日本血吸虫肺期童虫与其宿主细胞共培养体系建立的研究
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摘要
在终宿主体内的肺期童虫阶段,被视作宿主免疫系统攻击的最佳靶标,其抗原可诱导Thl介导的保护性免疫反应。故肺期童虫的特异性靶分子被认为是血吸虫病新一代疫苗的最佳候选分子,其筛选对血吸虫病疫苗的研制极为重要。然而,肺期童虫的材料来源较为困难,目前国内外研究者常使用体外培养的童虫替代。研究显示,体外培养的日本血吸虫童虫,尽管在超微结构上与宿主体内的相差甚微,但在基因表达上存在显著差别。据此推测,宿主的内环境影响血吸虫的生长、发育与基因表达。为此,本文将日本血吸虫机械断尾童虫与其宿主细胞共培养,摸索共培养的适合条件,研究体外培养童虫、共培养童虫以及宿主体内发育童虫之间形态学、基因表达谱以及可溶性蛋白的生化与免疫特性等方面的差异,建立日本血吸虫肺期童虫的共培养体系,为血吸虫病疫苗、转基因、基因组学与后基因组学等研究提供更接近于宿主体内状态的童虫材料。现概述如下：
一、共培养条件筛选
(一)宿主细胞种类筛选：将机械断尾法转变的日本血吸虫童虫以400-500条/ml的密度分别与接种密度为2.0×105个/ml、传代24h的人肝静脉内皮细胞(ED25)、人肺腺癌细胞(H1299)、鼠成纤维细胞(3T3)共培养72h,以童虫的体长、体宽、长宽比、体表面积和体积为指标,分别以“841”培养基常规培养(Conventional culture, CC)童虫和从宿主小鼠体内收集的感染72h的童虫(Host, H)为对照,比较与宿主细胞ED25(Co-culture1, CoE)、H1299(CoH)、3T3(CoT)共培养童虫和对照间的差别,筛选出与H组童虫差别最小的共培养的最佳宿主细胞种类。结果显示,H组童虫的体长、长宽比的值最大,分别为196.94±27.14gm和6.80±1.74；体宽、体表面积和体积最小,分别为30.13±5.32gm、(1.99±0.30)×104μm2和(1.33±0.37)×105μm3。CC组童虫体长、长宽比的值最小,分别为154.19±22.76μm和3.83±0.81；体宽、体表面积和体积最大,分别为40.88±3.78μm、(2.22±0.25)×104μm2和(1.82±0.30)×105μm3。共培养组童虫的各指标值居中；其中,体长、长宽比与H组童虫最接近的是CoE组童虫,分别为185.07±35.81μm、5.57±1.72；其次是CoiH组童虫,分别为179.57±31.37μm、5.46±1.38。体宽、体表面积和体积与H组童虫最接近的是CoH组童虫,分别为33.72±3.40μm、(2.06±0.23)×104μm (1.47±0.22)×105μm3;其次是CoE组童虫,分别为34.40×4.40μm、(2.15×0.23)×104um2、(1.57×0.24)×105μm3。差别最大的是CoT组童虫,其体长、体宽、长宽比、体表面积和体积分别为172.52×29.26μm、39.30×5.65μm、4.54×1.28、(2.34×0.26)×104μm2、(1.89×0.35)×105μm3。统计分析发现,CoE组和CoH组童虫的体长和体表面积与H组童虫的均无显著性差异(P>0.05)；体宽、长宽比与H组童虫的均有显著性差异(P<0.05)；CoE与CoH组童虫的各指标,其差异均无显著性(P>0.05)；共培养组与常规培养组比较,除CoE组与CC组童虫的体表面积间无显著性差异外(P>0.05), CoE、CoH组与CC组童虫各项指标间其差异均有显著性(P<0.05)。比较三种宿主细胞,认为ED25是日本血吸虫童虫体外共培养的最佳宿主细胞。
