- 學位論文
膽鹽依賴性脂肪酶的純化及其與人類腸道細胞株Caco-2之交互作用
Purification of bile-salt dependent lipase (BSDL) and its interaction with human intestinal cell line Caco-2
摘要
雖然目前已有許多蛋白質藥物於臨床上使用，但其給藥方式仍以侵入性給藥為主，其醫療花費成本高，容易引起病人的不適，並造成順醫矚性較低，若改以口服給藥方式投與蛋白質藥物，可以改善這些缺點，但蛋白質藥物的口服給藥仍處在研發階段。蛋白質藥物口服給藥最困難處是在於其低身體可用率，而低身體可用率主要是因為蛋白質藥物在腸胃道的低吸收率、蛋白酶的分解和吸收之後在血中的半衰期短。因此，研究增加蛋白質口服給藥之效率的策略，並期望將來可以於臨床上使用。 膽鹽依賴性脂肪酶 (Bile-salt dependent lipase, BSDL, EC 3.1.1.13)，主要是在胰臟細胞中合成，再從胰臟細胞分泌到小腸內來幫助日常飲食中脂質的消化和吸收，已有文獻指出，膽鹽依賴性脂肪酶可以經由受體媒介穿胞運輸來穿過腸道細胞，並保持其完整酵素活性。因此，本研究論文將：1. 驗證膽鹽依賴性脂肪酶與腸道細胞作用之專一性。2. 建立膽鹽依賴性脂肪酶和蛋白質藥物之接合體，並評估膽鹽依賴性脂肪酶作為蛋白質藥物的腸道載體之可行性。 我們選用AR42J大鼠胰臟上皮細胞株，作為膽鹽依賴性脂肪酶的純化來源，並採用Caco-2人類腸道上皮細胞株，作為體外實驗的模式。首先，利用無血清培養基來收集AR42J cultured medium，以簡化膽鹽依賴性脂肪酶的純化流程。純化方法一是使用實驗級切向流濃縮純化透析系統 (Millore® Pellicon XL cassettes and labscale TFF system) 來濃縮，並配合Sephacryl® S-200 High Resolution分子過濾膠體來分離，可以得到純度近90% 且蛋白活性為47.13 Units/mg的膽鹽依賴性脂肪酶，而其回復率約為67.32%；純化方法二是先用Q-sepharose® fast flow陰離子交換膠體進行粗分，再利用Sephacryl® S-200 High Resolution分子過濾膠體來分離，可以得到純度近90~95% 且蛋白活性為17.15 Units/mg的膽鹽依賴性脂肪酶，而其回復率約為41.74%。 給予Caco-2細胞125I-膽鹽依賴性脂肪酶和125I-過氧化酶來進行結合和攝取實驗，結果顯示，在0.1~1 μM的濃度之下，Caco-2細胞對膽鹽依賴性脂肪酶和過氧化酶的細胞內攝取分別為：-0.03±0.09, 1.23±0.21, 1.91±0.04 pmole和0.08±0.1, 0.05±0.02, 0.1±0.14 pmole，膽鹽依賴性脂肪酶的細胞內攝取量隨濃度增加而增加；而過氧化酶則否，僅能結合停留在Caco-2細胞表面。Caco-2穿胞運輸實驗結果顯示，膽鹽依賴性脂肪酶和過氧化酶的穿胞運輸和Papp為分別為1.39±0.11 pmole, 6.53±0.55*10-6 (cm/s) 和1.69±0.41 pmole, 7.88±1.88*10-6 (cm/s)，其總量和速率是相近的，但穿胞運輸後具有酵素活性的膽鹽依賴性脂肪酶為131.12±36.62 fmole，佔其穿胞運輸總量的10%，並沒有偵測到任何過氧化酶之酵素活性。 以雙反應性化學連接試劑SPDP，將過氧化酶與膽鹽依賴性脂肪酶以共價鍵接合，並藉由在波長280 nm的吸光值和SDS-PAGE來分析合成之產物，實驗結果發現，膽鹽依賴性脂肪酶-過氧化酶接合體之產物以非單一特定 (heterogeneous) 的鍵結方式連接。給予相同酵素活性的膽鹽依賴性脂肪酶-過氧化酶接合體、膽鹽依賴性脂肪酶-過氧化酶混合物和過氧化酶進行Caco-2穿胞運輸試驗，利用酵素活性和西方墨點法 (Western blot) 分析其底部培養基，均無任何可被偵測到的HRP訊號。所合成的非單一特定膽鹽依賴性脂肪酶-過氧化酶接合體無法攜帶過氧化酶經由穿胞運輸至Caco-2細胞的另一端。 綜和所有實驗結果，除了建立膽鹽依賴性脂肪酶的純化流程之外，膽鹽依賴性脂肪酶於Caco-2細胞的細胞內攝取和穿胞運輸後的酵素活性均優於過氧化酶，顯示其作為蛋白質藥物腸道載體之發展潛力。雖然本研究中合成的非單一特定的膽鹽依賴性脂肪酶-過氧化酶接合體無法攜帶氧化酶穿過Caco-2細胞，但在未來，可更進一步探討化學連接試劑之長度、蛋白藥物大小和特定單一性鍵結對於膽鹽依賴性脂肪酶作為蛋白質藥物腸道載體穿胞運輸效率之影響。
並列摘要
Recombinant insulin, the first commercially available therapeutic product, was approved by FDA in 1982. Since then, more and more proteins or peptides are the products of DNA recombinant technology and applied in pharmacotherapeutics to treat various diseases of cancers, blood dyscrasia, and infections, etc. Most of them are administered by injection which causes patients’ inconvenience and non-compliance. Oral delivery of biological therapeutics may be another alternative solution of these problems. Although oral administration of bioactive therapeutics is very attractive, there are some limitations of oral route, such as enzymatic degradation, poor absorption and short plasma half-life. Bile-salt dependent lipase (BSDL, EC 3.1.1.13) is synthesized in the pancreas and secreted into intestine to digest the dietary lipids. Recently, BSDL was reported the ability to across the intestinal cell monolayer by receptor-mediated transcytosis without degradation and maintained its enzyme acticity. Therefore, the specific aims of this study would like to confirm the specific interaction between BSDL and intestinal cells. In addition, conjugate the the BSDL and model protein drug by chemical covalent bond and evaluate the possibility of BSDL as a protein drug carrier in the intestine. In our study, BSDL was collected