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A method for microrna-181c-5p to promote the directed differentiation of human iPS into islet β cells
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A microRNA-181c-5p, 1.microRNA-181c-5p technology, applied in pancreatic cells, retroRNA viruses, biochemical equipment and methods, etc., can solve problems such as restricting clinical applications, and achieve the promotion of pancreatic islet beta cell differentiation. , the effect of improving expression and improving efficiency
Active Publication Date: 2022-02-11
SHENZHEN PEOPLES HOSPITAL
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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
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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
[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 microRNA‑181c‑5p to promote the directional differentiation of human iPS into pancreatic islet β cells. The present invention provides the application of microRNA-181c-5p, or its encoding gene, or a recombinant vector, recombinant virus or transgenic cell expressing said encoding gene in promoting the directed differentiation of stem cells into pancreatic islet β cells; also provides microRNA Application of ‑181c‑5p, or its coding gene, or a recombinant vector, recombinant virus or transgenic cell expressing the coding gene in the preparation of a product that promotes the directional differentiation of stem cells into pancreatic islet β cells. The present invention adopts the method of overexpressing microRNA-181c-5p, which greatly improves the efficiency of directional induction of human iPS differentiation in vitro, and the differentiation efficiency of pancreatic islet β cells in the present invention 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...
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