BackgroundCorneal neovascularization (CNV) is a common eye disease.The researches on the pathogenesis and treatment of CNV are focus in Ophthalmology.ObjectiveThis study was to investigate the effects of culture supernatant from human amniotic epithelial cells (AECs) on CNV in vitro and its mechanism.MethodsHuman AECs were obtained from a placenta and cultured in serum-free medium for 48 hours,and the supernatant was collected.The levels of interleukin-1 receptor antagonist (IL-1 Ra) and pigment epithelium-derived factor (PEDF) in the human AECs culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).Rabbit corneal epithelial cells (CECs) were obtained and cultured in different concentrations of human AECs culture supernatant for 48 hours,serum-free medium was used as the control group.The expression of vascular endothelial growth factor (VEGF) mRNA and basic fibroblast growth factor (bFGF) mRNA in rabbit CECs were measured by quantitative real-time PCR (QRT-PCR).Human umbilical cord vein endothelial cells (UVECs) were cultured in the three mediums above,and the proliferation of human UVECs (absorbance value,A value) was tested by the cell counting kit 8 ( CCK8 ).Migration assay was performed by the wound healing method for the human UVECs.The membrane ultra-structure of human UVECs was examined under the atomic force microscope (AFM).ResultsCultured and passaged human AECs showed a positive response for keratin.The expression of VEGF mRNA (1.00±0.22) and bFGF mRNA (1.00±0.36) in rabbit CECs was suppressed by the human AECs culture supernatant,with a significant reduction in comparison with the serum-free DMEM group (2.98±0.46,2.55±0.48 )(P=0.001,0.002).The A value was significantly lowered in the human AECs culture group for 72 hours compared with the serum-free DMEM group ( 1.941 ± 0.036 versus 2.144 ± 0.059 ) ( P =0.000 ),and the bFGF-induced migration rate of human UVECs was strongly inhibited by the culture supernatant of human AECs in comparison with serum-free DMEM.The plasma membrane of human UVECs cultured with the human AECs culture supernatant was full of bumps,and decreased intercellular connection and cellular pseudopodia were found on the AFM image.The concentration of IL-1Ra was (153.56±0.36)ng/L and that of PEDF was (70.41 ±0.68 )μ,g/L in the human AECs culture supernatant.Nothing was deteched in serum-free DMEM group.Conclusions Human AECs culture supernatant suppressed the expression of VEGF and bFGF in CECs as well as the migration and proliferation of vascular endothelial cells.The effect may be associated with IL-1Ra and PEDF secreted by human AECs.These results suggest that human AECs may be a potential therapy for the inhibition of CNV.

Background Mitomycin C (MMC) has an inhibitory effect on the growth and proliferation of human pterygium fibroblasts,however,there is little literature about its influence on plasma membrane. Objective This study was to investigate the influence of MMC on the physical and chemical features and ultrastructures of plasma membrane. Methods Human pterygium tissues were obtained during the surgery.Human pterygium fibroblasts were primarily cultured and passaged using explant cultured method and identified by Vimentin staining.The third generation of cells were incubated to 96 well plate at a density of 5 × 103 cells/well,and 0,50,100,200,300 and 400 mg/L MMC was added in the culture well respectively to act for 12 hours.Cell viability was assayed using cell counting kit-8 ( CCK-8 ),and cellular apoptosis was detected using annexin V-FICT/PI.The changes of cell membrane structure were examined under an atomic force microscope.Malondialdehyde( MDA ) content in cell supernatant and level of lactate dehydrogenase ( LDH ) in extracellular fluid were detected to assess the lipid peroxidation level and permeability of cell membrane.Intracellular Ca2+ changes and membrane surface topography were assessed by Fluo-3/AM mark and flow cytometry( FCM ).This study was approved by Ethic Commission of Affiliated First Hospital of Jinan University.Informed consent was obtained from each patient. Results A lot of cells grew with the shape of spindle 1-2 weeks after culture.Positive response was seen in cultured cells for Vimentin.Growth and proliferation of the cells reduced 12 hours after action of MMC with the increase of MMC concentrations.The apoptosis rate of human pterygium fibroblasts was 4.2％,4.2％,5.4％,19.3％ and 25.8％ in 0,50,100,200 and 300 mg/L MMC groups respectively.Different degrees of abnormalities of cells membrane were found in various concentrations of MMC groups.The elevated contents of LDH and MDA in extracellular fluid were detected in various concentrations of MMC groups compared with the control group,and the LDH and MDA levels were gradually ascended as the increase of MMC concentrations,with a significant difference between any two groups(P＜0.05).The disturbance of intracellular Ca2+ homeostasis was also been seen after MMC acted. Conclusions MMC leads to the changes of physical and chemical features in human pterygium fibroblasts at a dose-dependent manner.Cell membrane may be the acting target of MMC.

