Developed Single Strand RNA Amplification System In Vitro And Study On The Interaction Between BTV-10 And Different Cells

In order to establish a high-efficient single strand RNA (ssRNA) amplification system with which ssRNA can be amplified sensitively and specifically even for the direct experimental uses, the serum from chronic Hepatitis C patients was used as specimens. Based on the nucleic acid sequence-based amplification (NASBA) technique, single step reverse transcription (SRT-PCR) was performed. As a result, by ultraviolet spectrophotometry, comparing with the nested-PCR, the quantity of the final product was 20-fold higher. Also, the specificity was verified by the electrophoresis on agarose gel and the dot hybridization with biotin-labeled probe.Following, the natural distinct susceptibility of BTV-10 to 4 cell lines was investigated. The cell lines included Human hepatic carcinoma (Hep-3B) ; Human lung carcinoma (A549); Normal mouse f ibroblasts (NIH 3T3) ; Human embryonic lung fibroblast (HEL). Comparisons between the cell lines were made on the basis of time to observed cytopathic effects (CPE), ultra structure changes, titer in 50% tissue culture infectious doses, and the alterant distribution of DNA content in cell cycle. The results showed that, under identical conditions, various degrees of CPE could be found in the Hep-3B, A549 and NIH 3T3 36h post infection of BTV-10, while there was no CPE could be observed in HEL. Ultra structure under electron microscope revealed that, inside the susceptive cells, BTV-10 could proliferate efficiently and induce apoptosis. Combined the analysis of cellular surviving rate 36h post infection by MTT method and detection of DNA content by flow cytometry, BTV-10 has the most susceptibility to Hep-3B and A549 among the 4 cell lines,what's more, it did not affect the normal cell life cycle. The A549 cell line produced the highest BTV TCID50 of all cell lines tested. From these experiments, we can conclude that BTV-10 did not infect HEL cells, while it can selectively kill the other three cell lines, especially, the tumor cells. It tend to kill A549 by rapid proliferation in the cytoplasm, whereas tend to kill Hep-3B by inducing apoptosis. This differentiation in killing mode maybe relative to the espcial genetic background of the different tumor cells. For a example, the Ras signal transduction pathways inside A549 is activated, while it is not in the Hep-3B.Discovery of the unique susceptibility of BTV-10 to the tumor cells gave us the theoretic bases to use it as an oncolytic agent; Concurrently, establishment of the high-efficient ssRNA amplification system could provide us with the sufficient sourse of specific ssRNA or dsRNA for the direct experimental uses even from the serum. Based on which, we will have the chance to advance the study of BTV's targeting mechanism on molecular level, and realize the virion's genetic modification for the safer application in human body in the future.