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標題: | 金絲桃素於人類肝癌細胞的攝取路徑與細胞內分佈之探討 A study of the internalization pathway and subcellular distribution of hypericin in human hepatoma cells |
作者: | In-Chi Young 楊穎奇 |
指導教授: | 何?芳(Yunn-Fang Ho) |
關鍵字: | 金絲桃素,膽固醇,微脂粒,低密度脂蛋白,細胞內分佈, hypericin,Hep 3B,liposomes,low density lipoprotein,subcellular distribution, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 研究背景
金絲桃素(hypericin, Hyp)是由Hypericum perforatum萃取而得的親脂性螢光物質。由於特定波長的光源照射能激發hypericin產生光學活性(photoactive properties),並生成氧化能力極強的活性態氧(reactive oxygen species,ROS),因此被歸類為第二代光敏劑,運用於光動力治療(photodynamic therapy,PDT)研究,供腫瘤診斷及治療。PDT主要為光敏劑經局部或全身性給藥後,再於腫瘤部位投予適當光源以激發照射部位之光敏劑,並藉由局部生成的ROS,造成氧化壓力而達腫瘤組織破壞。然而,由於ROS的半衰期極其短暫,因此只能於其生成部位之鄰近處進行作用,因之,推測PDT所造成的腫瘤組織傷害程度及型式,將取決於光敏劑於生物體或細胞內的分佈位置;而光敏劑在細胞內的分布傾向應會受到光敏劑的物化性質與細胞攝取路徑的影響。光敏劑進入細胞之攝取路徑,與其後續在細胞內分布位置間關聯探討,將有助相關分子機制之瞭解,提昇PDT療效與安全性。 研究目的 本研究運用人類肝癌Hep3B細胞,培養於無血清(serum-free)、添加低密度脂蛋白(LDL)或胎牛血清(FBS)的基質中,加入free-form hypericin及liposomal hypericin,探討:(1)不同hypericin製劑是否會影響其進入細胞之路徑及攝取量;(2)hypericin進入細胞後,其於細胞內各類胞器間之運送或分佈,是否隨劑型及時間而異。簡言之,本研究藉由比較兩種劑型之hypericin於三類細胞培養基質中之細胞攝取及胞內傳送路徑,期釐清脂質分子對細胞攝取hypericin與對hypericin於胞內分佈路徑的影響。 研究方法 研究中之liposomal hypericin製備,係採用薄膜水合法製作成大粒徑的liposomes顆粒後,再藉由奈米擠壓器利用外力擠壓將粒徑大小控制在約200 nm的均一粒徑,最後再以管柱層析法去除未包覆的hypericin。製備之liposomal hypericin再以粒徑分析儀及螢光光譜儀分別進行粒徑與包覆率的測定。在liposomes的粒徑穩定性測定方面,分別以保存穩定性、培養環境穩定性以及光照穩定性檢定,供確保後續細胞攝取或分佈實驗期間中,liposomal hypericin是完整且均一的。以上製備之liposomal hypericin將供做下述實驗之用途。 在Hep3B細胞攝取實驗中,則藉由hypericin經光源激發後會產生紅色螢光的特性,分別將cholesterol含量不等的liposomal hypericin [cholesterol比例分別為0%、10%及50%(molar ratio)]與free-form hypericin,各以相當於0.1 μM之hypericin加入不同培養基質[如:無血清培養液、添加2.5 μg/mL LDL的培養液及添加10%(v/v)FBS的培養液]中培養Hep3B細胞,經0.5至24小時培養後,以流式細胞儀分析細胞攝取之螢光強度,並比較各類培養基質及劑型對細胞攝取hypericin效率影響。 後續在細胞內分佈的探討,則是利用搭載著控溫箱(37℃)及恆定濃度CO2(5%)之螢光顯微鏡,經特異性染劑針對胞器如內質網、粒線體、高基氏體與溶酶體進行染色後,再加入liposomal hypericin與free-form hypericin各0.1 μM,並以曠時攝影方式觀察hypericin被同一細胞攝取入胞內後,在細胞內各胞器間傳遞路徑之變化,並嘗試量化各胞器hypericin螢光強度,以估量hypericin於細胞中的分布傾向。 研究結果 Liposome粒徑穩定性測定結果顯示,當liposome中的cholesterol組成比例達總莫耳數的50%時,可顯著提高liposomal hypericin的保存穩定性,減緩liposomal hypericin經培養液中LDL與FBS所引起的粒徑脹大的現象,並能有效抑制光照所造成的粒徑變化。而在細胞攝取研究中,發現free-form hypericin的細胞攝取量會受到培養液成分的影響。Free-form hypericin在無血清培養液中藉由被動擴散能夠最快速地進入細胞;如在培養液中添加2.5 μg/mL LDL,則得以輔助hypericin進入細胞,並在9小時後,其hypericin攝取量明顯高於無血清培養環境下的hypericin攝取量,或許與內包作用(endocytosis)相關;在添加FBS的培養環境下時,hypericin的細胞攝取速率最慢,可能與FBS中含有之大分子物質與hypericin間相互作用,致延緩了hypericin進入細胞的速率有關。至於liposomal hypericin製劑在細胞攝取量及攝取速率變化上,皆慢於free-form hypericin,推測在運送過程中因牽涉到hypericin於磷脂質與細胞外膜間的分配作用(partition),降低了hypericin與細胞接觸的機會,因而減緩其細胞攝取的量及速率。 在細胞內分佈實驗中發現,被動擴散的free-form hypericin主要集中於內質網,而藉由LDL協助進入細胞的hypericin則傾向分布於高基氏體。由liposome運送的hypericin主要出現在溶酶體,但於內質網或高基氏體的分布表現,仍分別可藉由無血清或添加LDL培養液的影響而增加。 結論 實驗結果證實free-form hypericin進入細胞的路徑,將影響其後續於細胞內的分布路徑。由於hypericin的高親脂性,其被動擴散速率明顯快於由LDL、liposomes或其他蛋白分子所導引的運輸機制,且在進入細胞後主要分佈至內質網區域;而在添加LDL的基質中,推測藉由hypericin與cholesterol的高度親和性,使得hypericin進入細胞後主要集中於高基氏體區域,且hypericin在粒線體的分佈也相對提高。本實驗所使用的liposome劑型,雖能提昇hypericin在溶酶體的分佈,但在短時間培養下仍不影響hypericin分布於內質網的傾向。未來若能針對hypericin與脂質或蛋白分子間的作用進行詳細探討,並研究在細胞毒性上的影響,便能夠在PDT的開發與運用上有所增益。 Background Hypericin , extracted from Hypericum perforatum, is a lipophilic fluoresecnt chemical with photoactive properties. Reactive oxygen species (ROSs) will be produced when hypericin is illuminated by light of proper wavelength. Hypericin is classified as a second generation photosensitizer with potential for photodynamic therapy (PDT) in the treatment or diagnosis of malignant diseases. PDT encompasses topical or systemic administration of a photosensitizer, followed by delivery of an excitation light to the site of lesion, to induce a locally generated ROS with high oxidative stress which leads to eradication of the target tissue. Since the extremely shortly lived ROSs can only act closely to its site of generation, the extent and type of photodamage in a cell would greatly depend on the exact subcellular localization of the photosensitizer. It is possible that the intracellular distribution of a photosensitizer might be associated with its physical and chemical properties and by its cellular uptake pathway. As a result, to identify the relationship between cellular uptake pathway and the following subcellular localization of a photosensitizer is crucial for the future development of PDT. Objectives The human hepatoma Hep3B cells were cultured in three kinds of media (serum-free, 