Primary human osteosarcoma cell strain NEO217-luc from spine and construction method and application of cell strain

A neo217-luc, osteosarcoma cell technology, applied in the field of biomedicine, can solve problems such as difficult biological behavior of spinal osteosarcoma, and achieve the effects of good tumorigenicity, fast and stable growth

Pending Publication Date: 2019-05-14
SECOND AFFILIATED HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, few people have established spinal osteosarcoma cell lines, which makes it difficult to study the biological behavior of spinal osteosarcoma

Method used

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  • Primary human osteosarcoma cell strain NEO217-luc from spine and construction method and application of cell strain
  • Primary human osteosarcoma cell strain NEO217-luc from spine and construction method and application of cell strain
  • Primary human osteosarcoma cell strain NEO217-luc from spine and construction method and application of cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Primary Culture and Establishment of Spinal Osteosarcoma Cell Lines

[0032] Part of the sterile fresh tumor specimens from patients with spinal osteosarcoma after surgical resection were sent to the pathology department for pathological examination to confirm osteosarcoma. room for follow-up processing.

[0033] Pour the specimen in the centrifuge tube into a 10cm sterile petri dish in the cell ultra-clean workbench, wash it repeatedly with Hanks solution to remove the red blood cells adhered to the surface of the specimen, and cut the tissue block repeatedly to mince shape with sterile tissue scissors , move the tissue into a 50ml sterile centrifuge tube containing 10ml DMEM medium (Gibco, USA), add 10μl of type II collagenase, add penicillin (100U / ml) and streptomycin (100mg / ml) at the same time, put it in a constant temperature Oscillate at a speed of 200 times / min in an oscillator at 37°C, take it out after 2 hours, blow and mix it with a Pasteur pipette...

Embodiment 2

[0039] Embodiment 2 Human primary osteosarcoma cell line growth cycle

[0040] Collect 1×10 cells in the logarithmic growth phase 6 , resuspend the cells with 300μl PBS, add the cells dropwise to 700μl pre-cooled absolute ethanol, fix overnight at 4°C in the dark, centrifuge at 800g for 10min, remove the supernatant, wash twice with PBS, and resuspend the cells in 500μl containing 100u / ml RNaseA in PBS buffer, incubate at 37°C for 30 minutes in the dark, add 2 mg / ml PI to a final concentration of 50 μg / ml, and incubate in the dark for 30 minutes, take the PI fluorescence reading as the abscissa, and the cell number as the ordinate, in the flow cytometer The number of cells in each phase of the cell cycle was counted on a cytometer.

[0041] Experimental results: image 3 For the cell growth cycle of the primary human osteosarcoma cell line, the image 3 It can be seen that the cells G1 = 42.9%, G2 = 9.69%, and S = 47.42% as determined by the flow cytometer.

Embodiment 3

[0042] Example 3 Cell Proliferation Experiment

[0043] This experiment adopts the CCK8 method, and the operation steps are as follows: inoculate the cells in the logarithmic growth phase in a 96-well plate at a concentration of 5000 / 200 μl, set 5 controls in each well, and co-culture for 7 days. On day 2, day 4 and day 7, add 20 μl of CCK8 to each well and incubate together for 3.5 hours; then shake well with a horizontal shaker to fully dissolve Formazane; use an automatic microplate reader at a wavelength of 450 nm to measure The A(OD) value of each well was taken as the average value to trace the growth curve of each cell. This experiment was repeated 3 times to obtain the cell growth curve as follows: Figure 4 shown.

[0044] Experimental results:

[0045] The osteosarcoma cell that this embodiment builds the line grows rapidly, and the cell value is by 1 * 10 4 Proliferated rapidly to 7.8 × 10 on day 8 4 , with an average doubling time of 48h.

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Abstract

The invention relates to a primary human osteosarcoma cell strain NEO217-luc and a construction method and application thereof. The cell strain NEO217-luc is preserved in China Center for Type CultureCollection, the address is Wuhan University, Wuhan province, China, and the preservation number is CCTCC NO:C2018179. The cell of the cell strain NEO217-luc has the overlapping growth capacity, the cell atypia is obvious, the cell strain has the morphological characteristics of osteosarcoma cells and is a newly-constructed human osteosarcoma tumor cell strain, in-vitro culture can be conducted ina culture medium, during in-vitro culture, quick and stable growth can be achieved, continuous passage can be achieved, and accordingly an osteosarcoma immunodeficient mouse in-vitro model is conveniently constructed; the immunodeficiency mouse has good subcutaneous tumorigenicity, the tumor grows quickly and is high in wettability along with time prolonging, important guarantee is provided for in-vivo tests of medicine, and the cell strain can be used as a cell material for in-vitro study on osteosarcoma pathogenesis and individual treatment and can be applied to preparation, screening and evaluation of anti-spine-osteosarcoma medicine.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a spine-derived human primary osteosarcoma cell line NEO217-luc and its construction method and application. Background technique [0002] Osteosarcoma is one of the most common bone malignancies. However, spinal osteosarcoma is very rare, accounting for only 3% of all primary osteosarcomas. Although bone tumor surgeons recommend extensive resection for any primary osteosarcoma, the prognosis for osteosarcoma of the spine is poor compared with osteosarcoma of the extremities. Over the past few decades, there have been several reports on the establishment of human osteosarcoma cell lines. However, few people have established spinal osteosarcoma cell lines, which makes it difficult to study the biological behavior of spinal osteosarcoma. Therefore, establishing spinal osteosarcoma cell lines as a clinically relevant spinal osteosarcoma model to study its biologic...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/02A01K67/027C12R1/91
Inventor 周振华肖建如胡硕王旭东张薇薇匡牧宇龚德军贾奇李焱曹佳实
Owner SECOND AFFILIATED HOSPITAL SECOND MILITARY MEDICAL UNIV
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