Induction and molecular mechanism of placental trophoblast cell autophagy in preeclampsia / 中华围产医学杂志

Objective To investigate the induction and regulatory mechanism of placental trophoblast cell autophagy in women with preeclampsia (PE).Methods Twenty gravidas with severe PE who underwent cesarean section in the Department of Obstetrics and Gynecology of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University from August 2016 to November 2016 were enrolled in PE group.An equal number of normotensive gravidas without proteinuria who also underwent cesarean section during the same period were randomly selected as control group.Placental tissue samples were collected from all gravidas.Ultrastructure of placental trophoblast cells and changes in autophagosome formation were observed by transmission electron microscope.Expressions ofmicrotubule associated protein 1 light chain 3B (MAP1LC3B,or LC3B) and Beclin 1 in placental tissue samples were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot.Activities of protein kinase B (PKB,also known as Akt)/mammalian target of rapamycin (mTOR) pathway in placental tissue samples were detected by Western blot.Two independent samples t-test or Mann-Whitney U test was used for statistical analysis.Results Sparse and disordered villi and many typical autophagosomes were observed in placental trophoblast cells from patients with severe PE.Significantly enhanced expression of LC3B at mRNA and protein levels and increased ratio of LC3-Ⅱ/LC3-Ⅰ were observed in the PE group as compared with the control group [3.37 (2.37-6.11) vs 0.62 (0.25-4.15),1.40±0.17 vs 1.00±0.13,1.57±0.25 vs 1.00±0.31,Zor t=--4.440,3.274 and 3.113,all P＜0.05].No significant difference in the expression ofBeclin 1 at mRNA or protein level in placental tissues was found between the two groups (both P＞0.05).Furthermore,Akt and mTOR phosphorylation in the PE group was significantly suppressed as compared with that in the control group (1.00±0.29 vs 0.64±0.21,1.00±0.32 vs 0.60±0.22,t=--3.672 and-2.895,both P＜0.05).However,the two groups showed no significant difference in the expression of Akt or mTOR protein (both P＞0.05).Conclusions Suppressed activity of Akt/mTOR pathway and enhanced induction of trophoblast cell autophagy are detected in placental tissues of patients with severe PE,indicating that excessive trophoblast cell autophagy,induced by decreased activity of Akt/mTOR pathway,may be the pathogenesis for PE.

Isolation and identification of placental exosomes from maternal serum / 医学研究生学报

Objective During pregnancy , exosomes can be released from the placenta into maternal circulation and play im-portant roles in normal pregnancy or placenta-related diseases .We aimed to establish a simple and efficient method for isolating and i-dentifying placental exosomes from maternal serum and lay a foundation for the studies of pregnancy -related diseases . Methods Using sucrose gradient centrifugation with 8% PEG6000 precipitation twice , we isolated and purified placenta-derived exosomes from normal maternal serum and detected their molecular markers CD 63 , CD81 and PLAP by Western blot , followed by silver staining anal-ysis of the protein profile of the exosome pellet .We identified the morphology of the placenta-derived exosomes by transmission electron microscopy ( TEM) and measured the size and distribution of the particles by dynamic light scattering ( DLS) . Results Silver stai-ning of the protein profiles of the exosomes after sucrose gradient centrifugation clearly revealed the bands of the protein molecules . Western blot showed the expressions of CD 63, CD81, and PLAP in the 21-34%density layer, which demonstrated the presence of serum placental exosomes mainly in the 1.09-1.16 g/mL density layer.TEM exhibited that the placenta-derived exosomes were round or oval cup-shaped, specifically expressing PLAP, and the particles were uniform in size, with a mean diameter of (41.79 ±11.94) nm. Conclusion A simple, fast, and efficient method was successfully established for isolating placenta-derived exosomes from ma-ternal serum, which provides a basis for studying the roles of placental exosomes in normal pregnancy and placenta -related diseases.

AntiEGFRnano inhibites proliferation and migration of estrogen-dependent Ishikawa cells of human endometrial cancer cell line / 药学学报

Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.