Genetically Modified Stem Cells and Their Applications

A stem cell and cell technology, applied in the field of genetic engineering, can solve the problems of low stem cell induction efficiency and inability to meet clinical applications.

Active Publication Date: 2020-10-09
长春万成生物电子工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current technology has a low induction efficiency for stem cells, which cannot meet the requirements of clinical application.

Method used

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  • Genetically Modified Stem Cells and Their Applications
  • Genetically Modified Stem Cells and Their Applications
  • Genetically Modified Stem Cells and Their Applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Artificially synthesize the DNA fragment shown in SEQ ID NO: 2, and connect it into the vector pRRLSIN.cPPT with BamH I, Sal I, restriction site BamH I: G^GATCC, 4021, Sal I: G^TCGAC, 4049. PGK-WPRE ( figure 1 -a), construct and obtain the plasmid vector expressing fusion protein Pdx-1-Linker-IGF-1 ( figure 1 -b, the full sequence is shown in SEQ ID NO:3).

Embodiment 2

[0039] Experimental Materials:

[0040] Items required for virus production: 293T cells are virus packaging cells; Dulbecco’s modified Eaglemedium (DMEM medium); FBS (fetal bovine serum); 1× penicillin-streptomycin antibiotic; 3 plasmids; 2.5MCaCl 2 ; 2×HeBS buffer.

[0041] Virus production process:

[0042] A: 293T cells are cultured in DMEM medium (complete medium) containing 10% FBS, + penicillin-streptomycin antibiotics, taking a 15ml culture dish as an example, add 8×10 6 Cells, 22.5ml complete medium, 37°C, 5% CO 2 To cultivate.

[0043] B: After overnight culture, replace with serum-free medium DMEM.

[0044] C: The three plasmids (pMD2G, pCMVR8.74, pRRLSIN.cPPT.PGK-WPRE-Pdx-1-Linker-IGF-1) were mixed according to a certain ratio, and the buffer was HeBS.

[0045] D: Transfection method using CaCl 2 Transfection protocol, CaCl 2 Mix with HeBS buffer containing 3 plasmids in a certain proportion, mix thoroughly, add to 293T culture dish, 37°C, 5% CO 2 To cultivate...

Embodiment 3

[0052] Centrifuge to extract MNC (monocytes) from bone marrow tissue, after washing with PBS, suspend the cells in 8-10ml normal saline (2.2~3.8×10 8 cells). Use complete medium, the formula is as follows:

[0053] Stem cell medium containing 10% FBS (fetal bovine serum) (Beijing Dakowei Biotechnology Co., Ltd.: Mesenchymal Stem Cell Basal Medium (MSCBM), human mesenchymal stem cell basal medium), plus gentamicin and penicillin, 37 °C, 5% CO 2 Adhesive culture was carried out under certain conditions, and after 2 times of expansion and passage, when the cell expansion efficiency was stable and the viability reached 98%, it was used for cell transfection.

[0054] The cultured stem cells were re-adhered to the wall, added complete medium, and cultured overnight. The medium was aspirated the next day, and a certain amount of virus suspension and complete medium were mixed and added to a petri dish, and incubated at 37°C. After 4 hours, add a certain amount of complete medium,...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a genetically modified stem cell and an application thereof. The genetically modified stem cell can express fusion protein Pdx-1-Linker-IGF-1 and be efficiently differentiated into islet-like cell clusters. An experiment shows a conversion rate after induction for 5 days can reach 90%, and an insulin secretory volume can reach 109-141 pg / mL. Compared with other groups, the genetically modified stem cell has a stronger islet cell transformation effect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to genetically modified stem cells and applications thereof. Background technique [0002] Diabetes Mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. The long-term high blood sugar in diabetes leads to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. [0003] According to the classification recommended by the World Health Organization, diabetes can be divided into two types: type I diabetes with absolute insulin deficiency and type II diabetes with relative insulin deficiency and insulin resistance. Among them, about 10% of patients belong to type I diabetes, and the importance of genetic factors in this type of diabetes is as high as 50%, and the onset is common in children and ado...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867C12N7/01A61K35/28A61P3/10
CPCA61K35/28A61P3/10C07K14/575C07K14/65C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
Inventor 王立坚赵钢王竑婷
Owner 长春万成生物电子工程有限公司
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