Stem cells with enhanced immunoregulation capability, preparation method thereof, composition and application thereof
An immune regulation and stem cell technology, applied in the field of biomedicine, can solve problems such as ineffective treatment of autoimmune diseases
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[0047] [Preparation method of stem cells with enhanced immune regulation ability]
[0048] The second aspect of the present invention provides a method for preparing stem cells with enhanced immune regulation ability (sometimes referred to as "preparation method of the present invention" or "method of the present invention" for short in the present invention), including using, for example, genetic engineering to reduce The expression level of the Id3 gene in the cell or the step of reducing the activity of the ID3 protein.
[0049] In some embodiments, the preparation method of the present invention includes the step of reducing the expression level of the Id3 gene in the cells by using RNA interference technology. RNA interference technology is a technology in which exogenous and endogenous double-stranded RNA induces RNA-specific degradation of homologous target genes in vivo, resulting in post-transcriptional gene silencing. A technique using siRNA or shRNA is preferred. ...
Embodiment
[0084] 1. BMMSCs derived from human stem cell bank were modified by Id3
[0085] Preparation of Id3 siRNA knockout BMMSCs
[0086] 1) Synthesize Id3 siRNA, the sequence is GGAGCTTTTGCCACTGACT, or GCTTGCTGGACGACATGAA, or GCCAGGTGGAAATCCTACA
[0087] 2) Shop 5×10 5 Put BMMSCs cells per well into a 24-well plate, with 150 μL medium in each well, which is α-MEM complete medium;
[0088] 3) On the second day, 50 μL / well of OPTI-MEM was mixed with 1 μL Lipofectamin 2000, and incubated for 5 minutes;
[0089] 4) Mix 4 μL of synthetic si-Id3, siRNA control with another 50 μL / well of OPTI-MEM;
[0090] 5) Mix the above two mixtures and incubate at room temperature in the dark for 20 minutes;
[0091] 6) Suck off the original complete medium, and slowly add the mixture to the cell culture plate;
[0092] 7) After 6 hours of transfection, suck off the mixed solution, and replace with preheated complete culture medium without antibiotics. Three days later, collect the cells, extract ...
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