Name: xenograft skin model to manipulate human immune responses in vivo
Uploaded: 2022-06-29T13:30:00
Description: Watch this Scientific Journal Video about Xenograft Skin Model to Manipulate Human Immune Responses In Vivo at JoVE.com

这项工作部分由TRex Bio的赞助研究协议和NIH的资助（1R01AR075864-01A1）资助。JMM得到了癌症研究协会（拨款26005）的支持。我们承认Parnassus流式细胞术核心部分由NIH P30 DK063720，S10 1S10OD021822-01和S10 1S10OD018040-01支持。

本研究已获得批准并按照UCSF IACUC（AN191105-01H）和IRB（13-11307）协议进行。作为常规选择性外科手术（如疝气修复）的一部分丢弃的皮肤样本用于本研究。皮肤样本要么被去识别化并被认证为非人类受试者研究，要么，如果下游分析需要临床识别信息，则根据IRB协议13-11307提供患者书面同意。没有使用其他纳入或排除标准。研究中雇用了8-10周龄的男女NSG小鼠。小鼠是从商业来源获得的（见 材?...

<ol>
	<li>Zomer, H. D., Trentin, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+wound+healing+in+humans+and+mice:+Challenges+in+translational+research.">Skin wound healing in humans and mice: Challenges in translational research.</a> Journal of Dermatological Science. 90 (1), 3-12 (2018).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 969618 (2011).</li><li>Mestas, J., Hughes, C. C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+skin+organoids:+potential+opportunities+and+challenges.">Generation of skin organoids: potential opportunities and challenges.</a> Frontiers in Cell and Developmental Biology. 9, 3176 (2021).</li><li>Crist&#243;bal, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+human+skin+transplantation:+a+systematic+review.">Mouse models for human skin transplantation: a systematic review.</a> Cells Tissues Organs. 210 (4), 250-259 (2021).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD/SCID/&#947;cnull+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/&#947;cnull mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Meraz, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+patient-derived+xenograft+humanized+mouse+model+for+evaluation+of+lung+cancer+immune+responses.">An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses.</a> Cancer Immunology Research. 7 (8), 1267-1279 (2019).</li><li>Racki, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD-scid+IL2rgamma(null)+mouse+model+of+human+skin+transplantation+and+allograft+rejection.">NOD-scid IL2rgamma(null) mouse model of human skin transplantation and allograft rejection.</a> Transplantation. 89 (5), 527-536 (2010).</li><li>Meehan, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+xenograft+model+of+human+vasculature+in+the+mouse+ear+pinna.">Developing a xenograft model of human vasculature in the mouse ear pinna.</a> Scientific Reports. 10 (1), 2058 (2020).</li><li>Gokkaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+graft+storage+in+platelet+rich+plasma+(PRP).">Skin graft storage in platelet rich plasma (PRP).</a> Dermatologic Therapy. 33 (1), 13178 (2020).</li><li>. The Humeca D42 and D80 battery operated cordless dermatomes Available from: <a target="_blank" href="https://www.youtube.com/watch?v=YCRowX-TdA">https://www.youtube.com/watch?v=YCRowX-TdA</a> (2021)</li><li>Rodriguez, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+regulatory+T+cells+reside+in+human+skin.">Memory regulatory T cells reside in human skin.</a> The Journal of Clinical Investigation. 124 (3), 1027-1036 (2014).</li><li>Hoogstraten-Miller, S. L., Brown, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+rodent+aseptic+surgery.">Techniques in rodent aseptic surgery.</a> Current Protocols in Immunology. 82 (1), 12-14 (2008).</li><li>Karim, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+ischemia+and+neovascularization+in+a+murine+model+of+full+thickness+human+wound+healing.">Evolution of ischemia and neovascularization in a murine model of full thickness human wound healing.</a> Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. 28 (6), 812-822 (2020).</li><li>Ali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+graft-versus-host-disease+in+NOD-scid+IL-2R&#947;null+mice+display+a+T-effector+memory+phenotype.">Xenogeneic graft-versus-host-disease in NOD-scid IL-2R&#947;null mice display a T-effector memory phenotype.</a> PloS One. 7 (8), 44219 (2012).</li><li>Souci, L., Denesvre, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+skin+models+in+domestic+animals.">3D skin models in domestic animals.</a> Veterinary Research. 52 (1), 21 (2021).</li><li>Holtkamp, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clocks+guide+dendritic+cells+into+skin+lymphatics.">Circadian clocks guide dendritic cells into skin lymphatics.</a> Nature Immunology. 22 (11), 1375-1381 (2021).</li></ol>

小鼠异种移植皮肤移植模型是在 体内 环境中机械解剖人类皮肤免疫反应的关键技术14。成功的皮肤异种移植依赖于小鼠和皮肤标本和小鼠的适当制备以及坚持无菌啮齿动物手术方法15。在低温下快速冷却和适当储存皮肤样本在培养基（如无菌盐水）中对于确保移植前持续的组织健康非常重要12。使用诸如皮节之类的仪器收集分离厚度的?...

