湖北釘螺被目平外睪吸蟲與日本血吸蟲不同間隔時間感染后分泌物的檢測與分析

唐崇惕,黃帥欽,彭午弦,盧明科,彭文峰,陳 東

湖北釘螺(Oncomelaniahupensis)被外睪吸蟲(Exorchisspp.)感染后很快產生大量血淋巴細胞和分泌物并能殺害再進入其體內的日本血吸蟲(Schistosomajaponicum)幼蟲[1,3,11]。外睪吸蟲和日本血吸蟲先后雙重感染釘螺間隔時間愈長，血吸蟲被殺害的效果愈強，但釘螺血淋巴細胞的數目卻是逐漸減少[2,4]，釘螺體內還有什么東西在起作用？近年許多學者在南美洲和非洲大力開展曼氏血吸蟲(Shistosomamansoni)與其中間宿主水生雙臍螺(Biomphalariaspp.)的相互關系問題的研究[5]。探討感染曼氏血吸蟲的雙臍螺體內血漿中的蛋白質組學(proteomics)，有關它們的多態性、多態粘蛋白(polymorphic mucins)[8-9],與血纖維源有關的蛋白質(fibrinogen-related proteins) 等的特性、免疫應答基因家族情況[13-14],有關軟體動物防御細胞中激活酶(kinase)多方面作用情況[10]。有學者研究棘口吸蟲(Echinostomacaproni)和曼氏血吸蟲幼蟲在雙臍螺中的排泄和分泌的蛋白質組(proteome)[7]，棘口吸蟲寄生的雙臍螺血漿及血淋巴細的不同蛋白質的特性[6,12]。有關曼氏血吸蟲與雙臍螺相關的螺體血漿中物質的研究報告無數，可見其中復雜程度。筆者從外睪吸蟲和日本血吸蟲雙重感染的釘螺體上觀察到有復雜的分泌物增多，它們在不同間隔時間感染的釘螺體內外表現情況有所不同，茲將觀察結果簡單介紹于下。

1 材料與方法

用從湖南西洞庭湖鯰魚腸管收集的目平外睪吸蟲(ExorchismupingensisJiang,2011)的蟲卵拌以少量面粉飼食從西洞庭湖采集的湖北釘螺，外睪吸蟲感染后的釘螺分為5組，分別在感染后21 d、37 d、55 d、70 d、85 d，每粒釘螺與實驗室日本血吸蟲陽性小白鼠肝臟血吸蟲蟲卵孵化的毛蚴(40～90個)接觸；各組釘螺均在感染血吸蟲毛蚴后4～82 d之間不同時間，用10%福爾馬林溶液固定。釘螺單獨感染外睪吸蟲后20 d，也用10%福爾馬林溶液固定。所有實驗釘螺均經石蠟連續切片及用蘇木精與洋紅染色制片。顯微鏡油鏡檢查各切片所有斷面，比較觀察各組無其它吸蟲天然感染的釘螺其體內外分泌物的數量及它們的結構情況，并數碼相機顯微照相儲存于電腦。

2 結 果

在不同間隔先后雙重感染目平外睪吸蟲和日本血吸蟲的釘螺體內分泌物詳細情況的本實驗觀察中，同樣見到間隔時間長短與螺體殺害血吸蟲幼蟲的效力成正比，而與在螺體及異常血吸蟲幼蟲體內的3種血淋巴細胞數量成反比的現象。但釘螺體內外的分泌物密度沒有減少，有增多并且更加復雜化。情況分述如下。

2.1單獨感染外睪吸蟲的釘螺體表分泌物結構 作為吸蟲類的中間宿主貝類，其體表都有一層分泌物。切片觀察單獨感染日本血吸蟲的釘螺，其體表的分泌物僅一層粘液性薄膜，而被外睪吸蟲感染的釘螺其體表分泌物增厚[11]。本實驗觀察單獨感染外睪吸蟲后21 d的釘螺，其體表也有較厚的分泌物(圖1)，在油鏡下可見它們由排列不規則、大小和形狀各異、透明或不透明的晶體狀結構所密布，其中及邊緣有些微粘液樣物質(圖2～3)。

