Separation method and application of placenta-derived stem cells

A technology derived from placenta and stem cells, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve problems such as poor therapeutic effect and peripheral blood vessel occlusion

Inactive Publication Date: 2017-05-31
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ischemic disease Large arterial lesions can be treated with vascular bypass and intravascular intervention, but for patients with peripheral vascular occlusion and who cannot tolerate surgery, the effect of drug therapy alone is not good

Method used

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  • Separation method and application of placenta-derived stem cells
  • Separation method and application of placenta-derived stem cells
  • Separation method and application of placenta-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Isolation and culture of placental mesenchymal stem cells

[0053] Using a new compound enzyme culture system, including 0.05mg / mL trypsin, 1mg / mL collagenase and 1mg / mL dispase (neutral enzyme), the digestive enzyme combination solution was pumped into the placenta through the umbilical vein and arteries at a speed of 50mL / hour to achieve the effect of digesting and separating cells. Digest at 37°C for 2 hours. Flushed cells were collected, washed and cultured. The culture conditions are DMEM containing 15% FBS to separate and culture placental mesenchymal stem cells PSCs are fibroblast-like, and the cells are in good condition ( figure 1 ).

[0054] 1. Reagent preparation

[0055] a) Dispase (neutral protease Ⅰ) stock solution (5mg / mL)

[0056] Dissolve the dispase in ultrapure water. The freeze-dried enzyme can be stored at 2-8 degrees until the shelf life. The aqueous solution is stored at -15 to -25 degrees. When opening the lid, be careful not to...

Embodiment 2

[0095] Example 2: Ad-HGF modified PSCs promote HGF expression

[0096] PSCs were infected with Ad-HGF at a multiplicity of infection (MOI). After 48 hours of infection, the cells were collected, and then the expression level of HGF was determined by QPCR. Moreover, after the angiography, the ischemic hindlimb muscle tissue of the rabbit was taken, and then the expressions of VEGF, bFGF and HGF in the limb muscle tissue were determined by QPCR.

[0097] Materials: DMEM medium and Ad-HGF were produced and prepared by our laboratory, and VEGF, bFGF, and HGF primers were synthesized by Aoke Company. QPCR instrument 7500 (ABI company, the United States), Green Real-time PCR Master Mix (TAKARA company, Japan).

[0098] Method and steps

[0099] 1). Extraction of tissue RNA: After the imaging, take the rabbit ischemic hindlimb muscle tissue, add liquid nitrogen to freeze and grind, add TRIZOL to lyse and extract tissue total RNA, the steps are as follows:

[0100] ① Cut out 0....

Embodiment 3

[0132] Example 3: HGF Gene Modified PSC Transplantation Promotes Angiogenesis

[0133] 3.1 Production of animal models and stem cell transplantation:

[0134] Materials: 10 New Zealand white rabbits, ordered by the Experimental Animal Center of the Academy of Military Medical Sciences (animal license number: SCXK (Beijing) 2015-0005), weighing 2-3 kg, male and female, 6 weeks old.

[0135] Method and steps

[0136] 3.1.1 Rabbits were given general anesthesia by injecting 3% pentobarbital sodium (50mg / kg) through the ear vein, and the left and right lower limbs of the experimental rabbits were used to make models.

[0137] 3.1.2 After depilation and disinfection of the left and right hind limbs, a long incision was made from the midpoint of the inguinal ligament to the knee, and a longitudinal incision was made from the inguinal ligament on both sides to the knee joint to separate the femoral artery and its branches. The femoral artery and its branches were cut off, and 1 m...

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Abstract

The invention relates to a novel separation method of placenta-derived stem cells and an application of the placenta-derived stem cells to treatment of ischemic diseases. According to the separation method, the placenta-derived stem cells are effectively separated by the aid of novel compound enzyme liquid. In vitro genetic modification of the placenta-derived stem cells is achieved, vessel regeneration can be induced in an animal model, and collateral circulation is improved, so that effect for treating ischemia is achieved.

Description

technical field [0001] The invention relates to the field of separation of placenta-derived stem cells, gene modification method and treatment of ischemic diseases. Specifically, the present invention relates to a new method for isolating placenta-derived stem cells and the application of the genetically modified cells in the treatment of ischemic diseases. [0002] technical background [0003] Ischemic diseases include coronary heart disease, lower extremity arterial ischemia, diabetic arteriosclerosis, etc. Ischemic diseases and large arterial lesions can be treated with vascular bypass and intravascular intervention, but for patients with peripheral vascular occlusion and who cannot tolerate surgery, the effect of drug therapy alone is not good. The combination of stem cells and gene therapy can effectively improve the ischemic tissue of the body. [0004] Stem cell transplantation offers new therapeutic approaches for ischemic diseases of the limbs. The placenta conta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/10C07K14/515A61L27/38A61L27/54
Inventor 王立生肖凤君王华杨月峰黄玉香高宏
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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