BACKGROUND: Neural axon regeneration is one of the difficulties that must be overcome in treatment of injury of central nerve system. Significant therapeutic effects have been obtained in transplantation of neural stem cells (NSCs), embryonic stem cells (ESCs) and Schwann cells. But the bottleneck situation of insufficiency of cell provider has limited the development on it.OBJECTIVE: To observe directional-differentiation of retinoic-acid induced ESCs so as to find optimal condition for neuronal differentiation.DESIGN: Non-randomized controlled experiment was designed.SETTING: Teaching-Research Room of Histology and Embryology, Department of Basic Medicines. Third Military Medical University of Chinese PLAMATERIALS: The experiment was performed in Staff Room of Histology and Embryology, Third Military Medical University of Chinese PLA from January to May 2000. Eighteen Kunming mice in disoestrus were employed, of which. 12 mice were female and 6 mice male. They were placed in same cage at ratio of 2:1 for mating. The date of pregnancy was recorded. MESPU35 ESC line was prepared.METHODS: Removed head. internal organs and four limbs, feeder-layer Feeder-layer adherent culture was used to proliferate MESPU35 ESCs.Classic 4-/4+ method [The embryoid body (EB) grew naturally for 4 days,without retinoic acid added. In the coming 4 days, retinoic acid was added to induce neural EB of high proportion] was applied to induce the directional differentiation of the nerve. EB was cultured with serum of different concentrations. Phase contrast microscope was used to observe nerve-like EB in serum of different concentrations and to count numbers. ②Immunocytochemical technique was used to observe cellular morphological charac ters at various differentiating phase spots (5th. 9th, 14th days) and with retinoic acid at various concentrations. Flow cytometer (FCM) was used to count the proportion of differentiated neurons.MAIN OUTCOME MEASURES: ①Estimated measurement of the length of process and cell body during formation of neural EB after retinoic-acid induced differentiation of MESPU35 ESCs. ② Observation of cell morphology with immunocytochemical staining and proportion of differentiated cells assayed with FCM.RESULTS: ①It was discovered with phase contrast microscope that serum of different concentrations affect neural directional differentiation after EB formation to certain extent. Excessively high and low concentrations of serum reduced the proportion of neural differentiation of EB. The differentiating proportion is high in serum with 5% concentration. ② It is observed with immunocytochemical technique that the proportions of NF200 positive cell and glial fibrillary acidicprotein (GFAP) positive cell in differentiation of MESPU35 ESCs induced by retinoic acid were increased with phase spots in differentiation and increased concentration of retinoic acid. NF200 positive cell is transformed as multipolar neurons from absence of process in morphology. The processes of GFAP positive cell became longer and linked among each other as reticular pattern finally. ③ It was assayed with FCM that the proportion changes of GFAP positive cell and NF200 positive cell manufactured in differentiation were similar to immunocytochemical one.CONCLUSION: Retinoic acid in combination with proper concentration of serum and differentiating phase spots can induce neural-differentiation of MESPU35 ESC at high proportion and its differentiating regulation is in the patterns of concentration dependence and time dependence.

Objective To detect the expression changes and location of the mouse developmental regulation brain protein(Dbn1)in the developmental mouse brain.Methods Monoclonal antibodies against drebrin protein were used to assess Dbn1 by immunohistochemistry and Western blot.The paternal expression of Dbn1 in developmental mouse brain(E14,P1,P7 and adult)was initially investigated by Western blot.Dbn1 was shown at various developmental stages(E14,P1 and P7)as well as in adult in different brain area of developmental mouse brain by immunohistochemical.Results Dbn1 protein was detected in developmental brain but a little in adult brain by Western blot,high at E14,decreased at P1,gradually increased at P7 and lowest in adult.Immunohistochemistry confirmed as follows:Dbn1 expressed mostly in cortex,hippocampus and ependyma areas at E14,and the positive signal was distributed at cells border;The expression of Dbn1 was decreased at P1,mainly distributed at the verge of cells or dendrites;The peak expression of Dbn1 appeared at P7,Dbn1 located at nucleus of hippocampus and cortex,and the positive signal located within cytoplasm and dendrites;Only a little positive Dbn1 cells were found in adult mouse brain.Conclusion Dnb1 may be involved in regulating the differentiation and migration of neurons during the development of mouse brain.

