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Method for preparing immortalized hepatocytes with reversible liver functions and application of immortalized hepatocytes

A liver cell and immortalization technology, applied in the field of biomedicine, can solve the problems of difficult clinical transformation, low biological safety, xenograft rejection, etc., and achieve the effect of simple and easy preparation method, easy clinical transformation, and promotion of reversible function

Active Publication Date: 2022-02-25
GUANGDONG UNISUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these hepatocytes have certain liver functions in vitro, they each have application obstacles such as insufficient maturity, limited expansion, unstable function, xenograft rejection, low biological safety, and risk of tumorigenesis.
[0003] The Chinese invention patent application with publication number CN110438157B and publication number CN105521482A discloses methods for further inducing hepatocyte-like differentiation using different hepatocyte-specific transcription factors alone or in combination, but these methods or seed cells are derived from liver cancer cells, Or the cost of the culture system used is high, and it is difficult to carry out clinical transformation

Method used

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  • Method for preparing immortalized hepatocytes with reversible liver functions and application of immortalized hepatocytes
  • Method for preparing immortalized hepatocytes with reversible liver functions and application of immortalized hepatocytes
  • Method for preparing immortalized hepatocytes with reversible liver functions and application of immortalized hepatocytes

Examples

Experimental program
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Embodiment 1

[0048] This example provides a method for preparing immortalized hepatocytes with reversible liver function. The Tet-on system used to regulate hepatocyte transcription includes a regulatory expression cassette that includes a liver-specific promoter and an antisense tetracycline activator, and a responsive expression cassette that includes a tetracycline-inducible promoter and a hepatocyte transcriptional cassette. Factor-encoding genes; hepatocyte transcription factor-encoding genes include FOXA3 gene, CEBPA gene and GATA6 gene. Vector map reference for the regulated expression cassette Image 6 As shown, the sequence refers to SEQ ID NO.1, and the vector map of the reaction expression cassette refers to Figure 7 As shown, the sequence is shown in SEQ ID NO.2. The above lentiviral sequences were handed over to Yunzhou Biotechnology (Guangzhou) Co., Ltd. for virus packaging.

[0049] The method for preparing immortalized hepatocytes with reversible liver function comprise...

experiment example 1

[0055] Microscope Brightfield Reference figure 1 as shown, figure 1 After the successfully transfected target cells were cultured in a doxycycline-free medium for 7 days, the cells were in a normal state, with epithelial cell-like morphology under the microscope, and island-like aggregation growth, marked as C3A-FOXA3 / CEBPA / GATA6- CTRL. After the successfully transfected target cells were cultured in a medium containing doxycycline (DOX) for 7 days, the cell morphology under the microscope was a typical hepatocyte shape (polygonal), uniformly tiled and grown, labeled as C3A-FOXA3 / CEBPA / GATA6-DOX1000ng-day7.

[0056] However, the target cells that were not successfully transfected were cultured in a doxycycline-free medium for 7 days, and the cells were in a normal state, with epithelial cell-like morphology under the microscope, growing in an island-like aggregation, and labeled as C3A-CTRL. However, after the unsuccessfully transfected target cells were cultured in doxycy...

experiment example 2

[0059] In this experimental example, after 7 days of culture, the synthesis ability of albumin (ALB), α1-antitrypsin (AAT), urea (UREA) and coagulation factor 7 (FVII) of the four groups of cells was detected by ELISA. , the volume of culture supernatant, and the culture time are constant, and the single variable is cells).

[0060] The results showed that when the successfully transfected target cells were in a state of enhanced hepatic function (addition of doxycycline), normal hepatocyte markers were expressed, and ALB (refer to figure 2 shown), AAT (see image 3 shown), urea (see Figure 4 shown) and coagulation factor 7 (see Figure 5 Shown) the synthesis ability is improved. When doxycycline was removed, the synthesis ability of AAT, urea and blood coagulation factor 7 was not excellent.

[0061] This experimental example shows that the liver function-reversible immortalized hepatocytes prepared by the Tet-on system provided by the present invention have the effect ...

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Abstract

The invention discloses a method for preparing immortalized hepatocytes with reversible liver functions and application of the immortalized hepatocytes, and relates to the field of biomedicine. A Tet-on system is applied to the hepatocytes, and the functions of the hepatocytes are enhanced through the Tet-on system. The Tet-on system has the characteristics of high efficiency, accuracy, safety and reversibility. Therefore, a novel immortalized hepatocyte is prepared, reversible regulation and control of hepatocyte functions can be realized by adding or removing tetracycline analogues, and reversible enhancement of the hepatocyte functions is promoted. The preparation method is simple and easy to implement, the culture cost is low, seed cells are derived from immortalized hepatocytes, and clinical transformation is easy.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing immortalized hepatocytes with reversible liver function and its application. Background technique [0002] Appropriate hepatic cell lines play an increasingly important role in bioartificial liver technology, pharmacology and toxicology research, and liver tissue engineering research. An ideal source of hepatocytes should have complete liver function, good proliferation ability, and no risk of tumorigenesis or infection. Currently commonly used sources of hepatocytes include primary human hepatocytes, primary porcine hepatocytes, human hepatoma cell lines HepG2, HepaRG, immortalized cells, and stem cell differentiated hepatocytes. Although these hepatocytes have certain liver functions in vitro, they each have application obstacles such as insufficient maturity, limited expansion, unstable function, xenograft rejection, low biological safety, and tumorigenic ris...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N5/10
CPCC12N15/86C12N15/65C12N5/067C12N2740/15043C12N2800/107C12N2830/003C12N2830/008C12N2510/04
Inventor 高毅曾敏晏正明韩旭翁骏
Owner GUANGDONG UNISUN BIOTECHNOLOGY CO LTD
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