Name: establishment of the dual humanized tk-nog mouse model for hiv-associated liver pathogenesis
Uploaded: 2019-09-11T12:30:00
Description: Watch this Scientific Journal Video about Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis at JoVE.com

这项工作得到了国家卫生研究院的拨款R24OD018546（给L.Y.P.和S.G.）的支持。作者要感谢李伟哲博士，在外科手术方面的帮助，阿曼达·科科·伍兹，B.S.，严成免疫性组学，UNMC流式细胞学研究设施成员菲利普·赫克斯利主任，博士，维多利亚B.史密斯，B.S.和萨曼莎沃尔，B.S.，UNMC高级显微镜核心设施成员Janice A. 泰勒，B.S.和詹姆斯R.塔斯卡拉斯，B.S.，技术支持。作者承认CIEA的Mamoru Ito博士和Hiroshi Suemizu博士提供TK-NOG小鼠，约阿希姆·鲍姆加特博士提供硫磺范。作者感谢UNMC的阿德里安·科斯特斯博士对手稿的编辑贡献。

该协议已获得内布拉斯加大学医学中心机构动物护理和使用委员会（IACUC）的批准。
注：在动物实验前，先获得当地IACUC的批准。
1. 脐带血的处理与人体HSPC的分离
<ol><li>在层流柜的无菌条件下执行协议的所有步骤。</li>
	<li>取在肝化管中收集的脐带血（CB），通过添加磷酸盐缓冲盐水（PBS）使体积达到35 mL。如图 1所示?...

<ol>
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在HIV感染者24日肝脏受损。实验性小动物模型研究人类肝脏疾病在HIV-1的存在是极其有限的，尽管有一些联合移植的动物模型与CD34+ HSPCs和肝细胞7，12， 25.在体外实验中，肝细胞被证明有低水平的HIV-1感染26。携带这两种人类细胞的人类小鼠是理想的模型。在人类调节T细?...

自抗逆转录病毒疗法问世以来，与艾滋病毒-1单一感染有关的死亡人数已大大减少。然而，肝病已成为艾滋病病毒感染者发病的常见原因1，2。肝炎病毒感染与HIV-1感染比较常见，占美国艾滋病病毒感染者的10%-30%，3、4、5。
HIV-1和肝炎病毒的宿主特异性限制了小动物模型研究人类特定传染病或研究HIV-1相关肝脏发病机制的多个方面的效用。免疫缺陷小鼠，允许移植人体细胞和/或组织（称为人化小鼠模型）是可以接受的动物模型为临床前研究6，7，8。自21世纪初引入人化小鼠以来，多次对胆囊静物人类肝毒性、人类特异性病原体（包括HIV-1和HIV相关神经认知障碍、爱泼斯坦巴尔病毒、肝炎等）进行临床前研究传染病，已对这些小鼠进行了6、9、10、11的调查。CD34+ HSPCs和/或人类肝细胞移植的多种小鼠模型早已开发出来，并随着时间的推移得到了改进，以研究乙型肝炎病毒（HBV）相关肝病的疾病发病机制12，13,14.HSPC和人肝细胞移植的几个模型基于菌株，称为NOG（NOD）。Cg-Prkdcscid Il2rgtm1Sug/JicTac）8，13， NSG （NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ）15， 巴尔布/C-拉格2-/- -----（拉格2tm1.1Flv Il2rgtm1.1Flv/J）12，和fah-/- NOD rag1--il2ré_null鼠标16。但是，每个模型都有自己的优点和局限性;例如，AFC8 在 Balb/C-Rag2-/- ---c -/-背景上为 HEP 和人类干细胞 （HSCs） 提供双人机小鼠，使免疫细胞和 HSC 能够成功移植，但不存在抗原特异性 T 细胞和 B 细胞在此模型12中响应 。在重组双人性小鼠时，主要关切包括次优移植、缺乏支持不同组织的合适模型、不匹配的条件、免疫排斥或移植-宿主疾病（GVHD）和技术困难，如与新生儿的风险操纵和高死亡率由于代谢异常13。
虽然人类化小鼠已用于艾滋病研究多年17、18、19，但使用人化小鼠研究HIV-1对肝脏的损害已经有限20。我们之前曾报道建立了双重人性化的TK-NOG小鼠模型，并将其应用于与艾滋病毒相关的肝病8。该模型显示了肝脏和免疫细胞的强健移植，并重述了HIV感染的发病机制。本讨论提出了详细的方案，包括人类肝细胞移植中最关键的步骤。还介绍了成功移植HEP和在TK-NOG小鼠中建立功能免疫系统所需的HSPCs。详细介绍了这些小鼠研究HIV相关肝脏免疫机原机制。使用TK-NOG雄性小鼠携带肝脏特异性疱疹单纯疱疹病毒类型1胸腺激酶（HSV-TK）转基因。表达这种转基因的小鼠肝细胞在短暂接触无毒剂量的GCV后，很容易被消融。移植的人类肝细胞在小鼠肝脏内保持稳定，没有外源性药物21。小鼠还预置了非骨髓剂量的三硫丹，为人类细胞8在小鼠骨髓中创造一个利基。免疫缺陷的TK-NOG小鼠在脾脏注射了HEP和多能的HSPCs。然后，通过血液免疫造影和分别测量血清人-白蛋白水平，定期监测小鼠的血液和肝脏重组。成功重组超过15%的小鼠为人体免疫细胞和HEP注射HIV-1。艾滋病毒对肝脏的影响最早可在感染后4-5周内评估。必须指出，由于使用了HIV-1，在处理病毒并将其注射到小鼠体内时，必须采取一切必要的预防措施。

