探讨呼吸道合胞病毒(RSV)感染SD大鼠的肾病病理模型中，RSV持续感染证据。

选取2个月龄、体重为180～200 g的清洁级雄性SD大鼠62只为研究对象。采用随机数字表法将其随机分为RSV组(n＝35)与对照组(n＝27)。①对RSV组SD大鼠，采用毒力疱斑形成单位(PFU)为6×10⁶ RSV液(0.4 mL/d腹腔注射＋0.2 mL/d鼻腔滴注)×3 d处理，建立RSV感染SD大鼠肾病病理模型。对照组采用磷酸盐缓冲液(PBS)(0.4 mL/d腹腔注射＋ 0.2 mL/d鼻腔滴注)×3 d处理，建立对照组PBS模型。两组均以造模前1 d计算为第0天，造模结束时计算为造模成功第1天。通过电子显微镜观察RSV感染SD大鼠肾病病理模型造模结束后第7与60天的肾小球超微结构改变，判断SD大鼠肾病病理模型造模是否成功。②RSV组SD大鼠肾病病理模型造模成功后，将35只SD大鼠平均分为7个亚组(RSV 0、7、15、30、60、90及120 d亚组)，每个亚组均为5只SD大鼠；将对照组27只PBS模型SD大鼠，按照上述时间点分为7个亚组(PBS 0、7、15、30、60、90及120 d亚组)，每个亚组分别为5、5、5、3、3、3、3只SD大鼠。分别测定各个RSV亚组与PBS亚组SD大鼠的24 h尿蛋白定量、血清白蛋白及血清肌酐水平等指标，并进行统计学分析。③于上述指标测定完毕后，分别于上述时间点处死RSV亚组与PBS亚组SD大鼠。同时，切除SD大鼠左肾下2/3、左肺下2/3和脾下2/3，以4%多聚甲醛固定24 h后，石蜡包埋后切片，采用苏木精-伊红(HE)染色，光镜下观察各个亚组SD大鼠肾、肺、脾组织是否发生病理学形态结构改变。④以RSV G蛋白单克隆抗体为一抗，制作RSV组SD大鼠肾、肺、脾组织免疫组化检测标本，于光镜下寻找各个RSV亚组RSV G蛋白存在证据。⑤以RSV G蛋白单克隆抗体为一抗，采用与绿色荧光试剂结合，同时采用4'，6-二脒基-2-苯基吲哚(DAPI)染料蓝染细胞核，制作各个RSV亚组SD大鼠肾组织荧光免疫检测标本，于荧光显微镜下寻找RSV G蛋白存在证据及其与细胞核关系。⑥切除剩余RSV亚组左肾上1/3，以3%戊二醛固定后，送至四川大学华西医院病理科制作电子显微镜标本，于电子显微镜下观察各个RSV亚组SD大鼠肾小球超微结构是否发生病理改变，同时寻找RSV颗粒。

①RSV组SD大鼠肾病病理模型造模成功，对照组PBS模型亦造模成功。②各个RSV亚组与PBS亚组SD大鼠的24 h尿蛋白定量水平检测结果显示，RSV 0与90 d亚组分别与相同时间点PBS亚组比较，差异均无统计学意义(P>0.05)；而RSV 7、15、30、60及120 d亚组，却分别较相同时间点PBS亚组均显著升高，并且差异均有统计学意义(t＝6.9、3.1、3.9、2.2、5.6，P<0.05)。血清白蛋白及血清肌酐水平检测结果显示，7个RSV亚组分别与相同时间点的7个PBS亚组比较，差异均无统计学意义(P>0.05)。③于光镜下观察各个RSV亚组SD大鼠的肾、肺、脾组织形态结构(HE染色)结果显示，除RSV 0 d亚组外，其余各个亚组肾、肺、脾组织形态结构均出现不同程度病理改变。④于光镜下观察各个RSV亚组SD大鼠的肾、肺、脾组织免疫组化技术检测结果显示，除RSV 0 d亚组外，其余各个RSV亚组肾、肺、脾组织中，均有特异性RSV G蛋白呈持续阳性表达。各个RSV亚组肾小球RSV G蛋白定量分析结果显示，RSV 0、7、15及30 d亚组RSV G蛋白表达水平呈逐渐增强趋势，并维持在高水平，但在RSV 60、90及120 d亚组又呈逐渐减弱趋势。⑤于荧光显微镜下观察各个RSV亚组SD大鼠肾组织RSV G蛋白荧光免疫检测结果显示，RSV G蛋白与DAPI蓝染的细胞核重叠。⑥透射电子显微镜下观察发现，RSV组肾组织中RSV颗粒持续存在，RSV 120 d亚组肾小球超微结构仍存在病理改变。

RSV G蛋白在RSV感染SD大鼠肾病病理模型造模成功后，大鼠肾、肺、脾组织中RSV G蛋白呈持续阳性表达，证明RSV呈持续感染状态。这一结果或提示，包括RSV在内的呼吸道病毒持续感染，是导致微小病变型肾病综合征(MCNS)患儿免疫功能紊乱的主要原因。

To study the evidence of persistent infection of respiratory syncytial virus (RSV) in SD rats pathological model of nephropathy which were infected by RSV.

