| Malaria is parasitic disease which causes serious damage to human health. In recentyears, occurrence and development of resistance are the main stumbling blocks to tacklingmalaria in worldwide. It is urgent to search alternative approaches and techniques to fightagainst this disease. The breakthrough in malaria prevention and treatment depends onprofound understanding of pathogen biology, discovery and application of functionalmolecules involved in host-parasite interaction. The complex life cycle of the malariaparasite is divided into three different phases: one in the female mosquito (the sporogoniccycle) and two in the vertebrate host: the erythrocytic cycle (in blood cells) and theexo-erythrocytic cycle (in liver cells). Plasmodium sporozoites are introduced into thevertebrate host through the bite of infected mosquitoes and first invade hepatocytes, wherethey undergo schizogony and differentiate into merozoites that subsequently infecterythrocytes and cause illness and the possible death of the infected hosts. Hepatocytesinfection is the first obligatory step in the establishment of malarial infection, giving rise tonone clinical symptoms. Therefore, Plasmodium’s disability to invade hepatocyte and theirdefect in complete liver-stage development can completely eliminate malarial infection,which is important target in developing novel controlling and preventing technology.Previous researches mainly focus on erythrocytic stage malaria parasites owing todifficulty in obtaining the infected liver cells. Recently, due to the variety of newtechnology, we can conduct intensive study in malarial pre-erythrocytic stage.The cellular and molecular mechanism involved in host-parasite interaction is alwaysthe research focus in the fields of malaria’s biology and pathogenesis, and the basis of thedevelopment of new drugs, vaccines and novel control strategy for malaria. The key factorsin host-parasite interaction and their effects on the parasites’ growth and development isalso the research focus. The related researches mainly concentrate on some significantlyimportant molecules derived from parasite and host, and their role in Plasmodium’sdevelopment and proliferation. Only a few studies explore the microRNA and their effectsto malaria. MicroRNAs (miRNA) are a class of non-coding small RNA with a length ofapproximately18-22nucleotide, important in posttranscriptional gene regulation, generallyinvolved in the regulation of cell proliferation, differentiation, apoptosis, signaltransduction, tumorigenesis and so on. Recent reports point to the possibility thatapicomplexan parasites have developed tactics to interfere with host miRNA populations,thereby identifying the RNA silencing pathway as a new means to reshape their cellular environment in favor of the needs of growth, development and resistance from the hostdefense response. Plasmodium cannot produce its own miRNAs. Previous researches haveexplored the effects of miRNA on malaria in erythrocytic stage. It has not been reportedwhether miRNA derived from host liver cell has effects towards exo-erythrocytic parasites,which is the objective of our research. At first, we presented green fuorescent protein(GFP)-tagged parasites through transfection, and established the experimental modelduring the complete parasite life cycle including liver, blood and mosquito stages. Weisolated the hepatocytes from the infected BALB/c mice through the flow cell sorting,which were inoculated i.v. into the tail vein with PyGFP sporozoites isolated from theinfected mosquitoes. Using multiplex real-time PCR assays, we detected the miRNAprofile of the isolated hepatocyte. Specific results and methods are as follows:1. We presented green fuorescent protein tagged Plasmodium yoelii rodent malariaparasite for the live isolation of the elusive liver stages. Using the recently developednon-viral Nucleofector technology, Linearized vector pL0016was introduced into theblood stages of P.yoelii BY265. This vector contains the gfp gene under the control of thepbef1αa promoter, the pyrimethamine-resistant form of Toxoplasma gondii (tgdhfr/ts)selection cassette and a genome fragment of the small subunit of ribosomal RNA genes(c-ssu-rrna) of P.berghei. Since the sequence of the c-ssu-rrna is96%identical to the oneof P.yoelii, this vector can integrate into the c-rrna gene unit of P.yoelii. Lastly, we obtaineda P.yoelii population containing both wild type and transgenic parasites with integratedvector, as determined by PCR analysis. We cloned this population and found that one clonewas analyzed for correct integration of the vector and no wild type parasites by PCR.2. We