小鼠输卵管的显微解剖和解离：个体片段鉴定和单细胞分离
Summary
本文提出了一种小鼠输卵管显微解剖方法，该方法允许在保持RNA完整性的同时收集单个片段。此外，描述了非酶促卵管细胞解离过程。这些方法适用于功能不同的输卵管节段和解离的输卵管细胞的后续基因和蛋白质分析。
Abstract
小鼠模型系统在疾病过程分析方面是无与伦比的，因为它们具有遗传可操纵性和实验性治疗的低成本。然而，由于它们的体型小，一些结构，如直径为200-400μm的输卵管，已被证明相对难以研究，除非通过免疫组织化学。最近，免疫组织化学研究揭示了输卵管段比以前认识到的更复杂的差异;因此，输卵管被分为四个功能段，具有七种不同上皮细胞类型的不同比例。上皮细胞类型的不同胚胎起源和比例可能使这四个功能区域对疾病有不同的易感性。例如，浆液性上皮内癌的前体病变来自小鼠模型中的内底和人类输卵管中的相应腓肠区域。这里描述的方案详细介绍了一种显微解剖方法，以产生下游分析所需的足够量和纯度的RNA，例如逆转录定量PCR（RT-qPCR）和RNA测序（RNAseq）。还描述了一种大部分非酶解组织解离方法，适用于流式细胞术或单细胞RNAseq分析的完全分化的输卵管细胞。所描述的方法将促进在生殖，生育，癌症和免疫学领域利用小鼠输卵管的进一步研究。
Introduction
小鼠输卵管在功能和形态上与人类输卵管相似1。两者都由假分层的纤毛上皮组成，由两个历史上描述的上皮常驻细胞组成：纤毛细胞和分泌细胞1，2。输卵管有三个经典公认的部分：腹腔，壶腹和峡部。在最近的一项研究中，Harwalkar等人。3 研究了输卵管形态和基因表达，导致将常驻上皮细胞的分类扩展到七个不同的群体。此外，他们建立了壶腹 – 地峡连接处作为输卵管3的独特部分。本文描述的方法侧重于腹足、壶腹和峡部，可以很容易地扩展到包括壶腹-峡部交界处2，3。腹股沟区域包含输卵管的开口，包括腓肠区域以及近端茎。向子宫移动，接下来是壶腹，然后是峡部。纤毛细胞在该区域的远端、卵巢近端或足底最为突出，而分泌细胞在近端或峡部最为突出1。与人类输卵管不同，小鼠输卵管是由腹膜宽韧带的延伸中脉支撑的盘绕结构1，4。此外，小鼠输卵管被包裹在滑囊中，这增加了卵母细胞转移到输卵管中的可能性4。壶腹被确定为受精的位置，发育中的胚胎在进入子宫之前从该位置进入地峡5。输卵管节段直径为200-400μm，较长的壶腹和峡部区域的长度约为0.5-1.0厘米4。输卵管在发情周期期间膨胀，腹扩和腹股沟比峡更易扩张1。
细胞（尤其是分泌细胞）的过度增殖是盆腔中发现的浆液性肿瘤的前体病变的特征6。这些前体浆液性上皮内病变仅在腓骨区域出现在输卵管上皮中;目前尚不清楚为什么病变形成仅限于该区域，其中通常主要细胞类型是纤毛的，而不是分泌的2，7，8。正常生理功能的区域性以及对卵巢癌输卵管起源的兴趣增加9，10，11，12，13，强调了单独评估输卵管段的重要性。
这里描述的方法详细介绍了单独输卵管片段的收集，用于随后对片段特异性基因表达和解离细胞的功能进行下游分析。传统上，许多组织按照苯酚：氯仿法或柱上完全提取法进行全RNA提取;然而，我们发现RNA质量得以维持，同时使用所述的组合方法产生了足够的产量。利用这种方法，可以处理输卵管的非常小的功能段以进行下游分析，而不是将输卵管作为一个整体进行研究，这可以掩盖代表不同段的结果14。
分离的小鼠输卵管细胞很少通过流式细胞术进行研究，很可能是由于该组织的细胞产量有限。克服这个问题的一种方法是解离细胞，在培养物中培养它们，然后在 体外 刺激再分化，以获得适当的细胞数用于下游细胞分析15，16，17，18。这种方法的局限性是培养物中 离体 时间和微环境的改变，这两者都可能改变基因表达。还有一种假设是，形态学上的再分化具有与完整动物相同的转录和蛋白质组学特征。目前的解离方法旨在实现异质性输卵细胞群中最高数量的上皮细胞，同时保持单细胞分化。此外，大多数非酶的方法可能会限制细胞表面蛋白的损失。
Protocol
Representative Results
Discussion
输卵管的三个节段在组织学上，形态学上和功能上是不同的1，2，3。上皮从输卵管的一端到另一端变化很大。纤毛细胞在腓肠/腹肌末端占主导地位，而分泌细胞在峡部区域占主导地位1。虽然这种总体梯度已经被认识到了一段时间，但最近的工作已经发现了输卵管节段之间的更多区别。因此，虽然纤毛细胞在?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国防部对AMW突破奖（BCRP W81XWH-14-1-0425）的部分支持。KCR部分得到了校内奖学金的支持：Pease癌症博士预科奖学金和Mary Galvin Burden生物医学科学博士前奖学金以及加州大学河滨分校，校内奖：研究生理事会奖学金委员会论文研究补助金和研究生部论文年度计划奖。作者感谢Gillian M. Wright和Alyssa M. Kumari在这种方法的早期故障排除方面的帮助。
Materials
| 0.5 mm Stainless steel bead mix | Next Advance | SSB05 | Mix 1:1 with 1.4 mm SSB14B, sterilized |
| 1.4 mm Stainless steel bead mix | Next Advance | SSB14B | Mix 1:1 with 0.5 mm SSB05, sterilized |
| 1X Dulbecco's Phosphate Buffered Saline A, pH 7.4 (DPBS) | Gibco | 21600-010 | Cold, sterile |
| 25G needle | BD | 305122 | |
| 60 mm sterile petri dishes | Corning | 430166 | |
| 70 μm cell meshes | Fisherbrand | 22-636-548 | |
| Agilent Eukaryote Total RNA 6000 Pico Chip kit | Agilent 2100 Bioanalyzer | 5067-1513 | |
