Application of OATP1B1 in preparation of magnetic resonance for in vivo tracking and/or monitoring transplanted cells
A kind of magnetic resonance imaging, cell technology, applied in the field of biomedicine
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Embodiment 1
[0033] 1. Cell culture and production of Dox-induced stable cell lines
[0034] The doxycycline (Dox)-inducible system is an effective avenue for functional studies by modulating gene expression to avoid long-term off-target effects of transgene products. Therefore, for the first time, we demonstrated the expression, localization and functional validation of the transporter OATP1B1 in HEK293T cells under the Dox-induced TRE3G promoter. The C-terminus of each transgene was 3X labeled and expressed in frame with mVenus fluorescent protein (separated from the self-cleaving p2A peptide) ( figure 1 ). Stable cell lines with the OATP1B1 transporter were established by transduction followed by puromycin selection ( figure 2 ). The specific method is as follows:
[0035] Human embryonic kidney cells (HEK 293T), mouse Trippel-negative breast cancer cells (4T1) were obtained from commercial suppliers (US-type culture specimens), and cultured at 37°C and 5% CO 2 Under normal condit...
Embodiment 2
[0048] in vitro MRI
[0049] HEK-293T or 4T1 of OATP1B1 and relative Mock control cells were seeded in 6-well plates, 0.8x10 per well 6 cells were seeded in complete medium containing 0.5 μg / mL Dox. 24 h after inoculation of stable cell lines, the medium was aspirated and calibrated for 30 min with HBSS buffer containing 12 mM HEPES, pH 7.4. Cells with indicated concentrations of Gd-EOB-DTPA (Bayer) or Gd-DTPA (Magnevist) in HBSS buffer were incubated at 37 °C and 5% CO 2 Under treatment for 90 minutes. Uptake was stopped by adding ice-cold HBSS buffer, followed by excess washing and trypsinization. Cells were centrifuged at 5000 RPM for 5 minutes in a 0.2 mL PCR tube. Carefully remove the supernatant and seal the cell pellet with 180 μl of 1% agarose gel. Finally, line up the tubes in a PCR tube rack and place them in a plastic container with water. MRI was performed on a 3-Tesla clinical MR scanner (UMR790, Shanghai United Imaging Medical Co., Ltd.) using a commercial ...
Embodiment 3
[0051] To assess the in vivo cellular dynamics of transplanted cells, we further introduced the reporter into the highly metastatic breast cancer mouse 4T1 cell line. The induction and function of 4T1-OATP1B1 were confirmed in a similar manner.
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