菘藍毛狀根之建立及其藥效成分靛玉紅之生產研究

Abstract

本論文之目的為建構菘藍(Isatis indigotica Fort.)之毛狀根單株細胞，預期用以生產臨床中藥板藍根之藥效成分靛玉紅。 菘藍為二年生草本，常見中藥材「板藍根」之基原植物，其為臨床常用之中藥，具有清熱解毒、涼血利咽、解熱、抗炎等作用，現代藥理試驗亦證明其具有抗菌、抗病毒、增強免疫力及抗內毒素等作用。菘藍中含有多種化學成分，其中靛玉紅(indirubin)具有抗腫瘤作用，亦為近期被熱烈研究之藥用成份。 本研究藉農桿根群菌(Agrobacterium rhizogenes)感染菘藍外植體，既而誘導基因轉型產生毛狀根單株細胞後，以PCR進行轉形基因之確認，篩選出生長快速之菘藍毛狀根細胞。 本研究依各單株化毛狀根之生長速度及目標成分累積量篩選出優良品系，再探討菘藍毛狀根之誘導與培養、轉形基因之確認、毛狀根之生長曲線與生長動力學。培養至56天後，毛狀根乾重增加68.5倍，靛玉紅產量增加33.7倍。之後探討最佳培養方式進行毛狀根之單株培養，可得乾重0.065克，靛玉紅產量0.197毫克，最後達到快速提升靛玉紅產量之目的。 激發因子(elicitors) 能夠誘導植物產生植物防禦素(phytoalexins) ，進而誘導植物二次代謝物之生合成，因此成為有效增加毛狀根二次代謝物產量的方法。本研究亦檢討不同類型之激發因子對菘藍毛狀根靛玉紅累積量之影響，結果以茉莉酸(jasmonic acid)的誘導效果最佳，其最適作用濃度為250 μM，毛狀根在添加茉莉酸之三天後可得到較對照組提高6.08倍之靛玉紅含量。 為了檢驗菘藍毛狀根之氯仿萃取物是否能夠對癌細胞產生細胞毒性(cytotoxicity) 之作用，以兩種人類癌細胞株Hela及HepG2進行測試，觀察其生存率及型態變化。結果顯示毛狀根萃取物能夠使細胞形態出現明顯變化，包括細胞形狀較不規則、細胞膜皺縮、細胞核裂解等，並且顯著抑制Hela細胞的生長，40μM靛玉紅含量之萃取液使Hela細胞生存率下降至55.1%。建構菘藍之毛狀根細胞，可預期具備生長快速、培養時間短、藥用成分累積快速、成本較低等優點，將可促進對菘藍藥用成分之研究並擴大其醫療應用之貢獻。

The object of this paper is to study on the hairy root cultures of Isatis indigotica Fort, and to anticipate the production of indirubin in Chinese herbal medicine, Ban Lan Gen. Isatis indigotica, a biennial herbaceous plant, is the botanical origin of the common herbal medicine, Ban Lan Gen. Ban Lan Gen is a common herbal medicine for clinical cure, and it has some therapeutic mechanism in antipyretic and anti-inflammatory properties.In recently pharmacological test, it proves working against anti-inflammatory, anti-bacteria, anti-virus, anti-endotoxin , and so on. (Chang, 2007) Isatis indigotica contains many chemical ingradients, and one component in the plant, indirubin, has been extensively investigated for its anti-tumor activities. Explants of Isatis indigotica were infected by Agrobacterium rhizogenes that hairy roots were induced on the explants. The gene transformation of monoclonal hairy-root cultures were identified by PCR. And then, fast-growing clones were screened and cultured. This study has screened the best hairy root cultures depending on the growth rate and accumulation of medical ingredients. And then, we investigate the induction, cultivation, identification of the gene transformation, growth curve, growth kinetics. In a 56 days’ culture, the dry biomass of the hairy root increases 68.5 times and the indirubin production increases 33.7 times. We investigate the optimum medium to culture the hairy root. The biomass is 0.065g and the indirubin is 0.197mg in 21 days’ culture. Therefore, we could cultivate the hairy roots in optimum cultural condition to increase the productivity of indirubin. The addition of elicictors to the hairy root cultures is an effective method for inducing the biosynthesis of plant secondary metabolites by increasing the accumulation of phytoalexins in plant cells. In this study, using different types of elicitors to investigate the effect of the indirubin content in Isatis indigotica hairy roots and found that the most effective one was jasmonic acid. Three days after the addition of jasmonic acid to the hairy root cultures by final concentration of 250μM, the indirubin content was 6.08-fold increase compared with the controls. To analyze the cytotoxicity of the hairy root chloroform extracts, we use two human carcinoma cell lines, Hela and HepG2 cells, to examine the viability and morphology.It results the morphological changes in Hela and HepG2 cells were observed when exposed to the hairy root extracts, including membrane shrinking, and nuclear fragmentation. Obviously, hairy root extracts can inhibit the viability of Hela cells. The extracts containing 40μM indirubin could decrease the viability to 55.1% of the Hela cell The hairy root of Isatis indigotica has many advantages including fast growing rate, short cultivating time, fast accumulation of medical ingredients, lower cost, and so on. It will advance in studies of medical components of Isatis indigotica and expand contributions of the medical application.