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Immortalized human liver cell line, preparation method and application thereof

A liver cell and immortalization technology, applied in the field of biology, can solve problems such as liver cell function defects, immune rejection, virus infection, etc., and achieve good application prospects, avoid immune reactions, and prolong survival time

Active Publication Date: 2010-03-03
苏州瑞徕生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been proven that there are two outstanding problems in the preparation of bioartificial livers using pig hepatocytes: 1. The occurrence of immune rejection due to species differences
2. Animals carry virus infection
However, so far, several immortalized liver cell lines established at home and abroad such as: OUMS29 (Kobayashi N, Noguchi H, Fujiwara T, et al. Establishment of a highly differentiated immortalized adult human hepatitis cell line by retroviral gene transfer.Transplant Pro.2000; 32(7):2368-2369), HepZ (Werner A, Duvar S, Muthing J, et al. Cultivation and characterization of a new immortalized human hepatitis cell line, HepZ, for use in anartificial liver support system. Ann N YAcad Sci, 1999; 875: 364-368) etc. all showed different degrees of hepatocyte functional defects, so there is no literature report on the application of humanized immortalized hepatic cell lines in the preparation of bioartificial livers

Method used

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  • Immortalized human liver cell line, preparation method and application thereof
  • Immortalized human liver cell line, preparation method and application thereof
  • Immortalized human liver cell line, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The establishment of embodiment 1 immortalized human liver cell line (IHH):

[0046] Acquisition of Primary Isolated Human Hepatocytes: Isolation of Hepatocytes from Hepatectomy Surgical Specimens by Collagenase Perfusion (Nguyen TH, Oberholzer J, Birraux J, et al. Highly efficient Lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes. Molecular therapy, 2002, 6(2): 199-209).

[0047] Vectors and plasmids: eg figure 1 As shown, the vector used included three plasmids: the pWPT-GFP gene transfer vector, containing the enhanced GFP gene (EGFP) driven by the CMV promoter; the viral packaging plasmid psPAX2, encoding the Gag and Pol precursors of HIV-1, and Regulatory proteins Tat and Rev; capsid-encoding plasmid pMD.G, which expresses the viral packaging protein VSV G. The plasmid Plox-Ttag-iresTK carrying the SV40 T antigen gene and the plasmid pBabe-hygro-hTert carrying the hTERT gene. (Vector plasmids were purchased from Addg...

Embodiment 2

[0059] The functional characteristic of embodiment 2 IHH:

[0060] To evaluate the functional properties of the immortalized human hepatocyte cell line obtained in Example 1, firstly, the RT-PCR method was used to detect the expression of adult hepatocyte specific molecular mRNA by IHH. Collect IHH cells, use Qiagen's RNA extraction kit to extract total RNA, then take 2ug of total RNA, and use Qiagen's reverse transcription kit to perform reverse transcription. Then use the Taq enzyme of Promega Company to carry out PCR amplification, wherein the primers used are synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the specific primer sequences of each gene are as follows in Table 1:

[0061] Table 1. PCR amplification primer sequences

[0062]

[0063] The amplification conditions were as follows: initial denaturation temperature of 94°C for 2min, cycle denaturation temperature of 94°C for 30s, annealing temperature of 55°C for 30s, extension temperature of 72°C f...

Embodiment 3

[0066] Safety evaluation of embodiment 3 IHH:

[0067] Get the IHH cell that embodiment 1 of logarithmic growth phase obtains, with Hank's solution (137.93mM NaCl, 5.33mMKCl, 4.17mM NaHCO , 1.26mM CaCl , 0.493mM MgCl , 0.441mM KHPO , 0.338mMNaHPO , 5.56mM D-glucose) After washing twice, resuspend in DMEM / F12 basal medium, inoculate the cells in subcutaneous and liver of 5 BALB / c nude mice, inoculate 5×10 6 cells. Another group of nude mice was simultaneously inoculated with liver cancer cell BEL-7402 subcutaneously and in the same part of the liver as a positive control, and each point was inoculated with 5×10 6 cells as a positive control. Continuous observation for 6 months after inoculation showed that there was no tumor formation in the subcutaneous and liver inoculation sites of the nude mice, while nude mice inoculated with the same number of liver cancer cells BEL-7402 at the same site had tumor formation in the subcutaneous and liver one month after transplantation. ...

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Abstract

The invention discloses an immortalized human liver cell line, a preparation method and an application thereof. The immortalized human liver cell line has the main function of primarily cultured humanliver cell. The preparation method comprises the following steps of: transferring SV40T antigen and human telomerase catalyzed subunit gene into primarily cultured separated human normal liver cell by slow virus carrier; and sieving in a cloning way to obtain the immortalized human liver cell line. The immortalized human liver cell line also can be extrinsically amplified in large scales in a microcarrier way. The immortalized human liver cell line can be used for preparing a biological artificial liver support system and preparing the medicament for curing the liver failure.

Description

technical field [0001] The invention relates to the field of biology, in particular to an immortalized human liver cell line and a preparation method thereof, and the application of the immortalized human liver cell line in preparing a bioartificial liver and preparing medicines for treating liver failure. Background technique [0002] Liver transplantation is the only effective treatment for end-stage liver failure. However, due to the very limited source of liver donor organs, most patients with acute liver failure often die while waiting for a donor for liver transplantation. Therefore, establishing a bioartificial liver support system to restore basic liver function is a new technology for the treatment of acute liver failure, which can not only improve the survival rate of patients with liver failure, but also buy time for patients to undergo liver transplantation. The bioartificial liver technology is to use some or all of the active cells of the liver cells in the bi...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61M1/14C12Q1/02C12R1/91
Inventor 娄晋宁
Owner 苏州瑞徕生物科技有限公司
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