established PyGFP experimental model during the complete parasite life cycleincluding liver, blood and mosquito stages. The mice were inoculated with PyGFP. Threedays later, mosquitoes bited the PyGFP infected mice. Infected mosquitoes were dissectedat days7and16for examination of fluorescent midgut oocysts and salivary glands. Thefluorescent sporozoites were introduced into BALB/c mice through the bite of infectedmosquitoes.7days later, we got the fluorescent erythrocytic parasites. Then we come to aconclusion: the PyGFP can grow and develop in the vertebrate host and in the mosquitovector, expressing green fuorescent protein constitutively. Using the above method, weoptimize the experimental model (1) Optimize malarial infection in mosqutioes. Micereceived doses of1×107，5×106，1×106,5×105PyGFP by introperitoneal injectfion.3dayslater, mosquitoes picked up the parasites from infected mice. Mosquitoes were dissected on day7after infection for oocysts.There was on significant difference among three groups(1×107，5×106，1×106), more than50%of the mosquitoes had100-500oocysts in eachgroup; most mosquitoes from the last group (5×105) had100oocysts, had no significantdifference compared with the other three groups. We come to a conclusion that the bestinoculation amount per mouse is1×107-1×106PyGFP.(2) Optimize the PyGFP sporozoiteliver infection. We collected sporozoites in the salivary glands at different time afterinfection. BALB/c mice were inoculated i.v. into the tail vein with the same dose of theabove sporozites. After40h, the malaria infection rate of mice hepatocytes was measuredby flow cytometry. we found that the sprozites from the infected mosquitoes on23dayshad have the greatest infectivity.3. The isolation of the PyGFP infected hepatocytes. Using the above mosquito androdent models, we isolated PyGFP sporozoites from the infected anopheles on23days bydiscontinuous density gradient purification method. The purifed sporozoites were countedusing a hemocytometer.BALB/c mice were inoculated i.v. into the tail vein with3millionsporozites per mouse, used as infectional group. Normal mice received the material fromthe normal mosquito were used as control group. Hepatocytes were isolated from the aboveBALB/c mice at time point40h by a two-step collagenase perfusion technique. Usingfuorescence activated cell sorting (FACS), we isolated the PyGFP infected hepatocytes.Forward scatter and side scatter plots were used to gate on the majority of the intacthepatocytes while eliminating the cellular debris. The forward scatter versus the FITCchannel was used to identify and sort the PyGFP infected hepatocytes by gating the normalhepatocytes. We could get4000-17000PyGFP infected hepatocytes per BALB/c mouse.4. We conduct the miRNA profile analysis of PyGFP infected host liver cell.Thelimited number of infected hepatocyte made it difficult to satisfy the request of microRNAarray and RNA-seq in total RNA, therefore we choose TaqMAN microRNA Array toanalyze the miRNA profile of PyGFP infected hepatocytes. BALB/c mice were inoculatedi.v. into the tail vein with3million sporozites per mouse, used as infectional group.Normal mice received the material from the normal mosquito were used as controlgroup.We got two inflectional group and one control group. We got the miRNA Ct valuefrom the TaqMAN microRNA Array.Using the mouse snoRNA as the reference gene,wehandle the miRNA Ct value by a2-ΔΔCTmethod. Compared with the control group，wefound28up-regulated miRNAs and1down-regulated miRNAs showing≥3-foldexpression level changes, and17up-regulated miRNAs changed by above5-fold in infected hepatocytes. There was significant difference between inflectional group andcontrol group. We proved10miRNAs using SYBR Green Real Time PCR method.Theresult is consistant with the TaqMAN microRNA Array, but the fold had changedSummarily, we had generated a P.yoelii yoelii BY265line that expressed high levelsof GFP during the complete parasite life cycle and established PyGFP experimental model.Optimize the method of establish experimental model, we can get more sporozoites andmore infected hepatocyctes. Using the flow cell sorting technology, we could isolate theinfected hepatocyte. Then, we nalyze the miRNA profile of PyGFP infected hepatocytes,finding that17up-regulated miRNAs by5fold, which were proved using SYBR GreenReal Time PCR method. Generating PyGFP, establishing its experimental models,analyzing the miRNA profile of the infected hepatocytes form the basis of the next step. so,we can explore the effects of the host liver cell miRNAs on the growth and development ofthe Plasmodium. |
PDF Full Text Request