Construction method of stable cell line realizing FTH1 gene controlled expression

Abstract

The invention provides a construction method of a stable cell line realizing FTH1 gene controlled expression. The construction method comprises the following steps: (1) acquiring FTH1 genes; (2) transferring the FTH1 genes into a Tet-On vector to obtain recombinant plasmids; (3) constructing the recombinant plasmids into a lentiviral vector; (4) inoculating human neuroblastoma cells, after the cell fusion reaches 40-50%, adding a safe medium containing lentivirus liquid 0.8 [mu]l / ml (the titer is 1X10<9>TU / ml) and polybrene 5 [mu]g / ml, and polyethylene glycol 0.5-1 [mu]g / ml, and placing the safe medium into an incubator for culturing for 20-24 h; and (5) after cell passage, and the cell fusion reaches 50-60%, adding a medium containing 8 [mu]g / ml puromycin and treating for 7d, then adding a medium containing 4 [mu]g / ml puromycin and screening for 3d, and thus obtaining the stable cell line capable of inducing expression of the FTH1 genes, and the stable cell line is named SK-N-SH / Tet-on / FTH1. With the adoption of the construction method, the expression level and the expression time of FTH1 are accurately regulated, so that FTH1 is expressed when MR imaging is needed, and is turned off when MR imaging is not needed.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to the construction of cell strains. Background technique [0002] Clinically, magnetic resonance imaging (MRI) is widely used in the diagnosis, staging, follow-up and prognosis of diseases; in the field of molecular imaging, MRI has great applications because of its high spatial resolution, good soft tissue contrast and no radiation damage. Potential, especially for studies on transplanted cell tracking. MRI tracks transplanted cells mainly through magnetic labeling imaging and reporter gene imaging. The former uses MRI contrast agents such as metal chelates or superparamagnetic iron oxide particles (SPIO) to directly label cells. Due to the high transverse relaxation rate of SPIO, it is widely used in cell tracking imaging. However, this method of using MRI contrast agent to directly label cells has a big defect, that is, the contrast agent will gradually degrade with the divi...

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Problems solved by technology

However, it has also been reported that in some specific cells such as cervical cancer Hela cells and nasopharyngeal carcinoma cells, the overexpression of FTH1 can significantly reduce the proliferation activity of cells.

In addition, the long-term and continuous expression of the reporter gene using iron as the imaging medium can also induce iron metabolism disorders

Although iron is an important cofactor for many bioactive enzymes in metabolism, too much iron will induce a large number of active oxygen free radicals, resulting in cell damage or apoptosis

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