(二)宿主细胞接种密度筛选：以日本血吸虫童虫的体长、体宽、长宽比、体表面积和体积为指标,将机械断尾法转变的童虫分别与接种密度为1.0×105个/ml (Co1)、2.0×105个/ml (CoE2)与3.0×105个/ml (CoE3)传代24h的ED25共培养72h,以从宿主小鼠体内收集的感染72h的童虫(Host,H)为对照,比较与CoE不同密度共培养的童虫与对照之间的差别,筛选出共培养ED25的最适细胞密度。结果显示,CoE2组童虫的体长最长、体宽最小、长宽比最大,分别为183.21×17.81μm、37.30×3.30μm和4.98±0.91,最接近于对照组H童虫的数值,其中体长与H组童虫差异无显著性(P>0.05),其余指标均与H组童虫差异有显著性(P<0.05)；CoEl组、CoE2组各指标与H组童虫差异均有显著性(P<0.05)。比较三种宿主细胞密度,2.0×105个/ml为与日本血吸虫童虫共培养的最佳宿主细胞密度。
二、共培养肺期童虫的形态学观察
将机械断尾法转变的日本血吸虫童虫与接种密度为2.0×105个/ml、传代24h的ED25共培养(Coc),分别以"841”培养基常规体外培养(CC)的和宿主体内获取(H)的童虫作对照。
(一)光镜观察：光镜下,培养72h的各组日本血吸虫童虫虫体透明,可进行伸缩运动及左右摆动。当童虫纵向伸缩时,虫体内部组织前后运动,肠管隐约可见。比较三组童虫体长发现,H组童虫最长,为196.94±27.14μm；其次是Coc组童虫,为185.07±35.81μm；CC组童虫最短,为154.19±22.76μm。可见,Coc组童虫比CC组童虫更相似于H组童虫。
(二)扫描电镜观察：扫描电镜下,H组童虫体型细长,体表可见皮孔与20～30余个环槽；体棘在前后两端分布较密、中间稀疏,呈尖齿状,排列较整齐；童虫末端未见磨盘状结构,可见排泄孔。Coc组童虫的体型也较细长,与H组童虫较为相似；体表可见较多皮孔与少量环槽,体表覆盖密集的体棘；体棘数量较H组童虫多,且较大,呈长舌状,排列整齐,均指向末端。CC组童虫体型粗短,体表亦可清晰地观察到皮孔与环槽,体棘数量与H组童虫相近,但大小形态不一,分布、指向较凌乱。可见,Coc组童虫的体表结构与H组童虫更相像。
(三)透射电镜观察：H组童虫组织的电子致密程度较高,Coc组童虫与H组童虫相似,而CC组童虫其电子致密程度较低。H组童虫体壁由体被、基膜及体被下层构成。其中,体被包括外质膜及外质膜与基膜之间的基质层,较厚,约500～700nm。外质膜、基质和基底膜各层界限清晰。外质膜凸起或延伸,形成皱褶,基质层内电子透明区和浅色区相间,基膜连续完整,体棘从基膜崛起。外环肌和内纵肌位于体被下层,外环肌与体被下层中的实质细胞交错在一起,内纵肌肌纤维排列整齐。Coc组童虫体壁结构与H组童虫的类似,体被比H组童虫稍薄,约400～600nm。外质膜由7层组成,并凹陷形成凹窝或孔；基质层内可见电子致密的指环体分布。体被下层外环肌呈电子致密的点状分布,且电子致密度较H组高,内纵肌肌纤维排列整齐。CC组童虫外质膜向外形成棘状凸起,未观察到7层外质膜结构,基质层靠近外质膜一侧分布有较多圆形电子透亮区,与H组和Coc组童虫相比,其基膜不完整,时断时续,导致体被各层分界不明显,并与体被下层交错。体被下层中外环肌和内纵肌不易辨认。总而言之,Coc组童虫的体被结构比CC组童虫更接近于H组童虫。
三、共培养肺期童虫基因表达差异
将机械断尾法转变的日本血吸虫童虫与接种密度为2.0×105个/ml、贴壁24h的ED25共培养(Coc),分别以“841”培养基常规体外培养(CC)和宿主体内获取(H)的童虫作对照,应用抑制性差减杂交和斑点杂交技术,建立H组和Coc组童虫以及Coc组和CC组童虫正向和反向差减文库,分析并筛选三组童虫差异表达的基因。结果共获得H组童虫与Coc组童虫正向差减文库1982个克隆,反向差减文库1824个克隆,Coc组童虫与CC组童虫正向差减文库1128个克隆,反向差减文库1680个克隆；PCR检测克隆的阳性率均超过80%。测序后分别获得童虫的差异表达基因序列H组与Coc组91个、Coc组与CC组107个；其中,H组童虫与Coc组之间的上调基因前者55个、后者36个；Coc组童虫与CC组之间的上调基因前者76个、后者31个。
