5′-溴-2′-脱氧尿嘧啶核苷标记人巨细胞病毒感染SD大鼠脑组织神经元模型的建立  

Establishment of SD rat model infected by BrdU-labeled human cytomegalovirus in brain neurons

作　　者：傅鹰[1] 沈波[1] 陈利玉[2] 

机构地区：[1]浙江省台州医院检验科,317000 [2]中南大学湘雅医学院医学微生物学教研室

出　　处：《中华生物医学工程杂志》2008年第2期131-135,共5页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金（30470088）

摘　　要：目的用5′-溴-2′-脱氧尿嘧啶核苷（BrdU）标记的人巨细胞病毒（HCMV）进行脑内定位注射，建立SD大鼠脑组织感染模型；研究大鼠脑组织海马部位神经元对HCMV的容许性及HCMV在其内的感染特点。方法运用BrdU标记HCMV基因组，并测定病毒滴度。将21只SD大鼠随机分为病毒感染组（n=18）及对照组（n=3），病毒感染组根据感染时间分为感染24、48及96h组。每个感染时间组再分为2μl及4μl两个剂量组，每个剂量组包括3只动物，均以脑内注射途径将BrdU标记的HCMV直接注入大脑海马部位；对照组大鼠注射不含病毒的细胞培养上清液（4μl）。感染大鼠术后分笼饲养，于各感染时间点处理大鼠取脑组织，冰冻切片；对海马周围部位脑组织分别运用抗BrdU及抗微管相关蛋白2（MAP-2）抗体进行免疫荧光双标染色检测标记病毒；抗pp65抗体免疫组化染色及Nissil复染检测病毒蛋白pp65。结果病毒感染后24h，两个剂量组均未在神经元内检测到标记病毒及pp65。病毒感染后48h和96h，两个剂量组在海马部位神经元中均检测到了标记病毒阳性荧光及pp65的阳性着色；而且2μl组的受感染神经元细胞数较4μl组少，但同一剂量组在48h和96h受感染神经元细胞数未见明显变化。结论成功建立了BrdU标记HCMV感染SD大鼠啮组织的模型。大鼠脑组织海马部位神经元是HCMV的半容许细胞，HCMV在其内仅表现为潜伏性感染。Objective To establish an SD rat brain infection model by using 5′-bromo-2′-deoxyuridine （BrdU）-labeled human cytomegalovirus （HCMV） for exploring the permissiveness and infective features of HCMV in SD rat brain neurons. Methods HCMV was labeled with BrdU and virus PFU （ plaque forming unit） was measured. Twenty one SD rats were randomly divided into two groups：viral infection group （n = 18） and control group （n =3）. Then the viral infection group was subdivided into three groups： 24 h,48 h and 96 h according to the infection time, and each infection time group had two dosage groups ： 2 μl and 4μl Each dosage group had 3 rats which were injected directly with BrdU-labeled HCMV AD169 into the hippocampal gyrus of SD rat brain. The control group was injected with cell maintenance media （4 μl） at the same place. All rats were respectively bred then killed at different infection time points, and then brain tissue slices were prepared and immunofluorescence double staining with anti-BrdU and anti-MAP-2 was done to examine BrdU-labeled HCMV DNA, meanwhile the slices were subjected to immunohistochemistry with anti-pp65 and Nissil staining in turn to examine HCMV pp65. Results The positive immunofluorescence of BrdU-labeled HCMV and the positive staining of pp65 was not detected in neurons at 24 h, but was found at both 48 h and 96 h in hippocampal gyrus. The infected neurons was less in 2 μl dosage group than that in 4 μl, but in the same dosage group,neurons numberdid not change between 48 h and 96 h. Condusions An SD rat model infected by BrdU-labeled HCMV in brain neurons is established successfully. Brain neurons in hippccampal gyrus are semipermissive to HCMV. And HCMV shows latent feature on these cells.

关 键 词：巨细胞病毒 感染 模型 动物 5′-溴-2′-脱氧尿嘧啶核苷 PP65蛋白 

分 类 号：R-332[医药卫生]