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Double-gene modified stem cell and application thereof

A stem cell and double gene technology, applied in the field of cell therapy, can solve the problems of drug treatment, drug resistance, restriction, etc., and achieve the effect of lowering blood sugar, reducing weight, and improving blood sugar

Active Publication Date: 2020-12-29
北京华奥玄德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current research on SphK1 is mainly based on its role in cells, and the drugs developed to target the protein mainly use its antibody or antagonist, and do not directly make the protein into a drug for treatment; the other is based on Viruses, etc. are used as carriers to introduce the SPHK1 gene into cells for gene therapy, such as gene therapy using adenovirus as a carrier, but this method is prone to drug resistance in the body, and the treatment of metabolic diseases such as diabetes requires long-term medication, so it is limited use of the method

Method used

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  • Double-gene modified stem cell and application thereof
  • Double-gene modified stem cell and application thereof
  • Double-gene modified stem cell and application thereof

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Experimental program
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Effect test

Embodiment approach

[0038] According to a preferred embodiment of the present invention, the adiponectin variant has one or more amino acid substitutions, insertions or deletions, or at least 80%, preferably more than 95%, of the sequence from which it is derived. Such as 99% sequence identity, and retain the activity of adiponectin;

[0039] The sphingosine kinase 1 variant has one or more amino acid substitutions, insertions or deletions, or at least 80% or more, preferably at least 95% or more, such as 99% sequence identity, compared with the sequence it is derived from , and retained the activity of sphingosine kinase 1.

[0040] Among them, the adiponectin variant retains the activity of adiponectin, that is, it retains its ability to increase fatty acid oxidation and sugar absorption in skeletal muscle cells, strengthen the inhibitory effect of insulin on gluconeogenesis, and reduce blood triglyceride and low-density lipoprotein levels Ability;

[0041] The sphingosine kinase 1 variant re...

Embodiment 1

[0126] Example 1 Obtaining of mesenchymal stem cells

[0127] The mesenchymal stem cells were isolated by the umbilical cord tissue block slide method, and the specific steps were as follows:

[0128] Put the isolated umbilical cord from normal delivery into PBS buffer solution containing 200 U / mL penicillin and 200 U / mL streptomycin. To ensure the activity of umbilical cord tissue, the fresh umbilical cord should be separated within 6 hours. Flush the residual blood in the umbilical vein and umbilical artery with a 20mL syringe, and cut the umbilical cord tissue into 1mm pieces with tissue scissors 3 Then filter the obtained small pieces of umbilical cord tissue with a 200-mesh filter, collect the umbilical cord tissue pieces on the 200-mesh filter, remove the too small umbilical cord tissue pieces, and obtain multiple umbilical cord tissues with a diameter of 1-1.5mm piece. Collect tissue pieces with a diameter of 1-1.5mm, inoculate the tissue pieces directly in culture ...

Embodiment 2

[0129] Example 2 Cloning and vector construction of Adiponectin and QSPHK1 genes

[0130] The DNA molecules encoding Adiponectin and QSPHK1 were synthesized from the whole gene of Zhongmei Taihe Biotechnology (Beijing) Co., Ltd., wherein Adiponectin (the amino acid sequence is SEQ ID NO: 1, and the coding sequence is SEQ ID NO: 3) and QSPHK1 (the amino acid sequence is SEQ ID NO:2, coding sequence is SEQ ID NO:4) connected by T2A sequence (amino acid sequence is SEQ ID NO:5, coding sequence is SEQ ID NO:6).

[0131] With primer EF1-BamHI-Adiponectin-F (nucleotide sequence is SEQ ID NO:9) and primer T2A-SPHK1-R (nucleotide sequence is SEQ ID NO:12) amplified Adiponectin-T2A fragment (851bp), With primer T2A-SPHK1-F (nucleotide sequence is SEQ ID NO:11) and primer SPHK1-SalI-WPRE-R (nucleotide sequence is SEQ IDNO:10) amplified QSPHK1 fragment (1278bp), by agarose The amplified products were analyzed by gel electrophoresis.

[0132] The recombinant lentiviral vector plasmid pC...

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Abstract

The invention discloses a double-gene modified stem cell as well as a preparation method and application thereof, and further provides a pharmaceutical composition containing the stem cell. The stem cell comprises a first exogenous nucleic acid and a second exogenous nucleic acid which are located in the same expression vector. The first exogenous nucleic acid encodes a nucleotide sequence comprising a first protein, and the first protein is selected from adiponectin, an adiponectin variant or a first fusion protein comprising the adiponectin or the adiponectin variant. The second exogenous nucleic acid encodes a nucleotide sequence comprising a second protein, and the second protein is selected from sphingosine kinase I, a sphingosine kinase I variant, or a second fusion protein comprising the sphingosine kinase I or the sphingosine kinase I variant. The stem cell modified by double genes provided by the invention shows a synergistic effect of an Adiponectin gene and a mutated SPHKI gene, and can obviously reduce the blood sugar and blood fat of a tested animal and reduce the weight.

Description

technical field [0001] The invention belongs to the technical field of cell therapy, and in particular relates to stem cells for treating diabetes, in particular to stem cells co-modified by adiponectin and mutated sphingosine kinase 1 (SPHK1) genes and uses thereof. Background technique [0002] Obesity and type 2 diabetes mellitus (T2DM) are one of the major public health problems plaguing modern society. Obesity and its accompanying insulin resistance are key factors in the pathogenesis of type 2 diabetes. According to surveys, 80-90% of type 2 diabetes patients are overweight or obese. Therefore, effective control of blood sugar and body weight has always been a focus topic in related research fields. According to statistics, the global incidence of type 2 diabetes has reached 400 million, accounting for 90%-95% of all diabetic patients. Insufficient secretion, decreased activity or functional defects of endogenous hormones, cytokines or enzymes are closely related to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/16C12N15/54A61K35/28A61P3/10A61P3/04A61P3/06A61P3/00A61P9/10
CPCC12N5/0665C07K14/575C12N9/1205C12N15/86A61K35/28A61P3/10A61P3/04A61P3/06A61P3/00A61P9/10C12Y207/01091C12N2510/00C12N2740/15043
Inventor 段海峰薛冰华于婷婷解晶庞如梦刘丽华
Owner 北京华奥玄德生物医药科技有限公司
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