RNA interference carrier used for osteoporosis and rat mesenchymal stem cells

A technology for RNA interference and osteoporosis, applied in the field of molecular biology, with good application prospects

Inactive Publication Date: 2009-10-14
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many reasons for osteoporosis, such as increasing age, long-term use of glucocorticoids, etc., and there is currently no ideal treatment

Method used

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  • RNA interference carrier used for osteoporosis and rat mesenchymal stem cells
  • RNA interference carrier used for osteoporosis and rat mesenchymal stem cells
  • RNA interference carrier used for osteoporosis and rat mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of an shRNA lentiviral expression vector that down-regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ)

[0031] 1. Isolation, culture, expansion and identification of rat MSC

[0032] Isolation, culture, and expansion of MSCs: adult rat femurs were washed out of the bone marrow, mixed thoroughly, centrifuged, and the precipitate was collected, and then the mononuclear cell layer was collected with Percoll separation medium with a density of 1.083, and cultured in DMEM containing 10% FCS and low sugar for 5 -7 days, replace the culture medium, and then change the medium every 4 days. After the cells were 80% confluent, they were digested with 0.25% trypsin and passaged.

[0033] Rat MSC identification: flow cytometry identification of cell surface marker antigens, mainly including CD29, CD44, CD45 (Pharmigen), in which CD29, CD44 are positive, CD45 is negative. 2. Establish an in vitro model for directional inducti...

Embodiment 2

[0047] Example 2: Preparation of Rat Bone Marrow Mesenchymal Stem Cells Down-regulating the Expression of PPARγ

[0048] 1. Virus packaging, collection:

[0049] Virus packaging was carried out according to the instructions of the lentiviral packaging kit (catalogue number K4950-00) of Invitrogen Company. The details are as follows: 293FT cells (product of Invitrogen Company, catalog number R700-07) were inoculated in a cell culture dish with a diameter of 100 mm, and when the cells reached 95% confluence, they were replaced with fresh high-glucose DMEM medium containing 10% fetal bovine serum . Use 36 μl of liposome lipo-fectamin2000 to transfect 3 μg of PPARγshRNA lentiviral expression vector plasmid and 9 μg of package mix plasmid (including VSVG, RSV-REV, pMDL g / p RRE, provided in the Invitrogen lentiviral packaging kit) into 293FT cells. Incubate in a carbon dioxide cell incubator for 24 hours, change 10 ml of complete medium, and observe the changes of 293FT cells and...

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Abstract

The invention relates to a RNA interference carrier used for osteoporosis and mesenchymal stem cells. The RNA interference carrier is constructed by introducing a DNA fragment which uses peroxisome proliferator-activated receptor Gamma (PPAR Gamma) as an action target and can express short chain RNA (shRNA) so as to build a slow virus expression carrier in a framework plasmid expressed based on the short chain RNA of slow virus; the constructed RNA interference carrier can express a special short chain RNA aiming at the PPAR Gamma, thereby reducing the expression of the PPAR Gamma. The invention also provides rat mesenchymal stem cells transfected by the RNA interference carrier. The invention not only provides a new way for preparing the medicine for treating the osteoporosis, but also provides a better experiment platform for the development of other stem cell-origin cell therapy and tissue engineering products.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a shRNA lentiviral expression carrier for down-regulating the expression of PPARγ and a gene-modified bone marrow mesenchymal stem cell formed after the carrier is transferred into rat bone marrow mesenchymal stem cells. Background technique [0002] Osteoporosis is a systemic abnormal bone metabolism disease characterized by decreased bone mass and degeneration of bone tissue microstructure, which leads to increased bone fragility and increased risk of fracture. There are many reasons for osteoporosis, such as increasing age, long-term use of glucocorticoids, etc., and there is no ideal treatment method yet. With the prolongation of life expectancy, the harm caused by the increase of the incidence of osteoporosis is becoming more and more serious, and it is imminent to find effective treatment methods. [0003] In addition to the hematopoietic system, bone marrow also contains bo...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N15/11C12N5/10A61K48/00A61P19/10
Inventor 张丽蓉
Owner GUANGDONG PHARMA UNIV
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