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Low-expression CYP7A1 hepatic cell and constructing method thereof

A liver cell and gene expression technology, applied to cells modified by the introduction of foreign genetic material, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of graft rejection as an obstacle to successful transplantation, and achieve the goal of maintaining biological learning function, improving the quality of life, and improving the effect of the microenvironment

Inactive Publication Date: 2009-12-02
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Liver transplantation is currently the most effective method for the treatment of end-stage liver disease, but the recipient's rejection of the graft is the main obstacle to the success of transplantation

Method used

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  • Low-expression CYP7A1 hepatic cell and constructing method thereof
  • Low-expression CYP7A1 hepatic cell and constructing method thereof
  • Low-expression CYP7A1 hepatic cell and constructing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Obtaining of L-02 hepatocytes with low expression of CYP7A1

[0051] Using RNAi technology and lentiviral vector system to obtain hepatocytes with low expression of CYP7A1 (taking human hepatocyte L-02 cell line as an example), the specific method includes the following steps:

[0052] 1. Subculture and expansion of L-02 hepatocytes

[0053] Inoculate L-02 hepatocytes (gifted by the Laboratory of Experimental Hematology, Academy of Military Medical Sciences) on 25cm 2 In a culture flask, use RPMI1640 medium (purchased from Sigma) containing 10% fetal bovine serum at 37°C, 5% CO 2 Plane conventional culture was carried out in the cell culture box, the culture medium was replaced every other day, digested and passaged with 0.25% (V / V) trypsin, and cultured under the same conditions.

[0054] 2. Design of small interfering RNA that inhibits CYP7A1 gene expression

[0055] According to the CYP7A1 mRNA sequence (NM_000780) published by GenBank and the use requi...

Embodiment 2

[0075] Example 2, semi-quantitative RT-PCR method to detect the expression of the mRNA level of biological characteristic genes of hepatocytes before and after lentivirus infection

[0076] The expression of the biological characteristic genes ALB, AFP, ck18, ck19 and CYP1b1 mRNA levels of the L-02 hepatocytes before and after the lentivirus infection obtained in Example 1 was detected by the semi-quantitative RT-PCR method. The specific method is: extract The total RNA of normal L-02 hepatocytes and hepatocytes (including L-02 / CYP7A1-1, L-02 / CYP7A1-2 and L-02 / CYP7A1-3) transfected with lentiviral interference vector pSicoR-CYP7A1 was expressed as This is a template, and RT-PCR detection is carried out under the guidance of each primer pair shown in Table 1 (the annealing temperature is shown in Table 1), and the β-actin gene is used as an internal reference at the same time (the forward primer sequence used is: 5'-GATCCACATCTGCTGGAAGG- 3', the reverse primer sequence is: 5'-A...

Embodiment 3

[0079] Example 3, semi-quantitative RT-PCR method and real-time fluorescence quantitative RT-PCR method to detect the expression of CYP7A1 gene in liver cells before and after lentivirus infection

[0080] Take the 1 × 10 obtained by sorting in Example 1 respectively 6 L-02 / CYP7A1 (L-02 / CYP7A1-1, L-02 / CYP7A1-2 and L-02 / CYP7A1-3 each 1×10 6 ) and L-02 / pSicoR, after extracting the total RNA of the cells, the expression of the CYP7A1 gene at the mRNA level was detected by RT-PCR (primers used were P1:

[0081] 5'-CCGATGGATGGAAATACCAC-3', P2: 5'-TTTCATTGCTTCTGGGTTCC-3', the length of the amplified fragment is 391bp), and the β-actin gene is used as an internal reference (the forward primer sequence used is: 5'-GATCCACATCTGCTGGAAGG-3', The reverse primer sequence is: 5'-AAGTGTGACGTTGACATCCG-3'), and the annealing temperature is 57°C. After the reaction, the PCR amplification products were detected by 1.2% agarose gel electrophoresis, and the detection results were as follows: F...

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Abstract

The invention discloses a low-expression CYP7A1 hepatic cell and a constructing method thereof. A method for inhibiting the CYP7A1 expression level of the hepatic cell comprises the step of introducing an encoding gene of small-interference RNA which is used for inhibiting the CYP7A1 gene expression into the hepatic cell so as to inhibit the CYP7A1 expression level of the hepatic cell. A slow virus vector system can be used for introducing the encoding gene of small-interference RNA which is used for inhibiting the CYP7A1 gene expression into the hepatic cell. An experiment proves that the method of the invention can obviously regulate the expression level of the CYP7A1 gene of the hepatic cell in an mRNA level and a protein level downwards and can further effectively reduce the secretoryvolume of total bile acid. The Low-expression CYP7A1 hepatic cell and the constructing method thereof can be used for the fundamental research and the clinical application of biologic artificial liver supporting treatment and have a wide application prospect.

Description

technical field [0001] The present invention relates to small interfering RNA and its coding gene and application, in particular to small interfering RNA and its coding gene for suppressing CYP7A1 gene expression and its application in suppressing CYP7A1 gene expression level in liver cells. Background technique [0002] End-stage liver disease caused by a variety of reasons (viruses, drugs, tumors and genetic diseases, etc.) seriously threatens human health. Liver transplantation is currently the most effective method for treating end-stage liver disease, but the rejection of the graft by the recipient is the main obstacle to the success of transplantation. In recent years, domestic and foreign focus on the basic research of bioartificial liver (bioartificial liversystem, BAL) supportive therapy, so that it can support the liver function in the short term, maintain and prolong the life of patients, help patients through the dangerous period, and improve the survival rate of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/79C12N15/867C12N5/10C12N15/113
Inventor 裴雪涛郑吉春岳文师伟
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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