Gene application in inhibition and apoptosis of glioma cell

Abstract

The present invention relates the application of a human source dcf1 gene in the inhibition of U251 glioma cell line proliferation and in the induction of U251 glioma cell line apoptosis. The present invention makes judgments for dcf1-induced U251 cell line apoptosis by the methods of immune electron microscope, atomic force microscope, CCK8 proliferation detection, JC-1 staining, and nude mouce tumor heterotopic transplantation. The results show that: the dcf1 causes mitochondrial structure lesion and membrane potential reduction by locating in mitochondria, thereby causing apoptosis-related gene expression change, and eventually leading to apoptosis. The nude mouce experiments show that the dcf1 can significantly reduce glioma tumor volume and inhibit tumor growth.

Description

technical field

[0001] The present invention relates to a human source dcf1 new uses. especially human source dcf1 Application of gene in inhibition and apoptosis of glioma cells. Background technique

[0002] Glioma is a tumor arising from glial cells (astrocytes, oligodendrocytes, ependyma, etc.) differentiated from the neuroectoderm. Glioma ranks first in the incidence of intracranial tumors, accounting for about 45%. The treatment of glioma is a difficult problem in neurosurgery. Due to its high mortality rate, poor prognosis, uncontrollable cell proliferation, slow cell apoptosis, and strong invasion of tumor cells, the treatment of glioma faces a huge challenge. The average survival period of surgery plus radiotherapy and chemotherapy after illness is only 8 to 11 months. At present, there are mainly the following methods for tumor treatment: surgical treatment, chemical drugs, therapeutic radiation therapy, and photodynamic therapy. But these four kinds of treat...

Examples

Embodiment 1

[0018] Embodiment one: dcf1 Gene amplification and enzyme digestion identification of recombinant plasmid pEGFP-N2-DCF1

[0019] 1. Using the cDNA of HeLa cells as a template for PCR amplification, in dcf1 EcoRI and BamHI restriction sites were introduced at both ends of the primers respectively. The upstream primer is (5'-CGGAATTCATGGCGGCGCCGAAGGGGAG-3'),

[0020] The downstream primer is (5'-CGGGATCCGTAAAATTTCAGAATGAGCA-3'), Tm is 53 degrees Celsius, and after 30 cycles, the target fragment of 972bp is obtained ( figure 1 A).

[0021]2. After the positive recombinant plasmid pEGFP-N2-DCF1 was obtained, the recombinant was identified by double enzyme digestion. Two enzymes, EcoRI and BamHI, were added to the enzyme digestion system at the same time. As a control, pEGFP-N2 EcoRI single enzyme digestion was used, and the reaction was performed at 37 degrees Celsius for 2 hours. The enzyme digestion results were identified by electrophoresis, and a band was released at 97...

Embodiment 2

[0022] Example 2: Transfection of plasmid pEGFP-N2-DCF1

[0023] After the U251 cell line was plated in a 24-well plate for 24 hours, the plasmid pEGFP-N2-DCF1 was transfected by Lipofectamine® 2000 Transfection Reagent cationic liposome transfection method. 1ul lipo2000 per well, 1ug ​​plasmid pEGFP-

[0024] N2-DCF1 was dissolved in 50ul serum-free DMEM medium respectively, and after standing at room temperature for 5 minutes, the two were mixed evenly, and standing at room temperature for 20 minutes. The medium in the well plate was replaced with 400ul serum-free DMEM, and 100ul of the above mixed product was added. After culturing at 37°C for 6 hours, the medium was replaced with DMEM containing 10% fetal bovine serum. Fluorescence was detected 48 hours after transfection ( figure 2 A-C).

