第5卷‧第4期,
198110
, pp. 375-384
廣東住血線蟲第一期、第三期幼蟲及成蟲之體外培養
- 作者:
陳秀男; 唐堂; 李筱禎
- 作者服務機構:
國立臺灣大學理學院動物學系
- 中文摘要:
本實驗的主要目的是嘗試發展一種體外培養系統,來培養廣東住血線蟲一、三期幼蟲及成蟲使之不僅能長時間的存活,而且可有進一步之發育。 實驗的結果顯示:最適合的pH值是在6至8之間。將第一期幼蟲培養於RPMI-1640,McCoy's,MEM,L-15,及TC199等培養基中,可存活27~29天。在36±1℃溫度下,幼蟲以MEM培養15天後,長度不增加,但寬度增加了13%。若以MEM和TC199分別添加10~40%牛胚胎血清,初生牛血清,1%MEM vitamin,1%酵母萃取液及蝸牛萃取液來培養一期幼蟲,則發現此等物質並不能增加幼蟲體外發育及延長存活的日數,幼蟲只能存活約29天。然而若將幼蟲培養在蒸餾水或生理食鹽水中,於36±1℃下培養12小時後,則呈現不太活動的現象;而於5天培養後,全部死亡。 關於廣東住血線蟲第三期幼蟲之培養,實驗結果顯示:利用不同添加物的培養基,對增加感染性幼蟲的存活率並無顯著的差異。在包括RPMI-1640,MEM,TC199及L-15的培養基內,第三期幼蟲平均可存活48~52天,但培養在初級培養細胞內之幼蟲則平均可存活85~90天。實驗結果亦發現,加入rat brain extract,CEE和BEE亦可延長第三期幼蟲存活天數。有關荷爾蒙及神經傳導物質之實驗則顯示出:僅serotonin creatine sulfate,serotonin hydrogen oxalate,γ-amino-N-butyric acid等三種人工合成神經傳導物質,可增進幼蟲的體外發育,並促使之脫皮。然而這些物質卻減低了感染性幼蟲在體外培養的存活天數。另外將第三期幼蟲培養在老鼠肺、腦膜初級培養細胞中,添加CEE,BEE或ratbrain extract經36±1℃60天培養後,亦可觀察到脫皮現象。 成蟲培養在L-15加入10%牛胚胎血清(GM L-15),並充以5%CO2,可存活7天。若將之培養在含GM L-15之鼠肺初級培養細胞內,則可延長成蟲存活率至21天。但不論培養在GM L-15培養基中或鼠肺初級培養細胞內,經20小時培養後,均可發現成蟲有排卵的現象。此現象持續至75小時後方停止,且培養在GM L-15中之成蟲所排的卵較多。實驗中並發現,所有成蟲於體外培養系統內所排出之蟲卵,均為一或二個細胞的階段;且不論在GM L-15或鼠肺初級培養細胞的蟲卵,經過10天以上之培養,均不能孵化成幼蟲。 以上結果顯示:廣東住血線蟲之幼蟲及成蟲體外存活培養系統的條件,已可確立。雖此系統僅能部份促進幼蟲的生長與發育,但長時間存活日數之體外培養系統已足以供給往後對廣東住血線蟲之生理、生化及藥物開發的研究之用。
- 英文摘要:
The present study attempts to develop a culture system which can maintain the 1st and infectivestage larvae and adult worms of Angiostrongylus cantonensis and support the development of these threestages. The most favourable condition for the maintenance of the 1st stage larvae in vitro was in a range ofpH value between 6 and 8. In these culture media including RPMI-1640, McCoy's medium, Eagle's minimalessential medium (MEM), Leibovitz's L-15 (L-15)and tissue culture medium 199 (TC 199) the 1st stagelarvae survived for 27 to 29 days. Following 15 days incubation in MEM at 36±1℃, the larvae obtainedincreases of 13% in width and no increase in length. No enhancement of the development nor increase ofsurvival time of the 1st stage larvae was obtained using MEM and TC 199 supplemented with 10-40%foetal calf serum and new born calf serum, 1% MEM vitamin, 1% yeast extract and snail extract. However,when the larvae were cultivated in distilled water or in normal saline, following 12 hours' incubation at36±1℃, they became very lethargic. All the larvae died after 5 days' incubation. Using different culture media, no significant differences were observed in the survival of the 3rdstage infective larvae. In contrast to the average larval survival of 48-52 days in tissue culture media alone,the larvae survived for 85-90 days in rat tissue cell cultures. The larval survival was prolonged when ratbrain extract, chicken embryo extract (CEE) and bovine embryo extract (BEE) were used. The resultsalso demonstrated that three synthetic neural transmitters including serotonin creatinine sulfate, serotonin-hydrogen oxalate and γ-amino-N-butyric acid enhanced the larval development and moulting. However,these compounds depreciate the survival of the infective larvae of Angiostrongylus cantonensis in vitroWhen the infective larvae were cultivated in rat lung and meninges cell cultures and CEE, BEE or rat brainextract medium, moulting was observed following approximately 60 days' incubation of the larvae at 36±1℃. In culture system consisting of L-15 plus 10% foetal calf serum (GM L-15) gassed with 5% CO2,A. cantonensis adult worms survived approximately 7 days. However, the rat lung cells in GM L-15 pro-longed the survival of the worms up to 21 days in vitro. Following approximately 20 hours' incubationeither in GM L-15 or in GM L-15 with rat lung cells, the eggs were extruded. The egg production ceasedwhen the worms had been incubated for 75 hours. Also, the egg protrusion in GM L-15 was higher thanthat in GM L-15 with rat lung cells. All the eggs extruded from the worms in vitro were in one or two-cell stage. During the course of egg cultivation in GM L-15 with lung cells, no hatched larvae wereobserved. In this study, conditions for the maintenance in vitro of 1st stage larvae, 3rd stage larvae and adultworms of A. cantonensis have been determined. Although the culture system could only support a partialgrowth of the larvae and no development of reproductive organs of adults, long time survival in vitro ofthese stages would provide valuable systems for the studies on the physiology, biochemistry and experi-mental chemotherapy of A. cantonensis.
- 中文關鍵字:
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- 英文關鍵字:
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