Objective To investigate the etiologic roles of apoptosis-associated genes,environmental factors and their interactions in lumbar disc herniation (LDH).Methods A case-control trial was conducted.We recruited 128 outpatients with LDH as case group and 132 normal people matched by age and gender as control group.Peripheral venous blood samples were collected and DNA was extracted from leukocytes.By using a modified Brucker Autoflex MALDI-TOF mass spectrometer,we analyzed 3 genes with 9 polymorphic sites,namely,Fas-1377G/A rs2234767,Fas-670G/A rs1800682,Fas rs2147420,Fas rs2296603,Fas rs7901 656,Fas rs1 57101 9,FasL-844C/T rs7631 10,CASP-9-1263A > G rs4645978,and CASP-9-712C > T rs4645981.The correlations between polymorphism of Fas,FasL and CASP-9 genes and the risk of LDH were evaluated by non-conditional Logistic regression model.Multiple Logistic regression model was performed to assess the interaction between apoptosis-associated genes and environment factors,such as lumbar vertebral loads,bed type,spare-time exercises and spare-time activities. Results There were preferable balances in case and control groups in age and gender without significant differences.However,the two groups differed significantly (P <0.05)in lumbar vertebral loads,body mass index, bed type, spare-time exercises and spare-time activities. Correlation analysis showed remarkable correlations between LDH and the polymorphisms of FasL-844C/T (rs7631 10)and CASP-9-1263A>G (rs4645978),and FasL-844C/T TT and CASP-9-1263A>G GG genotypes might be the high risk genotypes of LDH.The gene-environment interaction analysis revealed that super-multiplicative and sub-multiplicative interactions respectively between FasL-844TT genotype and lumbar vertebral loads (3-4 level),and between CASP-9-rs4645978 GG and lumbar vertebral loads (3-4 level).Conclusion FasL,CASP-9 genes and lumbar vertebral loads and their interactions play important roles in the pathogenesis of LDH.It suggests that the risk of LDH may be codetermined by environmental factors and inherited susceptibility genes,and that the mechanisms of interactions vary in different genotypes and the same or different environmental factors.

Objective:To investigate the developmental behavior of early term infants at 6 months and its difference from that of complete full-term infants.Methods:A healthy maternal and infant birth cohort in maanshan city, established in Maanshan Maternal and Child Health Hospital from June 2015 to June 2016. Birth outcomes were copied from the hospital electronic medical record system after delivery. The Chinese Ages and Stages Questionnaires was used to assess developmental behavior. The chi-square test and multivariate unconditional logistic regression model were used to analyze the differences in the behavioral development of early and full term infants.Results:The birth rate of early term infants was 24.74% (500/2 021). The detection rates of communication, gross motor, fine motor, problem solving and individual-social areas in early term infants were 2.1%, 3.2%, 6.1%, 6.3% and 2.7%, respectively. The detection rate of problem-solving area in early term infants was significantly higher than that in the full-term infants (6.3% vs. 3.7%, χ 2=5.42, P<0.05). After controlling for confounding factors, compared with full-term infants, the risk of problem-solving area in early infants was significantly increased ( OR=1.65, 95%CI:1.01-2.70, P<0.05). Conclusion:The risk of behavioral retardation in Early term infants is significantly higher than that in full-term infants, and long-term follow-up and appropriate early development promotion interventions are needed to improve their quality of life.

