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Preparation method and application of immortalized human cartilage endplate stem cell line

A stem cell line and immortalization technology, applied in the field of cell engineering, can solve the problems of poor vitality and difficulty in survival, and achieve the effect of strong osteogenic ability, strong osteogenic differentiation ability, and low price

Inactive Publication Date: 2016-08-31
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the increase of the number of passages, the viability of CESCs isolated from the primary culture is getting worse and worse, and they will eventually undergo apoptosis, making it difficult to survive. Provide standard cell lines for studying the biological behavior of human endplate stem cells, provide a platform for basic research on tissue engineering, provide sufficient seed cells for clinical applications, and provide an effective and reliable method for establishing human degenerative cartilage endplate stem cell lines

Method used

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  • Preparation method and application of immortalized human cartilage endplate stem cell line
  • Preparation method and application of immortalized human cartilage endplate stem cell line
  • Preparation method and application of immortalized human cartilage endplate stem cell line

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Embodiment 1

[0028] The acquisition, separation and identification of embodiment 1 CESCs

[0029] Inclusion criteria for cases from specimen sources: age younger than 65 and older than 30 years old with informed consent; no tuberculosis, hepatitis, tumor, diabetes and other infectious diseases and autoimmune diseases; cervical, thoracic and lumbar fusion surgery.

[0030] The cartilage endplate specimens discarded during the operation were separated with a dissecting microscope under sterile conditions, the cotton-like nucleus pulposus tissue and the white fibrous ring fibrous tissue were removed, and the adherent tissue was carefully scraped off with a sharp knife. The nucleus pulposus on the cartilage endplate, leaving the hyaline cartilage endplate, was confirmed by hematoxylin-eosin staining (Huang B, Liu LT, Li CQ, et al. Study to determine the presence of progenitor cells in the degenerated human cartilage endplates. Eur SpineJ, 2012, 21:613-622). The primary cells of cartilage endp...

Embodiment 2

[0033] Example 2 Construction of a lentiviral vector encoding the SV40T antigen and packaging of viral particles

[0034] Construction of a lentiviral vector encoding the SV40T antigen: The lentiviral vector plasmid pWPT-GFP (Shanghai Jikai Gene Chemical Technology Co., Ltd.) was double-digested with BamHI and SalⅠ enzymes from NEB Company to release GFP. The enzyme digestion conditions were 50 μl reaction system Add 1 μl of BamHI and SalⅠ, respectively, and incubate at 37°C for 30min. Finally, the digested product was subjected to 1% low-melting point agarose gel electrophoresis, and the carrier pWPT fragment was recovered with a gel recovery kit from Qiagen, Germany. The plasmid Plox-Ttag-iresTK (Shanghai Jikai Gene Chemical Technology Co., Ltd.) and the plasmid Plox-SV40-iresTK (Shanghai Jikai Gene Chemical Technology Co., Ltd.) were double-digested with SalⅠ and EcoRI, and the digestion conditions were 37°C for 30min , 65°C for 20min to inactivate the enzyme; then smooth ...

Embodiment 3

[0036] Example 3 Infection of Human Cartilage Endplate Stem Cells by Lentiviral Particles

[0037] The CESCs cells passed to the third generation were planted in T25 cell culture flasks, and 2ml of lentiviral particles containing 4μg / ml polybrene were added, and the transfected cartilage endplate stem cells were as follows: image 3 shown. After 2 days of infection, apply 0.4mg / ml hygromycin B to select for 14 days to form cell colonies after selection, such as Figure 4 shown. The screened stem cell colonies were expanded and passed continuously for more than 30 passages to obtain the immortalized human degenerative cartilage endplate stem cell line iCESCs, which were cryopreserved for future use.

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Abstract

The invention belongs to the technical field of cell engineering, and particularly relates to a preparation method of an immortalized human cartilage endplate stem cell line and use of the immortalized human cartilage endplate stem cell line. The invention aims to solve the technical problem of providing an effective preparation method for constructing the immortalized human cartilage endplate stem cell line. The technical scheme is as follows: the preparation method for constructing the immortalized human cartilage endplate stem cell line by adopting the lentiviral transfection technology comprises the following steps: a, constructing a SV40T antigen virus vector; b, carrying out gene transfection of a human endplate stem cell on SV40T antigen; and c, passaging and sieving to obtain the immortalized human cartilage endplate stem cell line. The invention further provides the use of the immortalized human cartilage endplate stem cell line prepared by the method in preparation of bone tissue engineering materials. The immortalized human cartilage endplate stem cell line provided by the invention can be applied to preparation of a tissue-engineered bone and clinically provides a seed cell of the bone tissue engineering low in price and strong in osteogenic capability for the patients with a series of long bone defects and bone nonunion.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a preparation method and application of an immortalized human cartilage endplate stem cell line. Background technique [0002] Clinically, open fractures, nonunion, and bone defects are often caused by certain reasons such as trauma, infection, and tumors. Solving long bone defects and nonunions requires a large number of bone graft materials with good biological and biomechanical properties. For this reason, many scholars have carried out relevant research and achieved some achievements, but the long bone defect is still a difficult problem in the movement system disease. With the rise of tissue engineering technology in recent years, the research on bone defect repair has entered the era of tissue engineering. The current tissue engineering theory believes that seed cells, scaffold materials, and growth factors are the three elements of tissue engineering...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867A61L27/38C12R1/91
Inventor 黄博刘铭汉王海刘兰涛周跃
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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