Controls The above immunohistochemical procedures controls were performed replacing the primary antibody by PBS. Further controls were performed omitting the secondary antibody. The controls were always negative.
Western Blot detection
It is to detect c-erbB-2, MMP-2 and MMP-9 expression levels. Referring to the instruction of kits, two groups of villous total protein were extracted and stored in fridge at -70°C. Bradford method was used to measure protein concentration. In every sample, 50 μg 10% SDS-PAGE was taken and wet transferred to PVDF membrane. 5% skimmed milk powder was sealed for 2 h at room temperature. Rabbit anti c-erbB-2, MMP-2 and MMP-9 polyclonal antibodies were respectively added, cultured overnight at 4°C. After TBST washing membrane, it was added into second antibody and cultured for 2 h at room temperature. TBST washes membrane and ECL reagent developed color. Exposure imaging was done under gel imager. Quantityone gel image analysis software detected different groups’ protein and their loading control β-actin protein’s absorbance value, showing target protein level by the ratio of target strip and average absorbance of loading control protein.
Statistical analysis
These experiments above were repeated 3 times. SPSS 13.0 statistical software is adopted. Value of number is expressed by the mean ± standard deviation. Differences among groups are conducted independent sample t testing, while P < 0.05 means the difference is of statistical significance.
Villus HE staining
In control group, ST and CT were visible on villous surface, and cell boundary was clear. Villous interstitial substance was loose primitive interstitial substance. In interstitial substance, small blood vessels can be seen. There was no obvious abnormality in villus of infected group.
Villus HCMV detection results
When infected group was added into HCMV and cultured for 48 h, HCMVpp65 signals showed almost all CT, EVT, ST, interstitial cells and around villous interstitial substance small blood vessels in villus, while there was no HCMVpp65 antigen signal in control group(Figure 2A). HCMV was added into early pregnancy villus in vitro culture, after 48 h, which also can cause villus infection. Virus can reproduce in CT, EVT, ST, interstitial cells and around villous interstitial substance small blood vessels in villus.
Western blot method detects the two groups’villus(Figure 3D, E), and c-erbB-2 protein aimed strips can be detected in 185kDa, where infection group’s expression level decreased(P < 0.05). The results above indicate that infected HCMV may lead to the decrease of villous c-erbB-2 protein expression.
MMP-2 and MMP-9 protein expression level
Immunohistochemistry staining showed that the two groups’ MMP-2 proteins both expressed in EVT endochylema in abundance, but little in VT, ST and interstitial substance(Figure 4A, B); MMP-9 protein expresseed largely in villous interstitial substance, EVT and VT endochylema and little in ST. The results above indicate that infected HCMV may lead to the decrease of villous MMP-2 and MMP-9 protein expression.