The Role of RNASE L in Type I Diabetes and Development of Quantitative LC-MS/MS Methods for the Pharmacological Studies of Anti-Cancer Drug Candidates
Date of Award
Doctor of Philosophy in Clinical-Bioanalytical Chemistry
PROJECT I: The cause of type I diabetes continues to be a focus of investigation. In this study, we found that 2-5A dependent RNase L (RNase L), an IFN-a-inducible enzyme that functions in IFN action against viruses and cell proliferation, played an important role in dsRNA-induced onset of type I diabetes. By using RNase L deficient RIP-B7.1 mice which are more vulnerable to environmental harmful factors such as viral infection, we demonstrated that deficiency of RNase L in mice resulted in a significant delay of diabetes onset induced by polyinosinic:polycytidylic acid (poly I:C), a type of dsRNA, and streptozotocin (STZ). Immunohistostaining showed that the population of infiltrated CD8+ T-cells was remarkably reduced in the islets of RNase L deficient mice, implicating RNase L may contribute to type I diabetes onset through regulating immune responses. Furthermore, RNase L was responsible for the expression of certain proinflammatory genes in the pancreas under the special condition.
PROJECT II: It has been found that 6-hydroximino-4-aza-A-homo-cholest-3-one (HyM3) is a steroidal derivative with a significant anticancer effect. To develop a method for analyzing its pharmacological kinetics, HyM3 and the internal standard (3E)-Hydroximinocholest-6-one were extracted from mouse plasma samples through a protein precipitation procedure by mixing with acetonitrile (1:4 v/v) and then centrifuging at 12,400 g for 15 minutes. The supernatant (10 µl) was injected onto the LC/MS system consisting of a Shimadzu Prominence HPLC and an AB Sciex QTrap 5500 with positive electrospray ionization. A MRM mode was chosen for sensitive and specific detection of the analyte and internal standard. The HPLC separation employed a Phenomenex Kinetex C8 column (50x2.1 mm, 2.6µ) with a gradient mobile phase of 0.2% formic acid in water and 0.2% formic acid in acetonitrile at a rate of 0.2 ml/min. Full mass spectrometric scans of HyM3 and the internal standard showed protonated molecular ions of 431 and 416 m/z, and fragmentation of these two ions revealed predominant product ions of 370 m/z and 344 m/z respectively. HyM3 was retained on the HPLC column for 6.4 minutes, while the internal standard was retained for 7.3 minutes. The method was calibrated for a concentration range from 0.500 to 200 ng of Hym3 per mL of plasma.
Zeng, Chun, "The Role of RNASE L in Type I Diabetes and Development of Quantitative LC-MS/MS Methods for the Pharmacological Studies of Anti-Cancer Drug Candidates" (2016). ETD Archive. 934.