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Novel method for constructing osteosarcoma pulmonary metastasis model and application thereof

A construction method and osteosarcoma technology, which is applied in the field of construction of a new type of osteosarcoma lung metastasis model, can solve the problem of inability to observe the characteristics of lung metastasis of osteosarcoma, and achieve the effect of obvious knockout effect.

Pending Publication Date: 2019-08-27
SHANGHAI CHANGZHENG HOSPITAL
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] Traditional osteosarcoma models are mostly injected through the tibial plateau or subcutaneously bearing tumors, and it is impossible to observe the lung metastasis characteristics of osteosarcoma, especially its lung metastasis characteristics

Method used

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  • Novel method for constructing osteosarcoma pulmonary metastasis model and application thereof
  • Novel method for constructing osteosarcoma pulmonary metastasis model and application thereof
  • Novel method for constructing osteosarcoma pulmonary metastasis model and application thereof

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Experimental program
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Embodiment 1

[0046] 1. Experimental method

[0047] 1. Establishment of stable osteosarcoma cell lines and transfection of fluorescent genes

[0048]Lentiviral expression vector name: Lenti-EF1α-puro Source: Donated by the plasmid bank of Baylor College of Medicine, USA.

[0049] The base sequence of REGγ transfected with osteosarcoma 143B cells:

[0050] shREGγ: "5'-CCGGGGTTCTGAGAGAGTAAAAATTATTCTCGAGAATAATTTACTCAGAACCTTTTTG-3'", shNC is an empty plasmid, and a stable line of osteosarcoma cells was constructed by lentivirus method.

[0051] 1) First, according to the sequence information of the REGγ gene, construct the corresponding shRNA onto the lentiviral expression vector, that is, construct the plasmid;

[0052] 2) psPAX2, VsVg / PMD2G (wherein psPAX2 and VsVg / PMD2G are lentiviral packaging plasmids), and the constructed lentiviral vector, according to the volume ratio psPAX2:VsVg / PMD2G:lentiviral vector = 2:1:2 total Transfect 293T cells;

[0053] 3) Change the medium 8 hours after...

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Abstract

The invention relates to a novel method for constructing an osteosarcoma pulmonary metastasis model and application thereof. The invention designs siRNA and shRNA sequences with significant knockout effect for REGgamma, and constructs a lentiviral vector containing the above sequences, so as to construct a nude mouse osteosarcoma pulmonary metastasis model through the cell tail vein injection method. The results confirm that constructs containing the siRNA and shRNA in the invention can significantly reduce the REGgamma mRNA and protein expression, and the constructed osteosarcoma pulmonary metastasis model can obviously observe cell growth and metastasis. The method provides a new research idea for exploration of targeted drugs for osteosarcoma in future.

Description

technical field [0001] The invention relates to the technical field of tumor molecular biology, in particular to a method for constructing a novel osteosarcoma lung metastasis model and its application. Background technique [0002] Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. The incidence rate is higher in men than in women. Osteosarcoma can occur in all age groups, especially in adolescents. It accounts for about 5% of adolescent tumors and occurs worldwide every year. The rate is 1-3 / 1 million people. It is highly invasive, and its malignancy is high, and the prognosis of patients is extremely poor, especially after the early occurrence of lung metastases, the survival period of patients is shortened sharply. There is currently no effective cure for the disease. Osteosarcoma tends to occur in fast-growing bone tissue. The disease progresses rapidly and can metastasize to the lungs in a short period of time. The treatment eff...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10A01K67/027
CPCC12N15/1135C12N15/86A01K67/0271C12N2310/141C12N2310/14C12N2310/531C12N2740/15043A01K2227/105A01K2267/0331Y02A50/30
Inventor 肖建如刘玉杰李林李磊万维罗静高欣许克寒李博高晓何韶辉杨明磊
Owner SHANGHAI CHANGZHENG HOSPITAL