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Immortalized telocytes system and construction method thereof

A construction method, immortalization technology, applied in the field of cell engineering

Active Publication Date: 2017-08-11
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is no report on the construction method of the immortalized special cell line of the present invention.

Method used

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  • Immortalized telocytes system and construction method thereof
  • Immortalized telocytes system and construction method thereof
  • Immortalized telocytes system and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of immortalized special cell line of the present invention

[0037] (1) The staged adherence method was adopted when extracting the primary special cells: the lungs of C57 mice were separated and cut into 1m in normal saline under sterile conditions. 3 The tissue pieces were digested in 2mg / ml type II collagenase at 37°C for 30 minutes, filtered through a 70-micron pore size filter, centrifuged at 1500rm for 5 minutes, and the cell pellet was collected and cultured in DMEM / F12 medium containing 10% FBS After 30 minutes, after the fibroblasts adhere to the wall, transfer the supernatant to another culture dish, change the medium after culturing for 12 hours, and observe the special cells under a microscope after continuing to culture for 3-5 days.

[0038](2) Multiple purifications to ensure purity: use a micropipette tip as a cell scraper, and use the characteristic structure of the cell as a standard to scrape off other cells under a microscope. ...

Embodiment 2

[0041] Example 2 Construction of SV40 stably transfected cell lines

[0042] Phase 1: Construction of overexpression lentiviral vector

[0043] First design synthetic primers, amplify the target fragment, and then connect it into the overexpressed lentiviral vector after digestion through the enzyme cleavage sites at both ends; transfer the ligated product into the prepared bacterial competent cells, for The grown monoclonal colonies are first identified by PCR, and the positive colonies identified by PCR are identified by sequencing. The correct clone is the successful construction of the target gene overexpression lentiviral vector.

[0044] 1. Experimental materials and methods

[0045] (1) Materials

[0046] 1. Reagents

[0047] Reagent name Reagent source PCR reagent primer(R&F) Sangon Bioengineering (Shanghai) Co., Ltd. Taq polymerase NEB QIAGEN Plasmid Pumping Kit QIAGEN BSA Sigma LB or SOB or SOC Alphaaeser CaCL2 ...

Embodiment 3

[0142] Embodiment 3 The usage method of the present invention

[0143] 1. Aseptic operation. An appropriate amount of penicillin and streptomycin can be added to the culture medium used for operation. Before the experiment, the aseptic room and the aseptic operating table were sterilized by ultraviolet light irradiation for 30-60 minutes, the aseptic operation surface was wiped with 70% ethanol, and the experimental operation was started after the aseptic operating table fan was turned on for 10 minutes . Only one cell line is processed for each operation, and even if the medium is the same, the same bottle of medium should not be used to avoid confusion or contamination between cells. After the experiment is completed, the experimental items are taken out of the workbench, and the aseptic operation surface is wiped with 70% ethanol. The staff should pay attention to their own safety and wear lab coats and gloves during the experiment.

[0144] 2. The cell line is cultured...

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Abstract

The invention relates to a construction method of an immortalized telocytes system. The construction method comprises the following steps: (1) digesting shorn off mouse lung tissue block in type-II collagenase, carrying out filtering and centrifuging, collecting cyte precipitate, culturing the cyte precipitate in a culture medium, after fibrocytes are attached to a wall, transferring supernate to another culture dish, culturing for 12 hours, then changing liquid, and continuing culturing for 3-5 days; (2) taking a trace spearhead as a cyte scraper to scrape off the fibrocytes, and after scraping off the fibrocytes repeatedly, observing a specificity structure telopode and identifying telocytes by an immune marker; and (3) taking the telocytes as host cells, infecting the host cells by using a retroviral vector which contains SV-40-large tumor antigen, and carrying out passage, screening and identifying. Even if the immortalized telocytes system is cultured to the 50th generation, the state is still stable, and therefore, the problem that the purity and the stability of the telocytes cannot be guaranteed by repeated separation and extraction in the prior art.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for constructing an immortalized specialized cell line. Background technique [0002] The current research on the function of the special cells is to use the special cells extracted from different organs (lung, kidney, heart, etc.) of different species of animals (such as mice, rats, pigs, etc.), the extraction process is time-consuming and expensive every time Extraction requires separation, purification and identification, which requires a lot of manpower and material resources. Since multiple separations and extractions of primary cells are performed by different personnel, it is difficult to guarantee the purity and stability of the extracted cells. Under normal circumstances, when the 100% purified primary T cells are cultured at low density, they all die within a week. [0003] The paper "Isolation of new type of mesenchymal cells-Teroid cells from rat k...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12R1/91
CPCC07K14/005C12N5/0688C12N15/86C12N2510/04C12N2710/22022C12N2740/15043
Inventor 王向东宋东莉
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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