Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method.Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution.The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test.Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker.Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body.Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity.After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype.Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.

Objective To investigate the neuroprotective effect of vagus nerve stimulation ( VNS) on a model rat of focal cerebral ischemia. Methods A total of 42 adult male Sprague-Dawle ( SD) rats were randomly divided into a sham operation group (n=10),a model group (n=16),and a VNS-treated group ( n = 16 ) . Each group was randomly redivided into 2 subgroups:left VNS subgroup and right VNS subgroup. A model of focal cerebral ischemia (2 h) in rats was induced by the intraluminal suture method. At 30 minutes after modeling, the VNS-treated group received cervical VNS, the stimulation intensity was 0. 5 mA,the interval was 0. 5 ms,and the frequency was 20 Hz. Stimulation was once every 5 min within 1 h and each lasted for 30 s. The model group did not give any stimulation. Neither blood vessels were embolized nor were the nerves stimulated in the sham operation group. The changes of somatosensory evoked potentials ( SEP) on the lesion sides during operation were monitored. At 24 h after modeling,the neurobehavioral scores were performed. The rats were sacrificed,and their brain infarct volume was measured. Results (1) During the stimulation of left VNS in rats,the neurobehavioral scores of the sham operation group,model group and VNS-treated group were 0. 4 ± 0. 2,9. 5 ± 0. 4,6. 4 ± 0. 3,respectively;during the stimulation of right VNS in rats,the neurobehavioral scores of the 3 groups were 0. 6 ± 0. 2,9. 3 ± 0. 4,and 6. 9 ± 0. 4,respectively. There were significant differences between the scores of the model group and those of the other 2 groups (P<0. 05). (2) Compared with the model group,the brain infarct volume of the VNS-treated group was reduced ( stimulating the left VNS of the 2 groups was 120 ± 7 and 56 ± 7 mm3 respectively;stimulating the right VNS was 115 ± 10 and 54 ± 8 mm3 respectively ) . There were significant differences ( P <0. 05). (3) Compared with the sham operation group and the VNS-treated group,the SEP N1 amplitude of the model group was decreased significantly and the P1 latency was prolonged significantly. There was significant difference (P<0. 05). (4) There were no significant differences in the stimulation of the left or right VNS in the VNS-treated group among the infarct volume, neurobehavioral scores, SEP amplitude,and latency (P>0. 05). Conclusion No matter whether to stimulate the left or right vagus nerves, they both have neuroprotective effects on ischemic brain injury, and there was no significant difference on the action effects.

Objectives To investigate the neuroprotective mechanism of vagus nerve stimulation ( VNS) by stimulating the vagus nerve in ischemic cerebral tissue in a rat model of transient focal cerebral ischemia. Methods Twenty-six adult male Sprague-Dawley ( SD ) rats were randomly divided into sham operation group (n=6),model group (n=10),and VNS-treated group (n=10) . The model of rat transient focal cerebral ischemia was induced by the intraluminal suture method. At 30 min after modeling,the right side neck VNS in the VNS-treated group was stimulated ( stimulus intensity 0. 5 mA, interval 0. 5 ms, frequency 20 Hz),once every 5 min within 1 h,and once for 30 s. The model group repeated the steps of the VNS-treated group,but did not stimulate. The sham operation group repeated the experimental steps,but it neither embolized the vessels nor stimulated nerves. The changes of cerebral blood flow were monitored with a laser Doppler flowmeter. The rats were sacrificed after 24 h. The expressions of interleukin 6(IL-6) and caspase-3 in brain tissue were determined by immunohistochemistry staining. The neuronal apoptosis was observed by the in situ end-labelling technique. Results ( 1 ) Compared with the sham operation group, the number of positive cells of IL-6,caspase-3,and the numbers of neuronal apoptosis in the model group were significantly increased (20. 7 ± 5. 0 cells/HP vs. 2. 3 ± 1. 0 cells/HP,44. 5 ± 9. 5 cells/HP vs. 0,30. 9 ± 9. 0 cells/HP vs.0).Thereweresignificantdifferences(P<0.05).(2)Comparedwiththemodelgroup,thenumber of positive cells of IL-6(10. 9 ± 3. 7 cells/HP),the caspase-3 (18. 9 ± 6. 7 cells/HP),and the numbers of neuronal apoptosis (14. 0 ± 5. 2 cells/HP) in the VNS-treated group decreased significantly. There were significant differences (P<0. 01). (3) Before and after modeling,there were no significant differences in cerebral blood flow in various periods between the model group and the VNS-treated group (P>0. 05). Conclusion The neuroprotective mechanism of VNS for cerebral ischemia may be associated with the inhibition of neuronal apoptosis and decreasing inflammatory response. It may not be associated with the changes of cortical cerebral blood flow.