小鼠模型经常被用来推断人类生物学和疾病，部分原因是它们的实验可重复性和遗传操作能力。然而，小鼠生理学并不能完全概括人体器官系统，特别是皮肤，因此在药物开发中用作临床前模型具有局限性1。小鼠和人类皮肤之间的解剖学差异包括上皮厚度和结构的差异，缺乏小鼠内分泌汗腺以及毛发循环的变化2。此外，免疫系统的先天臂和适应性臂在两个物种之间是不同的3。小鼠皮肤含有树突状表皮T细胞（DETCs）的独特免疫群体，具有更高的真皮γδ T细胞丰度，并且与人体组织相比，免疫细胞亚群定位不同4。因此，关于人类皮肤生物学和炎症的实验结果受益于人体组织的验证。虽然 体 外和类器官培养系统是研究人体组织的广泛使用的工具，但这些系统受到缺乏或不完全免疫重建以及与外周脉管系统缺乏联系的限制5。人源化异种移植皮肤移植模型旨在允许对 体内人体组织中的免疫和非免疫途径进行治疗或生物操作。
人体皮肤异种移植模型已被用于研究皮肤生理学和药理学，分析免疫排斥和反应，剖析人类皮肤癌机制，了解皮肤病和伤口愈合6。虽然适用于皮肤研究的多个领域，但异种移植模型的通量低于 体外 研究，并且缺乏小鼠模型中采用的遗传操作的便利性。该模型中的时间点可能从数周到数月不等，成功的移植需要适当的设施和设备来进行这些手术。然而，异种移植模型为实验提供了生物学和生理背景，而类器官培养系统（例如组织外植体）通常需要以特定的时间间隔复制无数的运动部件，例如外源性信号7。因此，该模型最适合用于进一步验证 在体外 和小鼠模型中观察到的发现，或者用于生物学上不可行的工作。适当使用异种移植模型为研究和操作 体内完整的人体组织提供了独特的机会。
异种移植皮肤移植模型的优化依赖于数十年的研究，以随着时间的推移保持移植物的完整性。这一过程的关键是利用非肥胖糖尿病（NOD）-scid白细胞介素-2γ链受体（NSG）小鼠，该小鼠缺乏B和T适应性免疫细胞，功能性NK细胞，并且在巨噬细胞和树突状细胞中存在缺陷8。这些NSG宿主的免疫缺陷性质允许移植人造血细胞，患者来源的癌症和皮肤8，9，10。尽管存在这种免疫抑制宿主环境，但通过抗GR1给药对小鼠中性粒细胞免疫反应的额外抑制对于移植成功是必要的10。移植完整组织的主要障碍是感染、排斥和难以重建流向移植物的血流，有时会导致真皮和表皮完整性的丧失11。包括施用抗FR1和使用适当移植深度在内的技术可提高移植物存活率10。细致的优化使得对NSG小鼠进行人异种移植皮肤移植成为可能，效率和存活率从90%-100%不等。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher</td>
			<td>SF100-20</td>
			<td>Fixative for histology</td>
		</tr>
		<tr>
			<td>3M Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>surgical glue</td>
		</tr>
		<tr>
			<td>Alexa 700 CD45 monoclonal antibody (Clone 30F11)</td>
			<td>Thermo Fischer</td>
			<td>56-0451-82</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Anti-GR1 clone RB6-8C5</td>
			<td>BioXcell</td>
			<td>BE0075</td>
			<td>Anti-rejection</td>
		</tr>
		<tr>
			<td>APC mouse anti-human CD25&#160; (Clone 2A3)</td>
			<td>BD Biosciences</td>
			<td>340939</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>APC-eFluor 780 anti-human HLA-DR (Clone LN3)</td>
			<td>eBioscience</td>
			<td>47-9956-42</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Autoclave pouches</td>
			<td>VWR&#160;</td>
			<td>89140-800</td>
			<td>For autoclaving tools and paper towels</td>
		</tr>
		<tr>
			<td>Brilliant Violet 60 anti-human CD4 antibody (Clone OKT4</td>
			<td>Biolegend</td>
			<td>317438</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Brilliant Violet 65 anti-human CD8a antibody (Clone RPA-T8)</td>
			<td>Biolegend</td>
			<td>301042</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Brilliant Violet 711 anti-human CD3 antibody (Clone OKT3)</td>
			<td>Biolegend</td>
			<td>317328</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Buprenex 0.3 mg/mL</td>
			<td>Covetrus</td>
			<td>059122</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Carprofen 50 mg/mL</td>
			<td>Zoetis</td>
			<td>NADA # 141-199</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>4188</td>
			<td>Skin digestion</td>
		</tr>
		<tr>
			<td>D42 Dermatome blade</td>
			<td>Humeca</td>
			<td>5.D42BL10</td>
			<td>dermatome (1 blade per sample)</td>
		</tr>
		<tr>
			<td>Dermatome D42</td>
			<td>Humeca</td>
			<td>4.D42</td>
			<td>dermatome</td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Bard-Parker</td>
			<td>371610</td>
			<td>skin preparation</td>
		</tr>
		<tr>
			<td>Dissecting T-Pins; 1-1/2 inch, 1000/CS 1.5</td>
			<td>Cole-Parmer</td>
			<td>UX-10915-03</td>
			<td>To pin skin specimen for dermatome</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>medicon</td>
			<td>02.04.10</td>
			<td>sample preparation and mouse dissection</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-1G</td>
			<td>Skin digestion</td>
		</tr>
		<tr>
			<td>eBioscience Foxp3 / Transcription Factor Fixation/Permeabilization Concentrate and Diluent</td>
			<td>eBioscience</td>
			<td>00-5521-00</td>
			<td>Flow cytometry analysis: Cell Fixation and Permeabilization</td>
		</tr>
		<tr>
			<td>eFluor-450 FOXP3 monoclonal antibody (Clone PCH101)</td>
			<td>eBioscience</td>
			<td>48-4776-42</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>Electric clippers</td>
			<td>Kent</td>
			<td>CL8787-KIT</td>
			<td>hair removal</td>
