Method for raising expression of carbamoyl phosphate synthetase in HepGL liver cancer cells

A technology of carbamoyl phosphate and liver cancer cells, applied to cells modified by the introduction of foreign genetic material, DNA/RNA fragments, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as the lack of expression of CPS1, and achieve mRNA and protein expression The effect of increased level, increased promoter activity, and improved clearance ability

Inactive Publication Date: 2014-04-09
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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Problems solved by technology

At the same time, the study found that when the CPS1 coding gene is intact in liver cancer cells, there is a loss of CPS1 expression

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The research materials in this example are HepGL liver cancer cells.

[0017] In this example, firstly, it is verified whether the methylation of CPS1 affects the expression of its mRNA and protein, and the specific verification method is as follows:

[0018] The HepGL liver cancer cells were divided into experimental group and control group. Decitabine (5umol / ml) was added to the culture medium of the experimental group, and PBS equivalent to that of decitabine was added to the control group, and the medium was changed every 24 hours. After continuous culture for 3 days, Q-PCR and western blot were performed to confirm whether there were differences in the expression of CPS1 mRNA and protein between the control group and the experimental group. Experiments have proved that the methylation of CPS1 affects the expression of its mRNA and protein, specifically, the expression of CPS1 mRNA and protein in the control group is lower than that in the experimental group.

[00...

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PUM

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Abstract

The invention discloses a method for raising expression of carbamoyl phosphate synthetase in HepGL liver cancer cells. The method comprises steps: CPS1 genome DNAs extracted from HepGL liver cancer cells are subjected to bisulfite treatment; the amplified CPS1 promoter area of the treated DNAs is subjected to methylation specific gene amplification; the amplified methylation sequences are subjected to sequencing; TALENs expression plasmids and non-methylation homologous plasmids which are homologous with the methylation loci are constructed according to base sequences around methylation loci, wherein, the homologous non-methylation sequence in the homologous plasmids is a mutation sequence wherein CG is mutated into CA; the TALENs expression plasmids and the homologous plasmids are subjected to transfection into HepGL liver cancer cells, HepGL liver cancer cells with transfection generation are screened, and subjected to Q-PCR and western blotting processing, and the expression amounts of mRNA of CPS1 and proteins in the cells are detected. The method can enhance the ammonia metabolism capability of HepGL liver cancer cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for increasing the expression of carbamoyl phosphate synthetase in HepGL liver cancer cells. Background technique [0002] At present, the treatment of hepatic encephalopathy is mainly to reduce the blood ammonia content of the patient, so as to reduce or prevent toxic substances from entering the blood circulation without being processed by the liver. The reason is that high blood ammonia levels can cause central nervous system dysfunction, resulting in a clinical syndrome characterized by central nervous system metabolic disturbances with changes in consciousness and behavior or coma. [0003] The existing treatment methods for hepatic encephalopathy mainly include drug control, liver transplantation and bioartificial liver. However, these three treatment methods have their limitations. For example, drug control needs to consider whether the patient's own body can withsta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/85C12N5/10
Inventor 高毅姜锡男李阳
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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