Objective: The aim was to explore the association between the HLA-DRB1 allele polymorphism and susceptibility to chronic myelogenous leukemia(CML)in Chinese Han population.Methods: The polymorphism of HLA-DRB1 alleles in 762 CML patients and 2 264 normal umbihcal cord blood samples were tested by polymerase chain reaction with sequence specific primer(PCR-SSP).Results: The most common allele in DRB1 locus in normal population was DRB1 * 15(17.25%), whereas DRB1 * 10 was the least frequent allele(1.39%).Compared with normal controls, the frequency of HLA-DRB1 * 08 was significantly increased in all CML groups(7.48%vs 5.39%, X~2 =8.963,OR= 1.023, P =0.004)and CML male groups(7.72%vs 5.39%,X~2 = 8.059,OR= 1.025,P = 0.007).But there was no significant difference between the normal controls and CML female patients.Conclusion: It should be concluded that DRB1 * 08 could be considered a susceptible allele for CML male patients.

In order to summarize and perfect the basic skill practicing method in acupuncture teaching and form a characteristic teaching, the research on skill training and technique improving of acupuncture and tuina major was carried out. The undergraduate students in acupuncture and tuina major from College of Acupuncture-Moxibustion and Tuina were randomly divided into an experimental group and a control group. Students from both groups received the basic courses of acupuncture-moxibustion and tuina as requested in the syllabus and the special teaching was added to the experimental group, namely "three practice" (strength practice, coordination practice, targeted practice of acupuncture basic skills) and "three enlightenment" (technique enlightenment, mind and qi enlightenment, efficacy enlightenment). During and after the courses, the teaching effectiveness was evaluated on the basis of needling manipulation. The peacetime score in the experimental group was superior to that in the control group (38.03 +/- 1.14 vs 33.25 +/- 1.31, P < 0.05), the total score in the experimental group was superior to that in the control group (84.03 +/- 6.30 vs 78.05 +/- 6.55, P < 0.05). The special teaching method of "three practice" and "three enlightenment" can improve the effect of skill training teaching method. It highlights the unique feature of central plains by integrating the essence of Shaolin internal qigong and Chen-style Taiji, it can also provide new ideas for the practical teaching of TCM professional courses and the construction of characteristic professionals.
Acupuncture ; education ; manpower ; Acupuncture Therapy ; methods ; Humans ; Students ; Teaching

During professor SHAO Jing-ming's academic research and medical practice, his academic opinion of focusing spirit is gradually developed. In terms of nurturing the spirit, attention should be paid on persistence as well as everyday health maintenance and exercise to nurture the spirit of physician. In terms of clinical diagnosis and treatment, patients' psychology, employment and life status should be observed and experienced, which could bring more methods to take essential care of patients' spirit. The treatment should work with psychological counseling, advocating that based on patients' qi and spirit, various forms of treatment methods should be properly used, such as acupuncture or moxibustion or combination of acupuncture and medicine, along with simple acupoint selection and harmony medication. Before clinical treatment of acupuncture, calming the mind is critically emphasized to make a clear diagnosis. During the acupuncture, calming and focusing the mind is necessary as well as emphasizing the details, so acupuncture could be integrated with Chi Gong to create a new warming-sensation technique. In a word, the academic opinion of focusing spirit is shedding an inspiring light upon further study.
Acupuncture ; education ; history ; Acupuncture Therapy ; history ; psychology ; China ; History, 20th Century ; History, 21st Century ; Humans