Objective:To explore the effect of distal veins on the survival of a dorsal four-territory perforator flap in rats.Methods:A total of 32 SD rats were randomly divided into the control group and the experimental group, with 16 rats in each group. The multi-territory perforator flap including the bilateral iliolumbar and bilateral posterior intercostal angiosomes was cut from the back of each rat, with the size of 6 cm×7 cm. The right iliolumbar artery and vein were preserved in the control group, while the right iliolumbar artery and the right posterior intercostal vein were preserved in the experimental group. In both groups, incisions were made between the right iliolumbar angiosome and the right posterior intercostal angiosome. Finally, the flap was sutured back to their orthotopic site. At 6 hours and 1, 3, 5, 7 days after surgery, the blood perfusion at the bilateral iliolumbar and the left posterior intercostal vascular territories were measured. On the seventh day after surgery, the percentage of the survived area of the flaps were evaluated, arteriography was performed to observe the dilation of arteries within the flap, the intraluminal diameter of the choke artery in the choke 2 area was measured using hematoxylin and eosin staining, and the relative expression of endothelial nitric oxide synthase (eNOS) was detected by Western blotting. SPSS 28.0 was used for statistical analysis, and measurement data were presented as Mean±SD. Independent sample t-test was used to compare data across two groups. P<0.05 was considered statistically significant. Results:(1) At 6 hours and 1, 3, 5, 7 days after surgery, the experimental group displayed higher blood perfusion than the control group at the bilateral iliolumbar and the left posterior intercostal vascular territories (all P<0.01). (2) On the 7th day after surgery, the artery dilation of the experimental group was more obvious than that of the control group; the percentage of the survived flap area in the experimental group was higher than that in the control group [(87.6±3.2)% vs. (65.3±3.0)%, P<0.01]; the intraluminal diameter of the choke artery was greater in the experimental group than that in the control group[(49.3±3.1) μm vs. (35.1±2.3) μm, P<0.01]; and the relative expression of eNOS in the experimental group was higher than that in the control group (0.87±0.07 vs. 0.50±0.05, P<0.01). Conclusion:The distal vein (right posterior intercostal vein) of dorsal four-territory perforator flap of SD rats directly guided the pedicle artery blood supply to promote the expression of eNOS, dilated the arteries in each zone of the flap, increased the blood supply to the distal artery of the flap, and ultimately enhanced the flap survival area.

Objective:To explore the effect of distal veins on the survival of a dorsal four-territory perforator flap in rats.Methods:A total of 32 SD rats were randomly divided into the control group and the experimental group, with 16 rats in each group. The multi-territory perforator flap including the bilateral iliolumbar and bilateral posterior intercostal angiosomes was cut from the back of each rat, with the size of 6 cm×7 cm. The right iliolumbar artery and vein were preserved in the control group, while the right iliolumbar artery and the right posterior intercostal vein were preserved in the experimental group. In both groups, incisions were made between the right iliolumbar angiosome and the right posterior intercostal angiosome. Finally, the flap was sutured back to their orthotopic site. At 6 hours and 1, 3, 5, 7 days after surgery, the blood perfusion at the bilateral iliolumbar and the left posterior intercostal vascular territories were measured. On the seventh day after surgery, the percentage of the survived area of the flaps were evaluated, arteriography was performed to observe the dilation of arteries within the flap, the intraluminal diameter of the choke artery in the choke 2 area was measured using hematoxylin and eosin staining, and the relative expression of endothelial nitric oxide synthase (eNOS) was detected by Western blotting. SPSS 28.0 was used for statistical analysis, and measurement data were presented as Mean±SD. Independent sample t-test was used to compare data across two groups. P<0.05 was considered statistically significant. Results:(1) At 6 hours and 1, 3, 5, 7 days after surgery, the experimental group displayed higher blood perfusion than the control group at the bilateral iliolumbar and the left posterior intercostal vascular territories (all P<0.01). (2) On the 7th day after surgery, the artery dilation of the experimental group was more obvious than that of the control group; the percentage of the survived flap area in the experimental group was higher than that in the control group [(87.6±3.2)% vs. (65.3±3.0)%, P<0.01]; the intraluminal diameter of the choke artery was greater in the experimental group than that in the control group[(49.3±3.1) μm vs. (35.1±2.3) μm, P<0.01]; and the relative expression of eNOS in the experimental group was higher than that in the control group (0.87±0.07 vs. 0.50±0.05, P<0.01). Conclusion:The distal vein (right posterior intercostal vein) of dorsal four-territory perforator flap of SD rats directly guided the pedicle artery blood supply to promote the expression of eNOS, dilated the arteries in each zone of the flap, increased the blood supply to the distal artery of the flap, and ultimately enhanced the flap survival area.

