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Construction method and application of cell strain for expressing NTNG1 gene

A cell line and gene technology, applied in the direction of genetically modified cells, applications, cells modified by introducing foreign genetic material, etc.

Pending Publication Date: 2020-11-06
梁嵘
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no report on the application of cell lines stably overexpressing NTNG1 gene and stably knocking down NTNG1 cell lines in human primary liver cancer

Method used

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  • Construction method and application of cell strain for expressing NTNG1 gene
  • Construction method and application of cell strain for expressing NTNG1 gene
  • Construction method and application of cell strain for expressing NTNG1 gene

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Experimental program
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Effect test

Embodiment 1

[0059] 1. qRT-PCR detection of NTNG1 expression

[0060] Fresh tissue specimens obtained during surgery were cooled in liquid nitrogen and stored in a -80°C freezer. After all the liver cancer tissues and paracancerous tissues were cut up, the total RNA was extracted with Trizol (TaKaRa, A-79061) reagent, and the total RNA was reverse-transcribed into cDNA according to the instructions of the kit. Reaction conditions: 95°C pre-denaturation, enzyme activation (30s), 95°C denaturation (5s), 60°C (30s), 40 cycles. With βactin as internal reference, with 2 -ΔΔCt The relative expression level of target gene NTNG1 mRNA was calculated by method. The upstream primer of NTNG1 is 5′-CTTGGAAGGAGTATCCCAAGC-3′, the downstream primer is 5′-TGTGGCATAATACTGATAGGGCT-3′; the upstream primer of β-actin is 5′-CACCATTGGCAATGAGCGGTTC-3′, and the downstream primer is 5′-AGGTCTTTGCGGATGTCCACGT-3′. The experiment was repeated three times. Conclusions such as figure 1 a.

[0061] Establishment of...

Embodiment 2

[0106] CCK-8 assay (cytotoxicity detection)

[0107] The specific steps of the CCK-8 experiment were performed according to the manufacturer's standard protocol. Cells were diluted in serum-free medium and seeded in 96-well cell culture plates at a density of 2000 cells / well. The plate was incubated at 37°C in 5% CO 2 incubator. To measure the growth rate of the cells, replace 100 μL of the spent medium with an equal volume of fresh medium containing 10% CCK-8. Cells were then further incubated at 37°C for 3 hours. Finally, a microplate reader (5082Grodig, Tecan, Austria) was used to measure the value of cell absorbance at 450 nm. The experiment was repeated 3 times and the results were recorded.

[0108] 1. Plate colony formation experiment

[0109] Cells in the logarithmic growth phase were taken from the cells in good growth state, digested with trypsin, resuspended with medium, inoculated into six-well plates with 500 cells per well, and cultured normally for 1520 da...

Embodiment 3

[0118] Flow Cytometry

[0119] Collect the HepG2 and SMMC7721 cells of each group transfected for 48 hours, digest with EDTA-free trypsin, wash twice with PBS, add 500 μL of binding buffer to resuspend the cells, and then add 5 μL of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), protected from light, incubated at room temperature for 15 min, and then detected the apoptosis rate of cells in each group on a flow cytometer (BD, USA). Then the cells were collected and fixed with 70% ethanol at 4°C for 2 hours, treated with PI (50 μg / mL) and RNaseA I (100 μg / mL), incubated at 37°C in the dark for 30 minutes, and placed in a flow cytometer The changes of DNA content in cells were analyzed in each period. Experiments were repeated three times.

[0120] Conclusion: NTNG1 inhibits apoptosis and regulates cell cycle progression in human hepatocellular carcinoma cells

[0121] The uncontrolled proliferation of tumor cells caused by apoptosis and cycle regulati...

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Abstract

The invention belongs to the technical field of cell biology, and particularly discloses a construction method and application of a cell strain for expressing an NTNG1 gene. With liver cancer cells asa target system, the liver cancer cells with stably overexpressed NTNG1 genes are obtained through NTNG1 overexpression of lentiviral infection. With the liver cancer cells as a target system, the liver cancer cells with stably knocked-down NTNG1 gene is obtained through NTNG1 interference of lentiviral infection. Through the application of the cell strain constructed by the invention, the influence of NTNG1 on liver cancer proliferation, growth, apoptosis, invasion, migration and mouse tumorigenesis is studied, an experimental basis is provided for molecular mechanism and prognosis judgmentof liver cancer occurrence and development, and a basis is provided for clinical application.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a construction method and application of a cell line expressing NTNG1 gene. Background technique [0002] Hepatocellular carcinoma (HCC, hereinafter referred to as liver cancer) is the sixth most prevalent cancer worldwide and the third most common cause of cancer-related death. According to cancer statistics in 2018, there are 841,000 new cases of HCC every year worldwide, and about 55% of HCC patients are from China. In recent years, liver cancer is also one of the main causes of cancer death in my country, and its mortality rate ranks second. Due to the lack of effective clinical diagnostic targets, and the high degree of malignancy, strong invasiveness, and rapid progression of liver cancer, most patients have reached the middle and late stages when they are diagnosed. Even if they receive standard systemic treatment, the median OS often does not exceed 14 months, resul...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886G01N33/574C12N5/10C12N15/867C12N15/12C12N5/09
CPCC12Q1/6886G01N33/57438C07K14/78C12N15/86C12N5/0693C12Q2600/158C12N2740/15043C12N2510/02
Inventor 梁嵘林燕叶甲舟李永强刘志辉黎倩骆敏黄宇
Owner 梁嵘
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