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An npm1 knockout human bladder cancer t24/ddp cell line

A technology for bladder cancer cells and cell lines, which can be applied to genetically modified cells, cells modified by introducing foreign genetic material, viruses/phages, etc., and can solve problems such as poor prognosis of bladder cancer.

Active Publication Date: 2022-07-01
张曼
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Abnormally highly expressed proteins in bladder cancer can indicate poor prognosis of bladder cancer. Using it to monitor the progression of bladder cancer and performing radical surgery in time can reduce the recurrence rate to 25%-40%. However, these markers are affected by the degree of tumor infiltration and Due to the influence of concomitant symptoms, the sensitivity and specificity of tumor monitoring are not enough to be a clear indicator of bladder cancer monitoring. Therefore, it is of great clinical significance to find bladder cancer biomarkers with good sensitivity and specificity

Method used

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  • An npm1 knockout human bladder cancer t24/ddp cell line
  • An npm1 knockout human bladder cancer t24/ddp cell line
  • An npm1 knockout human bladder cancer t24/ddp cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cell recovery, culture, passage, cryopreservation and maintenance of drug resistance

[0022] Take out the cell cryovial from the liquid nitrogen irrigation, quickly put it into a 37°C water bath and shake it to dissolve as soon as possible. Use a 75% alcohol cotton ball to sterilize the tube wall and mouth of the cryopreservation tube. Use a pipette to remove the cells. Transfer to a 15ml centrifuge tube, add 3ml of medium at the same time, centrifuge at 800rpm for 5 minutes, discard the supernatant, add 1ml of RPMI1640 medium containing 18% fetal bovine serum to resuspend the cells, and blow the cells into a single cell suspension. It was transferred into a culture flask for cultivation, 4 ml of culture medium was added, and cultured in a 37°C, 5% CO2 incubator. Passage when cells are observed to fill the flask. The human bladder cancer cell line T24 was cultured in RPMI1640 medium containing 15% FBS and 0.8ug / ml DDP in a 37 ℃, 5% CO2-saturated humidity ...

Embodiment 2

[0023] Example 2 Real-time fluorescence quantitative PCR detection

[0024] In the logarithmic growth phase of the cells, the total RNA of the cells was extracted using the steps in the Trizol instructions, the RNA was reverse transcribed to obtain cDNA, and the SYBR Green fluorescent dye was used to prepare the PCR reaction system according to the kit instructions. Three replicate wells were set in each group. PCR reaction: 95°C for 2 min, 95°C for 15 s, 60°C for 30 s, 68°C for 1 min, a total of 40 cycles. GAPDH was used as the internal reference, upstream primer: 5'-CTACAATGAGCTGCGTGTGGC-3', downstream primer: 5'-CAGGTCCAGACGCAGGATGGC-3', NPM1 upstream primer: 5'-TGGTGCAAAGGATGATGTTGC-3', downstream primer: 5'-GTCATCATCTTCATCAGCAGC-3', Record the Ct value of each experimental well and the relative expression level of the target gene. All experiments were repeated three times. F=2-ΔΔCt. ΔCt=ΔCt target gene-ΔCt internal reference gene; ΔΔCt=ΔCt experimental group-ΔCt con...

Embodiment 3

[0025] Example 3 T24 / DDP cells were infected with NPM1-shRNA lentivirus to establish a stable cell line T24 / DDP-NPM1 ko

[0026] Inoculate the T24 / DDP cells in log phase growth into 96-well plates at a concentration of 1×103 cells / ml. When the confluency reaches about 30%-50%, quickly add 100μl / well of diluted virus solution and culture. Change to normal medium after 8-16 hours. 72-96 hours after infection, the GFP expression was observed and passaged if necessary. The recombinant cell lines were added to RPMI-1640 medium containing 2 μg / mL Puromycin and 10% fetal bovine serum, and the screening medium was replaced every 2 to 3 days, and the screening was cultured for 2 months. After the successful stable transformation was confirmed by immunofluorescence, monoclonal screening, isolation and culture were carried out to establish the stable transformation cell line T24 / DDP-NPM1 ko . like figure 2 As shown in A, compared with the control and empty groups, the fluorescenc...

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Abstract

The invention provides a lentivirus-infected human bladder cancer cell line to knock out endogenous genes, and successfully establish a stable transfection cell line. Specifically, NPM1‑shRNA lentivirus infected T24 / DDP cell line to silence NPM1 (nucleolar phosphoprotein 1) expression, and successfully established T24 / DDP‑NPM1 ko cell line. The present invention confirms T24 / DDP-NPM1 through research ko Cell lines can be stably silenced NPM1 expression and passaged. T24 / DDP‑NPM1 after silencing NMP1 compared to negative control ko The expression level of NPM1 protein in the cell line was significantly decreased, the cell proliferation ability was significantly inhibited, the cell migration ability was increased, and the cell cycle was arrested in the S phase.

Description

technical field [0001] The invention relates to lentivirus-infected human bladder cancer cell lines to knock out endogenous genes and establish stable transfection cell lines, specifically NPM1-shRNA lentivirus-infected T24 / DDP cell lines to silence NPM1 (nucleolar phosphoprotein 1) expression, and successfully established Cell line T24 / DDP-NPM1 ko . Background technique [0002] Bladder cancer, also known as bladder urothelial carcinoma, is one of the most common malignant tumors of the urinary system, of which 90% are transitional cell carcinomas. In 2017, approximately 79,030 new cases and 16,870 deaths from bladder cancer are expected in the United States, respectively. The progression of bladder cancer is a process involving multiple factors, multiple genes, multiple steps, and multiple pathways. Bladder cancer is prone to drug resistance and recurrence, which are the two most critical characteristics affecting its clinical efficacy. The recurrence rate of bladder ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12R1/91
CPCC07K14/47C12N15/86C12N2740/15043C12N2510/00
Inventor 张曼罗陈烁赵嫚谢清雷婷
Owner 张曼
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