Glioma cell line and construction method thereof

A technology for glioma cells and construction methods, applied to cells modified by introducing foreign genetic material, botanical equipment and methods, and methods based on microorganisms, which can solve the problem of stimulants and inhibitors not having tissue specificity and glue Selectivity of tumor cells, side effects, etc.

Inactive Publication Date: 2014-01-15
SHENZHEN INST OF ADVANCED TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional methods of affecting the excitability of glioma cells and regulating the opening of ion channels in the cell membrane are mostly accomplished through agonists and inhibitors of ion channels. However, most of the agonists and inhibitors of ion channels are not tissue specific and glial. Selective for tumor cells, has disadvantages such as large side effects

Method used

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  • Glioma cell line and construction method thereof
  • Glioma cell line and construction method thereof
  • Glioma cell line and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]Construction of viral vectors and infection of glioma cells.

[0043] The fusion plasmid including sequentially connected ITR, promoter, photosensitive gene, green fluorescent marker gene and ITR, and lentiviral packaging plasmids pCMVΔR8.74, pMD2.G. (1.5 g) were used in liposomes (Invitrogen Corporation Products) were transfected together into 293FT cells (products of ATCC company, the cells need to be replaced with a new batch of cell lines after 25 passages) (6 mg / L liposomes per 200,000 cells), 24 hours later DMEM containing 5mM sodium pyruvate replaced the original culture medium of 293FT cells and continued to culture. After 16 hours, the ultracentrifuge was used to rupture the membrane at a speed of 50,000g, and the suspension was passed through a 20% sucrose filter column to collect the supernatant. The virus titer can reach 3×10 8 Above TU / mL, the produced virus particles were resuspended in sterile phosphate buffered saline at a volume ratio of 1:1000. The ob...

Embodiment 2

[0047] Photocurrent induction and recording.

[0048] In order to verify the functional expression of photogenes in glioma cell lines, we used single-pulse stimulation and multi-pulse stimulation of blue light to record the membrane excitability changes of glioma cells by patch clamp technique. Stimulation can induce current changes in glioma cells; 50ms, 20HZ continuous blue light stimulation can induce corresponding series of membrane current changes. This electrophysiological recording demonstrates the successful expression of light-sensing genes on glioma cell lines.

[0049] The comparison graph of the stimulated current and the inward photocurrent obtained is figure 2 , where, F represents the photocurrent induced by 500ms blue light, G represents the photocurrent induced by 50ms, 20Hz blue light stimulation.

[0050] Depend on figure 2 It can be seen that blue light stimulated glioma cell lines, and single pulse stimulation and continuous light pulse stimulation ca...

Embodiment 3

[0052] Blue light stimulation induces a decrease in the survival rate of glioma cells.

[0053] After the glioma cell line functionally expressed the light-sensing gene, we used blue light to continuously stimulate the glioma cells for 1 h (20 Hz), and analyzed the survival rate of the glioma after 24 h. image 3 .

[0054] Depend on image 3 It can be seen that the survival rate of tumor cells in the light-sensing gene and light combination group (ChETA+light) was significantly lower than that in other groups.

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Abstract

The invention relates to a glioma cell line. The glioma cell line is glioma cells infected by a virus vector. The virus vector comprises ITR (inverted terminal repeat), a promoter, a photosensitive gene, a green fluorescence labeling gene and ITR, which are sequentially connected. The glioma cell line is specifically marked by the light-sensing gene, and can generate stable light current under induction and stimulation of light, so that the glioma cell line can be selectively performed with specific regulation with light. The invention further provides a construction method of the glioma cell line.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a glioma cell line and a construction method thereof. Background technique [0002] Glioma, as a tumor of the nervous system, is derived from glial cells or immature precursor cells, and has special electrophysiological characteristics and membrane channel properties. The opening and closing of ion channels in the cell membrane can affect the polarization state and excitability of cells, and then It affects the molecular signaling pathways in cells, thus playing an important role in the survival, growth and metastasis of glioma. Existing studies have also confirmed that the degree of malignant proliferation of cancer cells is mostly related to changes in the excitability of the cells themselves; for example, changes in the membrane potential of tumor cells themselves will affect the efficiency of tumor stem cell proliferation and differentiation. Early studies have shown that voltage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12R1/91
Inventor 杨帆屠洁王立平
Owner SHENZHEN INST OF ADVANCED TECH
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