测序结果显示：共培养3d的日本血吸虫童虫与宿主体内获得的肺期童虫相比,其编码肌动蛋白(Actin-2). NADH脱氢酶1亚基(ND1)、翻译起始因子、视紫红质抑制蛋白(Putative beta-arrestin)等的基因呈上调表达,编码热休克蛋白(HSP90α)、细胞色素C氧化酶(Cox Ⅰ)、赖氨酸羟化酶(LH)、腺苷激酶(AK1)、翻译延长因子、5’-核酸酶等的基因呈下调表达；与体外常规培养3d的童虫相比,编码HSP5. Cox. Actin-5C、ND1、细胞程序性死亡蛋白、虫卵抗原、ATP合酶、类固醇脱氢酶、糖基转移酶等的基因呈上调表达,编码组织蛋白酶L酶(Cathepsin L)、组织蛋白酶L酶原(Cathepsin L precursor)、LH、ND5、GAPDH、泛素结合酶、细胞分裂周期和凋亡调控蛋白等的基因呈下调表达。与CC组相比,Coc组童虫HSP和Cox基因均为上调表达,Cathepsin L、Cathepsin L precursor基因均下调表达,表达水平与H组更接近。与Coc组相比,CC组LH基因上调表达,Acti、ND1基因均下调表达,与H组童虫相似。可见,Coc组童虫的基因表达比CC组更接近H组童虫。
采用斑点杂交比较同一基因在H组童虫、Coc组童虫和CC组童虫的表达水平,由显色结果可见,无论是在H组童虫呈上调表达的基因,还是在Coc组童虫体内呈上调表达的基因,同一基因在H组童虫与Coc组童虫间表达丰度较为接近,与CC组童虫间则差异较大。
四、共培养肺期童虫可溶性蛋白的生化与免疫学特性
将机械断尾法转变的日本血吸虫童虫与接种密度为2.0×105个/ml、传代24h的ED25共培养(Coc),分别以“841”培养基常规体外培养(CC)和宿主体内获取(H)的童虫作对照。分别制备Coc组、CC组、H组童虫以及ED-25宿主细胞的可溶性蛋白,进行SDS-PAGE电泳,用硝酸银染色。结果显示,H、Coc和CC等三组童虫均含4条分子量约为55、40、28和18KDa的主要蛋白条带外,与CC组相比,Coc组与H组童虫还共同含有分子量约为65和26KDa的蛋白条带；CC组童虫的可溶性蛋白的分子量在60-70KDa范围处出现的条带较明显,而H组和Coc组童虫可溶性蛋白在此范围处的蛋白条带均较模糊。ED25细胞可溶性蛋白图谱与三组童虫可溶性蛋白图谱差异较大,上样浓度相同条件下,分子量约为100、50、38和18KDa处可见与Coc组童虫蛋白相同条带。可见,Coc组童虫可溶性蛋白的SDS-PAGE电泳图谱比CC组童虫的更类似于H组童虫的图谱。
以感染日本血吸虫27d和42d的昆明系小鼠血清为一抗,经Western Blot分析三组童虫可溶性蛋白,结果表明,三组童虫中100和45～55KDa大小的蛋白分子均能与感染日本血吸虫42天的小鼠血清中的抗体结合,形成褐色沉淀条带；其中,100KDa处条带三组童虫显色均较弱；而45～55KDa处条带H组童虫和Coc组童虫显色较强、较一致,CC组童虫显色较弱。此外,Coc组和CC组童虫在分子量约为12.5KDa处有一显色较弱的条带。表明,Coc组童虫可溶性蛋白的免疫学特性介于宿主体内获得童虫与常规体外培养虫体之间,相对CC组童虫更近似于H组童虫。
综上所述,与CC组童虫相比,Coc组童虫不仅在形态学(包括超微结构)与基因表达水平上更接近于H组童虫,而且童虫可溶性蛋白的生化与免疫学特性与H组童虫也更为相似。由此可见,本研究建立的日本血吸虫童虫与宿主细胞共培养体系,是更接近于宿主体内环境的体系,有望应用于日本血吸虫的分子生物学与血吸虫病免疫学研究；并可利用其进行血吸虫与宿主的相互关系研究,为揭示血吸虫的生长发育以及保护性免疫机制奠定良好基础。
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 schistosomula
Under 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 schistosomula
Suppression 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 schistosomula
The 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.
