Intestinal cancer cell strain expressing hypoxia inducible factor (HIF)-1 alpha gene efficiently

A HIF-1, high-efficiency expression technology, applied in the field of cell lines, can solve problems such as difficulties in clinical application

Inactive Publication Date: 2013-06-05
SHANGHAI PUTUO DISTRICT CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] With the deepening of tumor mechanism research, there are more and more related studies on HIF-1α, and its mechanism of action is gradually being revealed, but there are still many deficiencies and problems that require further in-depth research and discussion: For HIF-1α Targeted therapy of the 1α gene, directly reducing the expression and transcriptional activity of HIF-1α from the mRNA level is a research hotspot at present, but it can only be confirmed from cell culture experiments, and clinical application is still very difficult. Chemical agents and natural Drug extracts can inhibit the expression of HIF-1α, but there is no direct and specific drug targeting HIF-1α, which requires the development of more selective HIF-1α inhibitors for use in basic and clinical research

Method used

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  • Intestinal cancer cell strain expressing hypoxia inducible factor (HIF)-1 alpha gene efficiently
  • Intestinal cancer cell strain expressing hypoxia inducible factor (HIF)-1 alpha gene efficiently
  • Intestinal cancer cell strain expressing hypoxia inducible factor (HIF)-1 alpha gene efficiently

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction and packaging of lentiviral vector (pGC-FU-HIF-1α) containing HIF-1α gene

[0046] refer to figure 1 and Figure 6 The construction and packaging method of the lentiviral vector containing the HIF-1α gene of the present invention is as follows:

[0047] Step 1 Linearization of pGC-FU vector

[0048] Digest the pGC-FU vector with Age I restriction endonuclease, the system is as follows:

[0049]

[0050]

[0051] Reaction conditions: enzyme digestion reaction for 1 hour at 37°C.

[0052] For enzyme digestion results, see figure 2 .

[0053] Step 2 Acquisition of HIF-1α gene (target gene) fragment

[0054] 1) PCR amplification of the target gene

[0055] The PCR reaction system is as follows:

[0056] Template reagent (10ng / μL) 1μL

[0057] Primer (+) 0.4μL

[0058] Primer(-) 0.4μL

[0059] 10-fold dilution buffer 2 μL

[0060] MgCl 2 0.5μL

[0061] pfu polymerase 0.2 μL

[0062] dNTP 0.8 μL

[0063] wxya 2 O 14.7 μL

[00...

Embodiment 2

[0111] Example 2 Preparation of intestinal cancer cells highly expressing HIF-1α gene

[0112] Step 1 Cell Recovery

[0113] Frozen colorectal cancer HTC-116 cells were quickly thawed at 37°C, centrifuged, and placed at 37°C, 5% CO 2 cultivated in the environment.

[0114] Step 2 Cell passage

[0115] The old medium was removed, D-Hank's solution was added, the cell growth surface was washed, and the solution was removed.

[0116] Add 1ml of trypsin digestion solution and digest at 37°C until the cells are completely digested. Add complete culture medium.

[0117] Step 3 Infect cells with lentivirus

[0118] HTC-116 cells were cultured to the logarithmic cycle, digested with trypsin, and made into cell suspension (cell number 5×10 6 indivual).

[0119] The cell suspension was seeded in a 6-well plate at 37°C, 5% CO 2 Cultured under ambient conditions until the cell confluency reached about 30%. They were divided into blank group, negative virus group, and HIF-1α group...

Embodiment 3

[0124] Example 3 Application of Intestinal Cancer Cells Highly Expressing HIF-1α Gene of the Present Invention (1)

[0125] Cultured in Mycoy’s 5A complete medium containing 10% newborn calf serum, 100 U / ml penicillin, 100 μg / ml streptomycin (37°C, 5% CO 2 , saturated humidity) human colorectal cancer HTC-116 cell line, press 1×10 6 Seed cells in exponential growth phase in 24-well plates at 37°C in 5% CO 2 Incubate overnight in an incubator until the cell density reaches 60%-80%.

[0126] The above conventionally cultured human colorectal cancer HCT-116 cells were randomly divided into control group, negative virus treatment group, and HIF-1α lentivirus treatment group, and different plasmids were added according to the following doses for transient transfection:

[0127] Vector plasmid control group: transfected with pGL3-Basic plasmid and pRL-SV40 internal reference plasmid

[0128] COX-2 promoter plasmid control group: transfected with pGL3-Basic-COX-2 plasmid and pRL-S...

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Abstract

Aiming at the difficult problem that clinical application of existing hypoxia inducible factor (HIF)-1 alpha is difficult to achieve, the invention provides an intestinal cancer cell strain expressing an HIF-1 alpha gene efficiently, wherein the bacterial strain is preserved in a common microorganism center of China microorganism strains preservation and management committee and the preservation number is CGMCC No. 5261. The intestinal cancer cell strain expressing the HIF-1 alpha gene efficiently can be used in the fields such as research of an intestinal cancer mechanism and sifting of cancer treatment medicine, and provides a quite valuable tool for the basic and clinic research.

Description

technical field [0001] The present invention relates to a cell line, in particular to an intestinal cancer cell line highly expressing HIF-1α gene, a preparation method thereof, and a lentiviral vector for preparing the cell line and a preparation method thereof. Background technique [0002] Hypoxia inducible factor 1 (hypoxia inducible factor 1, HIF-1) is the first discovered member of the HIFs family, a heterodimer composed of an oxygen-sensitive α subunit and a stably expressed β subunit, as A hypoxia-induced nuclear factor linked to the hypoxia response element of the EPO gene plays a key regulatory role in tumor cell proliferation and metabolism, tumor angiogenesis, invasion and metastasis, and response to drug treatment. [0003] HIF-1α is the regulatory and active subunit of HIF-1, which can be induced by hypoxia. After being secreted from the cytoplasm, it is transferred to the nucleus to bind with HIF-1β and activate downstream target genes. The biological effect o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/66C12R1/91
Inventor 周利红李琦范忠泽王炎殷佩浩慈书俊周宁刘宣隋华
Owner SHANGHAI PUTUO DISTRICT CENT HOSPITAL
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