在肠系膜淋巴管空心大鼠模型：应用肠淋巴药物运输的评估
Summary
Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.
Abstract
肠道淋巴系统在流体输送，脂质的吸收及免疫功能键的作用。淋巴流直接从小肠通过一系列的淋巴管和节点会聚在肠系膜淋巴管。的肠系膜淋巴管插管从而使肠系膜淋巴结从肠流出的集合。肠系膜淋巴结由免疫细胞（99％淋巴细胞），水相（流体，肽和蛋白质，例如细胞因子和胃肠激素）和脂蛋白组分（脂质，亲脂性分子和apo-蛋白）的细胞级分。的肠系膜淋巴管插管模型因此可用于测量通过淋巴系统的浓度和一系列从肠道因素的输送速率。改变这些因素以响应不同的挑战（ 例如，饮食，抗原，药物）和在疾病（ 例如，炎性肠病，艾滋病，糖尿病）也BË确定。扩大感兴趣的一个领域是淋巴运输中的口服亲脂性药物和药物前体，与肠脂质吸收途径关联的吸收的作用。这里，我们描述，详细地说，一个肠系膜淋巴管空心大鼠模型使通过淋巴系统的速率和脂质和药物输送的程度的评价为以下的肠释放数小时。该方法是很容易适应在淋巴其它参数的测量。我们提供了建立这种复杂的外科手术方法时可能遇到的困难的详细描述，以及从实验失败和成功的有代表性的数据，以提供指令，关于如何确认实验的成功和解释所获得的数据。
Introduction
从小肠通过一个单向过程，起源于所包含的每个小肠绒毛1内单乳糜淋巴流动。乳糜是相对可渗透的流体，大分子和细胞和淋巴形成从而开始与这些因素进入乳糜。在乳糜初始淋巴随后从肠经由淋巴微血管网络，收集（传入）淋巴管中，一系列的肠系膜淋巴结，并最终在后节点（传出）淋巴管流动。中的节点，淋巴通过一系列髓窦的其中交换发生在节点驻留的免疫细胞，以及材料进入从血液中的节点传递。所有淋巴从小肠流入最终收敛到传出肠系膜淋巴管，随后将乳糜池。该乳糜池还收集淋巴引流尾外周组织，INTEST伊纳勒，肝和腰部区域和一起加入胸淋巴导管与来自纵隔和身体的颅部位淋巴结。胸淋巴导管直接排空淋巴液进入静脉系统在左侧颈内静脉和锁骨下静脉的交界处。这里所描述的协议，使淋巴收集直接从肠系膜淋巴管，从而便于直接从通过肠道淋巴系统肠道全身（普通）循环过渡的各种因素进行分析。
分配到肠淋巴系统的主要生理功能是维持体液平衡，以促进脂质和亲脂性分子的吸收，以及使适当的免疫应答1。肿瘤细胞和病毒也可通过淋巴管肠道淋巴管内发生的几种炎症和代谢病症5-7 2-4，关键的变化传播。可以的肠系膜淋巴管nulation收集淋巴结内肠系膜使得能够经由肠淋巴散装流体流动分析的各种细胞和分子的浓度和输送速率，以及量化。改变浓度或这些因素的过境响应于各种挑战（ 例如，饮食，抗原，药物）和在疾病模型（ 例如，结肠炎，艾滋病，糖尿病）也可以评估。而这是不可能广泛描述其可以被分析和比较，这里的每个淋巴成分，肠系膜淋巴结简单化由含水，类脂和细胞相。的在水相中的兴趣的组件包括肽和蛋白质，例如抗原或耐受原8，免疫信使如细胞因子和肥大细胞介质9和代谢介质如肠降血糖素10。后节点肠系膜淋巴结的细胞部分包含lymphocyt的几乎完全（99％以上）上课11。各种免疫细胞（树突状细胞，肥大细胞等 ）进入预结肠系膜淋巴管，但仍然在节点12内。如果内输入淋巴细胞是感兴趣的，则可以通过去除肠系膜淋巴结的收集这些细胞中的肠系膜淋巴管12的插管前节点几天。以这种方式，传入和传出淋巴管直接连接和淋巴细胞传入淋巴直接传递到肠系膜淋巴管。各种免疫细胞通过淋巴管肠道的运输和表型可以这样进行检查。或许引用于收集肠系膜淋巴结迄今为止最常见的原因，但是，是研究肠处理，吸收和膳食脂质和亲油性分子10的运输。
