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Method for constructing in-situ primary lung cancer animal model

An animal and culture method technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, animal cells, etc., can solve the problems of long reproduction cycle, low tumor formation probability and high cost, and achieve the effect of shortening the construction period

Pending Publication Date: 2021-05-28
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The background of the genetic mouse model is very clear, and it is entirely possible that the location of tumor formation is in situ in the lung, but the probability of tumor formation is low, and it is easy to die before modeling, the cost is very high, and the production and reproduction cycle of genetic mice is too long
The carcinogen-induced model is the oldest lung cancer model, which largely depends on the genetic background of the mouse, and it is actually difficult for this model to simulate the tumor formation in patients

Method used

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  • Method for constructing in-situ primary lung cancer animal model
  • Method for constructing in-situ primary lung cancer animal model
  • Method for constructing in-situ primary lung cancer animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1 Construction of mouse lung cancer model of the present invention

[0070] The construction method of the mouse lung cancer model of the present invention is as follows:

[0071] 1. Organoid culture

[0072] In this section, lung organoids were prepared from fresh mouse lung tissue, the steps are as follows:

[0073] (1) Take fresh mouse lung tissue and cut it into pieces on ice;

[0074] (2) Collagenase (2mg / mL Collagenase I and 1mg / mL Collagenase IV) resuspended the shredded tissue pieces, and used the gentalMACS automatic tissue processor to run the Mouse Tumor program 1 in the C tube; the shredded tissue pieces The dosage is 1-2 grams, and the dosage of collagenase is 10mL;

[0075] (3) The collagenase-treated tissue block was digested on a shaker at 37° C. at a speed of 220 rpm for 30 minutes. Fully disperse the tissue cells;

[0076] (4) Transfer the digested solution to the automatic tissue processor gentalMACS. On gentalMACS, run the Mouse Lung...

experiment example 1

[0112] Experimental Example 1 Construction and Identification of Small Cell Lung Cancer Model

[0113] 1. Method

[0114] In this experimental example, a control group and an experimental group were set up, with 5 mice in each of the control group and the experimental group, and the "orthotopic primary lung cancer mouse model" was constructed according to the method in Example 1. The difference between the two groups lies in the genetic The modified genes are different.

[0115] In the control group, the Scr gene was knocked out using CRISPR / Cas9 technology;

[0116] The experimental group used CRISPR / Cas9 technology to knock out the tumor suppressor genes Trp53 and Rb1, and transferred the oncogenes Kras and Myc with lentivirus.

[0117] 2. Results

[0118] The results of live tumor imaging 60 days after transplantation were as follows: figure 2 As shown in the figure, it can be seen that the left lung of the mice in the experimental group has obvious fluorescent signals...

experiment example 2

[0123] Experimental example 2 In vitro drug intervention experiment of tumor organoids

[0124] 1. Method

[0125] Obtain fresh tumor (obtained in Experimental Example 1) and normal tissue cells, and carry out organoid culture. When passing passage, use TryplE to digest it into a single cell suspension within 30 minutes. Every 4000-4500 cells are mixed with 10ul Matrixgel ) were mixed and cultivated in a 96-well plate, adding 50ul organoid medium to each well, and replacing it with organoid medium containing different concentrations of inhibitors after 24 hours, observing the growth status of organoids after 72 hours of drug treatment, and counting the growth of organoids numbers and analyze statistics.

[0126] In the above-mentioned drug screening system, different concentrations of cisplatin, 5, 10, 20, and 40 μM, were mixed with the organoid culture medium and added to normal lung tissue and tumor cell organoids respectively, and the effect was continued for 72 hours, and...

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Abstract

The invention discloses a method for constructing an in-situ primary lung cancer animal model, and belongs to the field of tumor animal models. Mouse lung cells are cultured into organoids with a specific culture medium, and then the organoids are subjected to gene editing and injected back to the mouse lungs, so that the organoids are developed into tumors. Compared with a genetic engineering tumor animal model, the method disclosed by the invention is short in time consumption and high in tumor formation rate; and compared with a transplanted tumor animal model, the model has an in-vivo micro-environment for tumor generation and development, and is closer to the most real state of lung cancer.

Description

technical field [0001] The invention belongs to the field of tumor animal models. Background technique [0002] Lung cancer is a malignant tumor that seriously affects human health. It is mainly divided into non-small cell lung cancer (NSCLC, Non-small cell lung cancer) and small cell lung cancer (SCLC, Small cell lung cancer). The incidence and mortality of lung cancer rank first among all cancers. first place. [0003] Lung cancer cell model is the basic model for studying the mechanism of lung cancer and the activity of drug therapy for lung cancer. However, the lung cancer cell model usually undergoes many generations of culture, and the genetic information has shifted, lost or increased some specific chromosomal segments. Normal tissues or tumors have strong heterogeneity, a single type of cell line is very different from the state in the body, and the resistance to certain drugs and the influence of genes cannot reflect the real situation of the patient. [0004] In ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/12A01K67/027
CPCC12N5/0688C07K14/47C07K14/82A01K67/0271C12N2509/00C12N2510/00A01K2227/105A01K2267/0331Y02A50/30
Inventor 陈崇纳飞飞刘玉
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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