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Transgenic cell with in-vivo tracking and oncotherapy functions and preparation method thereof

A technology for transgenic cells and tumor treatment, which is applied in the field of cells with in vivo tracer and tumor treatment functions. It can solve the problems of difficult acquisition, limited source of endothelial precursor cells, and clinical difficulties, and achieve the effect of evaluating curative effect

Active Publication Date: 2014-04-02
NANKAI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, studies have used endothelial precursor cells isolated from bone marrow, embryos or umbilical cords for experimental targeted tumor therapy. Although it has been proven to be effective, the source of such endothelial precursor cells is limited and difficult to obtain. Difficult to apply clinically

Method used

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  • Transgenic cell with in-vivo tracking and oncotherapy functions and preparation method thereof
  • Transgenic cell with in-vivo tracking and oncotherapy functions and preparation method thereof
  • Transgenic cell with in-vivo tracking and oncotherapy functions and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of human umbilical cord-derived mesenchymal stem cells carrying three fusion proteins

[0043] (1) Construction of a Bsd-resistant lentiviral expression vector containing triple fusion genes of renilla luciferase, red fluorescent protein and suicide gene thymidine kinase: subcloning the thymidine kinase containing renilla luciferase, red fluorescent protein and suicide gene thymidine kinase The fragment of the glycoside kinase triple fusion gene was inserted into the multiple cloning site of the commercialized vector pLV-EF1α-MCS-IRES-Bsd to obtain the pLV-EF1α-TF-Bsd plasmid, such as figure 1 shown;

[0044] ⑵Pave a six-well plate with 293T cells at a density of 1.5×10 6 cells / well for transfection;

[0045] (3) Transfect pLV-EF1α-TF-Bsd lentiviral expression plasmid and lentiviral packaging plasmid into 293T cells plated the day before with lipo-2000;

[0046] (4) Change fresh DMEM medium 16 hours after transfection (DMEM is a medium containing various ...

Embodiment 2

[0054] In vivo tracking of human umbilical cord-derived mesenchymal stem cells carrying triple fusion proteins in Nude mouse model of breast cancer after intratumoral injection.

[0055] (1) Orthotopic injection of 1.0×10 human breast cancer MDA-MB-231 cells carrying firefly luciferase and green fluorescent protein into both sides of the abdomen of 6-8-week-old female Nude mice 6 indivual;

[0056] (2) After 12 days, the firefly luciferase substrate D-Luciferin (150mg / kg) was injected into the mouse intraperitoneally, and after 5 minutes, the chemiluminescent signal was detected by the Xenogen IVIS Lumina II in vivo imaging system, with an exposure time of 30 seconds, and both sides of the abdomen could be seen A fluorescent signal was emitted, confirming that the Nude mouse breast cancer model was successfully established;

[0057] (3) The human umbilical cord-derived mesenchymal stem cells carrying the three fusion proteins obtained in Example 1 were digested with trypsin, ...

Embodiment 3

[0061] Human umbilical cord-derived mesenchymal stem cells carrying three fusion proteins were traced in vivo in breast cancer model Nude mice after intratumoral injection and intraperitoneal injection of ganciclovir.

[0062] (1) Orthotopic injection of 1.0×10 human breast cancer MDA-MB-231 cells into both sides of the abdomen of 6-8-week-old female Nude mice 6 indivual;

[0063] (2) After 12 days, the firefly luciferase substrate D-Luciferin (150mg / kg) was injected intraperitoneally into the mouse, and after 5 minutes, the chemiluminescent signal was detected by the Xenogen IVIS Lumina II in vivo imaging system, and the exposure time was 30 seconds. Signals occurred on both sides of the abdomen, confirming that the Nude mouse breast cancer model was successfully established.

[0064] (3) The human umbilical cord-derived mesenchymal stem cells carrying the three fusion proteins obtained in Example 1 were digested with trypsin, and resuspended to 1×10 7 Cells / ml, intratumora...

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Abstract

The invention discloses a transgenic cell with in-vivo tracking and oncotherapy functions and a preparation method thereof. The transgenic cell is a mesenchymal stem cell from a human umbilical cord, is capable of performing oncotherapy, has a molecular imaging function, and is capable of monitoring the body distribution and survival condition in real time in a living body state after being subjected to intratumor injection. Particularly, a renilla luciferase-red fluorescence protein-suicide gene thymidine kinase fusion gene is transfected in the mesenchymal stem cell from the human umbilical cord, and a fluorescein signal is collected by means of a living body imaging system to monitor targeted therapy of a Nude mouse breast cancer model; a mouse injected with the mesenchymal stem cell transfected with the fusion gene is administrated with a substrate ganciclovir of thymidine kinase, and the death of tumor cells around the mesenchymal stem cell is induced through the bystander effect.

Description

technical field [0001] The invention belongs to the field of cell therapy, and relates to a cell capable of tracking and treating tumors in vivo. Background technique [0002] At present, breast cancer has become one of the malignant tumors with the highest incidence rate in women all over the world, seriously threatening the physical and mental health of women. Under the current technical conditions, the primary tumor lesion can be radically cured by surgery, but the surgery brings great pain to the patient. Chemotherapy is currently used for the treatment of breast cancer. Most chemotherapeutic drugs cannot specifically act on tumor cells. While killing tumor cells, they also have a certain killing effect on normal cells, resulting in serious side effects. Patients often Long-term survival cannot be obtained, and the treatment cost is expensive, which brings heavy economic burden to patients. At present, there is an urgent need for tumor treatment to find a carrier that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867A61P35/00
Inventor 李宗金韩忠朝冷良韩之波徐旸赵钱杰王悦冰
Owner NANKAI UNIV
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