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			<td style="width:135px;">Sigma-aldrich</td>
			<td style="width:127px;">A9576</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Buprenorphine</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Controlled substance and pain-killer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD14-PE</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">555398</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD19-BV605</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">562653</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:125px;">CD34 MicroBead Kit, human</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-046-702</td>
			<td style="width:167px;">For isoation of&#160;&#160; CD34+ HSPC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">CD34-PE, human</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-081-002</td>
			<td style="width:167px;">Antibody used for purity check of eluted CD34+ cells&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD3-AF700</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">557943</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD45-PerCPCy5.5</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">564105</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD4-APC</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">555349</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD8-BV421</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">562428</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cell counting slides</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">ChargeSwitch gDNA Mini Tissue Kit</td>
			<td style="width:135px;">Thermofisher scientific</td>
			<td style="width:127px;">CS11204</td>
			<td style="width:167px;">for extraction of genomic DNA from ear piece</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cobas Amplicor system v1.5&#160;</td>
			<td style="width:135px;">Roche Molecular Diagnostics</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">bioanalyzer to measure viral load</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cotton-tipped applicators&#160;</td>
			<td style="width:135px;">&#160;McKesson</td>
			<td style="width:127px;">24-106-2S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cytokeratin-18 (CK18)</td>
			<td style="width:135px;">DAKO</td>
			<td style="width:127px;">M7010</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:135px;">Sigma-aldrich</td>
			<td style="width:127px;">D2650-5X5ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:125px;">Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">30914</td>
			<td style="width:167px;">Attached to dispensing pippet and to load with HSPC and HEP suspesion</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">FACS Diva version 6</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">flow cytometer software required for&#160; acqusition of sample</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:127px;">10438026</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">FLOWJO analysis software 
			v10.2</td>
			<td style="width:135px;">FLOWJO, LLC</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">flow cytometry analysis software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Ganciclovir</td>
			<td style="width:135px;">APP Pharmaceuticals, Inc.</td>
			<td style="width:127px;">315110</td>
			<td style="width:167px;">Prescripition drug</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Greiner MiniCollect EDTA Tubes</td>
			<td style="width:135px;">Greiner bio-one</td>
			<td style="width:127px;">450475</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Hepatocytes thawing medium&#160;</td>
			<td style="width:135px;">Triangle Research Labs&#160;</td>
			<td style="width:127px;">MCHT50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Horizon Open Ligating Clip Appliers</td>
			<td style="width:135px;">Teleflex</td>
			<td style="width:127px;">537061</td>
			<td style="width:167px;">To hold the ligating clips</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Hospira Sterile Water for Injection</td>
			<td style="width:135px;">ACE surgical supply co. Inc.</td>
			<td style="width:127px;">001-1187</td>
			<td style="width:167px;">For dilution of Buprenorphine (pain-killer)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Human Albumin ELISA Quantitation Set</td>
			<td style="width:135px;">Bethyl laboratories</td>
			<td style="width:127px;">E80-129</td>
			<td style="width:167px;">For assesing human albumin levels in mouse serum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Human hepatocyte</td>
			<td style="width:135px;">Triangle Research Labs&#160;</td>
			<td style="width:127px;">HUCP1</td>
			<td style="width:167px;">&#160;Cryopreserved human hepatocytes, induction qualified&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Iris Scissors, Straight</td>
			<td style="width:135px;">Ted Pella, Inc.</td>
			<td style="width:127px;">13295</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">Lancet</td>
			<td style="width:135px;">MEDIpoint</td>
			<td style="width:127px;">Goldenrod 5 mm</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:125px;">LS columns&#160;</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-042-401</td>
			<td style="width:167px;">Used to entrap CD34+ microbeads (positive selection)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Lymphocyte Separation Medium (LSM)</td>
			<td style="width:135px;">MP Biomedicals</td>
			<td style="width:127px;">50494</td>
			<td style="width:167px;">For isoation of&#160;&#160; lymphocytes from peripheral blood</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">MACS MultiStand</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-042-303</td>
			<td style="width:167px;">holds Qudro MACS seperator and LS columns</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">McPherson-Vannas Micro Dissecting Spring Scissors</td>
			<td style="width:135px;">Roboz Surgical Instrument Co.</td>
			<td style="width:127px;">RS-5605</td>
			<td style="width:167px;">Used to make an incision on skin to expose spleen</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Micro Dissecting Forceps</td>
			<td style="width:135px;">Roboz Surgical Instrument Co.</td>
			<td style="width:127px;">RS-5157&#160;</td>
			<td style="width:167px;">to hold and pull out spleen from peritoneal cavity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">mouse CD45-FITC</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">553080</td>
			<td style="width:167px;">mouse-specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">PBS (Phosphate Buffered Saline)</td>
			<td style="width:135px;">Hyclone</td>
			<td style="width:127px;">SH30256.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Qudro MACS separator&#160;</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-090-976</td>
			<td style="width:167px;">holds four LS columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">RPMI 1640 medium</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:127px;">11875093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">StepOne Plus Real Time PCR&#160;</td>
			<td style="width:135px;">Applied Biosystems</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Instrument used&#160; to&#160; genotype</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">Stepper Series Repetitive Dispensing Pipette 1ml</td>
			<td style="width:135px;">DYMAX CORP</td>
			<td style="width:127px;">T15469</td>
			<td style="width:167px;">Used to&#160; dispense&#160; HSPC and HEP supension in controlled manner</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Suturevet PGA synthetic absorbale suture</td>
			<td style="width:135px;">Henry Schein Animal Health</td>
			<td style="width:127px;">41178</td>
			<td style="width:167px;">Suturing of skin and peritoneum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:135px;">Thermofisher scientific</td>
			<td style="width:127px;">4369016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">TC20 automated cell counter</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450102</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:125px;">TK-NOG mice&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Treosulfan</td>
			<td style="width:135px;">Medac GmbH</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Provided by &#160;Dr. Joachim Baumgart (medac GmbH)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">Trypan Blue</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450022</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Vannas-type Micro Scissors, Straight, 80mm L</td>
			<td style="width:135px;">Ted Pella, Inc.</td>
			<td style="width:127px;">1346</td>
			<td style="width:167px;">Used to make an incision on skin to expose spleen</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">Weck hemoclip traditional titanium ligating clips</td>
			<td style="width:135px;">Esutures</td>
			<td style="width:127px;">523700</td>
			<td style="width:167px;">To ligate the spleen post-injection</td>
		</tr>
	</tbody>
</table>

