| Malaria is one of the most severely epidemic infectious diseases,which is transmitted by anopheles mosquito vectors,with high incidence and fatality rate in human being.The disease threatens more than 100 countries,and there are about 20 hundred million people, which occupied approximately 40%of the world's population are at risk.The disease seriously holds back the economy and society developing of these regions,especially regions in tropical and subtropical zone.After long time exploration of scientists all over the world,there are still about 3～5 billion new cases annually and mortality of approximately 1～3 million,most of which are African children under the age of five.In the south of China,especially in the mountain area with specified environment,there still have a possibility to break out epidemic malaria in wide-bound.Control of malaria will be confronted with great challenge of the rapid spread of multi-drug resistant parasites,the emergence of insecticide-resistant anopheles mosquitoes and the lack of effective malaria vaccine fighting against malaria.Therefore,safely and effective means for malaria control are urgently needed.Malaria is transmitted through the bite of infected mosquitoes that introduces sporozoites into host.Sporozoites travel to the liver where they invade hepatocytes, undergo multiple rounds of nuclear division,sporozoites finally differentiate into schizonts containing thousands of merozoites.Then the subsequent release of merozoites into the circulation initiates blood infection.We presumed that inhibiting the development of liver stage plasmodium would control the symptoms of the malaria.Once the vaccine target to liver stage plasmodium manufactured,it could prevent not only the pathological damage of liver tissue,but also the gametophyte from releasing into blood circulation.However, relatively little information is available concerning the unicellular malaria parasites during liver stage,mostly because of low-level infection of host hepatocytes and lack of purification techniques for the liver stage parasites.With the developing of biochemistry and molecular biology,people began to pay more and more attention to the study of liver stage plasmodium.The antigen disparity and variation are very widespread in plasmodium,of which antigen structure is extremely complex,and the quantity of epitopes is very huge.Most of the epitopes have the characters of phase specificity in plasmodium life cycle.Besides weak antigenicity,plasmodium presents species and strain specificity.The infection of plasmodium will induce multiple immune responses in host body.In these immune responses,T cell immunity has significant position.Therefore,we can conclude that the study of costimulatory signal,which induce T cell activation in liver stage,is the base theory of malaria therapy and vaccine manufacture.For the study on the development of plasmodium in liver stage and host immunological responses,we infected rat via vena caudalis injecting with the Plasmodium yoelii sporozoites to establish mammal model,which included integrity life cycle of the plasmodium.On the one hand,we used DD-PCR(differential display PCR,DD-PCR) protocol to select liver stage cDNA of plasmodium.After comparing and analyzing the obtained genes,we cloned the difference genes into plasmid T-vector.According to the result of sequences homology analysis and function anticipation,we could presume genes, which have relative to plasmodium development in liver stage.On the other hand,we utilized RT-PCR,immunofluorescence stain and laser scaning confocal microscope for revealing the expression of costimulatory molecules and genes,which have relative to host immune defence.The experimental contents and main results are as follows:1.On the basis of observation the report that adenine(A) and thymidine(T)(＞70%) are rich in Plasmodium falciparum and Plasmodium yoelii genome,we applied DD-PCR protocol that incorporate primers matching the AT rich plasmodium genome,to amplify plasmodium parasite messages from infected liver.The modification of the DD-PCR protocol overcomes the obstacle to purify the unicellual parasite from hepatocytes,and successfully amplifies parasite messages from liver tissue infected by the sporozoites of Plasmodium yoelii.We selected 21 clones to retrieve the GenBank database by BLAST research.After analyzing sequence homology,we got eight new genes(PyHs4,PyHs5,PyHs6,PyHs7,PyHs8,PyHs9,PyHs10,PyHs11) have relative to the development of Plasmodium yoelii.In these genes,PyHs4 has homology with phosphoacetylglucosamine mutase(AGM).PyHs5 has homology with Plasmodium yoelii erythrocyte membrance prontein(EMP3).PyHs6,PyHs7,PyHs8,PyHs9,PyHs10,PyHs11 are genes with unknown functions.Finding and assessing these difference genes of plasmodium provides theory foundation for further study of the development molecular mechanism in liver stage of plasmodium.2.In order to investigate the changes of immunological genes in mammalian host infected by plasmodium,we used RT-PCR approach to carry out qualitative and quantitative analysis with the immune-related genes B7.1,B7.2,TGF-β,IFN-γ.During 2～72 hours after host infected with Plasmodium yoelii sporozoites,the transcription of B7.2,IFN-γ,TGF-βwere up-regulated(P＜0.05),except B7.1,the transcript level of which nearly had no change.The data manifested that Plasmodium yoelii sporozoites invading induced mammalian host immune defence,immunoregulation and other relative immune responses.At the same time,we combined direct immunofluorescence assay and laser scaning confocal microscope techniques to observe the expression and site of B7.1, B7.2 in mammalian host body infected by plasmodium.The result indicated that both B7.1 and B7.2 were up-regulated during the regular intervals.The expression of B7.1 was slowly induced and descended at 72h;B7.2 was rapidly induced and maximally expressed at 48h, then down-regulated at 72h.Through immune stimulation,we assaied that the expression of B7.2 on macrophagus was significantly higher than expression of B7.1 at all time(P＜0.05). In addition,the picture of laser scaning confocal microscope displayed that the expression of B7.1 and B7.2 transfered slowly from endochylema to nucleus in macrophages infected by sporozoites.The phenomena hinted that these costimulators were likely involved in triggering protective immunity and immunomodulation of mammalian host infected with Plasmodium yoelii sporozoite.In conclusion,we investigated the development-related molecules of plasmodium during liver stage on the both sides of parasite and host.Our study systematically described the molecules mechanism of plasmodium merozoites development in hepatocytes and the immunopathogenesis responses induced by parasite.In addition,we pilot studyed the cellular effectors,moleculars and cytokines involved in the activation of the immune responses during plasmodium infection.This will provide us a stronger theory foundation for the development of all potential new anti-malarial drugs and vaccines.
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