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Home » Archive » 2008

TDK conference 2008

Functional examination of therapy resistance proteins by flowcytometric method
Szendi Eszter - year 5
Department of Internal Medicine
Supervisor: Vajdovich Péter

Abstract:

Principles of flow cytometry is the illumination of cells by laser light, which are flowing in an ultrathin tube. These cells can be separated virtually according to their light scattering by computerised technique. Thus the similar cells can be examined separately from cells with other character. Such an instrument is suitable for routine haematology examinations, but can be used for other purposes. If the cells are signed with fluorescent molecules by immunological methods, the cell will change its light scattering, so it can be examined separately. This called as FACS (fluorescence activated cell sorting). This method can be used to detect the membrane transporter function of the cells. If we add fluorescent molecule (calcein) to the cells, and incubate them, then this molecule will penetrate into the cells. If the cells have membrane transporter proteins (P-glycoprotein /Pgp/, multidrug resistance associated protein-1 /mrp1/, etc.) and multidrug resistance function, than the cells will efflux calcein.

In our study we were interested in whether the membrane transporter function of separated lymphoid cells of 9 dogs with lymphoma receiving chemotherapy is different from the same function of lymphoid cells of control (n=6) dogs.

Lymphoid cells from blood samples of dogs were separated into three parts. One was incubated with calcein without inhibitors. Second part was inhibited by verapamil, which inhibits both, Pgp and mrp1. The third part was inhibited by MK-571 molecule, which inhibits mrp1, only. Both inhibited cell solutions were then incubated with calcein. We analysed the intracellular fluorescence of the cells by FACS method. In order to express the fluorescent activity of the inhibited and non inhibited cells we calculated the MAF- (multidrug resistance activity factor-) values. These will characterize the Pgp and mrp1 function of the cells. The summarized multidrug resistance values of the control dogs: 31.6 ± 5.9, the MAF-value of mrp1: 18.5 ± 9.7, the MAF-value of Pgp: 13.2 ± 5.3. The summarized multidrug resistance values of the dogs with lymphoma and leukemia: 45.9 ± 8.9, the MAF-value of mrp1: 29.9 ± 11.5, the MAF-value of Pgp: 15.9 ± 14.2. Our examinations are proving that both the summarized „multidrug” resistance function of the patients and the efflux pump function of mrp1 respectively (p<0,01, p<0,05) increased significantly compared to the control dogs.



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