2.5 μg/mL LDL, or 10% FBS) to study the impacts of different media on the cellular uptake and subsequent intracellular trafficking of free hypericin and liposomal hypericin over time. Furthermore, liposomal hypericin with various molar ratios of cholesterol was also employed to investigate the effect of cholesterol on the encapsulation efficiency, storage life, incubation and light irradiation stability of liposomal hypericin. Methods The multilamellar vecicles were made by thin-film hydration method and followed by extrusion and gel filtration to make uni-dispersed liposomes of particle size around 200 nm. The particle size and hypericin encapsulation rate were determined by a particle size analyzer and a spectrofluorometer, respectively. The storage life, incubation and light irradiation stability of liposomal hypericin were also assessed. The cellular uptake capacity of free hypericin and liposomal hypericin (with 0%, 10%, or 50% cholesterol, in molar ratio) was examined. Cells were incubated in different culture medium (serum-free, LDL-enriched, FBS-enriched) for 0.5 to 24 hours and, then, subjected to flow cytometry analyses. The subcellular trafficking of hypericin was observed by a fluorescence microscopy mounted with a thermostatted incubation chamber and CO2 supply. Subcellular organelles such as endoplasmic reticulum, mitochondria, Golgi apparatus, and lysosomes were all visualized by respective organelle-specific dyes. The intracellular localization of hypericin in a single cell was photographed by a time-lapsed microscopy, and the fluorescence intensity of hypericin within a certain organelle was estimated. Results The liposomal stability study shows that cholesterol content, by 50% of total lipids in molar ratio, helped stabilizing liposomal hypericin, prolonging its storage life, and preventing particle size enlargement induced by LDL/FBS and light irradiation. In cellular uptake study, the amount and rate of hypericin uptake were demonstrated to be dependent on the contents of cell culture media and its formulation. Free hypericin entered Hep3B cell rapidly, apparently through passive diffusion in a serum-free medium. With the addition of LDL, the uptake of hypericin by 9 hours of incubation was significantly increased, perhaps augmented by receptor-mediated endocytosis The slowest uptake was observed in medium containing 10% FBS, probably due to hypericin-macromolecule interaction that retarded the cellular uptake efficiency. As for liposomal hypericin, the uptake was much less and far more slower than the free-form hypericin in all media mentioned above. Partitioning of hypericin between liposomes and the cell membrane is suspected. The subsequent subcellular localization experiment revealed that trafficking pathways differs among incubation media and formulation. We found that, under the study condition, the passively diffused hypericin was mainly distributed into the endoplasmic reticulum, and the LDL-mediated hypericin uptake was geared to the Golgi apparatus. Although liposomal hypericin was localized mainly in lysosomes, an enhanced distribution of liposomal hypericin was found in endoplasmic reticulum and Golgi by serum-free or LDL-enriched medium, respectively. Conclusion The subcellular distribution of hypericin is associated with its cellular uptake pathway. The passively diffused hypericin entered cells faster than pathways mediated by LDL, liposomes, or other marcromolecules such as FBS. It mainly distributed into endoplasmic reticulum regions. In the presence of LDL, hypericin was guided majorly to the Golgi apparatus region and the mitochondria distribution was also increased. The high affinity between cholesterol and hypericin might play an important role in this observation. Although the liposome preparations could increase the hypericin distribution in lysosomes, the endoplasmic reticulum was still the primary localization target of liposomal hypericin. Following our study, in the future, if the mode of interaction and affinity propensity among hypericin, lipids, and protein molecules could be thoroughly investigated, along with cytotoxicity studies, there would be a great improvement in the applications and development in the field of photodynamic therapy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9017 |
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