肝细胞部分肝切除术后脾移植。小鼠
Summary
我们已经描述了一个协议, 执行部分肝切除 (phx-空港) 和细胞移植通过脾脏点头。(点头。CB17-Prkdc有缺陷的/J) 鼠标。在本议定书中, 切口是为了揭露和切除肝脏的左叶, 其次是另一个切口的脾移植细胞。
Abstract
部分肝切除术是研究肝再生和细胞基础疗法在各种病理条件下的作用的一种多功能和可再生的方法。部分肝切除也促进移植细胞的植入和增殖, 加速新生血管和细胞向肝脏迁移。在这里, 我们描述了一个简单的协议, 执行30% 肝切除和移植细胞在脾脏的非肥胖糖尿病/严重联合 immunodeficient 点头。(点头。CB17Prkdc的/J) 鼠标。
在这个过程中, 做了两个小切口。第一切口是暴露和切除肝脏的左叶, 另一小切口被做暴露脾脏为脾移植细胞。这个程序不需要任何专门的外科手术技能, 它可以在5-7 分钟内完成, 减轻压力和疼痛, 更快的恢复, 更好的生存。我们已经展示了从绿色荧光蛋白 (gfp) 中分离出的肝细胞移植, 表达小鼠 (转基因 C57BL/6-Tg (不列颠哥伦比亚) 30 scha/J), 以及人源性肝细胞 (NeoHep) 在部分 hepatectomized 点头。老鼠。
Introduction
目前, 肝细胞移植是一种替代全器官移植治疗严重肝病患者。据信, 它可以把病人移植到全身器官,1。除了同种异体肝细胞2外, 异体肝细胞3和干细胞 (4 ) 的肝细胞也正在动物模型中进行研究。在这一背景下, 受体移植细胞的归巢和植入电位是急性肝功能衰竭 (AHF) 细胞治疗的重要标准。
为研究肝细胞或肝样细胞的移植5, AHF 是由手术的6或药理7程序在动物模型中创建的, 其次是移植细胞。通过药理试剂制作 AHF 动物模型, 许多 hepatotoxins 如 d-乳糖8、乙酰氨基酚9、四氯化碳10、硫乙酰胺11、刀豆蛋白 12、脂多糖13已使用、等。从这个列表中, 每个试剂为 AHF 生成一组独特的功能, 但不幸的是, 没有单一试剂模仿人类 AHF。此外, hepatotoxins 诱发的 AHF 时间长, 使动物处于慢性应激状态, 难以获得重现性结果。
另一方面, 部分肝切除术 (phx-空港) 的手术方法是技能依赖性的, 在培养所需技能后, 可重现性结果容易获得。仅以手术干预诱发 AHF, 需要切除超过70% 的肝脏;然而, 70% 的肝切除术仍可用于研究肝脏移植细胞的植入和增殖, 以分析其在肝损伤期间的治疗能力14。肝细胞移植术后经腹膜切除后, 经腹腔15、尾静脉16、肝静脉17或脾18。目前, 肝静脉输液和肝细胞脾移植是首选的程序, 因为它们更容易繁殖。
在本文中, 我们已经描述了30% 部分肝切除在点头的程序。(点头。CB17-Prkdcscid/J) 肝左叶切除的小鼠。其次是移植 20万 GFP 表达小鼠 (C57BL/6-Tg (不列颠哥伦比亚) 30 scha/J) 肝细胞以及人类起源 NeoHep19在脾脏。这个过程导致肝脏移植细胞的植入。这个程序是最不具侵入性和轻微疼痛的技术。
Protocol
本议定书所载程序已由新德里国家免疫学研究所机构动物伦理委员会批准。审批的串行参考编号为 IAEC#319/13. 注: 一般手术程序有很好的资源20和针对啮齿动物手术的特定协议21。对于那些第一次做动物外科手术的人来说, 建议在对动物进行手术前, 广泛地练习使用假的程序。 1. 准备 在试验之前, 保持无菌磷…
Representative Results
30% 部分肝切除术后肝细胞增殖:用免疫组化 (IHC) 染色 Ki-67 细胞增殖标记物, 观察30% 肝切除术后剩余肝脏中肝细胞的增殖。一天后肝切除, 小鼠被安乐死, 其余肝叶切除, 石蜡切片获得。切片染色的 Ki-67 抗体, 其次是标签与辣根过氧化物酶 (HRP) 共轭二级抗体。以二氨基联苯胺为基质, 用于开发褐色色素以识别染色细胞。细胞核被用苏木精染色, 并在20X 物镜下观察。图 1</str…
Discussion
部分肝切除术是一种研究肝再生的方法, 据报道过度肝切除是模拟 AHF 模型。在 AHF 动物模型中, 啮齿动物, 特别是小鼠, 是研究最多的模型。为了获得小鼠肝脏损伤模型, 多达70% 的肝切除已报告具有良好的存活率25,26。然而, 在裸和其他 immunodeficient 鼠, 70% 例肝切除被报告为致命, 动物死亡24小时内27。
米切尔和 Willenbr…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了印度政府生物技术部向新德里国家免疫学研究所提供的核心赠款的支持。巴塔查尔吉博士目前的讲话是胃肠病学、肝病和营养科, 洛杉矶儿童医院。
Materials
| Gas Anesthesia System | Ugo Basile; Italy | 211000 | |
| Weighing machine | Goldtech ; India | Local Procurement | |
| Biological safety cabinet ( Class I) | Kartos international; India | Local Procurement | |
| Hair Trimmer | Panasonic ; Japan | ER-GY10 | |
| Straight operating scissor with sharp /sharp blades | Major Surgicals; India | Local Procurement | |
| Forceps with Serrations | Major Surgicals; India | Local Procurement | |
| Micro needle holders straight & curved | Mercian ; England | BS-13-8 | |
| 1 ml insulin syringe with 30G *5/16 needles | Dispo Van; India | ||
| 1 ml syringe with 26 G * 1/2 needle | BD ; US | REF 303060 | |
| Nylon Threads | Mighty ; India | (1-0) Local Procurement | |
| MERSUTURES 4-0 Sterilised Surgical Needled Suture | Ethicon, Johnson & Johnson, India | NW 5047 | |
