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Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof

A bone marrow mesenchymal and stem cell technology, applied in the field of cell engineering, can solve the problems of lack of dynamic balance, easy degeneration, and short cell survival time, and achieve strong in vitro proliferation ability, stable biological properties, and good biological safety Effect

Inactive Publication Date: 2011-11-23
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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Problems solved by technology

[0003] Although MSCs have strong proliferative potential in vivo and can divide and proliferate multiple times, in the process of isolation and culture in vitro, due to the loss of neurohumoral regulation and intercellular interactions, they live in a relatively stable environment that lacks dynamic balance. Short survival time, limited proliferative capacity and prone to degeneration

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  • Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof
  • Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof
  • Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof

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Embodiment Construction

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0031] The SSR69 plasmid and pAmhpo plasmid used in the preferred embodiment were donated by Professor He Tongchuan from the University of Chicago.

[0032] 1. Isolation, culture and identification of primary rat MSCs

[0033] SPF grade 4-8 week-old SD rats weighing 80-150 g (purchased from the Animal Center of Chongqing Medical University) were sacrificed by cervical dislocation. The femur and tibia were taken out under sterile conditions, and the bone marrow was washed out with DMEM / F12 medium, and centrifuged. Remove the supernatant and fat, press 3×10 per bottle 6 25cm 2 In a plastic culture bottle, use DMEM / F12 complete medium containing 10% fetal bovine serum at a temperature of 37°C and CO 2 Cultivate under the condition of 5% volume fraction, discard...

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Abstract

The invention discloses a recoverable immortalized rat bone marrow mesenchyme stem cell. The cell carries an SV40T (Simian Virus 40T) gene and a hygromycin resistance gene and two ends of the SV40T gene also have orthokinetic Loxp sites. The preparation method of the recoverable immortalized rat bone marrow mesenchyme stem cell comprises the following steps of: co-transfecting an HEK293 (Human Embryo Kidney 293) cell by SSR69 (Simple Sequence Repeat 69) plasmids and pAmhpo plasmids and screening by the hygromycin to obtain the recoverable immortalized rat bone marrow mesenchyme stem cell with hygromycin resistance; the cell has the same biological property with a primary rat and also has the advantages of strong external multiplication capacity, stable biological property, capability of being recovered to a previous state, good biological safety and the like; and the cell provided by the invention can be used as a seed cell induced and differentiated by nerves and a seed cell for treating hypoxicischemic brain damage of a newly-born rat.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a reversible immortalized cell, and also to a preparation method and application of the reversible immortalized cell. Background technique [0002] Bone marrow mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are a type of stem cells with self-renewal and transdermal differentiation potential existing in bone marrow, peripheral blood and umbilical cord blood. The introduction of exogenous genes has the characteristics of being able to settle or homing in specific tissues, low immunogenicity and multi-directional differentiation potential. MSCs can differentiate into nerve-like cells in a specific in vitro induction environment, and animal experiments have also shown that MSCs transplantation can survive in the central nervous system and can differentiate into functional nerve-like cells, which brings new opportunities for the treatment of nervous system diseases. h...

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61L27/38
Inventor 李廷玉毕杨龚敏陈洁江伟张赟魏小平
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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