| Bead Bullet Blender Tissue Homogenizer | Next Advance | BBY24M | |
| Bioanlyzer | Agilent 2100 Bioanalyzer | ||
| Bovine serum albumin | Sigma Aldrich | A7906 | |
| Cold plate/pack/surface of choice | N/A | N/A | Kept at -20C for dissection |
| Dental wax | Polysciences Inc. | 403 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM)/ Ham’s F12 | Corning | 10-090-CV | Prepare dissection medium: DMEM/ Ham’s F12, 10% FBS, 25 mM Hepes, 1% Pen-Strep |
| Fetal Bovine Serum | Corning | 35-015CV | |
| Fine point forceps of choice | N/A | N/A | |
| Glycine | Sigma Aldrich | G712b | Immunocytochemical validation images |
| Goat anti-mouse IgG Alexa Fluor 555 | Invitrogen | A-21422 | Immunocytochemical validation images |
| Goat anti-rabbit IgG Alexa Fluor 488 | Invitrogen | A-11001 | Immunocytochemical validation images |
| Hepes | Sigma Aldrich | H-3784 | |
| Hoescht 33342 | Cell Signaling Technologies | 4082S | Immunocytochemical validation images |
| Inverted compound microscope | Keyence BZ-X700 | ||
| Mouse anti-mouse Occludin | Invitrogen | 33-1500 | Immunocytochemical validation images |
| Non-enzymatic dissociation buffer | N/A | 5 mM EDTA, 1 g/L glucose, 0.4% BSA, 1X DPBS | |
| Nylon macro-mesh 1 mm x 1 mm | Thomas Scientific | 1210U04 | |
| Paraformaldehyde | Sigma Aldrich | P-6148 | Immunocytochemical validation images |
| Pen-Strep | MP Biomedicals | 10220-718 | |
| Prolong Gold Antifade Reagent | Cell Signaling Technologies | 9071S | Immunocytochemical validation images: antifade mounting medium |
| Pronase | Sigma Aldrich | 10165921001 | Prepare pronase digestion medium: 0.15% Pronase in DMEM/Ham's F12, sterile |
| Propidium Iodide | Roche | 11 348 639 001 | Viability validation images |
| Rabbit anti-mouse Acetylated-Tubulin | Abcam | ab179484 | Immunocytochemical validation images |
| RBC lysis buffer | BD Biosciences | 555899 | |
| RNeasy Mini Kit | Qiagen | 74134 | Utilized for on-column purification in text. |
| Spring form microdissection scissors | Roboz Surgical | RS-5610 | |
| Sterile 3 mL bulb pipettors | Globe Scientific | 137135 | |
| Toluidine blue | Alfa Aesar | J66015 | Prepare toluidine blue solution: 1% in 1X DPBS, sterile |
| Tris-Buffered Saline-Tween (TBST) | N/A | N/A | Immunocytochemical validation images; 0.1 N NaCl, 10 mM Tris-Cl pH 7.5, 1% Tween 20 |
| Triton-X-100 | Mallinckrodt Inc. | 3555 | Immunocytochemical validation images |
| Trizol RNA Extraction Reagent | Invitrogen | 15596026 | Referred to as RNA extraction reagent. Nucelase-free water, chloroform and isoproponal are required in supplement of performing Trizol extraction per manufacturer's guidelines |
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