2.2釘螺體內分泌物 在釘螺體中部、肉足和鰓的下方有一大團副腺組織(圖6)，許多副腺細胞游離在螺體各組織中。在所有雙重感染外睪和血吸蟲的釘螺，副腺細胞亦可見于闊張的血管中[3]。在不同間隔雙重感染外睪和血吸蟲的釘螺，所有被擊毀的血吸蟲幼蟲殘體內外都可見到這些細胞(圖7、9、22等)。

單獨感染外睪吸蟲的釘螺體內血淋巴細胞和分泌物也比單獨感染日本血吸蟲的多，明顯的金黃色顆粒團狀分泌物(圖4～5)出現在釘螺體內一些區域。雙重感染外睪和血吸蟲的釘螺，雙重感染間隔時間加長體內分泌物有增無減，分泌物顆粒和血淋巴細胞經常一起出現在被擊毀的異常血吸蟲幼蟲體內外(圖8、10、13、17、19等)。有時它們會呈褐色團狀大量地分布在螺體組織及異常血吸蟲體內，以間隔55 d雙重感染后56 d(即外睪吸蟲感染后111 d，血吸蟲殘體為6 d)的照片(圖14)為例，大團褐色物占據血吸蟲殘體大部，蟲體所在血腔外的螺體組織中密布許多小褐色團。如此情況時?？梢?。

前此在外睪和血吸蟲雙重感染的釘螺體內，已見到在金黃色分泌物顆粒每團邊緣都有一個比小血淋巴細胞核更小的細胞核，其直徑只有1.9～2.6 μm[3]。此次用油鏡觀察，仍然是此情況，而且在核的外圍都可見到有一圈白色外圍，它應該是其細胞質部位。說明它們確有細胞的結構，雙重感染時間愈長此極小細胞愈明顯(圖8、11～12、16、18～20)。

2.3釘螺體內分泌物顆粒在異常血吸蟲幼蟲體內的異樣產物 在外睪和血吸蟲雙重感染的釘螺體內，尚有奇怪現象：在很早期被擊毀的異常血吸蟲幼蟲體內會出現一個大紅色球團，它整個表面布滿顆粒小點，球團邊緣圍繞甚多與上述分泌物顆粒上極小細胞同樣大小的胞核和細胞樣的結構(圖8～10、17)。雙重感染間隔時間愈長的釘螺，異常血吸蟲幼蟲體內此紅球團數常會增到2～4個,而且歷久不退，如間隔70 d后的6 d和25 d異常血吸蟲幼蟲的樣品(圖15、21)。

2.4外睪吸蟲和日本血吸蟲雙重感染的釘螺體表分泌物結構 外睪吸蟲和日本血吸蟲雙重感染的釘螺，它們體表分泌物均有增無減，其厚度可達單獨感染外睪吸蟲釘螺體表分泌物厚度數倍(圖23)。兩吸蟲感染間隔時間增長，其體表分泌物不僅增多，而且其內容更加復雜化。其中晶體狀物體逐漸呈有規則地成條成片地排列(圖26～27)，在許多晶體樣物質之間除了有更多粘液樣物體之外還有許多原本是在釘螺體內的物質。兩吸蟲間隔85 d雙重感染，在血吸蟲感染后5 d～25 d的釘螺，它們體中都只能查到已完全解體的血細蟲幼蟲殘骸。在血吸蟲感染后5 d的釘螺體表分泌物(圖23)中在許多成片條狀晶體物質之間除有許多粘液狀物質之外還夾雜著許多原來在釘螺體內的血淋巴細胞和含極小細胞的分泌物顆粒(圖24～28)。

圖版箭矢數字說明(Arrownumbersillustrate):

