Application of stem cells or transgenic stem cells

A method for constructing tissue-engineered cartilage from stem cells, characterized by comprising:

• step 1:
• isolating and culturing stem cells;
• step 2:
• inducing differentiation of the stem cells using an active protein;
• and
• step 3:
• using the stem cells obtained in the step 2 to construct a type III collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed;
• wherein the inducing differentiation of the stem cells using an active protein in the step 2 comprises:
• adding an active protein to a differentiation culture medium containing the stem cells, culturing at 37° C. with 5% CO2, culturing for 3 to 5 passages for later use, and replacing with the same differentiation culture medium per passage;
• the constructing a type III collagen 3D culture scaffold in vitro using the stem cells until tissue-engineered cartilage is formed in the step 3 comprises:
• preparing a type III collagen 3D scaffold using the differentiated stem cells (2.5×106-5×106 stem cells per scaffold), adding a DMEM cell culture medium containing the active protein to the scaffold, culturing at 37° C. with 5% CO2, replacing the DMEM cell culture medium containing the active protein every 7 days, and continuously culturing for 20-24 days to obtain tissue-engineered cartilage;
• the active protein consists of TGFβ, platelet-rich plasma, and SOX9, with final concentrations in the differentiation culture system of:
• TGFβ, 4.0 to 10.0 μg/L;
• platelet-rich plasma, 1.0% to 2.0% (v/v);
• and SOX9, 2.5 to 5.0 μg/L;
• and the stem cells are umbilical cord blood mesenchymal stem cells.

A method for preparing a stem cell gel solution from transgenic stem cells, characterized by comprising:

• step 1:
• constructing a recombinant plasmid containing a gene of interest;
• step 2:
• isolating and culturing stem cells;
• step 3:
• transfecting the stem cells cultured in the step 2 with the recombinant plasmid constructed in the step 1 to obtain stem cells transfected with the gene of interest;
• step 4:
• inducing differentiation of the stem cells transfected with the gene of interest using an active protein;
• and
• step 5:
• preparing a stem cell gel solution from the stem cells obtained in the step 4;
• wherein the constructing a recombinant plasmid containing a gene of interest in the step 1 comprises:
• step 1-1:
• obtaining a pBlue-BMP7 plasmid;
• step 1-2:
• subjecting the pBlue-BMP7 plasmid to double digestion and extracting and purifying the digested product;
• step 1-3:
• subjecting a pcDNA3.1 vector to double digestion, and then extracting and purifying the digested product;
• step 1-4:
• ligating the digested gene fragment obtained in the step 1-2 with the digested DNA fragment obtained in the step 1-3 to obtain a pcDNA3.1-BMP7 vector plasmid;
• step 1-5:
• transforming the pcDNA3.1-BMP7 vector plasmid into DH5α competent cells, amplifying, and extracting and purifying the amplified product;
• and
• the inducing differentiation of the stem cells transfected with the gene of interest using an active protein in the step 4 comprises:
• adding an active protein to a differentiation culture medium containing the stem cells transfected with the gene of interest, culturing at 37° C. with 5% CO2, culturing for 3 to 5 passages for later use, and replacing with the same differentiation culture medium per passage;
• the active protein consists of TGFβ, platelet-rich plasma, and SOX9, with final concentrations in the differentiation culture system of:
• TGFβ, 4.0 to 10.0 μg/L;
• platelet-rich plasma, 1.0% to 2.0% (v/v);
• and SOX9, 2.5 to 5.0 μg/L;
• and the stem cells are umbilical cord blood mesenchymal stem cells.

A method for constructing tissue-engineered cartilage from transgenic stem cells, characterized by comprising:

• step 1:
• constructing a recombinant plasmid containing a gene of interest;
• step 2:
• isolating and culturing stem cells;
• step 3:
• transfecting the stem cells cultured in the step 2 with the recombinant plasmid constructed in the step 1 to obtain stem cells transfected with the gene of interest;
• step 4:
• inducing differentiation of the stem cells transfected with the gene of interest using an active protein;
• and
• step 5:
• using the stem cells obtained in the step 4 to construct a type III collagen 3D culture scaffold in vitro until tissue-engineered cartilage is formed;
• wherein the constructing a recombinant plasmid containing a gene of interest in the step 1 comprises:
• step 1-1:
• obtaining a pBlue-BMP7 plasmid;
• step 1-2:
• subjecting the pBlue-BMP7 plasmid to double digestion and extracting and purifying the digested product;
• step 1-3:
• subjecting a pcDNA3.1 vector to double digestion and extracting and purifying the digested product;
• step 1-4:
• ligating the digested gene fragment obtained in the step 1-2 with the digested DNA fragment obtained in the step 1-3 to obtain a pcDNA3.1-BMP7 vector plasmid;
• step 1-5:
• transforming the pcDNA3.1-BMP7 vector plasmid into DH5α competent cells, amplifying, and extracting and purifying the amplified product;
• the inducing differentiation of the stem cells transfected with the gene of interest using an active protein in the step 4 comprises:
• adding an active protein to a differentiation culture medium containing the stem cells transfected with the gene of interest, culturing at 37° C. with 5% CO2, culturing for 3 to 5 passages for later use, and replacing with the same differentiation culture medium per passage;
• the constructing a type III collagen 3D culture scaffold in vitro using the stem cells until tissue-engineered cartilage is formed in the step 5 comprises:
• preparing a type III collagen 3D scaffold using the differentiated stem cells (2.5×106-5×106 stem cells per scaffold), adding a DMEM cell culture medium containing the active protein to the scaffold, culturing at 37° C. with 5% CO2, replacing the DMEM cell culture medium containing the active protein every 7 days, and continuously culturing for 20-24 days to obtain tissue-engineered cartilage;
• the active protein consists of TGFβ, platelet-rich plasma, and SOX9, with final concentrations in the differentiation culture system of:
• TGFβ, 4.0 to 10.0 μg/L;
• platelet-rich plasma, 1.0% to 2.0% (v/v);
• and SOX9, 2.5 to 5.0 μg/L;
• and the stem cells are umbilical cord blood mesenchymal stem cells.