		</tr>
		<tr>
			<td>Epredia&#160;Shandon Instant Eosin</td>
			<td>Fisher Scientific</td>
			<td>6765040</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>Epredia&#160;Shandon Instant Hematoxylin</td>
			<td>Fisher Scientific</td>
			<td>6765015</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>FITC anti-human CD45 (Clone HI30)</td>
			<td>Tonbo Biosciences</td>
			<td>35-0459-T100</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Forceps&#160;</td>
			<td>medicon</td>
			<td>07.60.07</td>
			<td>sample preparation and mouse dissection</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td>Sample preparation</td>
		</tr>
		<tr>
			<td>Heating lamp</td>
			<td>Morganville Scientific</td>
			<td>HL0100</td>
			<td>Post-surgical care</td>
		</tr>
		<tr>
			<td>Heating pads 4&#34; x 10&#34;</td>
			<td>Pristech</td>
			<td>20415</td>
			<td>Surgical heat supply</td>
		</tr>
		<tr>
			<td>Insulin 1cc 12.7 mm syringes</td>
			<td>BD</td>
			<td>329410</td>
			<td>drug administration</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>United States Pharmacopeia (USP)&#160;</td>
			<td>NDC 66794-013-25</td>
			<td>Anesthesia&#160;</td>
		</tr>
		<tr>
			<td>Isoflurane machine</td>
			<td>VetEquip</td>
			<td>911103</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Nair for Men</td>
			<td>Nair</td>
			<td>&#8206; 10022600588556</td>
			<td>hair removal</td>
		</tr>
		<tr>
			<td>Neomycin and Polymyxin Bisulfates and Bacitracin Zinc Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>&#160;NDC 17478-235-35</td>
			<td>eye ointment to prevent drying</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td>
			<td>The Jackson Laboratory</td>
			<td>005557</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Kleenex</td>
			<td>100848</td>
			<td>May be autoclaved for sterile surfaces</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td>Semitransparent sealing film</td>
		</tr>
		<tr>
			<td>PE mouse anti-human CD127 (Clone HIL-7R-M21)</td>
			<td>BD Biosciences</td>
			<td>557938</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>PE-Cy-7 mouse anti-Ki-67 (Clone B56)</td>
			<td>BD Biosciences</td>
			<td>561283</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>PerCP-eFluor-710 CD152 (CTLA-4) monoclonal antibody (Clone 14D3)</td>
			<td>eBioscience</td>
			<td>46-1529-42</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer 10x</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td>Flow cytometry analysis: Intracellular protein staining buffer</td>
		</tr>
		<tr>
			<td>Petri Dish 150 mm</td>
			<td>Corning</td>
			<td>430597</td>
			<td>Sample storage</td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>Fisherbrand</td>
			<td>22-305-654</td>
			<td>Site preparation</td>
		</tr>
		<tr>
			<td>Providone-Iodine Swab stick</td>
			<td>PDI</td>
			<td>S41350</td>
			<td>Site sterilization</td>
		</tr>
		<tr>
			<td>Soft-Feed and Oral Hydration (Napa Nectar)</td>
			<td>Se Lab Group Inc</td>
			<td>NC9066511&#160;</td>
			<td>For supplementing poorly recovering mice post-surgery</td>
		</tr>
		<tr>
			<td>Specimen Collection Cups</td>
			<td>Fisher Scientific</td>
			<td>22-150-266</td>
			<td>sample storage</td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td>skin preparation</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Media for sample storage</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-02</td>
			<td>For drug dilution</td>
		</tr>
		<tr>
			<td>Tegaderm Film 4&#8221; x 43/4&#8221;&#160;</td>
			<td>3M</td>
			<td>1626</td>
			<td>transparent film wound dressing</td>
		</tr>
		<tr>
			<td>Vaseline Petrolatum Gauze 3&#8221; x 8&#8221;&#160;</td>
			<td>Kendall</td>
			<td>414600</td>
			<td>wound dressing</td>
		</tr>
		<tr>
			<td>Violet 510 Ghost Dye&#160;</td>
			<td>Tonbo Biosciences</td>
			<td>13-0870-T100</td>
			<td>Flow cytometry analysis: Viability dye</td>
		</tr>
	</tbody>
</table>

