- 學位論文
廣東住血線蟲從第三期幼蟲體外培養到第四期幼蟲之研究
Angiostrongylus cantonensis:In Vitro cultivation from the infective Third-stage larvae to the Fourth-stage larvae
摘要
本研究之目的欲探討適當培養基使廣東住血線蟲第三期幼蟲藉體外培養模式發育到第四期幼蟲，嘗試找出蟲體發育對養分成分的需求。 廣東住血線蟲的第三期幼蟲以MEM medium添加胺基酸、脂肪酸及碳水化合物的培養基(完整培養基)在37℃含有5％ CO2的無菌培養箱中，培養至第21天已發育至第三期幼蟲中期的階段，第24天已發育至第三期幼蟲末期的階段，第30天開始出現帶鞘的第四期幼蟲，第36天則觀察到蟲體明顯變長並蛻去外鞘及第三期幼蟲特有的眼點(rod-shaped)消失的第四期幼蟲。而比較添加五種不同濃度胎牛血清之完全培養基，第三期幼蟲在含20%之胎牛血清的完全培養基培養36天時發育到第四期幼蟲的發育率為41±5%最理想。 比較第三期幼蟲蟲體於含20%胎牛血清的完全培養基的體外培養與蟲體在BALB/c小鼠腦內的發育，發現體外培養蟲體至少需30天才發育至第四期幼蟲，而在小鼠腦內僅需5天，而且前者蟲體大小顯著小於後者( p < 0.01)。 為了能更進一歩鑑別出蟲體發育對所添加的胺基酸，脂肪酸或醣分的需求何者重要，本實驗以刪除一項或兩項添加條件的非完整培養基進行進行培養，結果顯示以六種非完整培養基培養20天，第三期 幼蟲的存活率均低於30%，而且都無法進一歩的發育。以完全培養基進行培養的幼蟲卻可以維持90%以上的存活率並且觀察到發育的情形，因此我們推論要以體外培養的方式使廣東住血線蟲第三期幼蟲發育到第四期幼蟲，完全培養基所含有的添加物都是它們生長發育與存活所必須具備的。
並列摘要
An attempt to cultivation of Angiostrongylus cantonensis from third-stage larvae to fourth-stage larvae in vitro were successfully reach in chemically defined medium. The third-stage larvae were developed to fourth-stage larvae (the most optimal develop rate was 41%) when cultured in defined complete medium, further, cultured in defined incomplete medium, was examined no develop and a poorly survival rate. When the third-stage larvae were cultured in the defined complete medium under 37℃ and 5% CO2 in air, the larvae were began to develop to the fourth-stage larvae in cultured for 30 days, being enclosed within the sheaths of the third molts of life cycle. Under these conditions, the larvae developed uniformly and the larvae reached to the fourth-stage larvae at 36 days later. When compared to the fourth-stage larvae from BALB/c mice , the larvae cultured in vitro is shorter. The additives were refer to 3 kinds of nutrient source: Amino acid, Fatty acid and Carbohydrates. The based medium used to addition was the Minimum Essential Medium Eagle(MEM)(Sigma), and it was successfully reached to fourth-stage larvae when cultured in defined complete medium in vitro. Therefore, for distinguish the requirement of development, we cultured in defined incomplete medium. The result of cultured by defined incomplete medium showed that can not be survive enough long time to develop, thus we infer only the defined complete medium can make the third-stage larvae develop to fourth-stage larvae in vitro.