Objective:To compare the effects of leukocyte-poor platelet-rich plasma (Lp-PRP) and leukocyte-rich platelet-rich plasma (Lr-PRP) on dorsal wound healing in rats.Methods:Thirty-six male Sprague-Dawley rats were randomly divided into Lp-PRP group, Lr-PRP group and control group, each containing twelve rats. Venous blood was drawn and the Lp-PRP and Lr-PRP were prepared separately using a centrifugal method. Circular full-thickness skin defect wounds (15 mm in diameter) were created on the backs of the rats in the three groups. The wounds were then treated with 100 μl Lp-PRP, Lr-PRP and saline, respectively. At 7 and 14 days post-operation, the wounds were photographed, and Image J software was used to calculate the wound area rate (postoperative wound area/wound area at modeling time × 100%). At 14 days post-operation, the total neo-epithelium length and collagen deposition rate of the wounds were evaluated using HE and Masson staining, respectively. At 7 days post-operation, the relative expression of vascular endothelial growth factor (VEGF) in the wounds was detected by Western blotting, and the number of CD31 positive microvessels in the wounds was examined by immunohistochemistry. Statistical analysis was performed using SPSS 28.0. One-way analysis of variance (ANOVA) was used to compare the three groups, and Tukey’s test was used for pairwise comparisons. A significance level of P<0.05 was considered statistically significant. Results:Blood analysis revealed that the platelet concentrations in the prepared Lp-PRP and Lr-PRP were 4.1 times and 4.5 times that of whole blood, respectively ( P<0.01), with no significant difference between the two PRPs ( P>0.05). The leukocyte concentration in Lp-PRP was undetectable, while in Lr-PRP, it was 3.5 times that of whole blood ( P<0.01). The wound area rate at 7 and 14 days post-operation, the total neo-epithelium length and collagen deposition rate at 14 days post-operation, as well as the relative expression of VEGF and the number of CD31-positive microvessels at 7 days post-operation in the Lp-PRP and Lr-PRP groups were superior to those in the control group (all P<0.01). However, there was no significant difference between the two PRP groups (all P>0.05). Conclusion:Both Lp-PRP and Lr-PRP promote dorsal wound healing in rats by enhancing re-epithelialization, collagen deposition, and angiogenesis. The impacts of Lp-PRP and Lr-PRP on promoting wound healing are comparable and not influenced by the presence of leukocytes in PRPs.

Objective:To compare the effects of leukocyte-poor platelet-rich plasma (Lp-PRP) and leukocyte-rich platelet-rich plasma (Lr-PRP) on dorsal wound healing in rats.Methods:Thirty-six male Sprague-Dawley rats were randomly divided into Lp-PRP group, Lr-PRP group and control group, each containing twelve rats. Venous blood was drawn and the Lp-PRP and Lr-PRP were prepared separately using a centrifugal method. Circular full-thickness skin defect wounds (15 mm in diameter) were created on the backs of the rats in the three groups. The wounds were then treated with 100 μl Lp-PRP, Lr-PRP and saline, respectively. At 7 and 14 days post-operation, the wounds were photographed, and Image J software was used to calculate the wound area rate (postoperative wound area/wound area at modeling time × 100%). At 14 days post-operation, the total neo-epithelium length and collagen deposition rate of the wounds were evaluated using HE and Masson staining, respectively. At 7 days post-operation, the relative expression of vascular endothelial growth factor (VEGF) in the wounds was detected by Western blotting, and the number of CD31 positive microvessels in the wounds was examined by immunohistochemistry. Statistical analysis was performed using SPSS 28.0. One-way analysis of variance (ANOVA) was used to compare the three groups, and Tukey’s test was used for pairwise comparisons. A significance level of P<0.05 was considered statistically significant. Results:Blood analysis revealed that the platelet concentrations in the prepared Lp-PRP and Lr-PRP were 4.1 times and 4.5 times that of whole blood, respectively ( P<0.01), with no significant difference between the two PRPs ( P>0.05). The leukocyte concentration in Lp-PRP was undetectable, while in Lr-PRP, it was 3.5 times that of whole blood ( P<0.01). The wound area rate at 7 and 14 days post-operation, the total neo-epithelium length and collagen deposition rate at 14 days post-operation, as well as the relative expression of VEGF and the number of CD31-positive microvessels at 7 days post-operation in the Lp-PRP and Lr-PRP groups were superior to those in the control group (all P<0.01). However, there was no significant difference between the two PRP groups (all P>0.05). Conclusion:Both Lp-PRP and Lr-PRP promote dorsal wound healing in rats by enhancing re-epithelialization, collagen deposition, and angiogenesis. The impacts of Lp-PRP and Lr-PRP on promoting wound healing are comparable and not influenced by the presence of leukocytes in PRPs.

