以類胡蘿蔔素穩定之微脂粒包覆去氧核醣核酸水解酶之研究

Research of DNase encapsulated by carotenoids stabilized liposomes

楊政峯(Cheng-Feng Yang)

指導教授 ： 王鐘毅

大同大學/工程學院/生物工程學系所/碩士(2007年)

摘要

DNase（Deoxyribonuclease）是一種去氧核醣核酸水解酵素，目前被使用在囊胞性纖維症的治療，能分解黏液中因多核球被破壞而釋出的 DNA，降低肺部黏液的黏稠度，進而減少細菌孳生所造成的肺部發炎。目前的劑型是利用壓縮氣體將 DNase 溶液霧化後送至肺部，DNase 可能因接觸空氣而造成活性降低，同時氣霧微粒也容易聚集成較大的顆粒，無法順利送達肺部深處。另外，DNase 溶液在 37℃ 下的安定性不佳，可能影響 DNase 溶液在室溫保存的期限，因此本研究的目的是利用微脂粒作為藥物載體，開發適合包覆 DNase 之新劑型。
在本論文中，我們利用基因重組菌種 E.coli W3110 來發酵生產 DNase，純化後所獲得的 DNase 產量為 27 mg/L。另外我們也藉由在微脂粒中添加不同的類胡蘿蔔素來提高微脂粒的穩定性，在微脂粒中添加蝦紅素能有效的降低膜的滲透性，而菌紅素則可降低膜的流動性並且能延緩脂質的過氧化。
在數種不同的 EYPC（L-α-Phosphatidylcholine） 微脂粒配方中，則以添加 10mM 的氯化鈣的微脂粒配方能得到最佳的蛋白質包覆率（44.2 ％）與活性包覆率（45.2 ％）。

關鍵字

微脂粒 ； 囊胞性纖維症 ； 去氧核醣核酸水解酶 ； 蝦紅素

並列摘要

Deoxyribonuclease（DNase）is currently used for the treatment of cystic fibrosis (CF). CF patients often suffer pneumonia which caused by very viscous sputum. The destruction of polymorphonuclear cells and the release of DNA lead to viscous sputum. DNase can digest excess DNA in sputum, which makes patients easier to clear sputum from the airways with coughing. DNase was formulated as solution and aerosolized for patient to inhale. However, the aggregation of aerosol and the inactivation of DNase during the inhaling process may need the extra dose of DNase, which is very expensive. In this thesis, recombinant DNase was purified from fermentate of cell line E.coil W3110. The T2A-DNase domain-Im7 complex was first obtained with Ni-NTA column. Im7 was then removed with ion exchange. The yield of purified DNase was 27 mg/L. Liposomes composed of egg yolk phosphatidyl- choline (EYPC) were stabilized with astaxanthin or bacterioruberin to develop a carrier system for DNase. Addition of astaxanthin or bacterioruberin into liposomes can effectively decrease permeability and fluidity of lipid bilayers, which may stabilize liposome formulation. The size of liposomes and the encapsulation efficiency were determined. Among the formulations, addition of 10mM calcium chloride resulted in the best DNase encapsulation efficiency.

並列關鍵字

liposome ； cystic fibrosis ； deoxyribonuclease ； astaxanthin

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