establishment of the dual humanized tk-nog mouse model for hiv-associated liver pathogenesis

尽管感染人体免疫机能丧失病毒-1（HIV-1）的患者预期寿命延长，但肝病已成为其发病的常见原因。HIV-1引起的肝脏免疫病理学仍然难以捉摸。具有人类肝细胞和人类免疫系统的小型异种移植动物模型可以概括人类生物学的发病机制。本文描述了一个方案，通过人类肝细胞和CD34——造血干细胞/祖细胞（HSPCs）移植建立双重人化小鼠模型，以研究HIV感染者观察到的肝脏免疫病理学。为了实现双重重组，男性TK-NOG（NOD）。Cg-Prkdcscid Il2rgtm1Sug Tg（Alb-TK）7-2/ShiJic）小鼠在腹内注射甘西洛韦（GCV）剂量，以消除小鼠转基因肝细胞，并结合三硫丹进行非骨髓性调理，这两种促进人类肝细胞 （HEP） 移植和人类免疫系统 （HIS） 发育。人白蛋白 （ALB） 水平用于肝脏移植，流式细胞测定检测的血液中存在人体免疫细胞，证实了人类免疫系统的建立。使用此处所述的协议开发的模型类似于HIV-1感染造成的肝脏损伤的多个组成部分。其建立对于肝炎病毒合并感染的研究以及抗病毒和抗逆转录病毒药物的评估可能至关重要。

通过非常简单的ELISA和流式细胞测定，在每一步都可以轻松监测与人类肝脏和免疫细胞的双重人性化小鼠模型。定期进行流式细胞测量，以评估功能免疫系统的发展，并观察艾滋病毒感染对免疫细胞的影响。在双人性小鼠中，功能免疫细胞的发育范围为淋巴细胞门的15%至90%。免疫细胞的代表性子集以点图显示（图3）。为了评估人类肝细胞的移植，ELIS...

Watch this Scientific Journal Video about Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis at JoVE.com

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

该协议提供了一种可靠的方法，建立人类免疫系统和肝细胞的人性化小鼠。通过注射人肝细胞和CD34实现的双重组免疫缺陷小鼠–造血干细胞易受人体免疫缺陷病毒-1感染和重述肝损伤的影响，如艾滋病毒感染者。