Anterior cruciate ligament(ACL)injury is caused by strong violence,which can destabilize the knee joint,cause joint cartilage degeneration,meniscus injury,and in severe cases,develop osteoarthropathy.The gold standard for the treatment of ACLR injuries at this stage is arthroscopic anterior cruciate ligament recon-struction(ACLR).In clinical practice,the LARS(Ligament advanced reinforcement system)artificial ligament made of polyethylene terephthalate(PET)as the material has a good effect in the short and medium term,but the long-term biological healing between the graft and the host bone is poor,and the real"ligamentization"requirement of the postoperative graft cannot be met.Coating-modified modification of artificial ligaments can improve their hydrophilicity and biocompatibility,which in turn can promote the healing of graft-bone tunnels.Tendon bone healing is a bone-derived progressive process from indirect insertion to direct insertion,which takes a relatively long time and is closely related to the prognosis and early rehabilitation effect of patients.This article reviews the progress of superficial modification of artificial ligaments to promote ACLR tendon bone healing.

Objective:To examine the relationship between pre-pregnancy BMI, gestational diabetes (GDM) and different indicators of childhood obesity at the age of 4.Methods:Based on Ma’anshan Birth Cohort Study, singleton children who were born in Ma’anshan of Anhui province from October 2013 to April 2015, were followed for 4 years, consecutively. During the first questionnaire survey, data including pre-pregnancy weight, height and socio-demography were collected. During 24-28 week of gestation, 75 g oral glucose tolerance test was conducted for them. Childhood height, weight, waist circumference and body composition were measured at the age of 4. Comparisons between groups were performed using chi-square test, analysis of variance or t-test. The relationship between pre-pregnancy overweight/obesity, GDM and childhood obesity-related characteristics were analyzed by logistic regression model and generalized linear model analysis. Results:The prevalence rates of overweight and obesity in children at the age of 4 were 13.08% and 6.03%, respectively. After adjustment for characteristics related to mothers and their children, significantly increased risk of obesity ( OR=3.27, 95% CI: 2.15-4.98), larger waist circumference ( OR=2.32, 95% CI: 1.72-3.14) and higher waist-to-weight ratio ( OR=2.29, 95% CI: 1.73-3.02) were seen in the offspring of women with pre-pregnancy overweight/obesity. Body composition (skeletal muscle, body fat, body fat percentage) of the offspring were strongly correlated with pre-pregnancy overweight/obesity of the mothers ( P<0.05). Maternal GDM was associated with higher risk of childhood obesity ( OR=1.78, 95% CI: 1.14-2.79), on mothers without GDM during pregnancy. However, neither larger waist circumference, or higher waist-to-weight ratio seemed to increase the risk. Moreover, maternal GDM was not associated with body composition measures (skeletal muscle, body fat, body fat percentage). Conclusion:Pre-pregnancy BMI and maternal GDM were independent risk factors for obesity in 4-year-old children, and pre-pregnancy BMI was correlated with various indicators of body composition in children.