Objective To summanrize the operative method and follow-up data of total aortic arch replacement combined with transaortic stented graft implantation into the descending aorta (Sun's procedure) for acute Stanford type A aortic dissection.Methods Between August 2004 and March 2012,73 patients with acute type A aortic dissection underwent this procedure.60 males and 13 females ranging in age from 26 to 79 years (mean age,49,6 years).Right axillary or femoral artery cannulation was routinely used for cardiopulmonary bypass.Cerebral protection was achieved by bilatero-antegrade or selected hrain perfusion.The stented elephant trunk was implanted throuugh the aortic arch under hypothermic circulatory arrest.The stented elephant trunk was a 10cmlong self expandable graft.Patent false lumina were evaluated using computed tomography 3 months and once each year after discharge to evaluate the postoperative time course of the residual false lumen.Results Mean cardiopulmonary bypass time was (248.1±69.8)min,and selected cerebral perfusion time was (38.2±10.5)min.Hospital morality was 6.85 ％ (5/73).Thrombus obliteration of the residual false lumen in the descending thoracic aorta was observed in 9 1.7％ of the aortic dissections 3 months postoperatively.The mean follow-up time was(36.4 ± 31.6)months (range,2 to91 months).Survival at 1,5,7 years was 97％,87％ and 81％,respectively.Conclusion Total aortic arch replacement combined with transaortic stented paft implantation into the descending aorta is an effective treatment and n more promising choice for acute type A aortic dissection.

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Organism ; veterinary ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors ; genetics ; Goats ; genetics ; Integrases ; chemistry ; metabolism ; Recombination, Genetic ; Transgenes ; genetics

OBJECTIVETo explore the NEK-6 expression in gastric cancer tissue and its relationship with clinicopathological features.

METHODSFluorescent quantification PCR and Western blotting were used to examine the NEK-6 expression in 36 samples of fresh gastric cancer tissues and para-cancer gastric mucosal tissues, human gastric cancer cell lines(BGC-823, MKN-28, SGC-7901, MGC-803, HGC-27, AGS), and human normal gastric epithelial cell line (GES-1). Gastric cancer cell lines with the highest expression level were selected to perform the invasion and migration tests, and the effect of down-regulated NEK-6 expression by siRNA transfection on above invasion and migration tests were observed. Meanwhile NEK-6 expression in 94 paraffin samples of gastric cancer tissues was examined by immunohistochemistry and its positivity was compared among different clinicopathologic features.

RESULTSFluorescent quantification PCR revealed gastric cancer tissues had significantly higher NEK-6 expression than para-cancer tissues(0.002 80±0.001 36 vs. 0.001 91±0.001 48, P<0.05), NEK-6 expression was up-regulated in 31 gastric cancer tissues (86.1%), and human gastric cancer cell lines had significantly higher NEK-6 expression than GES-1 cells, among whom BGC-823 and AGS cell lines were the highest. Invasion and migration tests showed that as compared to negative siRNA control group, ability of invasion and migration in BGC-823 and AGS cells after siRNA transfection was obviously decreased. In 94 paraffin samples, positive expression rate of NEK-6 was 60.6%(57/94), and NEK-6 expression was significantly associated with gastric cancer distant metastasis, lymph nodes metastasis and TNM staging(all P<0.05).

CONCLUSIONSNEK-6 expression is up-regulated in gastric cancer tissues, which is significantly associated with distant metastasis, lymph nodes metastasis and TNM staging. Down-regulation of NEK-6 expression can inhibit the ability of invasion and migration in gastric cancer cells.