and purified from the cultured medium of rat pancreatic epitheial cell line AR42J and human intestinal cell line, Caco-2, was used as our in vitro model to assess the transport of BSDL into the intstine. AR42J cells were maintained in medium containing 10% fetal bovine serum (FBS) under normal condition. When cells reached the confluence, the medium was changed to the serum free medium in order to simplify the downstream purification. Two different purification methods were used. Method 1: the AR42J cultured medium was concentrated by the TFF ultrafiltration system and purified by Sephacry® S-200 (size exclusion) resins. The specific activity of BSDL was 314-fold increase from 0.15 to 47.13 Units/mg. After the serial purification processes, the recovery rate was nearly 67.32% and the purity of the purified BSDL was about 90% by image intensity analysis of SDS-PAGE. Method 2: The cultured medium was collected and applied to the Q-sepharose® fast flow (anion-exchange) column and subsequently to the Sephacry® S-200 (size exclusion) resins. The specific activity of BSDL was 117-fold increase from 0.15 to 17.15 Units/mg. After the serial purification processes, the recovery rate was 41% and the purity of the purified BSDL was 90%~95%. BSDL and negative control, horseradish peroxidase (HRP), were incubated, respectively, with Caco-2 cells grown on 24-well plate. At 0.1 ~ 1 μM concentrations, the intracellular uptake amounts of BSDL and HRP were -0.03±0.09, 1.23±0.21, 1.91±0.04 pmole and 0.08±0.1, 0.05±0.02, 0.1±0.14 pmole. The amount of intracellular uptake of BSDL was increased in a concentration-dependent manner but that of HRP was not. 0.3 μM BSDL and HRP were added at the apical side of Caco-2 monolayer grown on the transwell and medium from the basolateral side were collected and analyzed. After six hours incubation, the transcytosis amounts and Papp of BSDL and was 1.39±0.11 pmole, 6.53±0.55*10-6 (cm/s), and that of HRP was 1.69±0.41 pmole, 7.88±1.88*10-6 (cm/s). However, only transcytosed BSDL processed enzyme activity, but the enzyme activity of HRP was not detectable. BSDL and HRP were conjugated by bi-functional crosslinker SPDP and the synthesized product was analyzed by the absorbace at 280 nm and SDS-PAGE. It was found that BSDL-HRP was heterogeneous conjugate. Dosed the same enzyme activity of BSDL-HRP conjugate, BSDL-HRP mixture and HRP alone to the Caco-2 monolayer for the transport assay and the medium from the basolateral side was collected and analyzed by the enzyme activity assay and Western blot. However, there was no any detectable signal of HRP. The synthesized heterogeneous BSDL-HRP conjugate did not carry HRP to the other side of Caco-2 monolayer in this stidy. From our experiment results, besides the establishment of model of BSDL purification, the intracellular uptake and specificity of BSDL to the Caco-2 cells are better than that of HRP. It indicated the promising potential of BSDL as the protein carrier in the intestine. Although the synthesized heterogeneous BSDL-HRP conjugate did not carry HRP to the other side of Caco-2 monolayer, the effects of length of crosslinker, molecular weight of protein drug and homogeneous conjugation on the BSDL as the protein drug carrier in the intestine should be further investigated in the future.