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Application of miRNA-20a and its inhibitors in the preparation of glioma stem cell invasion regulators

A technology of glioma stem cells, miRNA-20a, applied in the medical field to achieve the effect of high invasive properties

Inactive Publication Date: 2011-12-28
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of miRNA may be involved in the regulation of malignant glioma invasion, but the relationship with the biological behavior of GSCs needs further study

Method used

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  • Application of miRNA-20a and its inhibitors in the preparation of glioma stem cell invasion regulators
  • Application of miRNA-20a and its inhibitors in the preparation of glioma stem cell invasion regulators
  • Application of miRNA-20a and its inhibitors in the preparation of glioma stem cell invasion regulators

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Glioma Stem Cells

[0038] 1 material

[0039] Culture of primary human glioma cells

[0040] Fresh glioma tissues were obtained from the Department of Neurosurgery of Southwest Hospital and Daping Hospital (Chongqing Third Military Medical University). A total of 2 cases were taken successively, both of which were glioblastoma multiforme (WHO grade IV). The primary glioma tissue block was washed 3 times with 0.01M PBS, and the surface fibers and necrotic tissue were carefully removed; the tissue block was immersed in a small amount of DMEM medium (Gibco, USA), and the tumor tissue block was repeatedly cut to 1mm with ophthalmic scissors 3 fragments; add mixed collagenase (Gibco Company, USA) to digest for 20-30min; while digesting, blow with a pipette to obtain a single-cell suspension, and place it in DMEM medium (containing 10% fetal bovine serum by volume fraction) Cultivate; after 24 hours, change the medium to remove red blood cells, and use it to sort...

Embodiment 2

[0052] The establishment of embodiment 2 xenograft tumor animal model

[0053] 1 Source of experimental animals

[0054] Experimental Animals Female nude mice aged 4-6 weeks, weighing 15-20 g, were provided by the Experimental Animal Center of Third Military Medical University, and were bred under standard conditions.

[0055] 2 experimental groups

[0056] The experiment was divided into 3 groups, 5 in each group, the first group was U87-CD133 - cells and U87-CD133 + Cell group; the second group is primary Case1-CD133 - Cellular and primary Case1-CD133 + Cell group; the third group is: primary Case2-CD133 - cells and primary Case2-CD133 + cell group.

[0057] 3. Subcutaneous xenograft tumor model

[0058] The subcutaneous xenograft model was used to analyze the tumorigenic ability of GSCs. 5×10 4 CD133 + cells (derived from U87 cell line and primary glioma cells) and CD133 - The cells (derived from U87 cell line and primary glioma cells) were suspended in 100 μl o...

Embodiment 3

[0060] In vitro invasion of embodiment 3GSCs and GCs

[0061] To study the difference in the invasion ability of GSCs and GCs, we used Transwell in vitro invasion assay to observe the invasion ability of GSCs and GCs. The Transwell in vitro invasion test was divided into two groups, namely the GSCs group and the GCs group. The specific operation steps were as follows: the cells were discarded from the culture medium, and the cells were washed 3 times with PBS; the adherent cells were digested with trypsin + EDTA and then centrifuged. After the suspended cells were directly centrifuged, they were resuspended in the culture medium, and the cell concentration was adjusted to 5×10 5 / ml; unpack the transwell chamber (BD Company, USA), use sterile tweezers to clamp out the transwell chamber and put it into a 24-well plate; pipette 10 μl of Matrigel glue (BD Company, USA) mixture on the transwell membrane, pay attention to the injection Uniform, rapid and gentle action, Matrigel ge...

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Abstract

The present invention relates to the field of medicine, in particular to regulators for regulating the invasion behavior of glioma stem cells. The application of tumor stem cell invasion inhibitor; the expression of miRNA-20a in GSC was significantly higher than that in GC, and the expression of TIMP-2 in GSC was significantly lower than that in GC regardless of mRNA level or protein level; the inhibitor of miRNA-20a can significantly inhibit Invasion ability of GSC; after inhibiting the expression of miRNA-20a, the expression of TIMP-2 mRNA level and protein level was significantly increased, and the luciferase reporter gene experiment confirmed that miRNA-20a can not only negatively regulate the expression of TIMP-2, but also through and The 3'UTR of TIMP-2 directly regulates the expression of TIMP-2.

Description

technical field [0001] The invention relates to the medical field, in particular to a regulator for regulating the invasion behavior of glioma stem cells. Background technique [0002] Glioblastoma (GBM) is the most common primary malignant brain tumor of the central nervous system. Invasive growth of tumor cells is an important biological characteristic of malignant glioma, which makes it difficult to completely remove the tumor tissue by surgery, and the effect of chemotherapy and radiotherapy is poor, the recurrence rate after surgery is high, and the prognosis is poor. Mechanisms of aggressive growth and therapy resistance in malignant gliomas are unclear. [0003] A large number of recent studies have shown that cancer stem cells (cancer stem cells, CSCs) play an important role in the occurrence and development of tumors. Studies have found that glioma stem cells (glioma stem cells, GSCs) exist in malignant gliomas, and these GSCs have self-renewal ability, multilinea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02
Inventor 王喆王斌卞修武
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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