This study is to establish a simple and practical co-culture method of cortical neurons and astrocytes of rats. The cortex of the new-born SD rats was digested by 0.125% pancreatic enzyme, and the differential adherence was applied to obtain the mixed cell suspension of neurons and astrocytes. A low concentration of cytarabine was used to inhibit the astrocytes in a moderate way to get neuronal and astrocyte co-culture. The morphological characteristics of the cells in different times were observed under the inverted microscope. The cells began to adhere the wall 2 h after the inoculation. Neurons and astrocytes grew in a good condition under the inverted microscope 9 days after the inoculation. The results of the immunofluorescence staining and Rosenfeld's staining indicated that the co-culture of neurons and astrocytes was successful and the ratio of neurons and astrocytes was close to 1:1. A new neurons and astrocytes co-culture method, which is simple and convenient, was successfully established. It will be an efficient method for the related researches about neuronal and astrocyte co-culture in vitro.

Gravidas receiving epidural labor analgesia are at an increased risk for intrapartum fever due to unknown mechanism, which may be related to thermoregulation, non-infectious inflammatory, continued use of local anesthetic agents and trauma from epidural catheter insertion. Epidural labor analgesia-related intrapartum fever is associated with excessive obstetric interventions, higher rates of cesarean and assisted vaginal deliveries, unnecessary maternal and neonatal exposure to antibiotics, and adverse neonatal outcomes. Hence it is imperative to explore its etiology and shorten the labor with a safe and effective measure to reduce the occurrence of intrapartum fever.

Both phytase and endoglucanase are additives in feed for mono-gastric animal known for their effects. Recombinant vector pPICZalpha-EG was constructed and transformed to GS115-phyA, a Pichia pastoris strain that had integrated with phytase gene, generating GS115-phyA-EG. Both phytase and endoglucanase activities in the supernatant were determined after methanol induction of GS115-phyA-EG. Phytase and endoglucanase activity reached 39.4% and 56.2% activity compared to GS115-phyA and GS115-EG, respectively. Properties of the mixed enzyme suggest that the optimal temperature and pH value be 55 degrees C and 5.5 respectively. Both phytase and endoglucanase showed greater than 80% activity across temperature ranges 45 degrees C to 55 degrees C and pH ranges 4.5 to 5.5. Expressing more than one enzyme in one system could save time and money during induced expression, and the mixed enzyme might apply for treating forge before feeding with poultry.
6-Phytase ; biosynthesis ; genetics ; Cellulase ; biosynthesis ; genetics ; Genetic Vectors ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

We studied the effects of simulated microgravity on mouse oocytes maturation, and analyzed whether the tail-suspended model can be applied to investigate simulated microgravity effects on reproductive processes in female mice. Mouse oocytes were cultured in vitro with microgravity simulated by a rotating wall vessel bioreactor and by tail-suspended model, and the maturation rate of the mouse oocytes in the two models were examined in vivo. The maturation rate of mouse oocytes cultured in simulated microgravity was 8.93%, and that was 72.33% in 1g gravity. In ratio, oocyte maturation rate had no significant difference between the rotational group and control group. Microgravity simulated by the tail-suspended model inhibited mouse oocytes maturation and increased the rate of oocytes abnormity. The maturation rate of tail-suspended mouse oocytes was 14.54%, which was significantly lower than that of control group. Tail-suspended model should be an ideal model to investigate simulated microgravity effects on reproductive processes of female mice.
Animals ; Cells, Cultured ; Female ; Hindlimb Suspension ; Mice ; Oocytes ; cytology ; physiology ; Oogenesis ; physiology ; Weightlessness Simulation

Objective:To study the expression and clinical significance of microRNA-574-3p (miR-574-3p) in colon cancer.Methods:A total of 106 colon cancer patients who were admitted to the First Hospital of Qinhuangdao and Shijiazhuang Hospital of Traditional Chinese Medicine from June 2012 to June 2015 were selected as the research objects. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-574-3p in colon cancer tissues and normal adjacent tissues. The relationship between the expression of miR-574-3p and the clinicopathological characteristics and prognosis of patients with colon cancer was analyzed. Immunohistochemical staining was used to detect the relationship between the expression of miR-574-3p and the expression of CyclinA2 or E-cadherin.Results:Compared with normal tissues adjacent to cancer, the expression level of miR-574-3p in 106 cases of colon cancer was significantly lower ( P<0.01). The decreased expression of miR-574-3p was related to tumor diameter, Dukes stage, histological grade and lymph node metastasis (all P<0.05), but not to age and tumor location (all P>0.05). The patients with low expression of miR-574-3p, high Dukes stage and histological grade, and lymph node metastasis had poor survival (all P<0.05). The 5-year overall survival rate of patients with decreased miR-574-3p expression in cancer tissue was significantly lower than that of patients without decreased miR-574-3p expression ( P=0.007 6). Compared with patients with no decreased miR-574-3p expression, patients with decreased miR-574-3p expression had higher CyclinA2 protein integrated optical density (IOD) value and lower E-cadherin protein IOD value in colon cancer tissues (all P<0.05). Conclusions:The decreased expression of miR-574-3p is related to the poor prognosis of colon cancer patients, which may affect tumor recurrence and metastasis by regulating the expression of CyclinA2 and E-cadherin proteins.

