Gene medicine for promoting differentiation of tumor stem cells and applications thereof
A technology of tumor stem cells and gene medicine, applied in the field of gene medicine that promotes the differentiation of tumor stem cells, can solve the problems of recurrence and metastasis that plague surgeons, produce tumor tissue treatment resistance, and lack of differentiation of tumor stem cells, so as to improve drug sensitivity, Safe to use, low toxicity and side effects
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[0024] Example 1 Nell-1 gene applied to glioma
[0025] The Nell-1 gene sequence was obtained by RT-PCR. After the detection was correct, the full-length gene was amplified by PCR in vitro, and then the amplified product was cloned into an expression plasmid to obtain an expression plasmid (Nell-1) carrying the Nell-1 gene. 1 expression plasmid). The Nell-1 expression plasmid was used to transfect into glioma stem cell line (DA66), and the positive ratio of CD133 in this tumor stem cell line was about 65-70%.
[0026] After G418 screening, a stable and high Nell-1 expression cell line (DA66-Nell-1) was obtained, stained with CD133 antibody, and then analyzed by flow cytometry, such as figure 1 As shown (where DA66 is a complete blank control without any treatment, and DA66-Neo is a blank plasmid transfection), Nell-1 overexpression significantly reduces the expression of CD133, while the transfection of blank plasmid (DA66-Neo) significantly reduces the expression of CD133. ...
Example Embodiment
[0031] The preparation of the recombinant of embodiment 2 target gene is Nell-1
[0032] The Nell-1 gene sequence was obtained by RT-PCR. After the detection was correct, the full length of the gene was amplified by PCR in vitro, and the amplified product clone and adenovirus shuttle plasmid were cut with specific endonuclease to construct a Nell-carrying plasmid. -1 gene shuttle plasmid, then subcloned into pQB1-AdCMV5 (containing adenovirus E1A), co-transfected with QB1-viral DNA, positive virus clones were screened by PCR to obtain Ad-Nell-1 recombinant, amplified, transfected HEK293 cells were transfected, cultured, the Nell-1 gene recombinant adenovirus was roughly divided, purified by ultracentrifugation, sterilized, and obtained.
Example Embodiment
[0033] The preparation of embodiment 3 injection
[0034] The Ad-Nell-1 recombinant obtained in Example 2 was 2×10 15 Virus titer, add normal saline for injection to 1000mL, sterile filter, and dispense into 2mL vials.
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