Specimen and sources of the virus
The tissue of villus is from healthy pregnant women whose peripheral blood is HCMV antibody negative and whose gestational age are between 5 to 10 weeks, who are voluntarily conducted abortion because of planned parenthood, from department of gynecology and obstetrics of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from March 2010 to July 2010. The research procedure complies with the ethnical standard drew by Ethics Committee of Tongji Hospital of Tongji Medical College. HCMV AD169 strain is provided by Hubei Province Institute of Viruses, while TCID50 is 10-5.
Sources of main reagents
DMEM/Ham’s F 12 culture medium (Gibco), standard fetal bovine serum (Hyclone), rabbit anti HCMV pp65 polyclonal antibody (Santa), rabbit anti c-erbB-2 polyclonal antibody, rabbit anti MMP-2 and MMP-9 polyclonal antibodies (Wuhan Boster Bio-engineering Co., Ltd), SP immunohistochemistry kit (Zhongshan Goldenbridge Biotechnology Co., Ltd), total protein extraction kit (BestBio), β-actin loading control antibody (Beijing Biosynthesis Biotechnology Co., Ltd) and so on.
Methods
Villous explant culture and experimental grouping
Improvements are made by referring to Dong cultural method[5]. The specific method is as follow: villous tissue was rapidly taken to laboratory by being placed in sterile pre-cooling D-hank’s solution (containing 100 UI/ml penicillin and 100 μg/ml streptomycin), which were washed for three times for removing gore and non-cruor; it was put into serum-free DMEM/Ham’s F 12 culture medium, and the apical tissue of villus (wet weight 40~50 mg) was carefully cut and inoculated in 24-hole cell culture plate, being cultured overnight in DMEM/Ham’s F 12 culture medium containing 10% fetal bovine serum with 5% CO2 at 37°C; after throwing away the supernatant, it was changed to be DMEM/Ham’s F 12 culture medium containing 3% fetal bovine serum. In infection group, 1 ul HCMV virus solution was added in every hole, while equal amount of PBS was added into every hole in control group; culture medium was absorbed, threw away in the two groups above after 2 h culture and washed by PBS for twice and added into complete medium; after 48 h, villous explants were taken out. Total protein was extracted from part of villous tissue for Western blot detection. The rest of villous tissue was fixed for 24 h in formalin buffer solution and paraffin wax embedding sliced (thickness of slice was 3 um), for conducting pathological and immunohistochemical detection.
Hematoxylin eosin(HE) staining
Two groups of villous slice are being HE stained. Optical microscope is utilized to observe villus pathological changes.
Immunohistochemistry staining
Two groups of slices are taken to conduct conventional treatment. Immunohistochemistry streptavidin-perosidase(SP) method is used to detect HCMV pp65, c-erbB-2, MMP-2 and MMP-9, and the method please refers to reference. Paraffin embedded sections were prepared in a microtome and de-paraffinized in a series of ethanols and xylenes. Sections were blocked with methanol containing 3% H2O2, sequentially 10% goat normal serum, and incubated with rabbit anti HCMV pp65 polyclonal antibody, rabbit anti c-erbB-2 polyclonal antibody, rabbit anti MMP-2 and MMP-9 polyclonal antibodies (1:100 dilution) overnight at 4°C. They were then sequentially treated with a biotinylated goat anti-rabbit antibody and a horseradish peroxidase(HRP) labelled streptoavidin for 1 h at 37°C respectively. Development was performed by treating the sections with a Liquid DAB-Plus Substrate kit. After counter-staining with haematoxylin, immunostaining of these proteins on the tissue sections was detected by light microscope. Under optical microscope, positive signals were nankeen or brown grains. HMIAS-2000 high definition color medical image analysis system was adopted to process images. Under high magnification, 10 visual fields were randomly observed, testing positive expression cells’ average optical density for semi-quantitative statistical analysis.