ABSTRACT
Objective To study the effect of cumulus cell apoptosis on the structure of human oocyte cultured in vitro and to probe into the reason why cumulus cell apoptosis rate can be used clinically to predict the developmental potential of oocyte. Methods Cumulus-enclosed oocyte complexes (COCs) at the GV stage were cultured in vitro. The apoptosis rate of cumulus cells for every COC was evaluated with HE staining, DAPI staining and TUNEL labeling methods. Oocytes were divided into two groups according to their cumulus cell apoptosis rate. Structure of oocytes was observed under light microscope and transmission electron microscope. Mature oocytes at MⅡ stage were fertilized and cultured in vitro for two days. Then the embryos were evaluated according to their morphology under light microscope.Results The oocytes with low cumulus cell apoptosis rate had a normal structure and a promising developmental potential, while the oocytes with high cumulus cell apoptosis rate had deformed structures and a poor developmental potential. Some deformed oocytes had uneven perivitelline space. For oocytes with a small perivitelline space, the organelle development was slow compared with those with a large perivitelline space. Some had unevenly piled organelles. Secondary lysosomes and large space probably caused by the decopmounding of lysosomes were found in the ooplasm of these oocytes. The deformed oocytes appeared to have lots of swollen mitochondria with blurred cristae and membrane. Some of these oocytes had fractured first polar bodies and abnormally thick or thin zona pellucida. The cell junctions and microvilli of cumulus cells reduced as well. Conclusion The effect of cumulus cell apoptosis on the structure of oocyte cultured in vitro was revealed. The reason why cumulus cell apoptosis rate prognosticated the developmental potential of oocyte was demonstrated in the aspect of oocyte structure.
ABSTRACT
To express a gene of heat shock protein 70(HSP70) in E.coli , HSP70 gene was amplified by PCR. The PCR products were cloned into pUCm T vector and were sequenced; The HSP70 gene was subcloned into vector pET 21a(+) and expressed in E.coli. SDS PAGE and Western blot were employed to identify the expression of the HSP70 gene. Results showed that a fragment about 1 96kb was amplified by PCR. Sequence analysis revealed that the sequence of HSP70 was correct. The HSP70 gene was cloned into pET 21a(+) identified by enzyme digestion and PCR. SDS PAGE and Western blot showed that a M 72 000 protein was expressed and could be recognized by anti HSP70 antibody. Therefore,HSP70 gene has been successfully expressed in E.coli .
ABSTRACT
Objective To study the relationship between gastric bile reflux and the digestive symptoms. Methods The extent of bile reflux into the stomach during 24 hours was monitored by using ambulatory bilirubin monitoring technique in 76 persons among them 44 were complaining of digestive symptoms and 22 asymptomatic. Results The total time percentile of bile reflux,the total area of bile reflux and frequency of bile reflux in asymptomatic group and symptomatic group were 15 6%?14 0%, 20 6?18 7, 22 3 times?13 6 times, and 26 4%?21 3%, 48 7?60 8, 42 9 times?44 5 times. Conclusion Bile reflux into the stomach was significantly more frequent and intensive in symptomatic group compared with asymptomatic group
ABSTRACT
Objective To construct a fusion gene vaccine of gastric cancer MG7-Ag mimotope and heat shock protein 70, and to investigate the humoral and cellular immune response in mice induced by the vaccine. Methods The coding sequence of MG7 mimotope was incorporated with HSP70 gene at the 3' terminus by PCR amplification. Then the PCR product was cloned into pcDNA3.1(+) vector to construct the eukaryotic expression vector. The pcDNA3.1/MG7+hsp70 was sequenced to ensure the proper encoding. C57BL/6 mice were immunized with the fusion gene vaccine, and pcDNA3.1/hsp70 and empty pcDNA3.1 were used as controls. The serum was collected at the 3rd week after each immunization. Cellular ELISA was performed to evaluate the induced humoral immunity. The splenocytes were first separated at the 9th week for the assay of antigen specific CTL lysis activity by 51Cr release. Results A fragment about 2.0 kb was obtained by PCR amplification. Sequence analysis revealed that the sequence of mimotope was connected successfully to 3' terminus of hsp70 and the fusion gene was cloned into the pcDNA3.1(+) successfully. Cellular ELISA results suggested that the serum level of MG7-Ag antibody appeared in the vaccinated mice at 9th week, while no MG7-Ag antibody was detected in the controls. The results of the specific lysis rate of splenocytes showed no statistical difference between fusion gene vaccine group and control groups. Conclusion The fusion gene vaccine was constructed successfully and the specific humoral immune response was induced by the fusion gene vaccine.