A sejten kívüli szabad DNS felszabadulásának és degradációjának in vivo elemzése = n vivo analysis of circulating cell-free DNA release and degradation

Absztrakt: Bevezetes: A sejten kivuli szabad DNS-t mar az 1940-es evekben kimutattak. Eredeteről tobb elmelet is letezik: lehetseges folyamat a tumoros sejtekből, valamint ezzel parhuzamosan az egeszseges sejtekből tortenő felszabadulas is. Celkitűzes: Munkank celja a szabad DNS felszabadulasi utemenek vizsgalata volt SHO-eger/HT-29 human colorectalis adenocarcinoma sejtvonal xenograftmodellben, valamint celul tűztuk ki egeszseges es C38 tumorral oltott C57BL/6-os egerek veraramaba juttatott mestersegesen folszaporitott metilalt es nem metilalt DNS-szakaszok lebomlasanak nyomon koveteset. Modszer: SHO-egerekre HT-29 sejteket oltottunk subcutan, majd vert vettunk 8 heten keresztul. A plazma szeparalasa utan DNS-t izolaltunk, majd mitokondrialis es genomialis RT-PCR-probakkal megallapitottuk a human/eger DNS-aranyt. A szabad DNS lebomlasanak vizsgalatahoz egeszseges es C38 tumorsejttel oltott C57BL/6-os allatok verebe 3000 bazispar (bp) meretű in vitro metilalt es nem metilalt DNS-fragmentumot juttattunk. Az amplikonok degradaciojat 19 valos idejű PCR-probaval mertuk, a bomlas utemere a relativ amplikonkoncentraciok alapjan kovetkeztettunk. Eredmenyek: A tumorbol szarmazo human DNS mennyisege a 2. hetig a kimutathatosagi hatar alatt volt, majd a 3. hettől folyamatos emelkedest tapasztaltunk, amely a 8. hetre 18,26%-ot ert el. A veraramba juttatott DNS-szakaszok lebomlasanak sebessegeben kulonbseget mutattunk ki a nem metilalt es a metilalt fragmentumok kozott. Az egeszseges allatokban a nem metilalt DNS 6 ora utan eltűnt a verplazmabol, mig a metilalt fragmentum szakaszai 24 ora mulva is kimutathatok voltak. Tumoros allatokban a degradacio merteke lelassult, es mindket forma kimutathatova valt 24 ora elteltevel. Kovetkeztetes: A szabad DNS szerepenek es hatasmechanizmusanak vizsgalatat egyre nagyobb erdeklődes ovezi. Munkank segitseget nyujthat a DNS felszabadulasanak es degradaciojanak pontosabb megismeresehez. Orv Hetil. 2018; 159(6): 223–233. | Abstract: Introduction: Cell-free DNA (cfDNA) was first detected in human plasma in the 1940s, but the knowledge on its regulation and rate of release is incomplete. CfDNA can originate from both normal and tumour cells. Aim: Our aims were to investigate the rate of cfDNA’s release in SHO mice/HT-29 colorectal adenocarcinoma cell line xenograft model and to define the decay of methylated and non-methylated DNA fragments in C57BL/6 bloodstream. Method: SHO mice were xenografted with human HT-29 cells, than blood samples were collected over 2 months. CfDNA was isolated, then quantified by real-time PCR with highly specific genomic and mitochondrial human and mouse primer sets. This method permitted to define the ratio of human/mouse DNA. To assess the degradation rate of cfDNA, 3000 bp sized methylated and non-methylated DNA fragments were injected into healthy and C38 tumour-cell vaccinated C57BL/6 mice’s bloodstream. The decay of amplicons was measured with 19 PCR assays. Results: The amount of human DNA until the 2nd week was below the limit of detection. From the third week, a continuous growth was experienced, which reached 18.26% by the 8th week. Moreover, it was found that in healthy animals the non-methylated DNA disappears from the plasma after 6 hours, while the methylated fragment was detectable even after 24 hours. In animals with tumour, both amplicons were detectable after 24 hours. Conclusion: The examination of the role and mechanism of cfDNA shows an increasing level of interest. This work can contribute to a better understanding of the release and degradation of cfDNA. Orv Hetil. 2018; 159(6): 223–233.