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Heterogeneous CIC cell model of targeted adhesion molecule and preparation method thereof

A cell model and target cell technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and cells modified by the introduction of foreign genetic material, etc., can solve the problem of lack of targeted specific gene models, and achieve the promotion of immune killing. , the effect of inhibiting tumor growth

Pending Publication Date: 2022-07-01
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Current research still lacks a model that targets specific genes to controllably regulate the formation of heterogeneous CIC

Method used

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  • Heterogeneous CIC cell model of targeted adhesion molecule and preparation method thereof
  • Heterogeneous CIC cell model of targeted adhesion molecule and preparation method thereof
  • Heterogeneous CIC cell model of targeted adhesion molecule and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Establishment of Inhibitory Heterogeneous CIC Cell Model

[0057] The target cell in this example is PLC / PRF / 5 liver cancer cell monoclonal line F6ft knockdown CD44 gene cell line shCD44-F6ft (high CIC formation rate), Western blot can detect N-cadherin and β-catenin genes Expression of the encoded protein in this cell line ( figure 1 B, C), please refer to Western blot in step one 2 of this example for details.

[0058] 1. Knock down the expression of N-cadherin and β-catenin genes

[0059] 1. Knock down N-cadherin and β-catenin genes by siRNA

[0060] 1) Design of siRNA: Two siRNA sequences targeting N-cadherin and β-catenin genes were designed and synthesized by Suzhou Gemma Company.

[0061] 2) Transfection of siRNA: using a transfection kit RNAiMAX Reagent (Invitrogen, #13778) was used for siRNA transfection, and the reaction system and conditions were operated according to the instructions of the kit. The details are as follows: Prepare 1×10 5 He...

Embodiment 2

[0096] Example 2. Establishment of induced heterogeneous CIC cell model

[0097] The target cell in this example is the PLC / PRF / 5 liver cancer cell monoclonal line F6ft. Western blot can detect that the expression of N-cadherin and β-catenin gene-encoded proteins is low ( figure 2 B, C), please refer to Western blot in step one 2 of this example for details.

[0098] 1. Establishment of F6ft-Ncadherin and F6ft-βcatenin overexpressing cell lines

[0099] 1. Construction of a plasmid overexpressing N-cadherin

[0100] 1) PCR amplification of N-cadherin fragment: PCR amplification of the N-cadherin gene coding sequence fragment CDS with Xho I and EcoR I restriction sites using cDNA as a template, and a gel DNA fragment recovery kit (Tiangen, #DP208-02) for purification.

[0101] 2) Enzyme digestion: The pQCXIP-mCherry-N1 backbone plasmid and the PCR-amplified N-cadherin fragment were mixed with restriction enzymes Xho I, EcoR I (New England Biolabs) and reaction buffer, and p...

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Abstract

The invention provides a heterogeneous CIC cell model targeting N-cadherin or beta-catenin and a preparation method of the heterogeneous CIC cell model. The heterogeneous CIC cell model is prepared by regulating and controlling the expression of an N-cadherin or beta-catenin gene in an external target cell. The inventor of the invention finds for the first time that improvement of expression of an N-cadherin or beta-catenin gene in liver cancer cells can promote the ability of internalizing immune cells CCRF and promote formation of a heterotypic CIC structure; on the contrary, the formation of a heterotypic CIC structure can be inhibited by reducing the expression of the N-cadherin or beta-catenin gene in the liver cancer cell; according to the present invention, the formation of the heterotypic CIC can be regulated and controlled by targeting N-cadherin and beta-catenin genes; according to the method for regulating and controlling the formation of the heterogeneity CIC structure by intervening the expression of the N-cadherin and beta-catenin genes, the heterogeneity CIC mediated immune killing is expected to be promoted, and the purposes of killing tumor cells and inhibiting tumor growth are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a heterogeneous CIC (heterotypic cell-in-cell) cell model targeting adhesion molecules (N-cadherin or β-catenin gene) and a preparation method thereof. Background technique [0002] Cell-in-cell (abbreviation: CIC) structure refers to the cell stacking structure formed by one or more living cells existing inside another cell, and the nucleus of the outer cell (target cell) is squeezed by the inner cell (effector cell) into Crescent-shaped, the overall morphological features resemble a bird's eye. According to the type of cells involved in the formation, it is divided into homogeneous CIC, that is, the CIC structure formed between the same tumor or epithelial cells; heterogeneous CIC, that is, the CIC structure formed between cells from different sources, which is commonly found in immune cells. into the CIC structures formed inside tumor cells. [0003] Heterogeneous CIC structures a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N5/078C12N5/09C12Q1/02
CPCC12N5/0693C12N5/0634C07K14/47G01N33/5011C12N2510/00
Inventor 孙强王晨曦苏艳高丽华
Owner ACADEMY OF MILITARY MEDICAL SCI
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