建立与艾滋病毒相关的肝脏发病机制的双人性化TK-NOG小鼠模型

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

该协议的总体目标是利用人类造血干细胞和肝细胞位点生成具有功能性人类免疫系统和肝脏的人性化小鼠模型。这种方法可以帮助回答遗传性肝人性化小鼠的关键问题。这种方法为研究艾滋病毒在人中观察到的免疫病理学提供了有力的工具。病理学和微生物学部的主管孙一民和研究技术专家王伟民和埃德德·马卡罗夫将展示这个过程。首先，将肝素管中收集的脐带血转移到无菌层流柜。添加 PBS，直到体积增加到 35 毫升。将样品层对淋巴细胞分离介质顶部，将样品离心400倍G，在4摄氏度下35分钟，无间断。接下来，小心地移除顶部等离子层，并使用转移移液器将白色薄漆界面转移到新管中。将抛光衣重新悬浮在 30 到 40 毫升的冰冷缓冲液中。使用移液器，将细胞悬浮液的20微升与20微升0.4%的蓝光混合。将这种混合物的10微升移液插入计数幻灯片的两个腔室的外开口，并将幻灯片插入自动细胞计数器以计算细胞。在300倍G和4摄氏度下将细胞离心10分钟。小心吸上清液，加入300微升的冰冷缓冲液。加入100微升的人类FC受体阻断试剂和100微升的单克隆小鼠抗人CD34抗体结合微珠，可产生多达1亿个细胞。在4摄氏度下孵育30分钟。加入10毫升的冰冷缓冲液清洗细胞，并将其离心300倍G和4摄氏度10分钟。小心地去除上清液，并在 500 微升的冰冷缓冲液中重新悬浮颗粒。在此之后，在磁激活细胞分拣场中放置一个正选择LS柱，然后用三毫升的冰冷缓冲液通过。将样品加载到 LS 柱中，将微珠捕获到样品中与人类 CD34 阳性细胞绑定的微珠，并允许它在重力的影响入收集管。用冰冷缓冲液清洗柱子三次，并在同一收集管中收集洗涤液。然后，用五毫升的冰冷缓冲液将柱子插入一个新的收集管中，将CD34阳性细胞插入。重复该过程以达到纯度超过90%Next，使用锥蓝染料和血细胞计来计数的CD34阳性细胞。计数后，将300次G的细胞离心5分钟。丢弃上一级，将PBS125微升中的细胞重新悬浮，用于移植。要检查CD34阳性吸液的纯度，服用50微升悬浮液，并在4摄氏度下用10微升PE结合的PE结合抗人CD34抗体孵育30分钟。在此之后，清洗和重新悬浮PBS中的细胞，然后继续进行流式细胞测量。获取后，分析文本协议中概述的数据。首先，检索和解冻文本协议中概述的冷冻保留的肝细胞。然后，取下生物帽，将解冻的肝细胞倒入含有加热解冻介质的50毫升锥形管中。用手摇动管子几秒钟，使细胞悬浮。在100倍G和室温下将细胞取出8分钟。用 0.1% BSA 清洗 PBS 中的颗粒细胞，并在 PBS 中用新鲜或解冻的 HSPCs 将它们混合到每只小鼠 80 微升的最终体积。首先，将无菌延长管的一端连接到 30 量针上，另一端连接到一毫升注射器。将注射器加注池状 HEP 和 HSPCs 的悬浮液。将注射器放入重复点胶移液器的槽口中，并调整分配器以在每压机中分配 10 微升。按照文本协议中概述的每只小鼠进行剃须，用 10%的 povidone 碘在 70% 异丙醇下擦洗每只小鼠身体的左侧。使用Vannas型剪刀，在腹膜壁左侧的皮肤、肌肉和腹膜上做一个小切口，进入肋骨笼下边缘下边缘约5毫米的腹腔。找到脾脏，并使用钳子将其稍微拉到操作区域，以便于检修，并将 30 量表针插入脾脏的下极。接下来，解锁点胶移液器的柱塞，一次分配 10 微升的体积，每个脾脏限制为 60 到 80 微升。