Objective To explore the effect of excessive fluoride on expression of mRNA and protein of Wnt3a and β-catenin in rats' osteoblasts and its correlation with pathogenic mechanism of fluorosis.Methods Thirty-six healthy SD rats,weighting 100-120 g and according to body mass,were randomly divided into three groups(twelve in each group).The rats of control were fed wich tap water(fluoride ＜ 1 mg/L) and the experimental rats were exposed to NaF(low-fluoride group:5 mg/L,high-fluoride group:50 mg/L) added to the drinking water to establish the chronic fluorosis model.After fed for eight morth,all rats were killed and metaphysic of femoral was collected.Rat dental fluorosis was observed and bone fluorine was detected by ashing-fluorin ion selective electrode method.The content of bone alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b(TRACP 5b) in rats' serum was detected by enzyme-linked immunosorbent assay (ELISA).The morphologic changes of the bone were observed by microscopy.The expression of mRNA and protein of Wnt3a and β-catenin in osteoblasts of rats was analyzed with gray scale by hybridization in situ and immunohistochemistry methods,respectively.Results Detection rate of dental fluorosis,fluoride contents of urine and bone were significantly increased [control group:0.0％,(1.26 + 0.17)mg/L,(305.58 ± 91.26)mg/kg; low-fluoride group:66.7％,(2.06 ± 0.64)mg/L,(632.33 ±123.21)mg/kg; high-fluoride group:91.7％,(7.69 ± 1.96)mg/L,(1088.75 ± 156.16) mg/kg] in the rats treated with fluoride,the difference between groups was statistically significant(χ2 =21.6; F =36.57,467.02; all P ＜0.05).The contents of BALP and TRACP-5b in rats' serum were significantly different between groups(F =89.57,7.68; all P ＜ 0.05).Compared with control group[(16.24 + 1.57)U/L],the contents of BALP in rats' serum of the low-fluoride and high-fluoride groups[(31.47 ± 5.30) and (54.61 ± 2.27)U/L] were increased gradually(all P ＜0.05).Compared with the low-fluoride group,the value in the high-fluoride group decreased significantly (P ＜ 0.05).The contents of TRACP-5b in rats' serum of low-fluoride group[(3.45 ± 1.85)U/L] were elevated significantly(all P ＜ 0.05) compared with the control group[(1.26 ± 0.23)U/L] and the high-fluoride group[(2.74 ± 1.85)U/L].The bone cortices were thickened and the bone trabecula was broadened,arranged closely together in chronic fluorosis rats with significant difference compared with the control group.In the low-fluoride and high-fluoride groups,the expression levels of Wnt3a and β-catenin mRNA (low-fluoride group:132.87 ± 5.72 and 132.57 ± 9.56; highfluoride group:135.60 ± 6.64 and 137.87 ± 9.16) were markedly elevated with significant difference,respectively (F =12.47,5.96; all P ＜ 0.05) compared with those in control groups(119.86 ± 5.04 and 120.58 ± 7.84) by hybridization in situ(P ＜ 0.05),but there was no statistical significance (P ＞ 0.05) of the level of Wnt3a and β-catenin mRNA between low-fluoride and high-fluoride groups.In the low-fluoride and high-fluoride groups,the protein expression of Wnt3a and β-catenin (low-fluoride group:137.50 ± 4.32 and 140.85 + 3.54; high-fluoride group:142.65 ± 11.84 and 152.52 ± 4.64) were markedly elevated with significant difference,respectively (F =10.07,53.82; all P ＜ 0.05) compared with those in control group (124.01 ± 2.63 and 126.75 ± 4.65) by immunohistochemistry(all P＜ 0.05),Wnt3a protein production in the low-fluoride group was increased without statistical significance compared with the high-fluoride group (P ＞ 0.05).But the protein production of β-catenin in the lowfluoride group was elevated with significant difference compared with the high-fluoride group(P ＜ 0.05).The mRNA and protein production of Wnt3a were positively correlated with the mRNA and protein production of β-catenin (r =0.731,0.658; all P ＜ 0.05).Conclusions Rat bone tissue lesions caused by excessive fluoride may be associated with an increased expression of Wnt3a and β-catenin mRNA and protein in osteoblasts.In chronic fluorosis,fluoride stimulates the overexpression of Wnt3a and β-catenin in the Wnt signal transduction pathway,enhances bone osteogenesis and causes skeletal fluorosis.

Objective To explore the role of serum S-100B concentration of umbilical cord blood and blood on the 24 h after admission in the early diagnosis and development of newborn hypoxic-ischemic encephalopathy(HIE).Methods Forty-six HIE newborns(31 cases with mild HIE and 15 cases with moderate and severe HIE)were selected as HIE group,and 43 normal full-term newborns were selected as control group.The umbilical cord blood sample and blood sample were aquired on the 24 h after admission.The serum S-100B concentration was detected by enzyme-linked immunosorbent assay(ELISA)analysis.Results 1.There was no significant difference of serum S-100B concentration between the male sub-group and female sub-group of normal group and their birth weight had no significant relative to the serum S-100B concentration.2.The serum S-100B concentration of umbilical cord blood of control group and HIE group were(1.03?0.32)and(2.53?1.1)?g/L,respectively,there was significant difference between two groups(t'=8.848 P

