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Method for promoting human iPS to be directionally differentiated into pancreatic Beta cells through microRNA-181c-5p

A technology of microrna-181c-5p, 1.microrna-181c-5p, which is applied in the direction of pancreatic cells, retroRNA viruses, biochemical equipment and methods, etc., can solve the problems that restrict clinical application, and achieve the promotion of islet β cell differentiation , increase the amount of expression, improve the effect of efficiency

Active Publication Date: 2019-03-19
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology has not made breakthroughs in the two key technical problems of "functional maturity" and "differentiation efficiency", which seriously restricts its clinical application

Method used

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  • Method for promoting human iPS to be directionally differentiated into pancreatic Beta cells through microRNA-181c-5p
  • Method for promoting human iPS to be directionally differentiated into pancreatic Beta cells through microRNA-181c-5p
  • Method for promoting human iPS to be directionally differentiated into pancreatic Beta cells through microRNA-181c-5p

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Establishment and identification of human iPS cell lines expressing microRNA-181c-5p (miR-181c-5p)

[0091] 1. Establishment of a human iPS cell line overexpressing microRNA-181c-5p

[0092] 1. Lentivirus overexpressing microRNA-181c-5p

[0093] The lentivirus overexpressing microRNA-181c-5p is a lentivirus expressing microRNA-181c-5p (marked as microRNA-181c-5p up) by transfecting the recombinant expression vector into 293T cells, which expresses microRNA-181c -5p (sequence 2).

[0094] The lentivirus that overexpresses the empty vector is the CV-130 empty vector (Shanghai Jikai Gene Chemical Technology Co., Ltd., such as figure 1 (shown) was transfected into 293T cells, and a lentivirus expressing an empty vector (marked as scramble) was packaged.

[0095] The recombined overexpression vector is to insert the microRNA-181c-5p precursor sequence (sequence 1) into the CV-130 vector (Shanghai Jikai Gene Chemical Technology Co., Ltd., such as figure 1 The DNA...

Embodiment 2

[0119] Example 2. Directed differentiation of human iPS cell lines overexpressing microRNA-181c-5p (hiPSCs-181c up) into pancreatic islet β cells in vitro

[0120] 1. A method for directed differentiation of human iPS cell lines overexpressing microRNA-181c-5p (hiPSCs-181c up) into pancreatic islet β cells

[0121] The configuration of the main reagents in the following method is as follows:

[0122] (1) Preparation of complete human-derived iPS medium: dissolve mTeSR overnight in a refrigerator at 4°C TM 1 Reagent component 2 (5×addition), add basal medium mTeSR TM 1 Reagent component 1 (basic medium), mix well and divide. It can be stored for 2 weeks in a refrigerator at 4°C for short-term storage, and can be stored for 6 months in a refrigerator at -20°C for a long-term storage.

[0123] (2) 0.1% BSA aqueous solution: Weigh 0.01g of BSA dry powder and dissolve it in 10mL of sterile ddH2O, filter through a 0.22μm PVDF membrane and store at 4°C.

[0124] (3) 0.1% BSA in P...

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Abstract

The invention discloses a method for promoting human iPS to be directionally differentiated into pancreatic Beta cells through microRNA-181c-5p, and provides an application of the microRNA-181c-5p, oran encoding gene thereof, or a recombinant vector for expressing the encoding gene, a recombinant virus or a transgenic cell for promoting a stem cell to be directionally differentiated into the pancreatic Beta cells, and further provides an application of the microRNA-181c-5p, or the encoding gene thereof, or the recombinant vector for expressing the encoding gene, the recombinant virus or the transgenic cell for preparing a product of promoting the stem cell to be directionally differentiated into the pancreatic Beta cells. The method is capable of, through using a mode of the overexpression microRNA-181c-5p, greatly improving efficiency of in-vitro directionally inducing human iPS differentiation. Differentiation efficiency of the pancreatic Beta cells in the method reaches 35.8%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for microRNA-181c-5p to promote directional differentiation of human iPS into pancreatic islet β cells. Background technique [0002] Diabetes has become a global frequently-occurring and refractory metabolic disease. According to the latest data released by the World Health Organization, in 2014, about 9% of adults over the age of 18 worldwide suffered from diabetes. In my country, the number of diabetic patients has reached 110 million, ranking first in the world. Diabetes can be divided into type I and type II. Type I diabetes is an autoimmune disease in which autoimmune cells are overactivated to attack pancreatic β cells, resulting in massive death of pancreatic β cells and insufficient insulin secretion. Traditional drug therapy or recombinant insulin therapy cannot delay the progression of diabetes, and many patients still suffer from acute or chronic co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0676C12N15/86C12N2501/117C12N2501/11C12N2501/115C12N2500/38C12N2500/40C12N2501/16C12N2501/155C12N2501/405C12N2501/335C12N2506/45C12N2506/02C12N2501/85C12N2501/998C12N2740/15043
Inventor 李富荣李宁蒋豆蔻邓春艳
Owner SHENZHEN PEOPLES HOSPITAL
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