小鼠自身免疫性糖尿病过继转移模型中移植物存活的生物发光监测
Summary
该协议描述了一种直接和微创的方法，用于在非肥胖糖尿病（NOD）严重联合免疫缺陷小鼠中移植和成像NIT-1细胞，这些小鼠受到从自发糖尿病NOD小鼠纯化的脾细胞的挑战。
Abstract
1型糖尿病的特征是胰腺产生胰岛素的β细胞的自身免疫性破坏。这种疾病的有希望的治疗方法是移植干细胞衍生的β细胞。然而，为了保护移植的细胞免受持续的自身免疫，可能需要进行基因改造。糖尿病小鼠模型是初步评估保护移植细胞免受自身免疫攻击的策略的有用工具。这里描述的是一种在小鼠糖尿病过继转移模型中移植和成像细胞移植物的微创方法。在该协议中，来自表达萤火虫荧光素酶转基因 luc2 的鼠胰腺β细胞系NIT-1的细胞被皮下移植到免疫缺陷的非肥胖糖尿病（NOD）严重联合免疫缺陷（scid）小鼠中。同时向这些小鼠静脉注射来自自发糖尿病NOD小鼠的脾细胞以转移自身免疫。通过非侵入性生物发光成像 定期 对移植物进行成像，以监测细胞存活。将突变细胞的存活率与移植到同一只小鼠中的对照细胞的存活率进行比较。
Introduction
1型糖尿病（T1D）是由胰腺产生胰岛素的β细胞的自身免疫性破坏引起的。β细胞质量的损失导致胰岛素缺乏和高血糖。T1D 患者每天依赖多次注射外源性胰岛素，并终生经历严重高血糖和低血糖发作。与这些发作相关的并发症包括糖尿病视网膜病变、肾功能下降和神经病变1。
胰岛素注射是T1D的治疗方法，但不能治愈。然而，替换丢失的β细胞团有可能通过使患者产生自己的胰岛素来逆转疾病。然而，尸体供体胰岛的供应有限2。干细胞来源的胰岛（SC-胰岛）可以为移植提供几乎无限的β细胞供应。几个小组已经证明，人类胚胎干细胞（ESC）和诱导多能干细胞（iPSCs）可以分化以产生功能性β样细胞3，4，5。有希望的早期临床试验数据表明，这些细胞在移植后仍能维持其功能，并可能使患者成为胰岛素非依赖性6。然而，需要慢性免疫抑制，从而增加他们对癌症和感染的易感性。此外，免疫抑制剂长期可能对移植物具有细胞毒性7。为了消除免疫抑制的需要，可以对SC胰岛进行基因改造，以保护它们免受复发性自身免疫以及移植后的同种异体免疫。
干细胞研究对成本和劳动力的要求很高。小鼠细胞系和动物模型是用于初始鉴定和实验验证保护移植细胞免受自身免疫的策略的有用工具。NOD小鼠发展为自发性自身免疫性糖尿病，与人类T1D8有许多相似之处，NIT-1胰岛素瘤细胞系与该小鼠品系9共享遗传背景。糖尿病可以通过从NOD小鼠注射糖尿病脾细胞来过继转移到相关的免疫缺陷NOD-scid小鼠品系，以便在复制实验小鼠10中暂时同步糖尿病的发作。该模型可用于相对快速且廉价地识别遗传靶标，以便在SC胰岛中进行进一步验证。最近，该方法被用于鉴定和验证RNLS，RNLS是一种靶标，被发现可以保护原代人胰岛免受体内自身免疫和iPSC衍生胰岛免受体外β细胞应激的影响11。这里描述的是移植基因工程NIT-1细胞并在小鼠自身免疫性糖尿病的过继转移模型中非侵入性监测其存活率的简单方案。
Protocol
Representative Results
Discussion
T1D是一种毁灭性的疾病，目前尚无治愈方法。β细胞替代疗法为患有这种疾病的患者提供了一种有希望的治疗方法，但这种策略的关键障碍是针对移植的β细胞的复发性自身免疫攻击的可能性。SC-β细胞的基因工程以降低其免疫可见性或易感性是这个问题的一个潜在解决方案。这里描述的是用于对移植的β细胞进行非侵入性成像的方案，以测量它们在小鼠自身免疫性糖尿病的过继转移模型中的存活?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢Erica P. Cai博士和Yuki Ishikawa博士开发本协议中描述的方法（见参考文献11）。S.K.和P.Y.实验室的研究得到了美国国立卫生研究院（NIH）（R01DK120445，P30DK036836），JDRF，哈佛干细胞研究所和Beatson基金会的资助。T.S.得到了国家糖尿病，消化和肾脏疾病研究所（NIDDK）（T32 DK007260-45）的博士后奖学金的支持，K.B.得到了Mary K. Iacocca基金会的部分奖学金支持。
Materials
| 0.05% Trypsin, 0.53 mM EDTA | Corning | 25-052-CI | |
| 293FT | Invitrogen | R70007 | Fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen |
| ACK Lysing Buffer | Gibco | A10492-01 | |
| Alcohol prep pads, 70% Isopropyl alcohol | Amazon/Ever Ready First Aid | B08NWF31DX | |
| BD 5ml Syringe Luer-Lok Tip | BD | 309646 | |
| BD PrecisionGlide Needle 26G x 5/8 (0.45 mm x 16 mm) Sub-Q | BD | 305115 | |
| BD 1 mL TB Syringe Slip Tip | BD | 309659 | |
| Blasticidin S HCl | Corning | 30-100-RB | |
| Cell strainer premium SureStrain, 70 µm, sterile | Southern Labware | C4070 | Or use similar sterile strainer with 40-70um pore size |
| CellDrop automated cell counter | Denovix | CellDrop BF-PAYG | Or use similar cell counter device |
| Corning 100 mL Penicillin-Streptomycin Solution, 100x | Corning | 30-002-CI | |