1=大血淋巴細胞(bighemo-lymphocyte)；2=中血淋巴細胞(mediatebighemo-lymphocyte)；

3=小血淋巴細胞(smallhemo-lymphocyte)；4=螺體外分泌物(snailbodyoutersecretion)；

5=螺體內分泌物及小核(snailinnersecretionandverysmallnucleus)；

6=螺副腺細胞(snailaccessoryglandcell)。

圖說明(Em=Exorchismupingensis;Oh=Oncomelaniahupensis;Sj=Schistosomajaponicum)

圖1～6被Em感染的Oh其分泌物及副腺細胞

Figs.1-6SecretionsandaccessoryglandcellsofOhsnailsinfectedbyEmtrematodes

圖7間隔21d雙重感染Em和Sj的Oh示其體內分泌物和血淋巴細胞，及其對Sj幼蟲的反應

Fig.7OhsnailduallyinfectedbyEmandSjfor21dinterval,showingthesnail’ssecretions,lymphocytesandtheirreactionstoSjlarvae

圖8～11.間隔37d雙重感染Em和Sj的Oh示其體內分泌物和血淋巴細胞，及其對Sj幼蟲的反應

Figs.8-11OhsnailsduallyinfectedbyEmandSjfor37dinterval,showingthesnail’ssecretions,lymphocytesandtheirreactionstoSjlarvae

圖12～14間隔55d雙重感染Em和Sj的Oh示其體內分泌物和血淋巴細胞，及其對Sj幼蟲的反應

Figs.12-14OhsnailsduallyinfectedbyEmandSjfor55dinterval,showingthesnail’ssecretions,lymphocytesandtheirreactionstoSjlarvae

圖15～22間隔70d雙重感染Em和Sj的Oh示其體內分泌物和血淋巴細胞，及其對Sj幼蟲的反應

Figs.15-22OhsnailsduallyinfectedbyEmandSjfor70dinterval,showingthesnail’ssecretions,lymphocytesandtheirreactionstoSjlarvae

圖23～28.間隔85d雙重感染Em和Sj的Oh示其體內分泌物和血淋巴細胞，及其對Sj幼蟲的反應

Figs.23-28OhsnailsduallyinfectedbyEmandSjfor85dinterval,showingthesnail’ssecretions,lymphocytesandtheirreactionstoSjlarvae

圖1Oh體外分泌物 (Scale bar=0.15 mm)

Fig.1 Outer secretions ofOhsnail

圖2Oh體外分泌物 (Scale bar=0.03 mm)

Fig.2 Outer secretions ofOhsnail

圖3Oh體外分泌物(Scale bar=0.023 mm)

Fig.3 Outer secretions ofOhsnail

圖4Oh體內分泌物(Scale bar=0.034 mm)

Fig.4 Inner secretions ofOhsnail

圖5Oh體內分泌物(Scale bar=0.030 mm)

Fig.5 Inner secretions ofOhsnail

圖6Oh體內副腺細胞(Scale bar=0.022 mm)

Fig.6 Accessory gland cells ofOhsnail

圖7Em感染后37 dOh體中3種血淋巴細胞在16 d異常Sj幼蟲體內外(Scale bar=0.034 mm)

Fig.7 Three species hemo-lymphocytes in 16-d-old abnormalSjlarva and tissue of snail post-infected byEmfor 37 d

圖8 含極小細胞核的分泌顆粒散布在感染Em后42 d的Oh體組織中，具分泌物顆粒的球形紅團在5d異常Sj幼蟲體內(Scale bar=0.022 mm)

Fig.8 The secretion granules with very small cell nucleus spreading in the tissue ofOhpost-infected byEmfor 42 d and a spherical red mass with secretions granules in 5-d-old abnormalSjlarva

圖9Em感染后42 d的Oh示其體中5d異常Sj幼蟲含副腺細胞及具分泌物顆粒的紅團(Scale bar=0.027 mm)

Fig.9 Body ofOhpost-infected byEmfor 42 d showing the 5-d-old abnormalSjlarva containing the accessory gland cells and a spherical red mass with secretions granules

圖10Em感染后42 d的Oh示其體中血淋巴細胞及具紅團的5d異常Sj幼蟲 (Scale bar=0.030 mm)

