The Detection Of HCMV DNA In Patients With Atherosclerotic Ischemic Cerebrovascular Diseases

Objective: As the incidence of cerebral infarction (CI) increasing year by year, it is becoming a problem needed solving badly in clinic what urges occurrence and development of CI. Atherosclerosis (AS) as a chief pathologic foundation of CI has been undstood clearly. Recently, to investigate other risk factors of AS and atherosclerotic ischemic cerebrovascular diseases and to develop intervention measures correspondingly is drawing attention more and more in academic circles. Especially about microbial pathogen of AS, there are various opinions on it. In our previous study on atherosclerotic ischemic cerebrovascular diseases patients and healthy volunteers in Shijiazhuang region, Hebei province, we found that human cytomegalovirus (HCMV) had a close relationship with AS and CI, it may be an independent risk factor of atherosclerotic ischemic cerebrovascular diseases. In this study, all objects were divided into cerebral atherosclerosis (CAS) group, complete stroke (CS) group and progressive stroke (PS) group, and then nested PCR was used to detect HCMV DNA in preparations of blood, some bone marrow, atherosclerotic plaque and smooth muscle under the plaque. To provide theoretical foundation for preventing atherosclerotic ischemic cerebrovascular diseases due to infection of HCMV by application of antivirals, we investigated the relationship between active infection of HCMV and occurrence and progressing of AS and CI. Methods: In this study, the direct evidence of HCMV active infection was got through detecting conserved sequence from the immediate early (IE) gene of HCMV with nested PCR (NT-PCR). The primers locating in EcoRI J fragment of IE gene were used, outer pair was used to amplify a 761bp fragment, and inner pair was used to amplify a 244bp fragment (primer sequence and other parametes in Tab.1). The results of amplification of HCMV AD169 strain and positive specimens of HCMV DNA by nested PCR showed that one DNA band could be found at 244bp site in gel. After PstⅠendonuclease analysis, two DNA products (116bp and 91bp) were generated as designing. The nested PCR had highly specificity and sensitivity, and moreover no cross-reaction with human DNA or with DNA of human embryonic lung fibroblast. The multiple proportions dilution at 10^-1 was performed on 106/ml TCID50 of AD169 viral culture medium. The positive result still appeared at 10^-3 concentration, but negtive at 10^-4. With common one-off PCR, the negtive result was at 10-3.The blood specimens were collected from 80 CAS, 83 CS and 48 PS patients, then the positive rate of HCMV DNA in blood was compared among three groups. To exploit infection status of AS patients, 5 marrow specimens (3 cases from CAS group, 2cases from CS group) and 10 specimens (3 cases of carotid atherosclerosis) were got from atherosclerotic plaque and smooth muscle under the plaque at different parts of arterial walls from arteriosclerosis obliterans patients (peripheral blood specimens were collected at the same time). Results: In CAS,CS and PS groups, the positive cases of HCMV DNA were 33, 20 and 22 respectively, the positive ratio were 41.25%,24.10% and 45.83% respectively. The positive ratio of HCMV DNA had a significant difference among three groups (χ2=8.103, P<0.05; Tab.4), between PS and CAS group (χ2=5.463,P<0.05; Tab.5), and between PS and CS group (χ2=6.597,P<0.05; Tab.7), but no difference between PS and CAS group (χ2=0.257,P>0.05, Tab.6). These results suggested that there was a correlation between active infection of HCMV and atherosclerotic ischemic cerebrovascular diseases, especially between PS and cerebral arteriosclerosis with instable AS plaques. Basing on 2 positive marrow specimans of HCMV (both from CAS group), we could suspect active infection of HCMV in marrow had a same condition with peripheral blood. The positive expression of HCMV DNA was found in 8 specimens of atherosclerotic plaques and smooth muscle under the plaques (2 from carotid atherosclerosis patients), and in 4 specimens of peripheral blood collected at the same time. These re...