Artificial Cultivation Of Ophiocordyceps Sinensis Phorozoon And Its Effect On HepG2Cell Apoptosis

Ophiocordyceps sinensis is very precious and its resources are scarce.The phorozoon of ophiocordyceps sinensis and its artificial cultivation were tesed in this study to develop cordyceps substitute. The biological characteristics of host insect includes worm-shaped sclerotium and weed-like stroma of ophiocordyceps sinensis was mastered through field investigation. The whole metamorphosis cycles of host insect of ophiocordyceps sinensis were four morpha included larva, pupa, adult and egg; larvae perch in soil perennially; it lived alone with habit of biting each other after3instar; it was phytophagous with wide recipe and sensitive to temperature; each age’s insect was sensitive to Hirsutella sinensis, plant diseases and insect pests were serious. Artificial simulated experiment confirmed that larvae overwintered in a dormant state with its body shrinked, and its body became yellow. Hypha in body of bombys batryticatus went through in clumps via the weak point of hair follicle, and formed membrane by packing surrounding soil detected by pathological section and microscope; some bombys batryticatus have no membrane due to the skin in hair follies became more solid and thick so that hyphae couldn’t get through.We found the penetration pegs in the early time of colony through the monospore culture of ascospore.Tianzhu ophiocordyceps sinensis in Gansu had lower altitude and smaller size, the complete sequence of isolate rDNA ITS and5.8S RNA was analyzed by the methods of PCR and DNA sequencing, the result showed that the isolates had similar characteristics with Hirsutella sinensis over98%.Ophiocordyceps sinensis grew very slow,.However,the current study indicated that breaking hypha artificially increased growth point and shallow cultivation could vastly improve the production of agamotype hypha. Furthermore, industrialization result confirmed the conclusion.Crystal violet staining assay was used to observe the cell viability, which indicated that HepG2cells could be inhibited obviously by ophiocordyceps sinensis hyphae solution with a dose-dependent manner. Then apoptosis of HepG2cell were assessed by Annexin V-FITC/PI staining and flow cytometry analysis, the results showed that HepG2cells could be induced to apoptosis by ophiocordyceps sinensis hyphae solution, also with a dose-dependent manner. Further conformation detected by fluorescent chemosensors was that ophiocordyceps sinensis hyphae solution could induce HepG2cell to produce ROS, meanwhile ROS inhibitor NAC could significantly prevent the cell from apoptosis mediated by ophiocordyceps sinensis, and subsequently, western-blot was used to analyse the expression of p-JNK. and Bcl-2proteins. With ascending concentrations,, the expression of p-JNK increased, while Bcl-2decreased. It revealed that the pro-apoptosis of ROS may be due to the activation of JNK pathway and down-regulation of Bcl-2expression, and their correlation needed further research.According to the result of artificial cultivation contrast test, agar can replace the soil to cultivate the host larva of ophiocordyceps sinensis and it had an advantage over the soil.The result of the host larva of ophiocordyceps sinensis infection test showed that the infection form of ophiocordyceps sinensis included ascospore and conidium. The routes of infection of ascospore and conidium were trauma and digestive tract,respectively.