| Disposable Aspirating Pipets, Polystyrene, Sterile, Capacity=2 mL | VWR | 414004-265 | Or use similar aspirating pipette |
| D-Luciferin, Potassium Salt , Molecular Biology Grade, Powder, >99% | Goldbio | LUCK-100 | |
| DMEM, high glucose, pyruvate, no glutamine | Gibco | 10313039 | |
| Falcon BD tubes, 50 mL | Fisher Scientific | 14-959-49A | |
| Fetal Bovine Serum | Gibco | 10437-028 | |
| Forceps premium for tissues, 1 x 2 teeth 5 in, German Steel | Fisher Scientific | 13-820-074 | |
| Glucose urine test strip | California Pet Pharmacy | u-tsg100 | Or use similar test strip for glucose measurments in urine/blood |
| GlutaMAX–1 (100x) | Gibco | 35050-061 | |
| Infrared heating lamp | Cole Parmer | 03057-00 | Or use similar infrared lamp |
| Insulin syringe 0.5 mL, U-100 29 G 0.5 in | Becton Dickinson | 309306 | |
| Isoflurane, USP | Piramal Critical Care | 6679401725 | |
| IVIS Spectrum in vivo imaging system | Perkin Elmer | 124262 | Instrument for non-invasively collecting bioluminescent images of transplanted cells |
| Living Image Analysis Software | Perkin Elmer | 128113 | Software for collecting and quantifying bioluminescent signal |
| Microcentrifuge tubes seal-rite, 1.5 mL | USA Scientific | 1615-5510 | Or use similar sterile microcentrifuge tubes |
| NIT-1 | ATCC | CRL-2055 | Pancreatic beta-celll line derived from NOD/Lt mice |
| NOD.Cg-Prkdcscid/J | The Jackson Laboratory | 001303 | Mice homozygous for the severe combined immune deficiency spontaneous mutation Prkdcscid, commonly referred to as scid, are characterized by an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment. |
| NOD/ShiLtJ | The Jackson Laboratory | 001976 | The NOD/ShiLtJ strain of mice (commonly called NOD) is a polygenic model for autoimmune type 1 diabetes |
| PBS, pH 7.4 | Thermo Fisher Scientific | 10010031 | No calcium, no magnesium, no phenol red |
| pCMV-VSV-G | Addgene | 8454 | |
| pLenti-luciferase-blast | Made in-house | Plasmid available upon request | See Supplemental File 1 |
| pMD2.G | Addgene | 12259 | |
| pMDLg/pRRE | Addgene | 12251 | |
| Polyethylenimine, Linear, MW 25,000, Transfection Grade (PEI 25K) | Fisher Scientific | NC1014320 | |
| pRSV-Rev | Addgene | 12253 | |
| Restrainer for rodents, broome-style round 1 in | Fisher Scientific | 01-288-32A | |
| Scissors, sharp-pointed | Fisher Scientific | 08-940 | Or use other scissors made of surgical-grade stainless steel |
| Tissue-culture treated culture dishes | Millipore Sigma | CLS430167-20EA | Or use other sterile cell culture-treated Petri dishes |
| Tweezers/Forceps, fine precision medium tipped | Fisher Scientific | 12-000-157 |
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