Mesenchymal stem cells with in vivo tracer and tumor treatment effects and preparation method thereof

A technology of therapeutic effect and quality stem cells, applied in the field of cell therapy, can solve the problems of difficult to obtain, limited source of endothelial precursor cells, difficult to clinical, etc., and achieve the effect of evaluating curative effect.

Active Publication Date: 2018-01-19
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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  • Mesenchymal stem cells with in vivo tracer and tumor treatment effects and preparation method thereof
  • Mesenchymal stem cells with in vivo tracer and tumor treatment effects and preparation method thereof
  • Mesenchymal stem cells with in vivo tracer and tumor treatment effects and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of human umbilical cord-derived mesenchymal stem cells carrying three fusion proteins

[0041](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;

[0042] ⑵Pave a six-well plate with 293T cells at a density of 1.5×10 6 cells / well for transfection;

[0043] (3) Use Lipo-2000 to transfect pLV-EF1α-TF-Bsd lentiviral expression plasmid and lentiviral packaging plasmid into 293T cells plated the day before;

[0044] (4) Change fresh DMEM medium 16 hours after transfection (DMEM is a medium containing various...

Embodiment 2

[0052] 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

[0053] (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;

[0054] (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;

[0055] (3) The human umbilical cord-derived mesenchymal stem cells carrying the three fusion proteins obtained in Example 1 were digested with trypsin, a...

Embodiment 3

[0059] 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

[0060] (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;

[0061] (2) After 12 days, inject the firefly luciferase substrate D-Luciferin (150mg / kg) into the mouse intraperitoneally, and after 5 minutes, use the Xenogen IVIS Lumina II in vivo imaging system to detect the chemiluminescent signal. The exposure time is 30 seconds, and it can be seen that Signals occurred on both sides of the abdomen, confirming that the Nude mouse breast cancer model was successfully established.

[0062] (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, i...

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Abstract

A cell with in vivo tracing and tumor treatment functions and a preparation method. The cell is a human umbilical cord-derived mesenchymal stem cell, which can be used for tumor treatment and has molecular imaging function, and can monitor its distribution and survival in vivo in real time after intratumoral injection. Specifically, the Renilla luciferase-red fluorescent protein-suicide gene thymidine kinase triple fusion gene was transfected into human umbilical cord-derived mesenchymal stem cells, and the luciferin signal was collected by an in vivo imaging system to monitor its effect on the mammary glands of Nude mice. Targeted therapy in cancer models; administration of the thymidine kinase substrate ganciclovir to mice injected with mesenchymal stem cells transfected with the triple fusion gene induces the death of tumor cells surrounding mesenchymal stem cells 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. At present, chemotherapy is often used for treatment. Most chemotherapy drugs cannot specifically act on tumor cells. While killing tumor cells, they also have a certain killing effect on normal cells, resulting in toxic and side effects, and often cannot obtain long-term results. Survive, and at great cost. Finding a carrier that can target multiple tumor lesions, and specifically transport anticancer drugs or genes to tumor lesions without affecting the surrounding ...

Claims

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Application Information

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Patent Type & Authority Patents(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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