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Immortalized alpha-1,3-galactosyltransferase gene knockout porcine hepatocyte line and preparation method and application thereof

A galactosyltransferase and gene knockout technology, applied in the field of organ transplantation medical biology, can solve the problems of complex operation of primary hepatocytes, apoptosis of hepatocytes, and little research on immortalization of hepatocytes.

Inactive Publication Date: 2019-01-15
窦科峰 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still the following problems in the use of primary hepatocytes: ① The extraction of primary hepatocytes from large animal GTKO pigs is complicated, and the operation of high-viability hepatocytes requires high technical requirements; ② The in vitro culture time of primary GTKO pig hepatocytes is short , the longest culture time is one week, the primary hepatocytes cultured in vitro will not proliferate, and due to the environment outside the body, the hepatocytes themselves will gradually undergo apoptosis and necrosis; ③Primary hepatocytes cultured in vitro are not easy to intervene at the gene level , and the reproducibility of the results is poor when used in scientific research
[0009] However, in the field of liver transplantation, there are still few studies on the immortalization of liver cells in GTKO pigs.

Method used

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  • Immortalized alpha-1,3-galactosyltransferase gene knockout porcine hepatocyte line and preparation method and application thereof
  • Immortalized alpha-1,3-galactosyltransferase gene knockout porcine hepatocyte line and preparation method and application thereof
  • Immortalized alpha-1,3-galactosyltransferase gene knockout porcine hepatocyte line and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0116] Example 2 Establishment of immortalized α-1,3-galactosyltransferase GTKO pig liver cell line (HepDT):

[0117] Host material: α-1,3-galactosyltransferase GTKO pigs were provided by Mr. Pan Dengke, Beijing Academy of Agricultural Sciences.

[0118] 1. Packaging and titration of lentiviral particles.

[0119] 3 Plasmid packaging system: pSPAX2 (purchased from Addgene, Switzerland), pMD2G plasmid (purchased from Addgene, Switzerland) and lentiviral packaging plasmid (SV40LT lentiviral packaging plasmid vector or h-TERT lentiviral packaging plasmid vector (purchased from Hanbio) Technology (Shanghai) Co., Ltd.)

[0120] The mass ratio of the three plasmids is 1 μg of pHBLV-CMV vector carrying SV40LT, 750ng of psPAX2 packaging plasma, and 250ng of pMD2.G envelope plasma.

[0121] (1) Transfect 293T cells with recombinant lentiviral vector pHBLV-CMV encoding SV40LT antigen

[0122] 293T cells were cultured in DMEM containing 10% FCS and 100ug / ml penicillin / streptomycin, an...

Embodiment 3

[0133] Example 3. Identification of cell morphology and biological function of immortalized GTKO pig liver cell line (please supplement the specific differences shown in A, B, and C in the accompanying drawings).

[0134] In this study, the immortalized α-1,3-galactosyltransferase GTKO pig liver cell line obtained from the α-1,3-galactosyltransferase GTKO pig cultivated by teacher Pan Dengke of Beijing Academy of Agricultural Sciences was used as the starting material for detection. In GTKO pigs, a gene sequence (SEQ ID NO.1) is inserted into the wild-type α-1,3-galactosyltransferase (α-1,3-galactosyltransferase, GGTA1) gene.

[0135] 1. Morphological observation by ordinary light microscope

[0136] Use OLYMPUS inverted fluorescent microscope (Olympus, Japan, model IX71FL+DP72) according to the operating steps shown in the instruction manual) to observe the high-viability immortalized GTKO pig liver cell line obtained in Example 2 co-cultured for 24 hours

[0137] Such as ...

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Abstract

The present invention discloses an immortalized alpha-1,3-galactosyltransferase gene knockout (GTKO) porcine hepatocyte line, a preparation method thereof, and application thereof in preparation of bioartificial live and preparation of medicament for treating hepatic failure. The immortalized alpha-1,3-galactosyltransferase GTKO porcine hepatocyte line retains main characteristics of primary alpha-1,3-galactosyl transferase GTKO porcine hepatocytes, including the function of synthesizing urea, the function of synthesizing albumin, the nearly unlimited in vitro expansion culture, the driving force of the key molecules and drug intervention targets in xenotransplantation rejection, the immortalized alpha-1,3-galactosyltransferase GTKO porcine hepatocytes can effectively solve the problem ofxenogeneic hyperacute immune rejection, reduce the use of immunosuppressive agents, prolong the survival time of transplanted recipients and the normal function of transplanted liver, and have an important medical application prospect.

Description

technical field [0001] The present invention relates to the field of medical biology of organ transplantation. It specifically relates to a preparation method of a liver transplantation pig liver cell line, especially an immortalized pig liver cell line. Background technique [0002] Liver transplantation is the most effective treatment for end-stage liver disease. [0003] However, due to the serious shortage of donors, a large number of patients lost their lives because they could not receive treatment in time. The core part of the bioartificial liver is the bioreactor. Bioreactors generally consist of biologically active cells and a support system that provides an environment for the growth and metabolism of biologically active cells. The nature and function of the cells used in the bioreactor are the main factors that determine the functional effect of the bioartificial liver. The bioactive cells currently used in bioreactors are mainly fresh pig liver cells. Becaus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/407A61P1/16A61L27/38C12R1/91
CPCA61L27/3804A61L27/3839A61P1/16A61K35/407C12N5/067C12N9/1051C12N15/86A61L2430/28C12N2510/00C12N2740/15043G01N2500/00C12N2510/04C12N2501/33C12N2501/11C12N2501/12C12N2320/10C12N2503/02C12N2503/04
Inventor 窦科峰王权成李霄张玄张虹白鸽黄敏王博杨佩军
Owner 窦科峰
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