xenograft skin model to manipulate human immune responses in vivo

人类皮肤异种移植模型将人类供体皮肤移植到免疫缺陷小鼠宿主上，是皮肤免疫学转化研究的重要选择。小鼠和人体皮肤在解剖学和免疫细胞组成方面存在很大差异。因此，传统的小鼠模型在皮肤病学研究和药物发现方面存在局限性。然而，成功的异种移植在技术上具有挑战性，需要最佳的标本和小鼠移植部位准备，以确保移植物和宿主存活。本协议提供了一种将人类皮肤移植到小鼠身上的优化技术，并讨论了下游实验目标的必要考虑因素。本报告描述了人类供体皮肤样本的适当制备、手术装置的组装、小鼠和手术部位的准备、皮肤移植和术后监测。坚持这些方法允许在手术后维持异种移植物超过6周。由于工程控制、无菌技术以及术前和术后调理的发展，下面概述的技术可实现最大的移植效率。异种移植模型的适当性能导致长寿命的人类皮肤移植样品用于人体皮肤的实验表征和 体内化合物的临床前测试。

在超级屏障动物设施内对NSG小鼠进行了人类皮肤异种移植。成功是由移植后小鼠的移植和小鼠存活时间延长以及行为健康来定义的。手术后一周内存活率低最初被认为是实验成功的最大障碍，高达50%的小鼠需要安乐死。在手术期间和手术后立即改进无菌技术和更好地支持小鼠体温，将手术存活率持续提高到80%以上，通常达到90%-100%。虽然试验了在小鼠饮用水中添加抗生素，但并未确定改善结局，?...

Watch this Scientific Journal Video about Xenograft Skin Model to Manipulate Human Immune Responses In Vivo at JoVE.com

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

本协议描述了如何将人体皮肤移植到非肥胖糖尿病（NOD）-scid白细胞介素-2γ链受体（NSG）小鼠上。报告中详细介绍了移植用人体皮肤的制备、移植用小鼠的准备、裂层人体皮肤的移植以及移植后的恢复程序。