缓慢地缩回针头，使用连接贴画夹夹住脾脏。然后，使用湿润的棉尖施用器与无菌 PBS 将脾脏推回体腔。使用中断的缝合图案关闭腹部壁的肌肉层。使用不可吸收的缝合线，使用中断的缝合图案完成皮肤闭合。将所有所需材料转移到指定的生物安全二级加设施。在使用病毒时，请时刻佩戴个人防护设备，包括一次性防护用品、鞋套、面罩和双手套，包括防割手套。选择重组超过15%的人类CD45阳性细胞且血清中存在人类白蛋白的小鼠，以治疗HIV-1感染。内向小鼠注射1000至10000个组织培养感染剂量50 HIV-1 ADA，每只小鼠100至200微升。对小鼠实施安乐死后，如文本协议中所述，从每只小鼠身上切除肝脏。收集和修复肝脏在4%的半成醛过夜。建立具有人类肝脏和免疫细胞的双重人性化小鼠模型，每一步都可以通过非常简单的ELIZA和流式细胞仪来轻松监测。定期进行流细胞学，以评估功能免疫系统的发展，并查看HIV感染对免疫细胞的影响。在双人性化小鼠中，功能免疫细胞的发育范围为淋巴细胞门的15%至90%。为了评估人类肝细胞的特异性，ELIZA每月在小鼠血清上进行人类特异性白蛋白水平评估。同时与HSPCs和 HEP一起感染的小鼠显示人类特异性白细胞素水平，从每毫升约7微克到每毫升377微克，在观察时间持续增长。HIV感染对二人化小鼠血液中人体免疫细胞的影响通过流动细胞学和肝脏的 HEP 通过人类特异性白化素 ELIZA 监测。到五个星期，HIV-1会导致血清中人类白蛋白水平下降，双人性化小鼠肝脏部分的人类CK18阳性肝细胞被耗尽。与感染前同一小鼠的血液和肝脏相比，通常观察到CD4与CD8的比例较低。血液应小心地分层在淋巴细胞分离介质上，以隔离布菲涂层。手术时，轻轻握住脾脏，将针头插入脾脏的下极。在分离造血干细胞后，检查CD34阳性造血干细胞的纯度，以避免CD3阳性细胞和不同供体急性注射肝细胞，这一点至关重要。由于人类化小鼠表现出人体免疫系统和肝脏的生理方面，可以利用发育中的小鼠研究HIV和物理感染和肝硬化的物理病理发生。

Research

JoVE Journal

Immunology and Infection

In vitro Biofilm Formation in an 8-well Chamber Slide

In vitro Biofilm Formation in an 8-well Chamber Slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

科研

免疫与感染

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

人源化的小鼠模型来研究细菌感染瞄准微血管

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Humanized NOD/SCID/IL2rÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³null hu-NSG Mouse Model for HIV Replication and Latency Studies

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

线虫模型中气单胞菌感染的毒力及发病机制评价

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...