Fig.10 Body ofOhpost-infected byEmfor 42 d showing the hemo-lymphocytes and 5-d-old abnormalSjlarva with hemo-cytes,red mass and secretion granules

圖11 帶有微小細胞核的分泌物顆粒團散布在Em感染后42 d的Oh組織中 (Scale bar=0.028 mm)

Fig.11 Secretion granules with small cell nucleus spreading in the tissue ofOhpost-infected byEmfor 42 d

圖12Em感染后66 d 的Oh和11 d 異常Sj幼蟲體中的具微小細胞核的分泌物顆粒(Scale bar=0.030 mm)

Fig.12 Secretion granules with small cell nucleus inOhpost-infected byEmfor 66 d and the body of 11-d-old abnormalSjlarva

圖13Em感染后79 d的Oh示24 d 異常Sj幼蟲殘骸內的血淋巴細胞和具微小細胞核的分泌物顆粒 (Scale bar=0.025 mm)

Fig.13Ohpost-infected byEmfor 79 d showing the hemo-lymphocytes and secretion granules with small cell nucleus in the 24-d-old abnormalSjlarva wreckage

圖14 大量分泌物團在Em感染后111 d的Oh及56 d異常Sj幼蟲殘骸內 (Scale bar=0.033 mm)

Fig.14Much secretions masses inOhpost-infected byEmfor 111 d and the 56-d-old abnormalSjlarva wreckage

圖15Em感染后76 d的Oh示6 d異常Sj幼蟲殘骸內的具分泌物顆粒2個紅團(Scale bar=0.016 mm)

Fig.15Ohpost-infected byEmfor 76 d showing two red masses with secretion granules and small cell nucleuses in the 6-d-old abnormalSjlarva wreckage

圖16 間隔70 d雙重感染Em和Sj后6 d的Oh體中散布許多具微小細胞核的分泌顆粒(Scale bar=0.028 mm)

Fig.16 Many secretion granules with small cell nucleus in theOhat the 6 d after dual infections ofEmandSjwith 70 d intervals

圖17Em感染后84 d的Oh示14 d異常Sj幼蟲殘骸內的紅團和微小細胞核(Scale bar=0.027 mm)

Fig.17Ohpost-infected byEmfor 84 d showing a red mass with small cell nucleuses in the 14-d-old abnormalSjlarva wreckage

圖18 間隔70 d雙重感染Em和Sj后25 d的Oh體中的許多具微小細胞核的分泌顆粒 (Scale bar=0.027 mm)

Fig.18 Many secretion granules with small cell nucleus in theOhat the 25 d after dual infections ofEmandSjwith 70 d intervals

圖19Em感染后84 d的Oh示25 d異常Sj幼蟲殘骸內外的具微小細胞核分泌物顆粒 (Scale bar=0.028 mm)

Fig.19Ohpost-infected byEmfor 84 d showing the secretion granules with small cell nucleus inside and outside of 25d old abnormalSjlarva wreckage

圖20 間隔70 d雙重感染Em和Sj后25 d的Oh體內許多具微小細胞核的分泌顆粒 (Scale bar=0.025 mm)

Fig.20Many secretion granules with small cell nucleus in theOhat the 25 d after dual infections ofEmandSjwith 70 d intervals

圖21Em感染后95 d的Oh示2個具分泌物顆粒的紅團在25 d 異常Sj幼蟲殘骸內 (Scale bar=0.027 mm)

Fig.21Ohpost-infected byEmfor 95 d showing two red masses with secretion granules in the 25-d-old abnormalSjlarva wreckage

圖22Em感染后95 d的Oh示副腺細胞在25d異常Sj幼蟲殘骸內 (Scale bar=0.030 mm)

Fig.22Ohpost-infected byEmfor 95 d showing the accessory gland cells in the 25-d-old abnormalSjlarva wreckage

圖23 間隔85 d雙重感染Em和Sj后5 d的Oh示體外分泌物(Scale bar=0.15 mm)

Fig.23Ohat the 5 d after dual infections ofEmandSjwith 85 d intervals showing its thick body secretion

