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A method for directed differentiation of autologous immune cells into islet cells

A directional differentiation, islet cell technology, applied in animal cells, pancreatic cells, vertebrate cells, etc., can solve the problems of long cell culture time, low transformation rate, low insulin secretion level of islet cells, etc., to reduce immune rejection. Effect

Active Publication Date: 2022-04-08
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Using autologous immune cells to differentiate into islet cells, it is first necessary to dedifferentiate autologous immune cells to obtain iPSCs, and then induce differentiation into islet cells. This process has long cell culture time, low conversion rate, and low purity of induced differentiation of islet cells. Defects in low levels of insulin secretion by islet cells

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  • A method for directed differentiation of autologous immune cells into islet cells
  • A method for directed differentiation of autologous immune cells into islet cells
  • A method for directed differentiation of autologous immune cells into islet cells

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Immune cells obtained from peripheral blood of patients were dedifferentiated into induced pluripotent stem cells in vitro

[0043] The method in this example refers to the method for inducing induced pluripotent stem cells in CN108642014 A patent. Dilute 50 mL of peripheral blood with normal saline at a ratio of 1:1. Carefully add the diluted blood to the same volume of lymphocyte separation medium to form obvious layers, and centrifuge horizontally at 900g for 25min at room temperature. At this time, four layers are formed in the centrifuge tube from top to bottom, and the buffy coat layer of the second layer is taken out as much as possible. Add 2 times of normal saline, wash the cells twice, discard the supernatant after centrifugation, and collect immune cells.

[0044] After the immune cells were cultured overnight in RPMI medium, the prepared lentivirus containing four genes of OCT4, SOX2, KLF4, and C-MYC was used to infect the immune cells, cultured, s...

Embodiment 2

[0045] Example 2 Directed differentiation of induced pluripotent stem cells to generate islet cells

[0046] The directed differentiation of induced pluripotent stem cells induces the generation of endoderm cells, the steps are as follows: separate the induced pluripotent stem cells with a separation medium, blow off the cell mass with a pipette, and inoculate in a culture medium coated with 0.1% fibronectin. When the cells plated to about 60%, they were cultured in S1 medium, and 14ug / ml of CHIR99021 compound was added on the first day to induce the cells. The next day, fresh S1 medium was replaced (without adding CHIR99021 compound), and cultured for 2 days to obtain definitive endoderm cells. The cells were collected, and the expressions of definitive endoderm marker genes SOX17 and FOXA2 were identified by RT-qPCR.

[0047] The endoderm cells continue to differentiate and induce protogut tubes, the medium is replaced with S2 medium every other day, and cultured for 3 days...

Embodiment 3

[0059] Example 3 RT-qPCR method to detect the expression of cell-specific markers at each stage

[0060] Collect the cells on the last day of S1, S4, and S6 stages, use conventional methods to extract total RNA, synthesize cDNA, and q-PCR to amplify the target gene. The specific markers detected include SOX17, FOXA2, PDX1, NKX6-1, INS , GCG, SST, the results are attached Figure 4-6 . The primer sequence was synthesized by Beijing Biomed Biotechnology Co., Ltd. The primer sequence and annealing temperature of the gene are shown in the table below.

[0061]

[0062] Depend on Figure 4-6 It can be seen that the endoderm-specific marker SOX17 is strongly expressed in the definitive endoderm stage, FOXA2 is also highly expressed in the definitive endoderm stage, and the transcription factor PDX1 related to pancreatic development and the transcription factor NKX6-1 related to pancreatic endoderm are in the original Expression is elevated when enterocytes are induced to the p...

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Abstract

The invention provides a method for using autologous immune cells to differentiate into pancreatic islet cells. The method is to obtain immune cells from peripheral blood of patients, dedifferentiate them in vitro into induced pluripotent stem cells, and then differentiate into pancreatic islet cells through directed differentiation. Through the method of cultivating islet cells in the present invention, immune cells isolated from autologous peripheral blood are selected to induce directional differentiation into islet cells, and then transplanted to the same patient to reduce the patient’s immune rejection. In addition, there is no Tissue matching and ethical issues. In the present invention, when induced pluripotent stem cells (iPSCs) are directedly differentiated into islet cells, after 20-28 days of induced differentiation, the purity of the finally induced islet cells identified by DTZ staining is more than 62.4%.

Description

technical field [0001] The invention relates to a method for directional differentiation of autologous immune cells into islet cells, which belongs to the technical field of biology and new medicine. Background technique [0002] The pancreas is an exocrine gland that secretes digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase, and an endocrine gland that secretes pancreatic hormones such as glucagon, insulin, somatostatin, and pancreatic polypeptide. Pancreatic hormones are secreted by a group of cells in the pancreas called "islets", which include four types of cells, namely alpha cells, beta cells, delta cells, and pp cells. [0003] Diabetes is a disease caused by insulin deficiency or insufficient work. Once this disease occurs in a patient, it is difficult to completely cure it. There are two main types of diabetes, insulin-dependent type 1 diabetes and insulin-independent type 2 diabetes. Type 2 diabetes is a chronic disease that...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/10
CPCC12N5/0676C12N2506/45
Inventor 刘明录强邦明韩庆梅王立新张传鹏金海锋卢永灿冯建海李希鹏
Owner SHANDONG XINRUI BIOTECH CO LTD
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