| The specific molecular targets of lung-stage schitosomula Schistosoma japonicum has been intensively investigated for being new vaccine candidates for schistosomiasis, because the antigens of the lung-stage schitosomula can induce Thl cell-mediated immune responses in infected hosts. It was difficult for the lung-stage schistosomula to be collected. Therefore researches generally culture schistosomula of the schistosomiasis in vitro rather than to collect from the lungs of infected hosts. However, there were significantly differences between their gene expression profiles in both of the above mentioned circumstances, though the observation of transmission electron microscope showed few differences in their ultra structures. It has been approved that the host factors affect the growth, development and gene expression of schistosome. So, in order to provide plentiful materials which were more similar or equivalent to those from the lung of the hosts for further researches on vaccine and genomics, the present paper has established a co-culture system between the mechanically transformed schistosomula of S. japonicum and its host cells. Summary of the major results is as follows.1. Conditions Screening of co-culture between schistosomula S. japonicum and its host cells(1) Specific host cell was screened out suitable for co-culture lung-stage schistosomula S. japonicum. Three types of host cells were used for co-culture with the junior, including the hepatic vein endothelial cells of human (ED25), human lung adenocarcinoma cells (H1299) and mouse fibroblast cells (3T3). Before use the cells were passed24hours in advance with a density of2.0×105/ml. Taking the length (L), width (W), length-width ratio (L/W), surface area and volume of schistosomula as the indexes, three groups co-cultured schistosomula (CoE, CoH, and CoT were named according to the feeder layer cells) were not only compared with those collected from the lungs of the mouse infected by cercariae for72hours (Host group, H), but also the ones cultured by conventional culture method (Conventional Culture group, CC) with "841" culture medium. The results showed that, a) The Host group schistosomula were196.94±27.14μm in length,30.13±5.32μm in width,6.80±1.74in L/W ratio,(1.99±0.30)÷104μm2in surface area and (1.33±0.37)×105μm3in volume, while the indicators mentioned above of CC group were separately154.19±22.76μm,40.88±3.78μm,3.83±0.81,(2.22±0.25)×104μm2and (1.82±0.30)×105μm3. Compared with the CC group, the co-cultured schistosomula were more similar to Host group larvae, especially the length and L/W ratio of CoE group and the width, surface area, volume of CoH group, b) The differences of length and surface area between CoE and H group, as well as CoH and H group, were not significant (P>0.05), though the differences between the widths, L/W ratios and volumes of them were opposite (P<0.05); c) There were not any significant differences between CoE and CoH group larvae, d) The CoE and CoH group schistosomula were significantly different from the CC group(P<0.05), except the surface area of CoE group which didn’t show significant difference with the CC group (P>0.05). Comparing the three types of host cells, ED25was the best host cell for the use of the co-culture with schistosomula.(2) This part intended to screen an optimized density of feeder layer cells for co-culture system. Taking the length (L), width (W), length-width ratio (L/W), surface area and volume of schistosomula as the indexes, the schistosomula co-cultured respectively with ED25cells of three different densities24hours passed in advance.1.0×105/ml (Co-culturel,CoEl),2.0×105/ml (Co-culture1,CoE2),3.0×105/ml (Co-culturel,CoE3) were compared with the H group larvae. Statistics analysis of the results showed that compared with the other two groups, the CoE2group schistosomula which were183.21±17.81μm in length,37.30±3.30μm in width and4.98±0.91in L/W ratio, were more similar to the Host group ones. And the lengths between them was not significantly different (P>0.05), while the other indicators were different significantly (P<0.05). Thus2.0×105/ml was the best one for the use of the co-culture with schistosomula among the three different densities.2. The observations on the morphology of the co-cultured schistosomulaUnder microscopy, the majority of the schistosomula with lucent body and well refracting surface could stretch and swing freely. The internal organs, cells as well as the indistinct intestine were seen flowing back and forth when the larva stretched. As the results mentioned above, the schistosomula co-cultured with ED25(Coc group) were as slim as those from host (H group), while the conventional cultured ones (CC group) are dumpier.Under scanning electron microscope,20～30grooves were seen clearly on the surface of the slim lung-stage schistosomula. The anterior and posterior parts of the larva were bestrewed regularly by some tiny and sharp spines, which were sparse on the middle part, as well as a few of pits. There were also a few lenticels on the surface of schistosomula. The discoid structure which can be seen at the posterior part of the skin-stage schistosomula was disappeared while the excretory pore was present. The Coc group schistosomula were observed to be similar to the Host group ones in the shape. It could be seen that the grooves and pits were lesser while spines were more and larger than what on the surface of Host group schistosomula. Besides this, the spines arrayed densely and pointed regularly to the posterior part. The CC group schistosomula were stubby with clear grooves on the surface, while the spines in different sizes arrayed and pointed disorderly.Under transmission electron microscope, the H group and Coc group shistosomula both presented high level electron intensity, while the CC group larvae showed lower intensity. The body wall of H group schistosomula could be identified into three parts, which were tegument, basal lamina and subtegument. Among them, the tegument approximately500-700nm thick, was consisted of outer plasma membrane and the matrix that between the outer plasma