In Vitro Experimental Study On The Natural History Of Hepatic Cystic Echinococcosis

Objective:In this study,we used the Echinococcus granulosus protoscoleces as the experimental material long-term in vitro culture to establish a more stable in vitro culture system culturing protoscoleces to producing hydatid cysts of Echinococcus granulosus.To observe the effects of bile and Escherichia coli on the growth and development of Echinococcus granulosus protoscoleces and hydatid cyst,and further explore the effect of biliary fistula and infection factors on the natural history of hepatic cystic echinococcosis.Methods:（1）The protoscolex of Echinococcus granulosus was aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Changji.After 1%（w/v）pepsin digestion to remove immature protoscoleces,the activity and number of protoscoleces were tested and then cultured in the single-phase system in a CO₂ incubator at 37 degree centigrade,and collected adequate amount of protoscoleces at different time points randomly and then observed using an inverted microscope and took photos and recorded the morphological changes and their volume growth situation,summarize the regularity of the growth and development of the protoscolex cultured in vitro.（2）Low concentration of bile and Escherichia coli were respectively applied to the in vitro cultured Echinococcus granulosus protoscoleces and its cysts.The morphological changes of the protoscoleces and its cysts with the extension of the action time were observed under the inverted microscope;and the microstructure changes of the protoscoleces and its cysts were observed under electron microscope.The histopathological changes were observed by HE staining.Results:（1）During the first 6 weeks of culture in vitro,the morphological features of protoscoleces were forming the vesicle at pedicle end.When cultivated for 7 days in vitro,they started to form the vesicle at pedicle end and the number is gradually increasing with the extension of the cultivation time.When cultivated for 6 weeks in vitro,they started to develop to cysts and the volume of protoscoleces gradually increased with the extension of culture time.The sizes of largest cyst could reach 1.2 mm.At the 17th week of in vitro culture,the cyst gradually went to aging and died,the stratum corneum was roughened,and the volume of the cyst was reduced,its internal structure became blurred and concentrated into clusters,and there was a clear boundary with the outer cuticle.After 7 months of culture,more than 90%of the cysts showed the above morphological changes.The rate of encystations was 11%.（2）After the low concentration of bile acts on the protoscoleces,the bile does not directly kill the protoscoleces in a short time.At this time,the activity of the protoscoleces is increased and evaginate.With the extension of culture time,a layer of cuticle was gradually formed around it,but its growth and development rate was significantly lower than that of the control group in the same period.With the extension of the action time,the growth of protoscoleces gradually stagnated;the internal structure was blurred,and gradually died.After E.coli acts on the protoscoleces,almost all of the protoscoleces lose their normal morphology,when they act for 48 hours,and the head hooks are scattered and the volume is reduced.Eosin staining also confirmed that all of the protoscoleces have lost their activity and rapid death.The morphological structure of the vesicles of the two groups was significantly changed compared with the control group in the experiment of bile and Escherichia coli acting on cysts.Most of the vesicles showed different degrees of shrinkage,the peripheral stratum corneum was severely damaged,the internal structure was pyknotic,and the stratum corneum and the germinal layer were separated.The results of transmission electron microscopy and HE staining also confirmed that the membrane structure of the vesicles had been significantly damaged.Conclusion:1 A stable in vitro culture system of Echinococcus granulosus from protoscoleces to cyst was successfully established,the volume of Echinococcus cyst cultured in vitro grows from small to large and then gradually shrinks with the prolongation of culture time and the development of the cyst has an important role in its long-term survival.2 Bile has an inhibitory effect on the growth and development of the protoscolex.Escherichia coli can cause the death of the protoscoleces and its vesicles rapidly.And both of them can damage the integrity of the cysts membrane structure and promote the aging and death of the hydatids.3 Biliary fistula and bacterial infection are important factors in the natural history of hepatic cystic echinococcosis,which promote the death of hydatid cysts.