【摘要】 目的通过经尾静脉或腹腔两种方式给NOD/SCID小鼠注射正常人外周血单核细胞(PBMCs)的方式,来探究对于建立HuPBMCs-NOD/SCID人鼠嵌合模型两种移植方式建模效果的比较,随后对该模型进行HIV病毒感染的鉴定。方法将9只雌性NOD/SCID小鼠给予亚致死剂量全身照射后,分为3组,实验Ⅰ组:3只雌性NOD/SCID小鼠,腹腔注射0.5 mLPBMC细胞悬液;实验Ⅱ组:3只雌性NOD/SCID小鼠,尾静脉注射0.2mLPBMC细胞悬液;实验Ⅲ组:3只雌性NOD/SCID小鼠,注射0.2mLRPMI 1640细胞培养液,作为Ⅰ组及Ⅱ组的实验对照。通过观察移植细胞后小鼠的存活时间与生活状态,并施以流式细胞技术鉴定小鼠眼眶外周血中CD45~+、CD4~+、CD8~+等人源化细胞的比例。另取4只NOD/SCID小鼠进行腹腔移植3天后,腹腔注射HIV感染的PBMCs细胞并以未移植的NOD/SCID小鼠作为对照,在感染后第5wk采集小鼠眼眶血,流式细胞技术及免疫荧光定量PCR方法分别检测其CD45~+、CD4~+、CD8~+等人源化细胞的比例及病毒滴度。结果两种方式注射正常人PBMC细胞至小鼠内2～10周后,实验组小鼠体内均检测到人源化免疫细胞的浸润,各细胞比例随小鼠移植时间而增加;但相比尾静脉注射,腹腔注射鼠内人源性免疫细胞比例明显较高;此外二种移植方式小鼠存活时间均达10wk,即成功建立HuPBMCs-NOD/SCID人鼠嵌合模型。随后对该人鼠嵌合模型进行腹腔注射HIV感染的PBMC,第5周免疫荧光定量PCR能检测到病毒滴度,发现病毒滴度上升,同时结合流式细胞技术分析随着病毒滴度上升,CD45~+分子比例下降且CD4~+/CD8~+<1。结论应用尾静脉或腹腔注射正常人外周血单个核细胞(PBMCs)的方法,均可建立人PBMCs-NOD/SCID人鼠嵌合模型;但我们发现相比尾静脉注射方式,腹腔注射对于建立HuPBMCs-NOD/SCID人鼠嵌合模型有着明显的优势。同时通过腹腔注射HIV感染的PBMC至该模型,HIV病毒可以成功地被感染。这为艾滋病体外潜伏机制的研究和抗病毒药物筛选提供了良好的动物模型。更多还原

【Abstract】 Objective To investigate which inoculation method was better established for setting up HuPBMCNOD/SCID human chimeric model by injecting normal human peripheral blood mononuclear cells(PBMCs) into NOD/SCID mice via the tail vein or the abdominal cavity. Afterwards,above model was identified for HIV infection. Methods Nine female NOD/SCID mice were given sublethal doses of whole body irradiation and divided into three groups. Group Ⅰ: 3 female NOD/SCID mice,0.5 mL PBMC cell suspension was injected intraperitoneally;Group Ⅱ: 3 female NOD/SCID mice,0.2 mL PBMC cell suspension in the tail vein;Group Ⅲ: 3 females NOD/SCID mice were injected with 0.2 mL RPMI 1640 cell culture medium as experimental controls. After transplantation,the survival time and living state of the mice were observed,and the proportion of humanized cells such as CD45~+,CD4~+and CD8~+in the peripheral blood of mouse eyelids was detected by flow cytometry.HIV-infected PBMCs were injected intraperitoneally in PBMCs transplantation NOD/SCID mice,untransplanted NOD/SCID mice as controls. After 5 wk infection,the ratio of humanized cells such as CD45~+,CD4~+,CD8~+and HIV virus titer weredetected by flow cytometry and immunofluorescence quantitative PCR. Results After injecting normal human PBMC cells into mice for 2 to 10 weeks,the infiltration of humanized immune cells was detected in the experimental group,and the proportion of each cell increased with the transplantation time of the mice;However,the proportion of humanized immune cells in the intraperitoneal injection groups was significantly higher compared with the tail vein injection groups;In addition,the survival time of both transplantedmethods mice was up to 10 wk,namely,the HuPBMCs-NOD/SCID human-chimeric chimeric model was successfully established. Subsequently,the humanied mice model was intraperitoneally injected with HIV-infected PBMC,the HIV-Virus Titer andproportion of humanized immune cells was respectively detected by RT-qPCR and flow cytometry. It was found that with the increase of virus titer,the ratio of CD45~+molecules decreased and CD4~+/CD8~+<1 by flow cytometry after 5 wk. Conclusion Humanized PBMCs-NOD/SCID chimeric model can be established via both tail vein and intraperitoneal injection of peripheral blood mononuclear cells(PBMCs). More importantly,we found that intraperitoneal injection for the establishment of HuPBMCs-NOD/SCID mouse chimeric model has obvious advantages,compared with tail vein injection. At the same time,the HIV virus can be successfully infected by intraperitoneal injection of HIV-infected PBMC into the model. This provides a good animal model for the study of AIDS latency mechanisms and screening for antiviral drugs in vivo.更多还原