异种移植皮肤模型在体内操纵人类免疫反应

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

我们概述了人类异种移植皮肤移植模型所需的技术，这是操纵体内人体组织中免疫和非免疫途径的重要工具。与体外方法相比，该技术更好地概括了人体皮肤生理学，允许在体内进行临床前实验操作。异种移植皮肤移植可用于体内靶向人体皮肤炎症的化合物的临床前测试。包扎小鼠可能特别具有挑战性，因此我们建议在进行手术之前对健康的麻醉小鼠进行包扎过程。首先，准备在灭菌解剖板上的灭菌负压组织培养罩中对人体皮肤样品进行皮节化。将表皮面朝上的皮肤样本放在解剖板上，然后用无菌酒精制备垫擦拭表皮，然后用PBS擦拭表皮。现在使用 1.5 英寸的解剖 T 销来固定皮肤的较近边缘。然后以400微米厚度的皮肤标本进行皮节，并施加稳定的压力，同时以30至45度的角度向前切割。将浸泡在无菌PBS中的无菌纱布放在培养皿底部，并将表皮一侧朝上的皮肤放在湿纱布上。用半透明的密封膜密封并覆盖板边缘，以确保样品不受污染，并在接枝前将样品储存在四摄氏度。准备无菌器械和手术站进行移植，并使用高压灭菌的纸巾作为器械和小鼠放置的无菌表面。麻醉诱导后，将鼠标转移到加热垫或其他热源上，并通过用戴手套的手指在眼睛上涂抹一小滴软膏来施用眼药膏。捏住皮肤并以与身体平行的角度皮下注射镇痛药。用尾巴抬起鼠标后，露出腹部，并使用一毫升胰岛素注射器以30度角腹膜内注射每100微升抗Gr111微克。然后使用动物安全的电动剪剃掉鼠标背侧的中部和上部。清除所有毛发后，将大量脱毛软膏涂抹在剃光的皮肤上。等待 30 秒到一分钟，然后用纸巾和 PBS 完全擦拭脱毛软膏。将小鼠转移到手术站后，对手术区域进行消毒，并在小鼠上放置无菌保鲜膜。在塑料中切一个窗口，略大于要嫁接的区域的大小。用镊子背面牢固地固定供体皮肤，并切下10×10毫米的供体皮肤的矩形部分，用手术刀与镊子一起移植。将皮肤从身体上拉开，以避免深深切入筋膜。要创建移植床，请剪下与供体皮肤片大小相匹配的小鼠皮肤矩形区域。然后将带有表皮的供体皮片朝上放在准备好的移植床上。使用镊子的背面，操纵皮肤来回滑动，直到供体皮肤完全平放在移植床上。在供体皮肤与小鼠皮肤相接的地方加入手术胶水组织粘合剂滴剂，并用镊子将小鼠和供体皮肤固定在一起一到两秒钟，使胶水粘附在组织上。完全密封移植物的边缘，让胶水彻底干燥。接下来，切开足够大的凡士林纱布以完全覆盖移植区域。用凡士林纱布覆盖移植物，并用镊子将纱布轻轻按压在皮肤上。纵向切一条透明薄膜敷料，使宽度足够大以覆盖小鼠的伤口。用力按压透明薄膜敷料，粘合剂面朝下盖在纱布上。快速滚动鼠标，将敷料完全包裹在躯干上，确保其紧密贴合而不会阻碍呼吸，并且所有肢体都可以自由活动。将鼠标放入恢复笼后，监视它，直到它发出警报并四处移动。恢复后，在笼子部分提供至少 15 分钟的热源。移植后10至21天的移植物分析表明，移植物仍然粘附在供体皮肤与小鼠皮肤相接的边缘，并且移植物仍然比健康人皮肤更厚，更发炎。流式细胞术分析显示，大多数移植物中存在人类免疫细胞，但异种移植物之间的计数不同。成功的移植物维持了人体免疫细胞。然而，尽管供体组织继续粘附和存活，但未能维持人类免疫细胞的移植物被认为是不成功的。35天后对苏木精和伊红染色的移植物切片的分析显示表皮完整，真皮为中度细胞，由丰满的椭圆形成纤维细胞组成，具有苍白的合胞细胞质和分散的淋巴细胞。50天后对移植物的分析显示，完整的表皮和含有椭圆形成纤维细胞的真皮和略微增强的细胞外基质沉积的结果相似。成功的皮肤异种移植依赖于皮肤标本的适当制备、通过抗 Gr1 给药抑制小鼠中性粒细胞免疫应答、坚持无菌啮齿动物手术方法以及安全包扎小鼠。人类异种移植皮肤移植模型允许研究人员研究体内完整器官中的人类免疫反应，为转化研究提供了新的途径。

Research

JoVE Journal

Immunology and Infection

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

Seven Steps to Stellate Cells

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...