High metastatic potential hepatoma cell line capable of steady autophagy indication, and establishment method and application method thereof

Abstract

The invention belongs to the field of biomedical science, and aims to provide a high metastatic potential hepatoma cell line capable of steady autophagy indication, and an establishment method and an application method thereof. Lentivirus expression vectors containing EGFP (enhanced green fluorescent protein)-LC3 (light chain 3) autophagy reporter genes are constructed and used to infect hepatoma cells with high metastatic potential, so that host cells can express the EGFP-LC3 steadily and efficiently and indicate autophagy changes of the hepatoma cells steadily. To apply the cell line, metastasis research models can be established in vitro and in vivo for the observation on autophagy changes during metastasis, and the cell autophagy changes can be analyzed rapidly and quantitatively by adopting an image analysis method based on Top-hat operators and morphological filter. The high metastatic potential hepatoma cell line has the characteristics that autophagy indication is conducted steadily, reliably and sensitively in a real-time manner, the metastasis occurs definitely, the autophagy is quantified rapidly and accurately, the operation is simple and convenient, and the like, and can greatly facilitate correlation study on autophagy and metastasis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a liver cancer cell strain capable of stably indicating cell autophagy activity and having high transfer potential, and methods for establishing and applying the cell strain. Background technique [0002] Primary liver cancer is a malignant disease that seriously threatens human health. Although its morbidity rate only ranks sixth in the incidence rate of malignant tumors, its mortality rate ranks third. China is a country with a high incidence of primary liver cancer, accounting for about 55% of the global incidence. The third national death cause retrospective sampling survey report shows that liver cancer is the second cause of death related to malignant tumors in my country (in cities and men it is the first cause of death). How to effectively prevent and treat liver cancer is a major problem that needs to be solved urgently in our country. [0003] Among the many factors affectin...

AI Technical Summary

Problems solved by technology

[0006] The current liver cancer cell lines can not meet the research needs

This is mainly because: (1) General-purpose liver cancer cell lines do not have high metastatic ability: currently widely used liver cancer research tool cells, such as ATCC HepG2, do not have high metastatic ability, and it is difficult to replicate the metastatic process well in vivo and in vitro ( Especially in vivo metastasis); (2) Insufficient autophagy indicator observation and rapid quantitative analysis methods: the autophagy observation methods used in current autophagy research have great limitations, and cannot complete the observation of autophagy changes during the transfer process

The application of human liver cancer cells to establish this model requires that the cells can stably indicate autophagy. The autophagy indicator cells produced by the current general transient transfection technology are used to grow tumors in vivo. After the cells divide multiple times and the tumor grows to a certain size (far Before cancer cell metastasis occurs), the GFP-LC3 autophagy indicator is weakened or lost, which makes it impossible to observe changes in autophagy during metastasis in vivo

In addition to the above-mentioned shortcomings in stable expression, the traditional GFP-LC3 research method also has serious shortcomings in application methods: lack of fast and reliable autophagy quantitative analysis methods

The manual counting workload is very heavy (the total number of counted cells in each treatment group is usually more than 200, and the total number of fluorescent bright spots is usually more than 1000), especially in multi-factor and large-sample experiments, the work is very heavy, and the counter is easy to see. Fatigue counting is wrong and the reliability is poor. At the same time, manual counting is subjective and arbitrary, and the quantitative analysis results have limited credibility due to lack of sufficient objectivity, etc.

In addition, the traditional GFP-LC3 method has some other shortcomings, including low transfection efficiency, cytotoxicity of transfection reagents and autophagy induction, etc.

Among them, the autophagy induction effect of the transfection reagent has a great influence on the related research of autophagy, because the transfection reagent can induce autophagy and maintain it for a long time after the transfection reagent is removed, resulting in a high background autophagy , in the case of high background autophagy, it will be difficult to accurately judge the occurrence and change of autophagy

[0007] In short, the current liver cancer cell lines and related research methods cannot meet the needs of the research on autophagy and metastasis, and there is an urgent need for liver cancer cell lines and related application methods that can overcome the current deficiencies

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