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Ischemia hypoxia-resistant human mesenchymal stem cells, and preparation method and application

A kind of stem cell and stem cell technology, which is applied in the directions of genetically modified cells, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effects of simple technology, improved survivability, and reduced death

Active Publication Date: 2016-09-28
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But whether ESE-3 can promote stem cell apoptosis has not been reported

Method used

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  • Ischemia hypoxia-resistant human mesenchymal stem cells, and preparation method and application
  • Ischemia hypoxia-resistant human mesenchymal stem cells, and preparation method and application
  • Ischemia hypoxia-resistant human mesenchymal stem cells, and preparation method and application

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Embodiment 1

[0025] A method for preparing human mesenchymal stem cells that stably interfere with ESE-3 gene ischemia and hypoxia resistance, comprising the following steps:

[0026] Isolation and culture of human placental mesenchymal stem cells, detection of ESE-3 gene expression and preparation of placental mesenchymal stem cells transfected with siRNA targeting ESE-3.

[0027] 1) Isolation of placental mesenchymal stem cells: the human placenta tissue was shredded mechanically, and the placental mesenchymal stem cells were obtained by enzymatic digestion method well known to professionals.

[0028] 2) Expansion and culture of placental mesenchymal stem cells: the above cells were divided into 0.5-5×10 5 / cm 2 Inoculate the culture flask at a density of 37°C, saturated humidity, 5% CO 2 Primary cell cultures were performed in an incubator. The medium used was serum-free medium. When cells are cultured to 80-90% confluence, use trypLE TM Digest and inoculate into new culture flasks...

Embodiment 2

[0033] A method for preparing human mesenchymal stem cells resistant to ischemia and hypoxia without exogenous gene insertion, comprising the following steps:

[0034] 1) Isolation and expansion of human mesenchymal stem cells: the umbilical cord tissue was cut into pieces mechanically, and the umbilical cord mesenchymal stem cells were isolated according to the conventional tissue block attachment method.

[0035] 2) Place the obtained umbilical cord tissue block at 37°C, saturated humidity, 5% CO 2Primary cell cultures were performed in an incubator. The medium used was DF12 medium containing 10% fetal bovine serum. When the cells were cultured to 80-90% confluence, they were digested with trypsin and inoculated into new culture flasks at a split ratio of 1:3. After several passages, the obtained mesenchymal stem cells can be used for subsequent operations;

[0036] 3) Transfect the synthesized siRNA interference fragment (targeting sequence: GCCGAGCTATGAGATATTA) (SEQ ID ...

Embodiment 3

[0039] Interfering with ESE-3 gene can significantly improve the survival of mesenchymal stem cells under severe hypoxic conditions.

[0040] We simulated a hypoxic environment through a three-gas incubator, with normoxia at 21% oxygen content and severe hypoxia at 1% oxygen content, and cultured cells with medium containing 2% serum. Mesenchymal stem cells were infected with a lentiviral vector carrying shRNA targeting ESE-3, the shRNA sequence was GCCGAGCTATGAGATATTA TCAAGAG TAATATCTCATAGCTCGGC (SEQ ID NO: 2), and the control group used a nonsense shRNA lentiviral vector, and the two groups of cells were respectively Inoculate into 6-well plate and 96-well plate, the normoxic environment is 21% oxygen content, 5% carbon dioxide, the hypoxic group is placed in 1% oxygen content, 5% carbon dioxide environment and cultured for 72 hours, and the cells in the 6-well plate are photographed to observe the cells Morphology, 96-well plate cells were subjected to MTT assay.

[0041] ...

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Abstract

The invention discloses ischemia hypoxia-resistant human mesenchymal stem cells, a preparation method and application. The preparation method comprises the following steps: (1) amplifying human mesenchymal stem cells; (2) inhibiting ESE-3 genes in the human mesenchymal stem cells through an RNA (Ribose Nucleic Acid) interference technology, wherein the ESE-3 genes are as shown in SEQ ID NO:1; obtaining the ischemia hypoxia-resistant human mesenchymal stem cells. An experiment shows that the survival ability of mesenchymal stem cells in an ischemia hypoxia environment can be obviously increased through inhibiting the expression of the ESE-3 genes of the human mesenchymal stem cells; according to the ischemia hypoxia-resistant human mesenchymal stem cells disclosed by the invention, the death caused by an ischemia hypoxia state can be obviously reduced. According to the ischemia hypoxia-resistant human mesenchymal stem cells, the preparation method and the application, disclosed by the invention, the technology is simple and is easy to realize; the ischemia hypoxia-resistant human mesenchymal stem cells can be used for tissue damage repairing and angiogenesis.

Description

technical field [0001] The invention belongs to the field of biotechnology, is related to cell therapy products, and specifically relates to a human mesenchymal stem cell resistant to ischemia and hypoxia, a preparation method and application thereof. Background technique [0002] Stem cells are the seed cells for the renewal of various tissue cells, and are the production factories of human cells. Through self-renewal, stem cells produce the same daughter cells and maintain the stability of the number of seed cells in the body; through multidirectional differentiation, they become a variety of different tissue cells that can perform certain physiological functions, replacing normal physiological metabolism or Damaged functional cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells under specific induction conditions in vitro, and they still have the ability to survive after continuous su...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/28A61P9/10
CPCA61K35/28C12N5/0668C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 韩之波池颖韩忠朝
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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