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A hpv-negative penile squamous cell carcinoma cell line carrying tp53 mutation and its use

A penile squamous cell carcinoma and cancer cell technology, applied in the field of HPV negative penile squamous cell carcinoma cell lines, can solve the problems of complex process, difficult to popularize and use, unclear pathogenesis, etc., and achieve the effect of stable nature

Inactive Publication Date: 2018-04-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the latest guidelines of the European Association of Urology (EAU) in 2014, HPV was identified for the first time as a risk factor for the onset of cancer, and it combined with other unknown factors to cause tumorigenesis, but its pathogenesis is still unclear
The former is often mixed with non-tumor cells, such as stromal cells, and the success rate is low, while the latter needs to rely on animals to form tumors, and the process is complicated and difficult to popularize. The success rate of the two methods reported abroad in the past is about 10%. %

Method used

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  • A hpv-negative penile squamous cell carcinoma cell line carrying tp53 mutation and its use
  • A hpv-negative penile squamous cell carcinoma cell line carrying tp53 mutation and its use
  • A hpv-negative penile squamous cell carcinoma cell line carrying tp53 mutation and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Establishment of cell lines

[0036] 1.1 Primary culture

[0037] The specimens were obtained from the Sun Yat-Sen University Cancer Center, and obtained from the metastatic inguinal lymph nodes of a 41-year-old male patient with squamous cell carcinoma of the penis. After the intraoperative lymph node specimens were isolated from the body, they were separated into small tissue pieces of about 5 mm, and immediately immersed in serum-free high-glucose DMEM medium ( Add 200 U / ml penicillin, 200 μg / ml streptomycin) to the EP tube. In the biological safety cabinet dedicated to cells in the laboratory, the tumor specimens were rinsed 3 times with phosphate buffered saline (PBS), and the tumor specimens were cut into small pieces with a diameter of 1-3 mm by sterile ophthalmic scissors, and placed evenly in 25 cm. 2Add about 1-3ml high-sugar DMEM medium (adding 10% imported fetal bovine serum (FBS), 200U / ml penicillin, 200μg / ml streptomycin, used as a growth mediu...

Embodiment 2

[0042] Example 2. Immunocytochemical staining and ultrastructural morphology observation of Penl1 cell line

[0043] 2.1 Cytochemical staining

[0044] When the cells were subcultured to the 8th generation, the Penl1 cells were grown on 24mm coverslips in a six-well plate. After the cells were completely attached to the wall and in a good growth state, the six-well plate was removed from the incubator, the culture medium was removed, and PBS was used to replace the cells. Rinse the coverslip, add 4% paraformaldehyde to fix the cells for 10 minutes, use 0.3% Triton X-100 to permeabilize the cells, then block the cells with 10% goat serum for 1 hour at room temperature, and incubate pan-CK and vimentin primary antibodies at 37 degrees For 1 hour, incubate the polymerized HRP-labeled anti-mouse IgG secondary antibody at 37°C for 30 minutes, develop color with DAB for about 5 minutes, and develop color with hematoxylin for 3 minutes, dehydrate and seal the slides. The above cytoc...

Embodiment 3

[0049] Example 3. Genetic characterization of the Penl1 cell line

[0050] 3.1 Short tandem repeat (STR) cell identification

[0051] The DNA of tissue samples and cells was extracted using the AllPrepDNA / RNA / Protein Mini kit from Qigen. The DNA of metastatic lymph nodes, normal lymph nodes, and Penl1 cell line of the patient from which the samples were collected was used to amplify 19 STR loci and 1 sex-determining locus with a total of 20 loci, and the PCR amplification products were ABI3730xl electrophoresis was performed, and GeneMapper 4.0 software was used to analyze, and the STR spectrum containing 9 commonly used sites for cell line identification was compared with the three major international cell databases (American Type Culture Collection (ATCC), German Bioresource Collection (DSMZ) and Japan Cancer Resource Bank (JRCB) STR database for comparison. As shown in Table 1, the STR profile of the metastatic lymph node is consistent with that of the normal lymph node, ...

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Abstract

The present invention "an HPV negative penile squamous cell carcinoma cell line carrying TP53 mutation and its application" discloses a human penile squamous carcinoma cell line. This HPV-negative penile squamous cell carcinoma cell line carrying a TP53 mutation was deposited in the China Center for Type Culture Collection on June 17, 2015, with the preservation number CCTCC NO: C201585. The cell line Penl1 has been stably passaged in vitro for 14 months. After tumor formation in animals, the tumor specimens are medium-differentiated squamous cell carcinoma of the penis, and cell morphology, genetic characteristics, protein expression, biological behavior, and chemotherapy sensitivity have been investigated. The cells were fully tested. This penile squamous cell carcinoma cell line can represent the tumor characteristics of HPV-negative penile squamous cell carcinoma. It can be used in vitro and in vivo to study the mechanism of occurrence, development, and metastasis of penile cancer, as well as the clinical diagnosis, chemotherapy and targeted drug screening of penile cancer. There are broad prospects in basic and clinical translational research.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, and in particular relates to an HPV-negative penile squamous cell line carrying TP53 mutation and its application. Background technique [0002] Penile cancer is a rare male malignant tumor with a very poor prognosis in the late stage. In some countries of Africa, Asia, and South America, the incidence rate of penile cancer is as high as 4 / 100,000, which can reach 10% of male malignant tumors, while in Western Europe and the United States, it only accounts for 0.4%-0.6% of malignant tumors. Penile cancer in my country accounts for 0.07% of male malignant tumors, which is similar to Western Europe and the United States. Risk factors for penile cancer include low hygiene, phimosis, lack of circumcision, smoking, and high-risk human papillomavirus (hrHPV) infection. The 2014 edition of the latest guidelines of the European Association of Urology (EAU) identified for the first time that H...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12Q1/02C12R1/91
Inventor 韩辉张振峰陈洁平尧凯
Owner SUN YAT SEN UNIV
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