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Small molecule compound combination and method for preparing vascular endothelial cells by using small molecule compound combination to induce differentiated cells

A small molecule compound, vascular endothelium technology, applied in the field of cell biology, can solve the problems of induced transdifferentiation of cells and low feasibility of transdifferentiation of similar cells

Pending Publication Date: 2018-05-25
深圳臻德济慈药品研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Since there are about 25% genetic differences between humans and mice, it is not feasible to apply the above-mentioned successful patent application technology scheme in mouse cell experiments to human cells of the same kind; on the other hand, due to the induction of similar cells The specific theoretical basis and technical means of transdifferentiation to obtain different target cells are not the same. The applicant used the technical solutions reported above to repeat the experiments, but failed to successfully apply the transdifferentiation technical solutions applied to mouse cells to similar human cells. Transdifferentiation, also failed to induce transdifferentiation of human differentiated cells into vascular endothelial cells

Method used

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  • Small molecule compound combination and method for preparing vascular endothelial cells by using small molecule compound combination to induce differentiated cells
  • Small molecule compound combination and method for preparing vascular endothelial cells by using small molecule compound combination to induce differentiated cells
  • Small molecule compound combination and method for preparing vascular endothelial cells by using small molecule compound combination to induce differentiated cells

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Experimental program
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Effect test

Embodiment 1

[0173] 1. Isolation of skin fibroblasts

[0174] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from any position on the donor body, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium. The basal culture medium refers to: 10% fetal bovine serum (Hyclone )+100 U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high sugar DMDM.

[0175] 1.2 Subsequent cell passage A large number of expansion, the number of cell generations between the 3rd and 12th passages, is used to induce transdifferentiation into vascular endothelial cells. The day before activation (Day-1), inoculate cell density 4~6x10 3 / cm2 in an incubator at 37°C with 5% CO2.

[0176] 2. Orientation induction of skin fibroblasts to vascular endothelial cells

[0177] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the induction medium, and culture the cells for 2 to 12 days. Culture medium A...

Embodiment 2

[0195] 1. Isolation of skin fibroblasts

[0196] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from any position on the donor body, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium. The basal culture medium refers to: 10% fetal bovine serum (Hyclone )+100 U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high sugar DMDM.

[0197] 1.2 Subsequent cell passage A large number of expansion, the number of cell generations between the 3rd and 12th passages, is used to induce transdifferentiation into vascular endothelial cells. The day before activation (Day-1), inoculate cell density 4~6x10 3 / cm2 in an incubator at 37°C with 5% CO2.

[0198] 2. Orientation induction of skin fibroblasts to vascular endothelial cells

[0199] When starting transdifferentiation (Day0), completely replace the basal culture medium with the induction culture medium, and culture the cells for 10 to 28 days. ml strepto...

Embodiment 3

[0216] 1. Isolation of skin fibroblasts

[0217] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from any position on the donor body, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium. The basal culture medium refers to: 10% fetal bovine serum (Hyclone )+100 U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high sugar DMDM.

[0218] 1.2 Subsequent cell passage A large number of expansion, the number of cell generations between the 3rd and 12th passages, is used to induce transdifferentiation into vascular endothelial cells. The day before activation (Day-1), inoculate cell density 4~6x10 3 / cm2 in an incubator at 37°C with 5% CO2.

[0219] 2. Orientation induction of skin fibroblasts to vascular endothelial cells

[0220] When starting transdifferentiation (Day0), completely replace the basal culture medium with the induction culture medium, and culture the cells for 10 to 28 days. ml strepto...

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Abstract

The invention discloses a small molecule compound combination and a method for preparing vascular endothelial cells by using the small molecule compound combination to induce differentiated cells. According to the method, differentiated cells are subjected to targeted induction so as to finally obtain vascular endothelial cells, wherein the targeted induction comprises: inhibiting the activity oflysine deacetylase activity, inhibiting the signaling pathway of TGF-beta, inhibiting the activity of DNA methyltransferase (DNMT), activating the signaling pathway of WNT / beta-catenin, and activatingthe signaling pathway of cAMP. According to the present invention, through the induction with the small molecule compound combination, the differentiated cells can be transdifferentiated into the vascular endothelial cells, each step can be subjected to precise quality control, and the standardized operation and the large-scale production can be conveniently achieved; and the donor source of themethod is wide, the patient himself can be served as the donor, and the vascular endothelial cells required by basic research, clinical treatment or tissue engineering production can be obtained within a short time.

Description

technical field [0001] The invention relates to the fields of cell biology, tissue engineering and regenerative medicine, in particular to a combination of small molecular compounds and a method for preparing vascular endothelial cells from cells induced by the combination of small molecular compounds. Background technique: [0002] Vascular endothelial cells (VEC), as the structural inner wall of blood vessels, play an important role in maintaining the normal shape and physiological functions of blood vessels. Vascular endothelial cells with metabolic activity have antithrombotic effect in vivo, which is the key to ensure the long-term patency rate after implantation of tissue engineered blood vessels, and also the basis of angiogenesis in clinical treatment. As the number of patients with trauma, organ transplantation, and coronary artery bypass grafting continues to increase, the market demand for vascular endothelial cells or their precursor cells as seed cells for tissu...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/069C12N2501/01C12N2501/15C12N2501/415C12N2501/72C12N2501/73
Inventor 胡敏李燕皎王兆杰
Owner 深圳臻德济慈药品研发有限公司
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