Analysis The Transcriptional Profiling Of Echinococcus Granulosus In Artemisinin Intervention

Objective:Using gene chip technique to research the effect of artemisinin （ART） intervention to Echinococcus granulosus protoscolex（EgPSC） survival gene regulatory networks, and speculate the molecular mechanism of ART in anti-echinococcosis. Methods:The protoscolex of Echinococcus granulosus were obtained from sheep liver cysts of an abattoir, after a short culturing in vitro, choose a cycle of10days for the intervention, set200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL as ART interfering concentration to determine ART concentration and action time of subsequent experiments. To screened ART and the positive control drug albendazole（ABZ） intervention to EgPSC, observed the ultrastructural changes using transmission electron microscope（TEM）. Extracted total RNA of EgPSC and reversed into cDNA, and detected differential gene expression of EgPSC after the intervention of ART, ABZ and DMSO in vitro using Echinococcus granulosus genomic microarray gene chip, on-line retrieved MAS3.0（Molecular Annotation System3.0, Beijing Boao company） database, functionally annotated of genes with differences, and classified genes in accordance with the gene function and pathway. The microarray data validation was confirmed by quantitative real-time reverse transcription PCR（qRT-PCR）. Results:The results showed that using ART to intervene EgPSC activity was better than ABZ in vitro, TEM observation indicated that ART mainly caused nuclear burst of protoscolex, nucleolus disappear, nuclear material color becomes deeper, heterochromatin margination phenomenon. Gene chip results of ART group showed changes of106genes in expression, of which100up-regulated and6down-regulated. Positive control group of ABZ, a total of42genes change in expression, of which7up-regulated,35down-regulated.1%DMSO group had a total of4changes in gene expression, all of them were up-regulated. In this study, there were9genes expression with common differences, of which there were8genes expression with common differences in ART and ABZ group. After ART intervention protoscolex in vitro, among16genes expressed differentially with the pathway annotation in KEGG,3genes are related with metabolism.10genes are related to the genetic information and process, of which4genes are related to DNA repair. Differences in gene function classification results displayed in the ART106differential genes, function of71genes were unknown,35genes were mainly related to cytoskeleton, cell cycle, metabolism, translation, transcription, signal transduction, apoptosis, oxidation, reduction. ABZ group had45differential genes, among which27genes were unknown,18genes remaining mainly related to cytoskeleton, metabolism, redox, cell cycle, biosynthesis, translation and transport. The differential gene annotation of ABZ group matched the mechanisms of ABZ in the known literature. qRT-PCR analysis indicated that among17differential genes,14genes matched screening results by gene chip, however results of384P10_M17,384P09_J05,384P17_O09gene by qRT-PCR did not match results of gene chip. Conclusion:ART own anti EgPSC fountion in vitro, we found it has a dependent relationship with dose and time. ART killed EgPSC by multitarget functions in vitro, the changes of gene expression involves multiple pathways, the main mechanism may exist for the influence of genetic information and metabolic process, in which DNA damage is one of the main mechanisms. The qRT-PCR results indicate that Echinococcus granulosus genomic microarray gene chip is credible in this research. This research helps to lay the foundation of clarification the mechanism of ART in anti Echinococcus granulosus.