| TRUGUT 76 cm 4-0 absorbable surgical suture | Sutures India Pvt. Ltd; India | SN 5048 | Sterilised Surgical Needled Suture Catgut Chromic |
| Cotton Buds | Pure Swabs Pvt Ltd ; India | Local Procurement | |
| Surgical Tape | 3M India ; India | 1530-1 | Micropore Surgical Tape |
| Microtome | Histo-Line Laboratories, Italy | MRS3500 | |
| Shandon Cryotome E Cryostat | Thermo Electron Corporation ; US | ||
| Confocal laser scanning microscope | Carl Zeiss ; Germany | LSM 510 META | |
| Bright Field Microscope | Olympus, Japan | LX51 | |
| Automated analyser | Tulip, Alto Santracruz, India | Screen Maaster 3000 | Biochemical analyser for liver functional test |
| Flow Cytometer | BD ; US | BD FACSverse | Assesment of presence of cells post transplantation |
| Veet hair removal cream | Reckitt Benckiser , India | ||
| FORANE | Abbott ; US | isoflurane USP 99.9% | |
| Taxim | AlKem ; India | cefotaxime sodium injection | |
| Povidone-Iodine solution | Win-Medicare; India | Betadine | |
| Paraformaldehyde | Himedia; India | GRM 3660 | |
| Iscove's Modified Dulbecco's Medium (IMDM) | Life technologies, Thermo Fisher scientific ; US | 12200-036 | |
| Sucrose | Sigma ; US | S0389 | |
| Tissue-Tek | Sakura; US | 25608-930 | O.C.T compound |
| DAPI | Himedia; India | MB 097 | |
| anti-Albumin goat Polyclonal | Thermo Scientific,Pierce, US | PA126081 | |
| anti-connexin 32/GJB1 Polyclonal | abcam, UK | ab64609-500 | |
| antiGFP rabbit polyclonal | Santa Cruz biotechnology; US | SC 8334 | |
| Alexa Fluor 594 donkey anti-goat | Molecular Probes , Thermo Fisher Scientific ; US | A11058 | |
| Alexa Fluor 488 donkey anti-sheep | Molecular Probes , Thermo Fisher Scientific ; US | A11015 | |
| Alexa Fluor 594 chicken anti rabbit | Molecular Probes , Thermo Fisher Scientific ; US | A21442 | |
| Goat anti rabbit IgG HRP | Invitrogen, Thermo Fisher Scientific; US | 65-6120 | |
| anti-Ki67 antibody | abcam, UK | ab15580 | |
| Antigen Unmasking Solution, Citric acid base | Vector laboratories, US | H-3300 | |
| ProLong Diamond antifade mountant | Life technologies, Thermo Fisher scientific ; US | P36966 | |
| SGOT (ASAT) KIT | Coral Clinical System, India | ||
| SGPT (ALAT) KIT | Coral Clinical System, India | ||
| Alkaline Phosphatase Kit (DEA) | Coral Clinical System, India | ||
| Hematoxylin Solution, Mayer's | Sigma ; US | MHS16 | |
| Eosin Y solution, alcoholic | Sigma ; US | HT110132 | |
| DPX Mountant | Sigma ; US | 6522 | |
| Melonex (Pain Killer) | Intas Pharmaceuticals Ltd; India | Meloxicam injection | |
| DAB enhanced liquid substrate system tetrahydrochloride | Sigma ; US | D3939 |
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Cite This Article
Das, B., Bhattacharjee, J., Preeti, Mishra, A., Jain, K., Iyer, S., Kesarwani, A., Sahu, P., Sinha, P., Nagarajan, P., Upadhyay, P. Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice. J. Vis. Exp. (132), e56018, doi:10.3791/56018 (2018).