以下摄取，膳食脂质被消化（例如，从甘油三酯脂肪酸和单甘油酯，phospholipid到脂肪酸和溶血磷脂，和胆固醇酯脂肪酸和胆固醇等 ），并经由加法从胆汁两亲物的肠腔成小胶束和囊泡结构中分散（磷脂，胆固醇和胆汁盐）和作用胰腺酶10,13。从这里，他们被吸收到肠。被吸收的组分的比例被重新酯化，以形成吸收细胞（肠）内的甘油三酯，磷脂和胆固醇酯。这些再酯化脂肪是由摄入外源性脂质成分和内源性脂质成分的自分泌胆汁，粘膜脂质池或肠道血液供应13的组合装配。从这里的酯化脂质要么存储肠内或组装成肠脂蛋白（乳糜微粒，极低密度脂蛋白（VLDL））连同各种脱辅基蛋白和其它亲脂性分子（ <eM>如维生素）10,13。退出后肠，脂蛋白是专门从肠道通过肠系膜淋巴系统运输到全身循环作为肠乳糜更可渗透其比肠毛细血管条目。吸收的脂质组分的比例还通过毛细血管和门静脉作为单独的，非相关的脂蛋白运送从肠道到体循环，分子14。在一般情况下，然而，门静脉输送路径是唯一的一个显著球员的短链和中链长度的脂质吸收。
肠系膜淋巴结的收集由此使从肠脂蛋白和相关的部件（脂质，亲脂性分子，脱辅基蛋白）的传输的评估。脂蛋白可定量和表征的优点，即肠系膜淋巴结脂蛋白，在一般情况下，是在一个nascen吨状态，因为它们没有被广泛地通过全身酶改性，如脂蛋白脂肪酶15。而肠系膜淋巴结插管的大鼠模型已经也许历来最广泛的脂质/脂蛋白运输从肠道的分析描述的，扩大的兴趣的区域是淋巴管中的亲脂性药物，前药和其他外源物13,16的传输中的作用这是在这里描述的模型的焦点。亲脂性药物（一般是那些与log P> 5和溶解度在长链甘油三酯> 50毫克/克，尽管异常是明显的）17,18，前药19和其他外源物13,16可以访问肠淋巴或者被动地或通过积极融入肠道脂蛋白运输通路19。
大鼠肠系膜淋巴结插管技术因此有许多应用。博尔曼等人首先描述一个techniqUE到导管插入大鼠的肠系膜淋巴管在1948年20，从此一数量的变型的模型已经描述。例如，收集可在昏迷状态，同时抑制15或自由活动23,24发生时，老鼠被麻醉的各种麻醉剂21,22，或。大鼠可以施用不同补液溶液和其他物质如脂质和药物制剂以不同的速率进入胃，肠或肠胃外（一般0 -为5毫升/小时）25。在一些研究中胸淋巴导管，而不是肠系膜淋巴管插管经由淋巴管来估计传输从肠道尽管这可能高估中转从小肠，取决于感兴趣的因素，因为胸淋巴导管还接收来自其它淋巴区22,26。淋巴插管车型也已经在其他几个物种，包括小鼠15,27，迷你PI描述GS 12，28,29羊，猪30和狗31。然而，在大鼠模型中是最广泛和一致引用。对肠系膜淋巴管随后采集淋巴液中有意识25或麻醉22大鼠和小鼠15,27的插管的详细方案先前已公布的和有兴趣的读者是针对这些协议。这个协议是第一次证明该技术在一个可视化格式。
淋巴空心大鼠模型拥有较大的动物模型的优势在费用方面，易用性的手术和伦理方面的考虑。相比，小鼠模型中，肠系膜淋巴结插管手术也是在大鼠容易虽然小鼠模型能够在转基因动物27更详细的研究。尽管如此，也有在大鼠模型中，特别是那些在生理差异有关，该限extrapolat的一些局限离子对其他临床前和临床情况。例如，在大鼠胆汁流量是恒定的，独立的食物摄取，而在更高的物种的食物或脂质刺激胆汁流32。这产生挑战获得代表前和餐后环境在大鼠反映了什么被认为是在较大的品种和人类。用于药物递送的研究，大的物质也可以评估淋巴运输时后的现实人类剂量的给药形式25优选的。在最近的研究中，在肠系膜淋巴结脂质转运率被发现是跨越一个等效质量和脂质的类型，它提供了一些信心跨物种27外插脂质转运数据给药后物种（小鼠，大鼠，狗）媲美。然而，模型的亲脂性药物，卤泛群运输，动物的大小（ 即狗>大鼠>小鼠）的顺序排名。比例因子可能因此被要求前trapolate大鼠淋巴药物传送数据到其他物种。
