Difference between revisions of "Team:Tongji Shanghai/Cell Experiments"

Overview

Photothermal therapy refers to use radiation(especially infrared wavelengths) for the treatment in many conditions, particularly in cancer.At temperatures greater than 42℃, protein denaturation and disruption of the cellular membrane is known to occur.As many cases have shown that there is an acceleration in both perfusion and reoxygenation of cancer cells while normal cells can bear it for a longer time.Despite of its non-invasive feature, the strategies are less selective and may cause damage to surrounding health tissues.By infecting plasmids hsp-P53 and hTert-P53, we are able to improve the therapy to more selective.

hcc1937 and 293T cells were harvested and were seeded onto a 96-well tissue culture plate at a density of 2X104 cells per well and were allowed to grow for 24h at 37 ℃, CO2 5%. All the equipment were sterilized by 75% ethanol and UV exposure for 30 minutes. Then the cells were incubates with GNRs(100μg/ml in terms of GNR) in the culture medium for 24h to allow cellular uptake. After incubation, the cells were exposed to NIR radiation(808nm diode laser, Power Technologies, 2.56~~~W/cm2) was irradiated on each well for 20 min. The cell viability was characterized by using cellTiter-Glo. Briefly, the cell were washed with PBS and 100 μl cellTiter-Glo was added to each well and was incubated in the dark at 37℃, CO2 5% for 20 min.

in vitro cytotoxicity test

To assess material toxicity, hcc1937 cells were exposed in decuplicate to a gradient of PEG-NRs ranging from 1 to 500 μg/ml(based on GNR amount).Then cells were further incubated for 12h at 37℃,and the cell viability was characterized by using cellTiter-Glo as mentioned.

in vitro photo thermal effect

To examine whether the laser works on the AuNRs to kill breast cancer cells, the cells which have been incubated in the AuNRs bearing medium were divided into two groups: group A (NIR exposure) and group B (lucifuge).

To compare the improvement of photo thermal after transfected with plasmids hsp70-P53 or hTert-P53, the cell were also divided into three groups: group C,D transfected by plasmids hsp70-p53 or hTert-p53 and group E transfected with control plasmids.

Result:

in vitro cytotoxicity test

The results shows that AuNRs do not affect the metabolic activities of the cells up to 500μl/mg..

And they are stably dispersed in serum-containing medium without aggregation. Therefore, the AuNRs used in this study do not cause any acute cytotoxic effect or aggregation during ciculaiton in in vivo applications.

in vitro photo thermal effect

The results shows that without NIR, the plasmids hsp-p53 and hTert-p53 is relatively harmless. But after exposed under NIR, the cellular viability reduces sharply. This experiment suggests that the plasmids actually improve the efficiency of killing cancer cells.