A total of 62 cases of specific pathogen-free male SD rats with 2-month-old, 180-200 g of body weight were selected and randomly divided into RSV groups (n=32) and control groups (n=27) by randomized digital table method. ①RSV groups were set up by dealing with RSV fluid, pock forming unit (PFU)=6×10⁶ (0.4 mL/d via intraperitoneal and 0.2 mL/d via nasal) for 3 days; control groups were set up by dealing with phosphate buffer saline (PBS) fluid (0.4 mL/d via intraperitoneal and 0.2 mL/d via nasal) for 3 days. The day before injection was regarded as 0 d, while the 1st day began when models were successfully set up. Ultrastructural changes of glomeruli in SD rats at 7th day and 60th day after being successfully set up of a pathological model of nephropathy were observed by electron microscopy, in order to judge whether the pathological models of nephropathy to SD rats were successfully built or not. ②A total of 35 cases of SD rats in RSV groups were randomly divided into 7 subgroups by randomized digital table method, such as RSV 0, 7, 15, 30, 60 d, 90, 120 d subgroups, 5 rats in each RSV subgroup; while 27 cases of SD rats in control groups were randomly divided into 7 subgroups by the same method, such as PBS 0, 7, 15, 30, 60, 90, 120 d subgroups, and 5, 5, 5, 3, 3, 3, 3 cases of SD rats in each PBS subgroup, respectively. Biochemical parameters including levels of 24 h urinary protein, serum albumin and serum creatinine were measured in each subgroup, and the parameters were analyzed by statistical methods. ③All SD rats in RSV and PBS subgroups were killed after biochemical parameters being measured, and then the inferior 2/3 of left kidneys, left lungs and spleens of each rat were harvested, fixed by 4% paraformaldehyde solution for 24 h, and embedded by paraffin to complete hematoxylin-eosin (HE) staining. Each slice was observed under light microscope to find out whether pathomorphological changes existed or not. ④With RSV G protein monoclonal antibody as primary antibody, immunohistochemical specimens of SD rats' kidneys, lungs and spleens in 7 RSV subgroups were made out to find evidence of RSV G protein under light microscope. ⑤With RSV G protein monoclonal antibodies as primary antibody combined with green fluorescence, blue stain nuclei by 4', 6-diamidino-2-phenylindole (DAPI) at the same time, all SD rats' kidneys in RSV subgroups were cut into fluoroimmunoassay slices to find out the evidence of RSV G protein and the relation between its location and the nucleus. ⑥Harvesting the remaining 1/3 of the left kidney, with 3% glutaraldehyde fixation before sent to the West China Hospital, Sichuan University, SD rat's glomerular ultrastructural pathology structure changes in each RSV subgroup were observed under electronic microscope, meanwhile RSV particles were searched.

①The pathological model of nephropathy SD rats in RSV group were duplicated successfully, as same as the model of SD rats in control group. ②Comparison of proteinuria in 24 h at the same point between RSV subgroups and PBS subgroups suggested that there were no statistical differences on 0 and 90th day (P>0.05), while the proteinuria in 24 h of RSV 7, 15, 30, 60, 120 d subgroups were all significantly higher than those in PBS subgroups at the same point (t=6.9, 3.1, 3.9, 2.2, 5.6; P<0.05). However, there were no significantly statistical differences in comparison of serum albumin and serum creatinine between RSV subgroups and PBS subgroups at the same point (P>0.05). ③HE stains showed that pathological changes at different levels of kidneys, lungs and spleens were observed in RSV 7, 15, 30, 60, 90, 120 d subgroups compared with RSV 0 d subgroup. ④Immunohistochemical stains showed that RSV G protein expressed positively in RSV 7, 15, 30, 60, 90, 120 d subgroups' kidneys, lungs and spleens while RSV 0 d subgroup had no positive expression. Quantitative analysis of RSV G protein suggested that the expression of RSV G protein increased gradually before day 30th and maintained at a high level, and then decreased after day 60th. ⑤Fluorescence staining suggested that RSV G proteins were located in the RSV infected cells' nuclei. ⑥RSV particles were observed in RSV subgroups' kidneys under electronic microscopy and glomerular pathogenesis persisted even on day 120th.

The expression of RSV G protein from 7th to 120th day successfully demonstrate that RSV could persist on a long time in RSV nephropathy rats, which may caused by immune dysfunction with RSV and it's persistence.