Expression of Annexin Ⅴ in hepatitis B infected placentas detected by double-labeled immunofluorenscence assay

Ailian YU; Yunbo QIAO; Yanling ZHANG; Danru LIU; Mingfeng YANG; Yu WANG; Guizhi SHI

ABSTRACT

BACKGROUND:

Recently, one focus of research has been Annexin Ⅴ (AnV) existing on hepatic cells membranes as a fundamental receptor related to hepatitis B virus (HBV) infection. Also its expression in placental tissues has been a matter of debate. The study of the relationships between placental cells infected with HBV and their AnV expression will be of great value in future prevention strategies and treatments.

OBJECTIVE:

To investigate the presence of AnV in HBV infected human's placental cells and its potential role in HBV intrauterine transmission.

DESIGN:

Randomized controlled experiment.

SETTING:

Taishan Medical College.MATERIALS Placental tissue was collected from HBsAg positive full term pregnant women (30 cases) admitted to Jinan Institute for Maternal and Child Health, Taian Central Hospital and Taian Institute for Maternal and Child Health from January 2003 to December 2004. Maternal serum was also obtained. Informed consents for participating in this study were obtained from all the involved pregnant women and this experiment was approved by the Hospital Ethics Committee. Rabbit-anti-human AnV purified affinity antibody (first antibody), rat-anti-human HBs mAb (first antibody),and biotinylated goat-anti-mouse IgG (secondary antibody) were supplied by Wuhan Boster Bioengineering Company.

METHODS:

Using SABC immunohistochemical staining reagent, 18 HBsAg positive placentas were obtained from 30HBsAg infected patients in full term pregnancy. These were considered as the positive group and the other 12 were used as negative controls. The staining process included dewaxing, dehydration of embedded slides and microwave antigen restoration. In the wet box, rabbit-anti-human AnV purified antibody (first antibody, 160, monoclonal antibody)was added on the slides and kept at 4 ℃ overnight. Rat-anti-human antibody HBs mAb(secondary antibody, 150) was added and kept at 4 ℃ ovemight, after this procedure, biotinylated goat-anti-mouse IgG(1100), the first fluorescent antibody such as FITC-goat anti-rabbit IgG (150) and the second fluorescent antibody (Avidin-Cy3) were used,respectively. The slides were sealed with buffered glycerol and examined under a confocal laser scanning microscope.The images on the slides were analyzed with IPP 4.5 image programs.MAIN OUTCOME

MEASURES:

Detecting the simultaneous existence and distribution of HBsAg/AnV in placental cells with HBV infection.

RESULTS:

Ten cases from the positive group were simultaneously detected for HBsAg/AnV by double-labeled immunofluorenscence assay and confocal laser scanning microscope. AnV expression was detected in the trophoblastic, interstitial cells and vascular endothelial cells of villi interstitial blood vessels, and the coexistence of HBsAg/AnV was found even in one cell.

CONCLUSION:

HBsAg combined with the receptor AnV in the same placental cells is a common finding in HBV infected full term pregnant women. This finding is very suggestive of a mechanism where AnV could promote hepatitis B virus to enter the placental cells and cause intrauterine infection.