Fish egg cell extract and use thereof for inducing human adult cell to differentiate into pluripotent stem cell

A technology of pluripotent stem cells and fish egg cells, which is applied in the field of biological preparations, can solve the problems of long experiment period, hidden safety hazards, complicated operation, etc., and achieve the effect of short experiment period, no damage to cells, and simple operation

Inactive Publication Date: 2009-07-15
成都军区昆明总医院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, retroviral vectors carrying genes will lead to tumors and the expression of the oncogene c-Myc gene in tissues developed from pluripotent stem cells, all of which have great safety risks, and the cost is high, the operation is very complicated, and the experiment The cycle is long

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1) Isolation and culture of adult cells

[0023] Such as the acquisition of human long fiber cells: surgically cut out the patient’s abdomen 2~3cm 2 Put the skin in the phosphate buffered solution (PBS) containing 100U / ml (U is the international unit) ampicillin and 100mg / ml streptomycin sulfate. After repeated rinsing with PBS, cut the fat And connective tissue. Cut the skin into 1mm 3 Add the left and right small pieces to DMEM medium containing 200U / ml collagenase and 300U / ml hyaluronidase, and place them at 4°C overnight. Add a PBS digestion solution containing 0.25% pancreatin and 0.02% ethylenediamin etetraacetic acid (EDTA) at a ratio of 1:1, and use an air shaker at 37°C for 180 revolutions per minute (rpm) for 20 minutes, The serum is terminated. The cells were collected by centrifugation at 1000 rpm for 10 min. After eluting the DMEM medium twice, adjust the cell density with DMEM medium containing 15% fetal bovine serum to a density of (2~4)×10 per ml. 5 , Cell s...

Embodiment 2

[0036] 1) Isolation and culture of human adult cells

[0037] Obtain human adult primary lymphocytes by conventional methods.

[0038] 2) Preparation of fish egg cell extract

[0039] Fresh fish eggs were purchased, except for freezing and thawing, centrifugation and filtration as in Example 1, and grinding for 3 minutes after freezing and thawing. Obtain 50 ml of the secondary supernatant, determine the protein content to be 1 mg / ml, and concentrate it by lyophilization to make the protein content reach 4 mg / ml, and store it at 4°C for later use, which is the fish egg cell extract of the present invention.

[0040] 3) Application of fish egg cell extracts in inducing human adult cells to differentiate into pluripotent stem cells

[0041] 1. Prepare human adult cells, obtain human lymphocyte primary cells according to Example 2, take the primary adult cells, and then induce culture;

[0042] 2. Induction culture, inoculate the above-mentioned primary lymphocyte cells in an inductio...

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PUM

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Abstract

The invention discloses a fish egg cell extract and application thereof in inducing human adult cells to differentiate into pluripotent stem cells. Egg cells are obtained from a killed fish, centrifugated, filtered until the protein concentration is 4mg / ml, thus obtaining the fish egg cell extract. Ripe somatic cell primary culture cells or second-generation or fourth-generation offspring cells are obtained from each tissue of an adult; the extract is used for inducing the adult cells to be reversely differentiated into pluripotent stem cells which can provide various resources for clinic and can be used for treating various diseases. Compared with the prior art, the invention has advantages of not damaging cells, simple culturing conditions, non-toxicity, high inducing efficiency and the like, and at the same time avoids hidden safety hazard by using gene recombination technology.

Description

Technical field [0001] The invention relates to a biological preparation, in particular to a fish germ cell extract and its application in inducing various mature adult cells to transform into immature pluripotent stem cells. Background technique [0002] Currently, a well-known technique for reversely differentiating adult cells into pluripotent stem cells is gene rearrangement technique. Gene rearrangement technology is a pluripotent stem cell obtained by introducing specific genes related to stem cells into cells using viral vectors. The gene-carrying retroviral vector can cause tumors and the expression of the oncogene c-Myc gene in the tissues developed from pluripotent stem cells. There are great safety risks, and the cost is high, the operation is very complicated, and the experiment The cycle is long and so on. [0003] In order to avoid destroying early embryos; in order to avoid the need to directly use human embryonic stem cells in research, to avoid long-term ethical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/074
Inventor 潘兴华朱向情
Owner 成都军区昆明总医院
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