一、共培养条件筛选
(一)宿主细胞种类筛选：将机械断尾法转变的日本血吸虫童虫以400-500条/ml的密度分别与接种密度为2.0×105个/ml、传代24h的人肝静脉内皮细胞(ED25)、人肺腺癌细胞(H1299)、鼠成纤维细胞(3T3)共培养72h,以童虫的体长、体宽、长宽比、体表面积和体积为指标,分别以“841”培养基常规培养(Conventional culture, CC)童虫和从宿主小鼠体内收集的感染72h的童虫(Host, H)为对照,比较与宿主细胞ED25(Co-culture1, CoE)、H1299(CoH)、3T3(CoT)共培养童虫和对照间的差别,筛选出与H组童虫差别最小的共培养的最佳宿主细胞种类。结果显示,H组童虫的体长、长宽比的值最大,分别为196.94±27.14gm和6.80±1.74；体宽、体表面积和体积最小,分别为30.13±5.32gm、(1.99±0.30)×104μm2和(1.33±0.37)×105μm3。CC组童虫体长、长宽比的值最小,分别为154.19±22.76μm和3.83±0.81；体宽、体表面积和体积最大,分别为40.88±3.78μm、(2.22±0.25)×104μm2和(1.82±0.30)×105μm3。共培养组童虫的各指标值居中；其中,体长、长宽比与H组童虫最接近的是CoE组童虫,分别为185.07±35.81μm、5.57±1.72；其次是CoiH组童虫,分别为179.57±31.37μm、5.46±1.38。体宽、体表面积和体积与H组童虫最接近的是CoH组童虫,分别为33.72±3.40μm、(2.06±0.23)×104μm (1.47±0.22)×105μm3;其次是CoE组童虫,分别为34.40×4.40μm、(2.15×0.23)×104um2、(1.57×0.24)×105μm3。差别最大的是CoT组童虫,其体长、体宽、长宽比、体表面积和体积分别为172.52×29.26μm、39.30×5.65μm、4.54×1.28、(2.34×0.26)×104μm2、(1.89×0.35)×105μm3。统计分析发现,CoE组和CoH组童虫的体长和体表面积与H组童虫的均无显著性差异(P>0.05)；体宽、长宽比与H组童虫的均有显著性差异(P<0.05)；CoE与CoH组童虫的各指标,其差异均无显著性(P>0.05)；共培养组与常规培养组比较,除CoE组与CC组童虫的体表面积间无显著性差异外(P>0.05), CoE、CoH组与CC组童虫各项指标间其差异均有显著性(P<0.05)。比较三种宿主细胞,认为ED25是日本血吸虫童虫体外共培养的最佳宿主细胞。
(二)宿主细胞接种密度筛选：以日本血吸虫童虫的体长、体宽、长宽比、体表面积和体积为指标,将机械断尾法转变的童虫分别与接种密度为1.0×105个/ml (Co1)、2.0×105个/ml (CoE2)与3.0×105个/ml (CoE3)传代24h的ED25共培养72h,以从宿主小鼠体内收集的感染72h的童虫(Host,H)为对照,比较与CoE不同密度共培养的童虫与对照之间的差别,筛选出共培养ED25的最适细胞密度。结果显示,CoE2组童虫的体长最长、体宽最小、长宽比最大,分别为183.21×17.81μm、37.30×3.30μm和4.98±0.91,最接近于对照组H童虫的数值,其中体长与H组童虫差异无显著性(P>0.05),其余指标均与H组童虫差异有显著性(P<0.05)；CoEl组、CoE2组各指标与H组童虫差异均有显著性(P<0.05)。比较三种宿主细胞密度,2.0×105个/ml为与日本血吸虫童虫共培养的最佳宿主细胞密度。
二、共培养肺期童虫的形态学观察
将机械断尾法转变的日本血吸虫童虫与接种密度为2.0×105个/ml、传代24h的ED25共培养(Coc),分别以"841”培养基常规体外培养(CC)的和宿主体内获取(H)的童虫作对照。
