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Mouse liver tumor cell line for highly expressing CD133 and preparation method thereof

A cell line and liver tumor technology, applied in biochemical equipment and methods, embryonic cells, animal cells, etc., can solve the problem of scarcity of tumor stem cells

Active Publication Date: 2011-09-14
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the technical problems to be solved by the present invention is to provide a mouse liver tumor cell line LPC-H12 with a high expression of CD133 in view of the scarcity of tumor stem cells expressing the stem cell marker CD133 in the current tumor cell lines. It was deposited in the China Center for Type Culture Collection (CCTCC for short) on December 8, 2010, with the preservation number CCTCC No.C2010115, the name of the biological material sample: mouse liver tumor cell LPC-H12, and the address of the depository unit: Wuhan, China. Wuhan University

Method used

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  • Mouse liver tumor cell line for highly expressing CD133 and preparation method thereof
  • Mouse liver tumor cell line for highly expressing CD133 and preparation method thereof
  • Mouse liver tumor cell line for highly expressing CD133 and preparation method thereof

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Experimental program
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Embodiment 1

[0036] Establishment of mouse liver tumor cell line LPC-H12 with high expression of CD133:

[0037] a) Isolation of p53- / - mouse embryonic liver cells (LPC cells): take p53- / - mouse embryos at E13.5d, rinse them in PBS for 2-3 times; use sterile ophthalmic scissors and tweezers to cut them completely liver. After cutting it into pieces, add liver digestion solution to digest at 37°C for 30 minutes to form a single cell suspension. Centrifuge to remove the supernatant, wash 2-3 times with PBS, and incubate the cells with rat anti-mouse E-cadherin monoclonal antibody at 4°C for 30min (the amount of antibody used is 4ug E-cadherin / 1×10 7 cells). Wash 2-3 times with PBS, centrifuge to remove the supernatant, and mix the cells with goat anti-rat magnetic beads (20ul / l×10 8 cells) were incubated at 4°C for 15 min. Finally, the E-cadherin+ embryonic liver cells were isolated by magnetic bead sorting system (MACS). The isolated cells were inoculated on a cell culture dish pre-coa...

Embodiment 2

[0042] LPC-H12 cell line highly expresses stem cell markers CD133 and EpCAM:

[0043] a) The conventional in vitro culture conditions of LPC-H12 cell line are: DMEM / F12 culture medium prepared by 10% FBS and 50 mM 2-mercaptoethanol, 37°C, 5% carbon dioxide / 95% air, saturated humidity.

[0044] b) LPC-H12 cells grown to the logarithmic growth phase were digested with 0.25% trypsin+0.1% EDTA, washed twice with PBS, and 1×10 5 ~1×10 7 Put the cells into flow tubes, centrifuge, remove the supernatant, add 100 μl of 10% goat serum to suspend the cells, and block at 4° C. for 10 min.

[0045] c) Add CD133-PE or EpCAM-PE fluorescent antibody 2.5ul (0.5mg / ml) in step b), and incubate at 4°C for 30min.

[0046] d) Add FACS buffer, mix gently, centrifuge at 300g for 5min, and discard the supernatant.

[0047] e) Add 300-500 ul of FACS buffer solution, and perform detection with a BD flow analyzer. Such as image 3 Shown: LPC-H12 has higher expression levels of stem cell markers CD1...

Embodiment 3

[0049] Expression of liver stem cell-related genes in LPC-H12 cell line:

[0050] a) The LPC-H12 cells that were routinely cultured in vitro and grown to the logarithmic growth phase were digested with 0.25% trypsin + 0.1% EDTA, and then the cells were collected. The cells were washed twice with PBS, and the total RNA extraction kit from TaKara was used for the cell pellet. Extraction of total RNA was performed.

[0051] b) Take 2ug of total RNA and reverse it into cDNA using the reverse transcription kit from TaKara Company.

[0052] c) PCR detection of expression of genes related to proliferation and self-renewal of hepatic stem cells. The amplified target gene and its primer sequences are shown in Table 1, and the primers were synthesized by Handsome Biotechnology Co., Ltd. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, and cycled 35 times. The cycle conditions were: denaturation at 95°C for 130 s, annealing for 30 s, the annealing temperature is sho...

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Abstract

The invention provides a mouse liver tumor cell line for highly expressing CD133 and a preparation method thereof. The mouse liver tumor cell line with high content of CD133+cell subpopulations is established by the following steps of: importing a cancer gene Hras into p53- / - mouse fetal liver cells, obtaining a single-cell clonal group derived from CD133+cells by utilizing a monoclonal patternmaking technology, and finally screening. The cell line LPC-H12 expresses related genes of various liver stem cells and the stem cells, namely Marker:CD133 and EpCAM, and has high in-vitro balling capacity and in-vitro tumorigenic capacity. The mouse liver tumor cell line provides a powerful tool for researching the action and the mechanism of CD133+liver cancer stem cell subpopulations in the occurrence and development process of liver cancer and screening medicaments used for liver tumor stem cells.

Description

technical field [0001] The invention relates to the field of tumor cell therapy, in particular to a rat liver tumor cell line highly expressing CD133 and a preparation method thereof. Background technique [0002] In recent years, more and more studies have shown that there is a very small proportion of stem cell populations in tumor cells, called cancer stem cells (cancer stem cells, CSCs), this group of cells plays a role in tumor treatment resistance, recurrence and malignancy. play a key role in transfer. Cancer stem cells have similar unlimited proliferation and self-renewal abilities as ordinary stem cells; express stem cell markers, such as: CD133, EpCAM, CD44, CD90, CD24, CXCR4, ALDH1, etc.; express and regulate stemness-related factors, such as: Notch, Bmi1 , Oct4, Nanog, etc. Although tumor stem cells are rare in number, they are highly tumorigenic and maintain tumor growth and metastasis. Among the primary isolated tumor cells, only a few cells expressing stem ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/073C12N7/00C12R1/91
Inventor 刘永忠杨兆娟马爱辉张力刘兰兰
Owner SHANGHAI INST OF ONCOLOGY
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