Objective To explore the application effects of clinical nursing pathway on recovery of upper-extremity functions during different stages after surgery of breast tumor. Methods A total of 120 patients who received the surgery of breast tumor in our hospital from January 2012 to December 2013 were selected as research subjects. They were randomly assigned to a research group and a regular group according to the method of random number table, with 60 in each. The regular group was given clinical regular nursing for the recovery of upper-extremity functions after the surgery, and the research group was given clinical nursing pathway for the recovery of upper-extremity functions after the surgery. Nursing effects were observed between the two groups. Results Arms bending forward and arms bending inward as well as abduction and rotation in the research group were significantly better than those in the regular group, and the differences were statistically significant (P<0.05). Satisfaction rate on clinical nursing in the research group was 93.3%, higher than that of 75.0%in the regular group(P<0.05). Life quality score in the research group was(56.1±5.9), significantly higher than that of(43.8±6.3)in the regular group, and the difference was statistically significant(P<0.05). Conclusion Clinical nursing pathway is feasible clinically for the recovery of upper-extremity functions during different stages after the surgery of breast tumor. It promotes the recovery of upper-extremity functions, as well as en-hances satisfaction rate on clinical nursing and life quality, which is worthy of clinical application.

Objective:To analyze the effects of a novel type of polydopamine (PDA)-coated porous titanium alloy scaffolds loaded with zoledronic acid-gelatin nanoparticles (ZOL-GNPs) for topical sustained drug release on osteoclasts in vitro. Methods:After porous titanium alloy scaffolds were fabricated using electron beam melting technique and ZOL-GNPs with different ZOL concentrations (0, 1, 10, 50, 100, 500 μmol/L) were prepared by desolvation method, PDA-coated porous titanium alloy scaffolds loaded with ZOL-GNPs were constructed by combining the two. The characteristics of the scaffolds were analyzed. The biomechanics of 3 different scaffolds (bare porous titanium alloy scaffolds, PDA-coated porous titanium alloy scaffolds, and PDA-coated porous titanium alloy scaffolds loaded with ZOL-GNPs) were investigated. Drug release detection was carried out by high performance liquid chromatography on the 1st, 4th, 7th, 14th, 21st, and 28th days respectively. The osteoclasts were inoculated into the novel scaffolds with different ZOL concentrations. The expression of osteoclast-related genes was detected by real-time quantitative (RT)-polymerase chain reaction (PCR); the expression of osteoclast-related proteins was detected by Western-blot.Results:The PDA-coated porous titanium alloy scaffolds loaded with ZOL-GNPs were successfully constructed. Electron microscope scanning showed that the GNPs were well spheroidized, smooth in surface, and uniformly dispersed, with a particle size of (243.6±63.4) nm. The ZOL-GNPs were uniformly compounded on the surface and in the pores of the scaffolds, and the spheres were regular in shape with no adhesion. The biomechanical experiments showed that the elastic moduli of the porous titanium alloy scaffolds under 3 different conditions were (1.81±0.12) GPa, (1.80±0.23) GPa and (1.81±0.15) GPa, showing no significant difference ( P> 0.05). The drug release percentage in the porous titanium alloy scaffolds was obviously high on the first day, and increased gradually and slowly in the subsequent 27 days. In the scaffolds with a low concentration ZOL, more osteoclasts adhered and proliferated; in the 50 μmol/L scaffolds, spheroid cells appeared; the spheroid cells increased and even apoptosis occurred with an increase in the ZOL concentration. RT-PCR showed that the expression of Ctsk gene and TRAP gene increased with the increased ZOL concentration, peaked in the 50 μmol/L scaffolds, and then decreased with the increased concentration, showing statistically significant differences ( P < 0.05). Western-blot showed that the expression pattern of Ctsk and TRAP was similar to that of their related genes. Conclusions:The novel PDA-coated porous titanium alloy scaffolds loaded with ZOL-GNPs demonstrate good mechanical properties and an anti-osteoporosis effect via their topical sustained drug release. The scaffolds with a ZOL concentration of 50 μmol/L may exert the best effect on inhibition of osteoclasts.

