【Objective】 To investigate the oxidative stress status in follicular fluid and granulosa cells obtained during oocyte retrieval cycles among women of different ages and to explore the relationship between the oxidative stress and the embryo quality. 【Methods】 From September 8, 2018 to January 18, 2019 in our reproductive center, 70 follicular fluid samples on the day of oocyte retrieval were collected from infertile women undergoing in vitro fertilization(IVF) or intracytoplasmic sperm injection(ICSI). We divided the samples into those at or above(aged group, n = 34) and below the age of 35(young group, n = 36) and compared seven oxidative stress markers in follicular fluid and gene expression of antioxidant enzymes in granulosa cells, and then analyzed their differences between groups and their relationship with embryo development parameters. 【Results】 We observed significantly higher concentration of hydrogen peroxide(H2O2) [155.84(137.11~199.30) vs. 66.94(51.05~95.42) μmol/L, P = 0.001] , significantly lower total superoxide dismutase (SOD) activity [0.69(0.55~0.85) vs. 0.83(0.76~0.94) U, P = 0.005] , decreased catalase(CAT) and glutathione reductase(GSR) expression(P<0.05) in women with advanced materal age. Total SOD activity in follicular fluid was statistically higher both in patients with normal fertilization rate(≥60%) and with blastocyst formation rate(≥ 60%) [0.82 (0.71~0.91) vs. 0.67(0.55~0.80) U, P = 0.007, 0.83(0.78~0.90) vs. 0.73(0.60~0.89) U, P = 0.049] . Multivariate logistic analysis revealed that higher total SOD activity correlated with normal fertilization rate(≥ 60%) and blastocyst formation rate(≥60%), with 5.478 and 3.383 for odds ratio(OR) values, 1.721~17.433 and 1.111~10.299 for 95% CI, 0.004 and 0.032 for P values, respectively. 【Conclusions】 The oxidative stress in follicular environment among women with advanced materal age is mainly characterized by increased level of reactive oxygen species(ROS), decreased SOD activity in follicular fluid and reduced expression of CAT and GSR in granulosa cells. Moreover, total SOD activity in follicular fluid was positively related to normal fertilization rate and blastocyst rate, which suggests that it may have a certain effect on the quality of oocytes.

OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Animals ; Antibodies ; Capsid ; Capsid Proteins/genetics* ; Dependovirus/genetics* ; Prokaryotic Cells ; Rabbits ; Recombinant Proteins/genetics*

Humans ; Mississippi/epidemiology* ; Influenza, Human/epidemiology* ; Meteorological Concepts ; Virus Diseases