圖24 間隔85 d雙重感染Em和Sj后5 d的Oh示體外分泌物中的體內血淋巴細胞(Scale bar=0.027 mm)

Fig.24Ohat the 5 d after dual infections ofEmandSjwith 85 d intervals showing hemo-lymphocytes in the body outside secretion

圖25 間隔85d雙重感染Em和Sj后5 d的Oh示體外分泌物中的體內小血淋巴細胞和分泌物顆粒微小細胞核(Scale bar=0.027 mm)

Fig.25Ohat the 5 d after dual infections ofEmandSjwith 85 d intervals showing small hemo-lymphocytes and secretion granules with small cell nucleus in the body outside secretion

圖26 間隔85 d雙重感染Em和Sj后5 d的Oh示體外分泌物中的結晶體和體內分泌物顆粒微小細胞核(Scale bar=0.019 mm)

Fig.26Ohat the 5 d after dual infections ofEmandSjwith 85 d intervals showing the opaque crystal structure of body secretion and inner secretion granules with small cell nucleus in the body outside secretion

圖27 間隔85 d雙重感染Em和Sj后5 d的Oh示體外分泌物中的結晶體和體內分泌物顆粒微小細胞核(Scale bar=0.019 mm)

Fig.27Ohat the 5 d after dual infections ofEmandSjwith 85 d intervals showing the opaque crystal structure of body secretion and inner secretion granules with small cell nucleus in the body outside secretion

圖28 間隔85 d雙重感染Em和Sj后5 d的Oh的體外分泌物中的結晶體和體內分泌物顆粒微小細胞核(Scale bar=0.019 mm)

Fig.28Ohat the 5 d after dual infections ofEmandSjwith 85 d intervals showing the opaque crystal structure of body secretion and inner secretion granules with small cell nucleus in the body outside secretion

3 討 論

實驗證明外睪吸蟲和日本血吸蟲雙重感染釘螺，其間隔時間愈長螺體殺害血吸蟲幼蟲的效力愈強，釘螺感染外睪吸蟲的開始時會增生大量血淋巴細胞，但在感染后一個月左右螺體增生血淋巴細胞性能就開始逐漸衰退，時間稍久后其數量逐漸顯著減少[2,4]；外睪吸蟲幼蟲期在釘螺體內無性生殖期很長，感染后105 d還處于原始胚細胞大量增殖期，6～7個月才成熟[1]。本實驗外睪吸蟲陽性釘螺同樣表現在其感染后37～85 d能逐漸地更有力地攻擊后侵入的血吸蟲幼蟲，使其致命解體，是否與螺體內外睪吸蟲胚細胞正在大量增生有關？螺體內分泌物逐漸大量增加是否也與其有關？但是為何螺體的血淋巴細胞卻在逐漸減少？其中奧秘機理需要繼續探究。

釘螺體內外的分泌物從那里產生？在外睪吸蟲和日本血吸蟲雙重感染的釘螺其體外分泌物中呈晶體結構是何物質？體內金黃色分泌物顆粒有增無減，每個顆粒團都更明顯出有細胞核和細胞質的極小細胞，這細胞和金黃色顆粒團是何關系？這些分泌物與血吸蟲幼蟲被擊毀有何關系？其中機理亦需繼續探究。

外睪吸蟲和日本血吸蟲雙重感染的釘螺，其體中被殺害的血吸蟲幼蟲體內經常有球狀紅團，兩吸蟲雙重感染間隔時間愈長這紅團數會從1個增加到4個。這紅團的產生與其外表分泌物顆粒及極小細胞核相仿的物體有何關系？與血吸蟲幼蟲的被殺有無關系？其中機理亦需繼續探究。

應用無害的外睪吸蟲作材料處理釘螺，所有實驗都證明可以百分之百殺死再侵入的日本血吸蟲幼蟲，有關此生物控制的機制都需要從多方面，包括免疫學、蛋白質組學和基因組學等等，進行深入研究，有利于更好地應用。當時研究生郭躍和王逸難同學協助感染工作，在此致謝。

參考文獻：

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