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Method for researching influence of targeted silence STAT3 on hypoxia induction of human brain glioma U251 cells and application of targeted silence STAT3

A human glioma, targeted silencing technology, applied in the field of biomedicine

Pending Publication Date: 2020-09-01
NINGXIA MEDICAL UNIV
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Problems solved by technology

Our previous studies have shown that the JAK-STAT signaling pathway plays an important role in the Crosstalk mechanism between glioma cells and human brain microvascular endothelial cells, but there are few reports on whether it can play a role by affecting the formation of glioma VM

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  • Method for researching influence of targeted silence STAT3 on hypoxia induction of human brain glioma U251 cells and application of targeted silence STAT3
  • Method for researching influence of targeted silence STAT3 on hypoxia induction of human brain glioma U251 cells and application of targeted silence STAT3
  • Method for researching influence of targeted silence STAT3 on hypoxia induction of human brain glioma U251 cells and application of targeted silence STAT3

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Embodiment Construction

[0037] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

[0038] 1 Materials and methods

[0039] 1.1 Main reagent materials

[0040] The human glioma cell line U251 was purchased from the Shanghai Cell Resource Center of the Chinese Academy of Sciences, a glioblastoma monoclonal line, and the WHO grading standard was grade IV. DMEM culture medium and trypsin were purchased from Gibco Company of the United States, and fetal bovine serum was purchased from HyClone Company of the United States. Rabbit anti-human STAT3, STAT3Phospho (pY705) and MMP9, MMP14, EphA2, Laminin5γ2 monoclonal antibodies were purchased from Cell Signal Technology, USA; rabbit anti-human GAPDH polyclonal antibody (AB-P-R 001) was purchased from Hangzhou Xianzhi Biotechnology Co., Ltd. , the fluorescent secondary antibody Goat anti-Rabbit 926-32211 was purchased from Li-cor, USA. Reagents related to Western blotting were...

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Abstract

The invention discloses a method for researching the influence of targeted silence STAT3 on hypoxia induction of human brain glioma U251 cells and application of the targeted silence STAT3. The methodis characterized by constructing ShRNA of a targeted STAT3 gene to transfect a U251 cell, observing the infection efficiency with a microscope, and carrying out real-time fluorescent quantitative PCRand Western blotting to detect the silencing efficiency of STAT3; detecting the influence of STAT3 gene silencing on angiogenesis mimicry formation and invasion ability of the U251 cell induced by cell hypoxia by virtue of in-vitro three-dimensional culture and Transwell cell invasion experiments; and detecting the expression level of the VM / invasion related gene / protein through real-time fluorescent quantitative PCR and Western blotting. The ShRNA lentiviral plasmids of the targeted STAT3 gene are successfully constructed, and the U251 cell is efficiently transfected.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a research method for the effect of targeted silencing of STAT3 on hypoxia induction of human brain glioma U251 cells and its application, in particular to a method for targeting the effect of silencing of STAT3 on human brain glial cells. Research methods and applications of angiogenesis mimicry and invasion effects induced by hypoxia in tumor U251 cells. Background technique [0002] Glioma is the most common primary tumor of the human central nervous system, which has the characteristics of high incidence, high recurrence rate, high mortality rate and low cure rate. Although comprehensive diagnosis and treatment such as surgery, radiotherapy and chemotherapy, the effect is still unsatisfactory. To study its pathogenesis and find new therapeutic targets is one of the urgent problems to be solved. As a complex process involving multiple factors, multiple gene changes, and mu...

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Application Information

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IPC IPC(8): C12N15/867C12Q1/02G01N21/64
CPCC12N15/86C07K14/82G01N33/5005G01N21/6458C12N2740/15043G01N2500/10
Inventor 顾金海顾珈榕孙涛牛建国王峰文玉军王鹏强媛媛和祯泉杨勇
Owner NINGXIA MEDICAL UNIV
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