以體外細胞培養法建立抗病毒中藥篩檢：大蒜與台灣金線蓮抽取液之評估   全文下載

Evaluation of an in Vitro Virus Culture System for Anti-Virus Study of the Chinese Herb

使用抗A型流行性感冒病毒的兩種單株抗體：抗病毒核殼蛋白（anti-NP）與抗病毒血球凝集素（anti-HA），做免疫螢光染色，可判定HL-CZ細胞受病毒感染的情況。在測知大蒜精溶液及台灣金線蓮抽取液的最低細胞毒殺濃度分別為10倍稀釋（10%w／v）與4.0 mg／ml之後，以稍低於此，濃度的藥液與病毒懸浮液一起加入細胞培養中，在數個特定培養時段分別收集定量細胞做成抹片，並給予免疫螢光染色。初步結果顯示，只接種病毒的陽性對照組及加入大蒜液、金線蓮液與三環胺（amantadine，西藥對照）的實驗組，其平均抗NP與抗HA螢光細胞百分率分別為59.7%、19.4%、33.6%及30.6%，據此推測，這些藥液在48小時內可部分抑制病毒抗原在細胞內的表現。如果以核酸聚合?連鎖反應（PCR）來偵測細胞內病毒RNA反轉錄成cDNA之質與量則可用來了解藥液對病毒基因在細胞內複製的影響，目前已知病毒的第四段RNA（HA1 基因）在感染細胞後12小時可用PCR方法測得。我們的這套實驗模式可做為未來發展抗病毒中藥體外篩檢研究之參考。

Influenza A virus was selected for anti-virus study with a garlic extract and a Chinese herb. Anoectochilus formosanus Hay. The virus was inoculated into the human promonocyte cell line. HL-CZ, which had been estabished in this laboratory. The infected cells have shown viral antigens detected by immunofluorescence staining with both monoclonal antibodies against virus nucleoprotein (NP) and hemagglutinin (HA) after 1-3 days of virus inoculation. This in vitro virus culture system was used as a tentative model for evaluating anti-virus activity of Chinese herbs. The relative minimum concentration of cytotoxicity (MCC) of the herb preparation to the HL-CZ cells was observed and determined during 7 days of cultivation. The MCCs tested for garlic and A. formosanus Hay extracts were I: I 0 dilution (from the original package of garlic essence) (10% w/v) and 4.0 mg/ml, respectively. In several experiments. the average number of cells infected with the influenza A virus was 59.7%; those of the cells simultaneously treated with sub-MCC of garlic. A. formosanus Hay and amantadine solutions were 19.4%. 33.6%. and 30.6%, respectively. These results suggest that the expression of influenza viral anitgens in cells was partially inhibited by the component in these herb extracts. In addition. the replication of viral RNA was measured by polymerase chain reaction (PCR) after the HA1 region of viral hemagglutinin gene was converted into cDNA with reverse transcription. It was shown that viral HA gene could be detected 12 hours post-inoculation even in the presence of Chinese herbs. This method could he applied to detect the replication of viral RNA when the virus-infected cells were treated with the extracts of Chinese herbs.