(一)光镜观察：光镜下,培养72h的各组日本血吸虫童虫虫体透明,可进行伸缩运动及左右摆动。当童虫纵向伸缩时,虫体内部组织前后运动,肠管隐约可见。比较三组童虫体长发现,H组童虫最长,为196.94±27.14μm；其次是Coc组童虫,为185.07±35.81μm；CC组童虫最短,为154.19±22.76μm。可见,Coc组童虫比CC组童虫更相似于H组童虫。
(二)扫描电镜观察：扫描电镜下,H组童虫体型细长,体表可见皮孔与20～30余个环槽；体棘在前后两端分布较密、中间稀疏,呈尖齿状,排列较整齐；童虫末端未见磨盘状结构,可见排泄孔。Coc组童虫的体型也较细长,与H组童虫较为相似；体表可见较多皮孔与少量环槽,体表覆盖密集的体棘；体棘数量较H组童虫多,且较大,呈长舌状,排列整齐,均指向末端。CC组童虫体型粗短,体表亦可清晰地观察到皮孔与环槽,体棘数量与H组童虫相近,但大小形态不一,分布、指向较凌乱。可见,Coc组童虫的体表结构与H组童虫更相像。
(三)透射电镜观察：H组童虫组织的电子致密程度较高,Coc组童虫与H组童虫相似,而CC组童虫其电子致密程度较低。H组童虫体壁由体被、基膜及体被下层构成。其中,体被包括外质膜及外质膜与基膜之间的基质层,较厚,约500～700nm。外质膜、基质和基底膜各层界限清晰。外质膜凸起或延伸,形成皱褶,基质层内电子透明区和浅色区相间,基膜连续完整,体棘从基膜崛起。外环肌和内纵肌位于体被下层,外环肌与体被下层中的实质细胞交错在一起,内纵肌肌纤维排列整齐。Coc组童虫体壁结构与H组童虫的类似,体被比H组童虫稍薄,约400～600nm。外质膜由7层组成,并凹陷形成凹窝或孔；基质层内可见电子致密的指环体分布。体被下层外环肌呈电子致密的点状分布,且电子致密度较H组高,内纵肌肌纤维排列整齐。CC组童虫外质膜向外形成棘状凸起,未观察到7层外质膜结构,基质层靠近外质膜一侧分布有较多圆形电子透亮区,与H组和Coc组童虫相比,其基膜不完整,时断时续,导致体被各层分界不明显,并与体被下层交错。体被下层中外环肌和内纵肌不易辨认。总而言之,Coc组童虫的体被结构比CC组童虫更接近于H组童虫。
三、共培养肺期童虫基因表达差异
将机械断尾法转变的日本血吸虫童虫与接种密度为2.0×105个/ml、贴壁24h的ED25共培养(Coc),分别以“841”培养基常规体外培养(CC)和宿主体内获取(H)的童虫作对照,应用抑制性差减杂交和斑点杂交技术,建立H组和Coc组童虫以及Coc组和CC组童虫正向和反向差减文库,分析并筛选三组童虫差异表达的基因。结果共获得H组童虫与Coc组童虫正向差减文库1982个克隆,反向差减文库1824个克隆,Coc组童虫与CC组童虫正向差减文库1128个克隆,反向差减文库1680个克隆；PCR检测克隆的阳性率均超过80%。测序后分别获得童虫的差异表达基因序列H组与Coc组91个、Coc组与CC组107个；其中,H组童虫与Coc组之间的上调基因前者55个、后者36个；Coc组童虫与CC组之间的上调基因前者76个、后者31个。
测序结果显示：共培养3d的日本血吸虫童虫与宿主体内获得的肺期童虫相比,其编码肌动蛋白(Actin-2). NADH脱氢酶1亚基(ND1)、翻译起始因子、视紫红质抑制蛋白(Putative beta-arrestin)等的基因呈上调表达,编码热休克蛋白(HSP90α)、细胞色素C氧化酶(Cox Ⅰ)、赖氨酸羟化酶(LH)、腺苷激酶(AK1)、翻译延长因子、5’-核酸酶等的基因呈下调表达；与体外常规培养3d的童虫相比,编码HSP5. Cox. Actin-5C、ND1、细胞程序性死亡蛋白、虫卵抗原、ATP合酶、类固醇脱氢酶、糖基转移酶等的基因呈上调表达,编码组织蛋白酶L酶(Cathepsin L)、组织蛋白酶L酶原(Cathepsin L precursor)、LH、ND5、GAPDH、泛素结合酶、细胞分裂周期和凋亡调控蛋白等的基因呈下调表达。与CC组相比,Coc组童虫HSP和Cox基因均为上调表达,Cathepsin L、Cathepsin L precursor基因均下调表达,表达水平与H组更接近。与Coc组相比,CC组LH基因上调表达,Acti、ND1基因均下调表达,与H组童虫相似。可见,Coc组童虫的基因表达比CC组更接近H组童虫。
