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Method for constructing immortalized pork liver stellate cell line, immortalized pork liver stellate cell line and application thereof

A stellate cell, immortalization technology, applied in the field of immortalized porcine liver stellate cell line and application, construction of immortalized porcine hepatic stellate cell line, can solve the problem of inability to construct immortalized porcine hepatic stellate cell and expand hepatic stellate cell line In order to avoid problems such as the source of shape cells, the effect of large fragments, clear cell outlines and complete cells can be achieved.

Active Publication Date: 2019-05-03
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the defect of being unable to construct immortalized porcine hepatic stellate cells at present, the present invention provides a method for constructing immortalized porcine hepatic stellate cells, which can obtain cell morphology and characteristics that are not significantly different from those of primary porcine hepatic stellate cells. Immortalized porcine hepatic stellate cell line, expanding the source of hepatic stellate cells, and providing convenient conditions for drug screening for liver therapy

Method used

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  • Method for constructing immortalized pork liver stellate cell line, immortalized pork liver stellate cell line and application thereof
  • Method for constructing immortalized pork liver stellate cell line, immortalized pork liver stellate cell line and application thereof
  • Method for constructing immortalized pork liver stellate cell line, immortalized pork liver stellate cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] 1. Isolation and culture of porcine hepatic stellate cells

[0081] 1) Pigs were bled to death from the carotid artery, and part of the liver tissue was taken, placed in PBS containing P / S,

[0082] 2) Remove the capsule, etc., then cut the liver tissue into small pieces, wash with PBS at least 3 times,

[0083] 3) Digest with 0.1% collagenase type IV at least 3 times the volume of the tissue in a water bath at 37°C for 1 hour,

[0084] 4) Pass through a 200-mesh sieve to obtain a whole liver cell suspension,

[0085] 5) Transfer the suspension into a centrifuge tube, centrifuge at 400rpm for 10min, and the precipitated cells are liver parenchymal cells.

[0086] 6) Transfer the supernatant to another centrifuge tube, and centrifuge at 1500rpm for 10 minutes. After the centrifugation is completed, discard the supernatant, and the obtained precipitate is porcine hepatic stellate cells;

[0087] 7) After resuspending the precipitate in step 6) with the culture medium, ...

Embodiment 2

[0140] Example 2: Using the immortalized porcine hepatic stellate cell line of the present invention to establish an endoplasmic reticulum stress model

[0141] Proceed as follows:

[0142] ICELL star containing 10% fetal bovine serum, 1% double antibody (penicillin / streptomycin, purchased from Saibakang (Shanghai) Biotechnology Co., Ltd.) and 1% growth factor PriMed cell basal medium (purchased from PriMed (Shanghai) Biotechnology Co., Ltd., product number: PriMed-iCELL-009), at 37 ° C, 5% CO 2 The immortalized porcine hepatic stellate cells constructed in Example 1 were cultivated in an incubator. Immortalized porcine hepatic stellate cells were seeded at 150,000 cells / well in a six-well plate, treated with tunicamycin at concentrations of 1, 2, 5, 10, and 15 μg / mL, and cultured. The time was 2h, 4h, 8h, 16h, 24h and 36h, respectively, and three replicate holes were made for each treatment. According to the cell viability, cell cycle and the expression of endoplasmic reti...

Embodiment 3

[0149] Example 3: Knockdown of GRP94 expression in immortalized porcine hepatic stellate cells using lentivirus-mediated shRNA

[0150] Proceed as follows:

[0151] Construct the GRP94 gene silencing lentiviral plasmid vector, and entrust Beijing Hopson Gene Technology Co., Ltd. for lentiviral packaging. The immortalized porcine hepatic stellate cells constructed in Example 1 were transfected with GRP94-shRNA lentivirus at 37°C, 5% CO 2 Cultivate in an incubator for 72 hours, and observe the brightness of red fluorescence under a fluorescence microscope. The expression of GRP94 in cells was detected by real-time fluorescent quantitative PCR technology, and the expression level of GRP94 protein in cells was detected by Western blot technology.

[0152] The specific operation is as follows:

[0153] (1) Construction, cloning and identification of GRP94-shRNA interference lentiviral vector

[0154]According to the general principle of shRNA design, three specific silencing si...

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Abstract

The present invention provides a method for constructing an immortalized pork liver stellate cell line, the immortalized pork liver stellate cell line and an application thereof, and relates to the field of immortalized cell technology. The normal pork liver stellate cells are obtain by separation from pork liver and the pork liver stellate cells are transfected with SV40LT overexpression lentivirus to obtain the immortalized pork liver stellate cell line. A lentiviral vector containing a SV40LT sequence is used to immortalize the pork liver stellate cells and has advantages of infecting non-dividing cells, accommodating exogenous gene fragments, and being capable of conducting long-term expression, etc. The immortalized pork liver stellate cell line constructed by the method grows in an extended state and has pseudopods, the cells have a clear cell outline, are complete, and have typical morphological characteristics similar to those of original pork liver stellate cells; and after passage of the pork liver stellate cell line for 10 generations, cell morphology and characteristics are not significantly different from those of the first generation cells. The constructed immortalized pork liver stellate cell line can be applied in drug screening.

Description

technical field [0001] The invention relates to the technical field of immortalized cells, in particular to a method for constructing an immortalized porcine hepatic stellate cell line, the immortalized porcine hepatic stellate cell line and applications. Background technique [0002] Various liver diseases are common and frequently-occurring diseases in the world, which seriously affect human health and bring huge economic burdens to governments of all countries. However, due to the difficulty in controlling the background conditions of human research samples and large-scale sampling, little is known about its pathogenesis at present. It is urgent to use suitable models to study the cellular and molecular mechanisms of its pathogenesis in order to provide scientific evidence for prevention, treatment and drug development. in accordance with. [0003] The metabolic function of porcine hepatocytes is the closest to that of human hepatocytes, and most basic and clinical studi...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/071C12N5/10C12Q1/02C12R1/91
Inventor 郝月顾宪红
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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