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Use of mesenchymal stem cell and preparation method thereof

A stem cell and transgenic technology, applied in the field of storage and release vectors and transgenic expression, can solve problems such as application limitation and body damage, and achieve the effects of convenient material collection, improved survival rate, and wide source.

Inactive Publication Date: 2013-07-10
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of these cells is limited due to reasons such as age and gender restrictions on obtaining these cells, or the possibility of damage to the body, or their immunogenicity

Method used

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  • Use of mesenchymal stem cell and preparation method thereof
  • Use of mesenchymal stem cell and preparation method thereof
  • Use of mesenchymal stem cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 for the preparation of transgenic mesenchymal stem cells

[0050] (1) Isolation, culture and identification of hair follicle mesenchymal stem cells:

[0051] Human hair follicle stem cells obtained by extracting or surgically obtaining human hair follicles or human hair follicle tissue blocks, and digesting hair follicles or hair follicle tissue blocks with proteases for culture, specifically:

[0052] Phosphate-buffered saline (PBS) was used to wash the hair follicles or hair follicle tissue pieces obtained from humans, and then digested with 0.25w / v% trypsin-0.02v / v%EDTA solution at 37°C for 3-10 minutes, then terminated with fetal bovine serum and inoculated Into a well plate containing 10v / v% fetal bovine serum and bFGF (10ng / ml) DMEM-F12 medium, at 37°C, 5%CO 2 cultivated in the environment.

[0053] Dispersed human hair follicle mesenchymal stem cells were digested with 0.25% trypsin / EDTA (see figure 1 ), after adding an equal volume of DMEM-F12 mediu...

Embodiment 2

[0066] Example 2 Lentiviral transfection of mesenchymal stem cells containing insulin gene

[0067] 293T cells (see Journal of Cell Biology [J], 2009,31(1):130) were plated one day before transfection (4×10 4 cells / cm 2 ). On the day of transfection, 6 μg of pSRV-rev plasmid, 10 μg of pMDL g / p plasmid, 3 μg of pMD2G / VSVG plasmid, and 20 μg of fusion protein plasmid containing proinsulin (the plasmids are all delivered by Professor Andreadis, Department of Chemical and Biological Engineering, State University of New York at Buffalo, USA) Gift) mix, add 120 μl transfection reagent HD (Promega), after mixing, was added dropwise to 293T cells. After 48 hours, the supernatant was collected and filtered through a 0.45 μm filter to obtain live virus particles. by As a transduction agent, live virus particles are used to transfect human hair follicle mesenchymal stem cells or umbilical cord mesenchymal stem cells obtained in Example 1. After 3 days of transfection, when cells ...

Embodiment 3

[0068] Example 3 Experimental Research on Insulin Release in Vitro

[0069] After the (insulin) gene transgenic hair follicle mesenchymal stem cells or umbilical cord mesenchymal stem cells were cultured to cover a single layer, Rapamycin was added to the culture medium to a final concentration of 5 μmol / L. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours after the addition of Rapamycin, the culture supernatant was collected. The content of C-peptide in the supernatant was detected by ultra-sensitive ELISA kit (human C-peptide ELISA kit, Millipore, Billerica, MA). The results showed that: 4 hours after adding Rapamycin, the content of C-peptide in the supernatant was the highest (see Figure 8 ).

[0070] After the (insulin) gene transgenic hair follicle mesenchymal stem cells or umbilical cord mesenchymal stem cells were cultured to cover a single layer, Rapamycin was added to the culture medium to a final concentration of 1, 2, 3, 4, 5, 6, 7, 8 , 9 and 10 μmol / l. Four hours after...

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Abstract

The invention discloses a mesenchymal stem cell, which is used as a target cell for gene treatment, and used for a carrier of expressing, storing and releasing a transgenic drug. An exogenous promoter and a target gene are led to the mesenchymal stem cell by a virus carrier; and the target gene is integrated into a genome of a host cell, so as to stabilize that the endogenously expressed target gene is not lost and used for gene treatment. It is fully verified that the mesenchymal stem cell can be used as a storage cavern for expressing, storing and releasing a target drug, and has the characteristics of being efficient, safe, reliable and the like by an insulin gene treatment experiment.

Description

technical field [0001] The invention relates to a transgene expression, storage and release carrier, in particular to a mesenchymal stem cell used for the expression, storage and release of a transgene drug and a preparation method thereof, which is used as a carrier for the preparation of a transgene drug. Background technique [0002] Currently, there are various treatments and medicines for diabetes. Among these methods, islet cell transplantation is the first choice for the treatment of diabetes. However, islet transplantation is not only a complicated operation process, but also the source of islets is limited, which is far from meeting the needs of a large number of diabetic patients. [0003] In recent years, stem cell therapy has gradually become a research hotspot, trying to repair diseased islet tissue through stem cell transplantation, so that it can reproduce the function of secreting insulin, or directly induce differentiation of stem cells or transfect genes in...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P3/10C12N5/10C12N15/867A61K31/436
Inventor 刘晋宇吴春铃赵桂芳刘菲琳
Owner JILIN UNIV
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