[0043] Example 1 Reprogramming Immortalized Lymphocyte Lines into iPSCs
[0044] (1) The growth state of the lymphocyte line is good, and the density reaches 1×10 6 / mL or so, take 2 mL of cells and centrifuge at 1000 rpm for 5 min at room temperature. Rinse the cells with pre-warmed 1×PBS and discard the supernatant. At the same time, preheat the reprogramming medium. The reprogramming medium is DMEM / F12 medium supplemented with 20% by volume Knockout serum substitute, the final concentration is 1× non-essential amino acids, the final concentration is 1× GlutaMAX, and the final concentration is 50 μg / mL magnesium ascorbyl phosphate , basic fibroblast growth factor at a final concentration of 10 ng / mL.
[0045] (2) Each sample was mixed according to the ratio of Cell Line Nuceofector Solution V 81.8 μL and Supplement 18.2 μL, and the plasmids pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, pCXLE- 2 μg each of EGFP and pCXWB-EBNA1, and mix well to obtain an electrotransfer sol...
Embodiment 2
[0055] Example 2 Identification of iPSC pluripotency gene expression
[0056] (1) Collect normal subcultured iPSCs, extract the total RNA of iPSCs by TRIzol method, then reverse transcribe the total RNA into cDNA, and perform PCR with primers of pluripotency genes SOX2, OCT4, C-MYC, KLF4, and NANOG, and the product Expression was analyzed by agarose gel electrophoresis.
[0057] (2) Using normal subcultured iPSCs, fix them with paraformaldehyde, rinse with PBS, and after blocking, add SOX2, OCT4, NANOG, SSEA4, TRA-1-60 antibodies to each well and incubate overnight at 4 degrees, wash with PBS After rinsing, add the corresponding species fluorescent secondary antibody and incubate at room temperature for 2 hours, then stain with DAPI, rinse with PBS, observe and take pictures under a fluorescence microscope.
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Abstract
The invention provides a method for reprogramming an immortalized lymphocyte line into induced pluripotent stem cells. The reprogramming method mainly comprises the step of introducing additional plasmids for expressing exogenous reprogramming factors into the immortalized lymphocytes through a nuclear transformation method, wherein the reprogramming factors comprise OCT3/4, shP53, SOX2, KLF4, LIN28 and L-MYC. According to the method, the immortalized lymphocyte line can be efficiently reprogrammed into the iPSC, and the additional plasmids cannot be integrated into the genome of host cells and can be gradually lost along with cell proliferation, so that exogenous expression can be timely silenced, and the cell tumorigenesis risk is reduced. The obtained iPSC has typical stem cell morphology and characteristics similar to ESC, expresses cell stemness genes, and has differentiation potential of differentiating into ectoderm, mesoderm and endoderm tissues. The method is high in reprogramming efficiency and low in tumorigenicity, the clinical research value of the immortalized lymphocyte line is expanded, and the method has application prospects.
Description
technical field [0001] The invention relates to the field of biomedicine, in particular to a method for reprogramming immortalized lymphocyte lines into induced pluripotent stem cells. Background technique [0002] In 2006, Yamanaka and Takahashi successfully obtained induced pluripotent stem cells (induced Pluripotent Stem Cell, iPSC) by using mouse cells. This method is to transfer four transcription factors Sox2 (sex determining region Y box protein 2), Oct4 (octamer bidding transcription factor 4), c-Myc (cellular myelocytomatosis oncogene) and Klf4 (kruppel like factor 4) were introduced into mouse fibroblasts. The iPSCs obtained after reprogramming have cell characteristics similar to those of embryonic stem cells (ESCs), such as the expression of stemness marker genes and the differentiation potential of three germ layers (Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126...
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