Cell strain capable of inducing and realizing dual-stabilization of P53 expression and construction method and application thereof

A cell line, stable technology, applied in the field of biomedicine

Inactive Publication Date: 2012-07-04
SECOND AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the active development of neoadjuvant chemotherapy and active surgical treatment such as limb salvage surgery, the quality of life and prognosis of osteosarcoma have been significantly improved. The five-year survival rate in foreign countries has reached more than 80%. The five-year survival rate of the center has reached about 60%, and there is still a big gap with foreign countries. The reasons are as follows: 1. The huge cost of treatment is unbearable for patients and they give up treatment or stop treatment halfway. The clinical promotion and application of these methods have been affected by the serious side effects and complications of chemotherapy and radiotherapy, local metastasis, local recurrence, and drug resistance.

Method used

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  • Cell strain capable of inducing and realizing dual-stabilization of P53 expression and construction method and application thereof
  • Cell strain capable of inducing and realizing dual-stabilization of P53 expression and construction method and application thereof
  • Cell strain capable of inducing and realizing dual-stabilization of P53 expression and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, construction of recombinant expression vector pTRE-HA-E6

[0024] The plasmid pHA-E6 containing E6 gene with HA tag was constructed. Firstly, the pCl neo plasmid was double-digested with restriction endonucleases BglⅡ and EcoRI, and the digested products were purified and collected by agarose gel electrophoresis to obtain the cDNA containing the early immediate enhancer / promoter of cytomegalovirus. At the same time, the pEGFP-1 plasmid that does not contain the promoter is also double-digested with restriction endonucleases BglII and EcoRI, and the digested products are also purified and collected by agarose gel electrophoresis, and the product obtained above is ligated with DNA ligase to obtain pEGFP-1 plasmid with insertion of CMV early immediate enhancer / promoter. HPV type 16 E6 gene was amplified from cervical cancer Siha cell genome by PCR (forward primer 5'-GAAACCGGTTAGTATAAAAGC-3'; reverse primer, 5'-CCATGGTAGATTATGGTTTCT-3'). The obtained cDNA ...

Embodiment 2

[0025] Example 2 Construction of cell line U-2OSE6tet6 inducible and dual stable P53 expression

[0026] Osteosarcoma U-2OS cells were seeded on 6-well plates at a density of about 300,000 cells / well. Take Lipofectamine TM In 2000, the pTet-Off plasmid of Clontech Company was transfected into U-2OS cells, and the cells were harvested after continuing to culture for 48 hours, and inoculated in multiple 10cm cell culture dishes with increasing concentrations from low to high (0, 50, 100, 200, 400, 800 μg / ml) G418 to select cell clones stably transfected with pTet-Off gene, and found that the cells basically died after 5 days of selection with G418 at a concentration above 200 μg / ml, and then cultured at 200 μg / ml for 2 weeks to obtain G418-resistant cells clone. Lipofectamine TM 2000 Transiently transfect the tetracycline-responsive element pTRE-luc expression plasmid containing the luciferase reporter gene into the above cells, and screen the cell clone with the highest ...

Embodiment 3

[0027] Example 3 Inducible, dual stable P53 expression U-2OSE6tet6 cell response analysis to doxycycline

[0028] The U-2OSE6tet6 cells obtained in Example 2 were cultured in the medium of McCoy's 5A+10% fetal calf serum, maintained with G418 100 μg / ml and hygromycin 50 μg / ml in the medium, and will be in the logarithmic phase of growth Count the cells after trypsinization, inoculate 200,000 cells / well in a 6-well plate, add the above-mentioned medium to a volume of 2ml, and store at 37°C with 5% CO 2 in the incubator. After 24 hours, replace the medium, take 5 wells of the well-growth plate and add doxycycline, so that the concentration of doxycycline is 0, 0.1, 1, 5, and 20ng / ml respectively, and the medium is adjusted to 2ml. Continue to culture the samples in the incubator for 48 hours. After 48 hours, the nuclear protein was extracted, and the changes in the protein content of P53 in the cells were analyzed by western blot, as shown in Figure 4 shown.

[0029] The ...

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Abstract

The invention relates to the field of biological medicine, in particular to a cell strain capable of inducing and realizing dual-stabilization of P53 expression and a construction method and application thereof. As for the cell strain capable of inducing and realizing the dual-stabilization of the P53 expression, the cell strain U-2OSE6tet6 was collected in China Center for Type Culture Collection on August 12, 2010, and the collection number is CCTCC No: C201075. Experiments prove that the cell strain U-2OSE6tet6 capable of inducing and realizing the dual-stabilization of the P53 expression can indirectly change the P53 content in U-2OSE6tet6 cells by regulating the expression of an E6 gene through doxycycline, and provide an experimental platform for studying relevant action mechanism of the P53 of screening medicaments for treating osteosarcoma.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a cell line capable of inducible and dual stable expression of P53 and its construction method and application. Background technique [0002] Osteosarcoma is a kind of disgusting bone tumor that tends to occur in teenagers. The age of onset is mainly concentrated between 10-20 years old. It accounts for 35%-44% of malignant bone tumor patients in hospital because of its high degree of malignancy and metastasis. It is early and the curative effect is poor, which has always been the difficulty, focus and hot spot of clinical orthopedic surgeons' research. With the active development of neoadjuvant chemotherapy and active surgical treatment such as limb salvage surgery, the quality of life and prognosis of osteosarcoma have been significantly improved. The five-year survival rate in foreign countries has reached more than 80%. The five-year survival rate of the center has reached about 6...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85A61K35/12A61P35/02A61K48/00
Inventor 陶惠民苗旭东杨迪生
Owner SECOND AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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