Objective To quantitatively explore the influence of knife sharpness on forearm wounds in knife slash cases. Methods The finite element models of the upper limb and knives with 3 degrees of sharpness (with sharp blade, blunt blade, wide blade) were developed based on human CT images and prototype of slash knife. The slash by 3 kinds of knives on the forearm at velocity of 4 m/s and duration of 10 ms was simulated, so as to analyze changes in contact forces, wound dimensions and energy. Results During the slash by knives with sharp, blunt, wide blade, the blades reached the ulna at about 65, 85, 95 ms, respectively. The corresponding slash forces were 846, 1 064 and 1 865 N; the wound lengths were 135.64, 105.47 and 99.23 mm; the wound depths were 38.77, 27.81 and 18.74 mm. With the sharpness of blade decreasing, the wound formation was slowed, the length and depth decreased and the slash force increased. The model system for slash knife with sharp blade had obviously greater total energy and inner energy, but smaller kinetic energy, compared with slash knife with blunt blade and wide blade. Conclusions The method for quantitatively assessing wound formation in knife slash upon the forearm was developed. The research findings deepen the understanding of biomechanical mechanism of wound formation by knife slash, and provide new scientific means for forensic investigation and court trial of knife slash cases.

Nerve growth factor (NGF) can promote the development, differentiation and regeneration of neurons. Recently, in order to efficiently produce human NGF (hNGF) drugs with better efficacy, we created transgenic mice expressing hNGF specifically in their salivary glands, and purified highly active hNGF protein from their saliva. Some studies reported that the NGF secretion in mouse saliva is affected by gender and age. Here, in order to select hNGF transgenic mice with high NGF secretion for saliva collection and hNGF purification, we divided transgenic mice into 4 groups, including 28-day-old young males and females, 63-day-old adult males and females. We compared their saliva volume, total salivary protein amount, salivary mNGF protein amount and salivary hNGF protein amount. The results showed that the saliva volume as well as amounts of total salivary protein, salivary mNGF protein and salivary hNGF protein secreted by 63-day-old transgenic mice were significantly higher than those secreted by sex-match 28-day-old transgenic mice, and the salivary hNGF protein amount secreted by male transgenic mice at the age of 63 days was significantly higher than that of female transgenic mice at the same age; Among 4 groups of mice, 63-day-old male transgenic mice secreted the highest salivary hNGF content, which was about 46 times higher than that secreted by the 28-day-old female transgenic mice. Therefore, 63-day-old male transgenic mice should be selected for saliva collection and hNGF purification.
Animals ; Cell Differentiation ; Female ; Humans ; Male ; Mice ; Mice, Transgenic ; Nerve Growth Factor ; Saliva

This study was aimed to investigate the effect of Forkhead Box G1 (FOXG1) on the epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells and the underlying mechanism. For this purpose, FOXG1 lentiviral interference (shRNA) plasmid and expression plasmid were constructed. Western blotting was used to analyze the expression of FOXG1 protein in five CRC cells, namely RKO, SW480, SW620, LoVo and DLD-1. The shRNA fragment of FOXG1 (shFOXG1) was designed and synthesized. Recombinant plasmids were obtained with the aid of DNA recombination technique. Double digestion and sequencing were used to identify the recombinant plasmids, and then lentivirus packaging, purification and stable transfection were carried out. Additionally, stable CRC cell lines were screened out. The changes of FOXG1 knockdown and overexpression efficiency, E-cadherin, Vimentin, Fibronectin, Snail, Twist mRNA and protein were investigated respectively by Western blotting and qRT-PCR analysis. Furthermore, the changes of cell morphology after knockdown and cell migration ability were evaluated respectively with optical microscopy, scratch test and Transwell assay. FOXG1 had the highest protein expression in RKO and the lowest in DLD-1 among the five CRC cells. Compared with those of the control group, the cell morphology in FOXG1 knockdown RKO group was changed from spindle into round or polygonal shape, cell polarization was enhanced and tight junction assembly was acclerated while cell migration distance was noticeably decreased. Moreover, the number of cells invaded and migrated through chambers was significantly reduced. Among these key factors of EMT, the expression of E-cadherin was increased while the expressions of Vimentin, Fibronectin, Snail and Twist were decreased. The opposite was the case in the overexpressed FOXG1 group. The overexpression of FOXG1 in CRC promoted the invasion and metastasis of CRC cells and played a crucial role in regulating the EMT. Thus, FOXG1 might be a novel therapeutic target in CRC treatment.