采用斑点杂交比较同一基因在H组童虫、Coc组童虫和CC组童虫的表达水平,由显色结果可见,无论是在H组童虫呈上调表达的基因,还是在Coc组童虫体内呈上调表达的基因,同一基因在H组童虫与Coc组童虫间表达丰度较为接近,与CC组童虫间则差异较大。
四、共培养肺期童虫可溶性蛋白的生化与免疫学特性
将机械断尾法转变的日本血吸虫童虫与接种密度为2.0×105个/ml、传代24h的ED25共培养(Coc),分别以“841”培养基常规体外培养(CC)和宿主体内获取(H)的童虫作对照。分别制备Coc组、CC组、H组童虫以及ED-25宿主细胞的可溶性蛋白,进行SDS-PAGE电泳,用硝酸银染色。结果显示,H、Coc和CC等三组童虫均含4条分子量约为55、40、28和18KDa的主要蛋白条带外,与CC组相比,Coc组与H组童虫还共同含有分子量约为65和26KDa的蛋白条带；CC组童虫的可溶性蛋白的分子量在60-70KDa范围处出现的条带较明显,而H组和Coc组童虫可溶性蛋白在此范围处的蛋白条带均较模糊。ED25细胞可溶性蛋白图谱与三组童虫可溶性蛋白图谱差异较大,上样浓度相同条件下,分子量约为100、50、38和18KDa处可见与Coc组童虫蛋白相同条带。可见,Coc组童虫可溶性蛋白的SDS-PAGE电泳图谱比CC组童虫的更类似于H组童虫的图谱。
以感染日本血吸虫27d和42d的昆明系小鼠血清为一抗,经Western Blot分析三组童虫可溶性蛋白,结果表明,三组童虫中100和45～55KDa大小的蛋白分子均能与感染日本血吸虫42天的小鼠血清中的抗体结合,形成褐色沉淀条带；其中,100KDa处条带三组童虫显色均较弱；而45～55KDa处条带H组童虫和Coc组童虫显色较强、较一致,CC组童虫显色较弱。此外,Coc组和CC组童虫在分子量约为12.5KDa处有一显色较弱的条带。表明,Coc组童虫可溶性蛋白的免疫学特性介于宿主体内获得童虫与常规体外培养虫体之间,相对CC组童虫更近似于H组童虫。
综上所述,与CC组童虫相比,Coc组童虫不仅在形态学(包括超微结构)与基因表达水平上更接近于H组童虫,而且童虫可溶性蛋白的生化与免疫学特性与H组童虫也更为相似。由此可见,本研究建立的日本血吸虫童虫与宿主细胞共培养体系,是更接近于宿主体内环境的体系,有望应用于日本血吸虫的分子生物学与血吸虫病免疫学研究；并可利用其进行血吸虫与宿主的相互关系研究,为揭示血吸虫的生长发育以及保护性免疫机制奠定良好基础。
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 schistosomula
Under 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 schistosomula
Suppression 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 schistosomula
The 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.
引文
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