Objective To explore the impact of silencing PLCε1 gene on proliferation and cell cycle of esophageal carci-noma Eca109 cells.Methods Three plasmid expression vectors (PLCε11, PLCε12 and PLCε13) were constructed to si-lence PLCε1 gene.A negative control plasmid expression vector (HK) was constructed at the same time to serve as a control .The plasmid expression vectors were transfected into esophageal carcinoma Eca 109 cells by cations liposome . The plasmid expression vector with the best interference effect ( PLCε12 ) was chosen .The study included Eca 109 group , HK group and PLCε12 group .Cell viability of Eca 109 cells was evaluated by MTT assay .The cell cycles were detected by FCM .The mRNA expression of P16 and CyclinD1 gene was measured by RT-PCR.Results The cell vi-abilitys of Eca109 cells in PLCε12 group were 80.73%and 75.88%at 48 and 72 h after transfection , which were significantly lower than that of Eca 109 cells in HK group (P<0.001).The percentage of S phase Eca109 cells in PLCε12 group was lower than that of Eca 109 cells in HK group ( P <0.01 ) , the cell cycle of PLCε12 group Eca109 cells was arrested in G0/G1 phase.The P16 gene mRNA expression of PLCε12 group Eca109 cells was higher than that of HK group Eca 109 cells ( P<0.01 ) .Conclusions Silencing PLCε1 gene may up-regulate P16 gene mRNA expression and then arrest the cell cycle at G 0/G1 phase and so inhibit proliferation of Eca 109 cells.

OBJECTIVEThe study was to describe the breastfeeding status of children under the age of three in counties of western China and to provide evidence to the government for decision-making on intervention.

METHODSA cross-sectional study with probability-proportional-to-size (PPS) sampling method was used. The information on breastfeeding was obtained through memory of the mothers. Fourteen thousand and seventy-seven children were studied. Data on breastfed status in counties of western China was compared with those of the children from the survey of the counties of western China in 2001.

RESULTSThe breastfeeding rate of children under 3 years old in western China was 96.5%. However, the overall breastfed rate of children under 6 months were only 33.4%, with rates of 11.4% and 22.0% on exclusively and predominantly breastfed groups respectively. Timely first-suckling rate was 43.5% with the continued breastfeeding rate (1 year) as 64.9%, but the continued breastfeeding rate (2 year) was only 9.7%. Reasons causing mothers to wean would include according to her own intention and to be able to attend the physical labor while exclusive breastfeeding under 6 months was for the growth and development of children, which might reduce the two-week prevalence of diarrhea. Major risks of exclusive breastfeeding of children under 6 months were seen as: level of education of the mothers, economic depression of the counties and mother's nationality (if as minority).

CONCLUSIONMost of the children were ever or being breastfed at the time of interview with timely first-suckling took place earlier than in 2001. However continued breastfeeding did not last long. During these five years, the exclusive breastfeeding rate had been at low level, especially at the economic depression and the minority area.

Breast Feeding ; epidemiology ; Child, Preschool ; China ; epidemiology ; ethnology ; Humans ; Infant ; Infant, Newborn ; Weaning

OBJECTIVETo probe into the effect of acupuncture at "Sishencong" (EX-HN 1) on sleep and the mechanism.

METHODSForty-eight white mice were randomly divided into a Sishencong group, a Zusanli group, a model group and a blank control group, 12 mice in each group. In the mice of the first 3 groups, sleep rhythm disorders was induced by repeatedly regular intraperitoneal injection of small dose of Caffeine, and the mice of the first two groups were treated by acupuncture at "Sishencong" (EX-HN 1) and "Zusanli" (ST 36), respectively. Living state and, circadian activities were observed, and nitric oxide synthase (NOS) activity and nitric oxide(NO) content in the brain were detected.

RESULTSAcupuncture at "Sishencong" (EX-HN 1) or "Zusanli" (ST 36) could significantly increase NOS activity and NO content in the brain, with acupuncture at "Sishencong" (EX-HN 1) being significantly better than acupuncture at "Zusanli" (ST 36) (P < 0.01).

CONCLUSIONAcupuncture at Sishencong (EX-HN 1) treats insomnia and regulates sleep and many mental and nervous symptoms, which are carried out partially through increasing NOS activity and NO content, promoting normal function of brain tissues.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Brain ; metabolism ; Disease Models, Animal ; Female ; Male ; Mice ; Nitric Oxide ; analysis ; Nitric Oxide Synthase ; metabolism ; Sleep Wake Disorders ; physiopathology ; psychology ; therapy