的淋巴插管模型的限制，一般来说，是直接被动淋巴收集从淋巴管可以修改淋巴流动和传输自淋巴管的工作对一个压力梯度被改变，一旦容器被插入套管33。淋巴插管模型也可以是难以建立在实验室中是不熟悉的技术。替代模式进行了说明。举例来说，经由肠道淋巴系统的因素，如脂蛋白和亲脂性分子的转运，已经间接通过采集血液的研究。一个这样的模型涉及口服给药后，在存在和不存在抑制剂的比较脂质和/或药物的血药浓度（ 例如，秋水仙碱，普朗尼克L81，放线菌酮）肠脂蛋白生产阻断淋巴运输34。优势的经由采集血样 量化淋巴运输间接模型是，它使淋巴运输的一些评估在人中作为侵入性手术，不需要35。然而，淋巴运输的抑制剂不特定且经由淋巴管输送因子稀释并修改在全身循环变得复杂这样的评估， 在体外替代物也被描述。例如，Caco-2细胞或分离的肠细胞培养物已被用于更详细地研究该进入淋巴管36-38分子的肠分泌。一种先进的体外模型是比较有代表性的人体肠道微环境也在最近描述39。在此模型中的淋巴管内皮细胞层是共培养Caco-2细胞使物质转移从肠进入淋巴管的更详细的分析。然而， 在VITRO单元系统缺乏交流流动和转移与肠腔和潜在血液和淋巴血管供应，即互联。在另一种方法，Kassis 等人建立了原位成像系统，使血管收缩，淋巴流动和肠系膜淋巴管荧光灯33血脂浓度之间的定量比较，双通道（高速明场视频和荧光）。该模型在上述的体外系统的一个优点是，它使免疫细胞通过淋巴管的通道的准确的跟踪。质量的脂质（或药物）的运输的绝对测量，但是，尚未建立使用的成像方法， 在体外和在硅片接近经由肠淋巴也已出版40-42特异性预测脂溶性药物输送的程度。例如， 离体数的c亲和力ompounds血浆乳糜微粒被发现关联相当好与它们在体内的41淋巴运输。接着，在同一组，在硅片模型建立一个预测药物亲和性基于多个物理化学性质40乳糜微粒。冬青等人还建立了在计算机芯片模型相对复杂，以彻底预测分子描述符42的基础上，亲脂性化合物的淋巴运输。这些模型可提供预测未知药物淋巴运输的程度上有用的方法。与广泛的药物和不同的实验室模型的验证将然而，需要确认其准确性和可重复性。
的肠系膜淋巴管插管因而仍然是唯一的手段来直接检查淋巴引流小肠和的因子（细胞，蛋白质的复数排列的传输速率的内容，肽在体内的情况淋巴液，脂类，药物）。这里我们描述了一个协议，用于在肠系膜淋巴管和颈动脉，使肠系膜淋巴结和从麻醉大鼠的全身血液的收集的插管。代表性的数据说明了如何在模型可用于通过肠系膜淋巴系统以检查脂质和药物输送从肠道。这之后，可以在建立模型和故障排除指南会遇到一个困难的讨论。一旦建立了模型是一个强大的工具，调查肠道淋巴运输。
Protocol
Representative Results
Discussion
大鼠肠系膜淋巴结插管模型使浓度和各种细胞和分子（例如脂质和药物）从肠进入淋巴和修改这些发生在响应的传输速率的直接量化挑战各种物质（饮食，抗原，药物，制剂等 ）10,27和疾病（癌症，病毒，大肠炎，胰岛素抗性等 ）5-7。收集在淋巴结的组件也可被进一步用于另外的实验。例如，细胞可以是培养的44，脂蛋白分馏和淋巴或它的组分，例如脂蛋白?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) is gratefully acknowledged.
