原位RT-PCR检测骨髓塑料切片中巨细胞病毒活动性感染的组织学依据探讨  

Histological evidence of active cytomegalovirus infection in plastic embedded bone marrow biopsy specimens:studies by RT-PCR in situ

作　　者：桂敏[1] 周东风[1] 孟浦[1] 邱奕宁[1] 金润铭[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院儿科,湖北武汉430022

出　　处：《中国实用儿科杂志》2008年第7期517-519,562,共4页Chinese Journal of Practical Pediatrics

摘　　要：目的寻找骨髓造血功能障碍性疾病中巨细胞病毒(CMV)活动性感染的组织学依据。方法华中科技大学同济医学院附属协和医院2006年2月至2007年10月筛选造血系统两系或三系减少患儿37例(再生障碍性贫血26例,骨髓增生异常综合征11例)。取患儿骨髓活检组织,塑料包埋制备薄切片;将切片置于Eppen-dorf管反应液内进行原位逆转录并扩增,原位检测骨髓组织中CMV mRNA表达。同时取患儿骨髓液行普通液相RT-PCR检测CMV mRNA作为对照。结果14例(37.8%)骨髓塑料切片检测到CMV mRNA表达;阳性细胞多为骨髓实质细胞,呈局灶性或片状分布,阳性信号主要位于细胞胞浆内,少量出现在部分细胞的胞核。普通液相RT-PCR检测16例(43.2%)骨髓液中有CMV mRNA表达,2种方法阳性率差异无统计学意义(P>0.05)。结论改良的原位RT-PCR方法可用于塑料包埋切片的检测,可定位诊断骨髓中CMV活动性感染,为CMV活动性感染致血液学改变提供了组织学依据,对骨髓造血功能障碍性疾病发病机制的探讨具有重要意义。Objective To looking for the histological evidence of active cytomegalovirus （ CMV ） infection in functional disorder of haematopoiesis. Methods All the 37 cases （26 children with aplastic anemia, 11 children with myelodysplastic syndrome ） underwent a clinical screening evaluation. The inclusion criteria were thrombocytopenia, neutropenia, and anemia. The bone marrow biopsies from the 37 children were embedded with plastics and cut into sections. Then these se tions were put into Eppendorf tubes for in situ reverse-transcriptase proliferation. Expression of CMV mRNA was detected by RT-PCR in situ technique. Routine RT-PCR technique was used to detect CMV mRNA expression in bone marrow aspiration sample from the same patients. Results The CMV mRNA was found to be positively expressed in 14 of 37 plastic embedded sections by RT-PCR in situ technique,the positive rate being 37. 8%. Most of the positive signal appeared in the endochylema of myeloid parenchymal cells, presenting focal or lamellar disposition. Little positive signal appeared in the nucleus of a few cells. The positive expression of CMV mRNA was detected in 16 of 37 bone marrow aspiration sample by routine RT-PCR technique, the positive rate being 43.2%. The difference of positive rate between two methods was not significant （ P 〉 0. 05 ）. Conclusion Our results show that the modified RT-PCR in situ technique can be applied to plastic embedded sections. This method is expected to provide level diagnosis of active CMV infection in bone marrow, which can find histological evidence of hematologic change influenced by CMV infection. It is important to the pathogenesis research of functional disorder of haematopoiesis in bone marrow.

关 键 词：RT-PCR 骨髓 巨细胞病毒感染 组织学 

分 类 号：R72[医药卫生—儿科]