membrane and basal lamina. The fluctuant membrane was observed to form some reductus on the surface of the larvae. And the matrix appeared to be mixed by electronic lucent zones and gray parts. The basal lamina, from which the spine generated, showed continuous integrity. In the subtegument, there was the outer circular muscle overlapped with the parenchymal cells, as well as the regularly arrayed inner longitudinal muscle. The structures of the body wall of Coc group schistosomula were quite similar to those of H group larvae, though there were still some differences. The400-600nm thick tegument was thinner than the former one. The hepta-outer plasma membrane formed some pits rather than reductus on the larval surface. In the matrix, there were some ring-like granules with electron intensity. The outer circular muscle appeared to be dense electronic in dark granules, which was different from H group. In CC group schistosomula, no hepta-membrane was observed. But there were some round-shape electronic lucent zones near the membrane in the matrix. Compared to the schistosomula in H and Coc group, the basal lamina of CC group schistosomula was lacking integrity, leading to a confusion of the tegument. As a result, both the outer circular muscle and inner longitudinal muscle were difficult to recognize. In a word, compared to the CC group, Coc group schistosomula shared more similarities on tegument structures with the H group schistosomula.3. The differential gene expression of co-cultured schistosomulaSuppression subtractive hybridization and dot hybridization technology were used to establish forward and reverse subtracted libraries between schistosomula collected from host and co-cultured with ED25feeder layer cells, as well as between schistosomula co-cultured and cultured conventionally. And the differential expressed genes of schistosomula from three groups were further screened and analyzed respectively.1982clones for forward subtracted library and1824clones for reverse subtracted library were obtained between schistosomula from H and Coc group, while1128clones for forward and1680clones for reverse subtracted library were obtained between Coc and CC group schistosomula. All of the positive rates of the clone were more than80%according to the PCR results. After sequencing by a corporation and matching with BLAST on NCBI web site,91significantly differential expressed genes between H and Coc group schistosomula were obtained. Among these genes,55were up-regulated in H group and36were up-regulated in Coc group. While among107differently expressed genes obtained between Coc and CC group,76of them highly expressed in Coc group and31highly expressed in CC group.BLAST analysis showed that the functions of the differential expressed genes were related to the stress response, metabolism of energy, electron transport, cell division and apoptosis, protein translation and formation of cytoskeleton, etc. a) Compared with CC group, the genes encoding Heat shock protein90(HSP90) and cytochrome c oxidase (Cox) were up-regulated while cathepsin L and cathepsin L precursor were down-regulated in Coc group schsitosomula, which was similar to H group larvae. b) Compared to Coc group, the genes encoding lysyl hydroxylase were up-regulated while actin and NADH dehydrogenase subunit1were down-regulated in CC group schsitosomula, which was similar to H group larvae.The comparison of the abundance of the same genes expressed in schistosomula of three groups was done by the dot blot hybridization. The coloring results showed the similarity of the same gene expression between the schistosomula from H and Coc group, but difference from the conventionally cultured group.4. The biochemical and immunological characters of soluble proteins of co-cultured schistosomulaThe soluble proteins of schistosomula from H, Coc and CC group were extracted separately and separated by SDS-PAGE. Silver staining results of SDS-PAGE showed that the maps of soluble antigens of schistosomula from host, co-cultured and conventionally cultured system were different from each other. The three groups all had four same clear bands which indicated that the molecular weights of proteins were55,40,28,18KDa, respectively. Additionally, proteins weighted65and26KDa were present in Coc and H group larvae compared to CC group. A band at60-70kDa was seen clearly in conventionally cultured schistosomula, but vaguely in host and co-cultured group. The profile of Coc group schistosomula proteins also possessed another4bands, weighted100,38,32, and12.5KDa, which were not available in H and CC groups. But the4bands could be seen in the map of ED25cells proteins with the same sampled concentration.Taking the serum collected from the mouse infected by S. japonicum cercarea for42days as the first antibody, Western blot was used to analyses the immunological characters of soluble proteins of schistosomula. The result of Western Blot showed that the soluble proteins of100and45～55KDa in three groups all could be combined by the antibodies in the serum. The bands appeared at100kDa were expressed analogously in larva from three groups, while the45-55KDa bands were higher expressed in Coc and H groups than in CC group. Moreover, vague12.5kDa bands could also be observed in both Coc and CC groups. All of the above indicated that the immunological properties of soluble proteins extracted from Coc group schistosomula were more similar to that of schistosomula from H group than CC group.To sum up, compared to the CC group schistosomula, not only the morphology and gene expression, but also the biochemical and immunological properties of the Coc group larvae were more similar to larvae from the host. It indicated that co-culture system between the schistosomula and its host cells established in present study could provide suitable conditions which imitate the host internal environment for the growth and development of schistosomula. And the biological and immunological characters of the co-cultured larvae were similar to the ones collected from the lung of the host. |
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