Materials
| Sterile saline | Baxter healthcare | AHB 1307 | Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50 |
| 70 % ethanol in water | Any | Any brand can be used | |
| Chlorhexidine gluconate solution (Microshield 4) | Livingstone International | JJ60243L | Any brand can be used. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =JJ60243L |
| Betadine solution | Livingstone International | BU0510 | Any brand can be used. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =BU0510 |
| Ilium Ketamil (Ketamine 100 mg/ml) | PROVET VICTORIA | KETA I 1 | http://www.provet.com.au/ |
| Ilium Xylazil (Xylazine 100 mg/ml) | PROVET VICTORIA | TRO-3828 | http://www.provet.com.au/ |
| ACP 10 Injection (Acepromazine 10 mg/ml) | PROVET VICTORIA | VTG-DACP010020 | http://www.provet.com.au/ |
| Sodium pentobarbitone | PROVET VICTORIA | 24529 | Any brand can be used. Example here is Lethabarb® 325 mg/ml sodium pentobarbitone, Virbac Animal Health. http://www.provet.com.au |
| Heparin (35000I.U. in 35 mL) | Sigma Pharmaceuticals | 337220 | http://sigmaco.com.au/ |
| Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate | Sigma-Aldrich | E1644 | Any brand can be used. Example here is disodium salt of EDTA from Sigma. |
| Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm | Microtube extensions | PE8050 | Any brand can be used. Example here is PE tubing 0.8×0.5 mm, 30 m |
| Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm | Microtube extensions | PE9658 | Any brand can be used. Example here is PE tubing 0.96×0.58 mm, 30 m |
| Ruler | Any | Any brand can be used | |
| Markers | Any | Any brand can be used | |
| Cigarette lighter | Any | Any brand can be used | |
| Cyanoacrylate glue | Any | Any brand can be used | |
| 23 gauge needles | Livingstone International | DN23GX0.75LV | Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&CAT= 6&search=DN23GX0.75LV |
| 25 gauge needles | Livingstone International | DN25GX1.0LV | Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&CAT=6&search= DN25GX1.0LV |
| 1 ml syringe | Livingstone International | T3SS01TA | Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =T3SS01TA |
| 10 ml syringe | Livingstone International | T3SS10SA | Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =T3SS10SA |
| Gauze swabs | Livingstone International | GSC075 | Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5×7.5 cm, 8 ply. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =GSC075 |
| Cotton buds | Livingstone International | CTAST075DP | Any brand can be used. Example here is Livingstone cotton applicator plastic double tipped. 75MM. 100/PK. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =CTAST075DP |
| Heating pad | Ratek | WT1 | Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray. |
| Surgical light | Harvard Apparatus | 72-0215 with 72-0267 | Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_ -1_HAI_ProductDetail and http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_ -1_HAI_ProductDetail___ |
| Surgical microscope | Zeiss | 495005-0014-000 | Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https://www.micro-shop.zeiss.com/?l=en&p=us&f=e&i=10143 |
| Silk suture | Livingstone International | DTSK163019F4 | Any brand can be used. Example here is * Email this item to my friend 3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&CAT=6&search =DTSK163019F4 |
| Scalpel blades | Fine Science Tools (FST) | 10020-00 | Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191 |
| Scalpel handle | Fine Science Tools (FST) | 10004-13 | Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&CategoryId=51 |
| 1 x Small surgical scissors | Fine Science Tools (FST) | 14060-09 | Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=40&CategoryId=17 |
| 2 x Forceps with serrated curved tip | Fine Science Tools (FST) | 11001-13 | Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8×1.4 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=405&CategoryId=32 |
| 1 x Iridectomy scissors | Fine Science Tools (FST) | 15000-08 | Any brand can be used. Example here is FST Vannas Spring Scissors – 2.5mm Cutting Edge, Straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=17&CategoryId=16 |
| 1 x Forceps with straight serated tip | Fine Science Tools (FST) | 11650-10 | Any brand can be used. Example here is FST Graefe 10 cm straight with serrated 1 x 0.99 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=390&CategoryId=32 |
| 1 x Forceps with smooth sharp straight fine tip | Fine Science Tools (FST) | 11251-10 | Any brand can be used. Example here is FST Dumont #5 forceps straight 11cm with 0.08 x 0.04mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=335&CategoryId=29 |
| 1 x Forceps with smooth fine curved forceps | Fine Science Tools (FST) | 11063-07 | Any brand can be used. Example here is FST Delicate Forceps 9 cm with smooth 0.4 x 0.3mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=360 |
| 2 x Hemostats | Fine Science Tools (FST) | 13010-12 | Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&CategoryId=33 |
| 1 x Suture needle holder | Fine Science Tools (FST) | 12001-13 | Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&CategoryId=70 |
| 1 x Artery clamp | Fine Science Tools (FST) | 18050-28 | Any brand can be used. Example here is FST Bulldog Serrefines straight, 28 mm long, 9×1.6 mm jaw dimension with medium clamp press. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=270&CategoryId=82 |
| Oleic acid | Sigma Aldrich | O1008 | When required, any brand can be used. Example here is 99% pure oleic acid. http://www.sigmaaldrich.com/catalog/product/sial/o1008?lang=en®ion=AU |
| 14C-oleic acid | Perkin | NEC317050UC | Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50µCi (1.85MBq). http://www.perkinelmer.com/Catalog/Product/ID/NEC317050UC |
| Sodium taurocholate | Sigma Aldrich | T4009 | Any brand can be used. Example here is taurocholic acid sodium salt hydrate ≥95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en®ion=AU |
| Halofantrine | Glaxo Smith Kline | Halofantrine was kindly provided as a gift from Glaxo Smith Kline | |
| Sodium phosphate monobasic | Sigma Aldrich | 71507 | Any brand can be used. Example here is sodium phosphate monobasic monohydrate, BioXtra, for molecular biology, >99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en®ion=AU |
| Sodium phosphate dibasic | Sigma Aldrich | 71643 | Any brand can be used. Example here is sodium phosphate dibasic dihydrate, BioUltra, for molecular biology, >99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en®ion=AU |
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