Use of cationic microbubbles targeted to P-selectin to improve ultrasound-mediated gene transfection of hVEGF165 to the ischemic myocardium / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection (TCUMGT) via ultrasound-targeted microbubble destruction (UTMD) to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles (MBs) with pcDNA3.1-human vascular endothelial growth factor 165 (pcDNA3.1-hVEGF165) plasmids targeted to P-selectin (MB+P+VEGFp) were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups: microbubble only (MB), microbubble+P-selectin (MB+P), microbubble+pcDNA3.1-hVEGF165 plasmid (MB+VEGFp), and microbubble+ P-selectin+pcDNA3.1-hVEGF165 plasmid (MB+P+VEGFp). The reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB+P+VEGFp delivery showed greater improvement than MB+VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA3.1-hVEGF165 recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.

Tauroursodeoxycholic acid enhances the development of porcine embryos derived from in vitro-matured oocytes and evaporatively dried spermatozoa.

Evaporative drying (ED) is an alternative technique for long-term preservation of mammalian sperm, which does not require liquid nitrogen or freeze-drying equipment, but offers advantages for storage and shipping at ambient temperature and low cost. However, the development of zygotes generated from these sperms was poor. Here, we demonstrated that the supplementation of tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, during embryo culture improved the developmental competency of embryos derived from in vitro matured pig oocytes injected intracytoplasmically with boar ED spermatozoa by reducing the production of reactive oxygen species, the DNA degradation and fragmentation, and the expression of apoptosis-related gene Bax and Bak, and by increasing the transcription of anti-apoptosis gene Bcl-XL and Bcl-2. Furthermore, TUDCA treatment promoted the blastocyst quality manifested by the total cell numbers and the ratio of inner cell mass. Taken together, our data suggest that evaporative drying would be a potentially useful method for the routine preservation of boar sperm in combination with further optimization of subsequently embryo culture conditions.

Glycine increases preimplantation development of mouse oocytes following vitrification at the germinal vesicle stage.

Ice-free cryopreservation, referred to as vitrification, is receiving increased attention in the human and animal assisted reproduction. However, it introduces the detrimental osmotic stress by adding and removing high contents of cryoprotectants. In this study, we evaluated the effects of normalizing cell volume regulation by adding glycine, an organic osmolyte, during vitrification of mouse germinal vesicle stage oocyte and/or subsequent maturation on its development. The data showed that glycine supplementation in either vitrification/thawing or maturation medium significantly improved the cytoplasmic maturation of MII oocytes manifested by spindle assembly, chromosomal alignment, mitochondrial distribution, euploidy rate, and blastocyst development following fertilization in vitro, compared to the control without glycine treatment. Furthermore, glycine addition during both vitrification/thawing and maturation further enhanced the oocyte quality demonstrated by various markers, including ATP contents and embryo development. Lastly, the effect of anti-apoptosis was also observed when glycine was added during vitrification. Our result suggests that reducing osmotic stress induced by vitrification could improve the development of vitrified mouse oocyte.

Developmental potential of prepubertal mouse oocytes is compromised due mainly to their impaired synthesis of glutathione.

Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca(2+) reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca(2+) storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca(2+) store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.

[A new method for integrated radical resection of uncinate process of the pancreas: a report of 306 cases].

OBJECTIVE: To investigate the methods and skills of integrated radical resection of uncinate process of the pancreas for patients with periampullary malignant tumor. METHODS: From March 2005 to March 2010, 306 cases of radical pancreaticoduodenectomy (RPD) of periampullary malignant tumor had been continuously performed. By exchanging superior mesenteric artery and controlling blood stream of pancreatic uncinate process, the integrated radical resection of uncinate process for these patients had been successfully completed. Deal with restitution of alimentary tract by all using Child method. The method of simplify binding pancreaticojejunostomy was carried out to pancreatico-jejunal anastomosis. The cases included 169 male and 137 female with 37 - 79 years old, and the mean age was 58 years. Tumor types included 151 pancreatic head and neck tumors, 48 distal bile duct tumors, 55 ampullary tumors and 52 duodenal papilla tumors. RESULTS: Among the 306 cases with RPD, operation time were 4 - 6 h and the blood loss were 200 - 600 ml with no intraoperative and postoperative bleeding of pancreatic uncinate process site. The incidence rates of postoperative bleeding and mortality were 3.3% and 0.9% respectively. The incidence rates of postoperative pancreatic fistula and biliary fistula incidence were 1.6% and 0.6% respectively. And patients with fistula had well recovered by expectant treatment of ultrasound-guided puncture and drainage. Follow-up to March 2010, there were no patients died from the recurrence of superior mesenteric vascular tumor. CONCLUSIONS: By exchanging superior mesenteric artery and controlling blood stream of pancreatic uncinate process, the integrated radical resection of uncinate process for those patients who have periampullary malignant tumor can be successfully completed. It can reduce the operating bleeding, operating time and the miscut of superior mesenteric vein and(or) superior mesenteric artery, it can avoid postoperative pancreas necrosis off, infection and hemorrhage caused by the pancreas uncinate process residues, and it also theoretically reduces the chance of tumor cells spreading.

Effects of puberty and gonadotropins on the molecular events controlling meiotic resumption of mouse oocytes.

Although studies suggest that the low competence of oocytes from prepubertal animals is due to their insufficient cytoplasmic maturation and that FSH improves oocyte maturation possibly by retarding meiotic progression and allowing more time for cytoplasmic maturation, the mechanisms by which puberty and gonadotropins regulate meiotic progression require additional detailed studies. For the first time, we observed that while meiotic progression was significantly slower, the maturation-promoting factor (MPF) activity of oocytes was significantly higher in prepubertal than in adult mice. To resolve this contradiction, we specified the molecules regulating the MPF activity and their localization during oocyte maturation in prepubertal and adult mice primed with or without gonadotropins. Our tests using corresponding enzyme regulators suggested that while activities of protein kinase A were unaffected, the activity of adenylate cyclase (ADCY) and phosphodiesterase increased while cell division cycle 2 homolog A (CDC2A) decreased significantly after puberty. While most of the adult oocytes had CDC2A protein concentrated in the germinal vesicle (GV) region, the majority of prepubertal oocytes showed no nuclear concentration of CDC2A. Maximally priming mice with equine chorionic gonadotropin brought the above parameters of prepubertal oocytes close to those in adult oocytes. Together, the results suggest that puberty and gonadotropin control oocyte meiotic progression mainly by regulating the ADCY activity and the concentration of the activated MPF toward the GV region.