Embodiment 3

[0025] Example 3: Cell Immunofluorescence

[0026] Forty-eight hours after transfection of U251 cells, discard the medium and wash three times with PBS. Fix with 4% PFA for 10 minutes and wash with PBS three times. Permeabilize with 1‰Triton-X100 for 10 minutes, wash with PBS three times. Block with 2% BSA-PBS for 1 hour at room temperature. Dilute DCF1 rabbit polyclonal antibody (1:700) with 1% BSA-PBS, incubate at 37°C for 1 hour, wash with PBS three times. Dilute TRITC fluorescent secondary antibody (1:300) with 1% BSA-PBS, incubate at 37°C for 40 minutes in the dark, wash with PBS three times, and observe under a fluorescence microscope ( figure 2 D-F).

technical field

[0001] The present invention relates to a human source dcf1 new uses. especially human source dcf1 Application of gene in inhibition and apoptosis of glioma cells. Background technique

[0002] Glioma is a tumor arising from glial cells (astrocytes, oligodendrocytes, ependyma, etc.) differentiated from the neuroectoderm. Glioma ranks first in the incidence of intracranial tumors, accounting for about 45%. The treatment of glioma is a difficult problem in neurosurgery. Due to its high mortality rate, poor prognosis, uncontrollable cell proliferation, slow cell apoptosis, and strong invasion of tumor cells, the treatment of glioma faces a huge challenge. The average survival period of surgery plus radiotherapy and chemotherapy after illness is only 8 to 11 months. At present, there are mainly the following methods for tumor treatment: surgical treatment, chemical drugs, therapeutic radiation therapy, and photodynamic therapy. But these four kinds of treat...

Examples

Embodiment 1

[0018] Embodiment one: dcf1 Gene amplification and enzyme digestion identification of recombinant plasmid pEGFP-N2-DCF1

[0019] 1. Using the cDNA of HeLa cells as a template for PCR amplification, in dcf1 EcoRI and BamHI restriction sites were introduced at both ends of the primers respectively. The upstream primer is (5'-CGGAATTCATGGCGGCGCCGAAGGGGAG-3'),

[0020] The downstream primer is (5'-CGGGATCCGTAAAATTTCAGAATGAGCA-3'), Tm is 53 degrees Celsius, and after 30 cycles, the target fragment of 972bp is obtained ( figure 1 A).

[0021]2. After the positive recombinant plasmid pEGFP-N2-DCF1 was obtained, the recombinant was identified by double enzyme digestion. Two enzymes, EcoRI and BamHI, were added to the enzyme digestion system at the same time. As a control, pEGFP-N2 EcoRI single enzyme digestion was used, and the reaction was performed at 37 degrees Celsius for 2 hours. The enzyme digestion results were identified by electrophoresis, and a band was released at 97...

Embodiment 2

[0022] Example 2: Transfection of plasmid pEGFP-N2-DCF1

[0023] After the U251 cell line was plated in a 24-well plate for 24 hours, the plasmid pEGFP-N2-DCF1 was transfected by Lipofectamine® 2000 Transfection Reagent cationic liposome transfection method. 1ul lipo2000 per well, 1ug ​​plasmid pEGFP-

[0024] N2-DCF1 was dissolved in 50ul serum-free DMEM medium respectively, and after standing at room temperature for 5 minutes, the two were mixed evenly, and standing at room temperature for 20 minutes. The medium in the well plate was replaced with 400ul serum-free DMEM, and 100ul of the above mixed product was added. After culturing at 37°C for 6 hours, the medium was replaced with DMEM containing 10% fetal bovine serum. Fluorescence was detected 48 hours after transfection ( figure 2 A-C).

Embodiment 3

[0025] Example 3: Cell Immunofluorescence

[0026] Forty-eight hours after transfection of U251 cells, discard the medium and wash three times with PBS. Fix with 4% PFA for 10 minutes and wash with PBS three times. Permeabilize with 1‰Triton-X100 for 10 minutes, wash with PBS three times. Block with 2% BSA-PBS for 1 hour at room temperature. Dilute DCF1 rabbit polyclonal antibody (1:700) with 1% BSA-PBS, incubate at 37°C for 1 hour, wash with PBS three times. Dilute TRITC fluorescent secondary antibody (1:300) with 1% BSA-PBS, incubate at 37°C for 40 minutes in the dark, wash with PBS three times, and observe under a fluorescence microscope ( figure 2 D-F).

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