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.
Animals ; Cattle ; Cloning, Organism ; veterinary ; Embryo Culture Techniques ; methods ; veterinary ; Embryo, Mammalian ; physiology ; Embryonic Development ; physiology ; Female ; Fibroblasts ; cytology ; Goats ; embryology ; genetics ; Humans ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology ; Pregnancy

Objective To explore the effect of carbon monoxide (CO) and ozone (O3) in the air on the myocardial infarction mortality in Ningbo,Zhejiang province,from 2011 to 2015.Methods The data of daily air quality surveillance and the causes of deaths in Ningbo from January 1,2011 to December 31,2015 were collected and the time series study using a generalized additive model was conducted to evaluate the relationship between the mortality of myocardial infarction and the air pollutants after adjustment for the long-term trend of death,weather conditions,"days of the week" and other confounding factors.Results The daily average concentrations of CO and O3 in Ningbo during 2011-2015 were 0.90 (0.02-3.31) mg/m3 and 82.78 (4-236) μg/m3,respectively.A total of 5 388 myocardial infarction deaths occurred,with a daily average of 3 deaths.In single-pollutant model,an increase of 0.1 mg/m3 in average concentration of CO could increase the risk of myocardial infarction mortality by 1.06％ (95％ CI:0.29％-1.93％) in general population,and by 1.26％ (95％ CI:0.28％-2.24％) in aged people aged ≥65 years in lagged 6 days,but the influence was not significant in people aged ＜65 years.The influence had no significant difference in males,but it increased the risk of myocardial infarction mortality by 1.77％ in females (95％ CI:0.44％-3.13％).In multipollutant model,CO did remain robust after adjusting for other co-pollutants.Whereas the effect of O3 had no significant influence.Conclusion These findings suggested that the increased risk of daily myocardial infarction mortality was associated with the increase of CO concentration,but no such association was found for O3 in Ningbo.

Objective To explore the NEK-6 expression in gastric cancer tissue and its relationship with clinicopathological features. Methods Fluorescent quantification PCR and Western blotting were used to examine the NEK-6 expression in 36 samples of fresh gastric cancer tissues and para-cancer gastric mucosal tissues, human gastric cancer cell lines ﹙BGC-823, MKN-28, SGC-7901, MGC-803, HGC-27, AGS), and human normal gastric epithelial cell line ﹙GES-1). Gastric cancer cell lines with the highest expression level were selected to perform the invasion and migration tests, and the effect of down-regulated NEK-6 expression by siRNA transfection on above invasion and migration tests were observed. Meanwhile NEK-6 expression in 94 paraffin samples of gastric cancer tissues was examined by immunohistochemistry and its positivity was compared among different clinicopathologic features. Results Fluorescent quantification PCR revealed gastric cancer tissues had significantly higher NEK-6 expression than para-cancer tissues ﹙0.002 80 ±0.001 36 vs. 0.001 91 ±0.001 48, P<0.05), NEK-6 expression was up-regulated in 31 gastric cancer tissues ﹙86.1%), and human gastric cancer cell lines had significantly higher NEK-6 expression than GES-1 cells, among whom BGC-823 and AGS cell lines were the highest. Invasion and migration tests showed that as compared to negative siRNA control group, ability of invasion and migration in BGC-823 and AGS cells after siRNA transfection was obviously decreased. In 94 paraffin samples, positive expression rate of NEK-6 was 60.6%﹙57/94),and NEK-6 expression was significantly associated with gastric cancer distant metastasis, lymph nodes metastasis and TNM staging ﹙all P<0.05). Conclusions NEK-6 expression is up-regulated in gastric cancer tissues, which is significantly associated with distant metastasis, lymph nodes metastasis and TNM staging. Down-regulation of NEK-6 expression can inhibit the ability of invasion and migration in gastric cancer cells.

Objective To explore the NEK-6 expression in gastric cancer tissue and its relationship with clinicopathological features. Methods Fluorescent quantification PCR and Western blotting were used to examine the NEK-6 expression in 36 samples of fresh gastric cancer tissues and para-cancer gastric mucosal tissues, human gastric cancer cell lines ﹙BGC-823, MKN-28, SGC-7901, MGC-803, HGC-27, AGS), and human normal gastric epithelial cell line ﹙GES-1). Gastric cancer cell lines with the highest expression level were selected to perform the invasion and migration tests, and the effect of down-regulated NEK-6 expression by siRNA transfection on above invasion and migration tests were observed. Meanwhile NEK-6 expression in 94 paraffin samples of gastric cancer tissues was examined by immunohistochemistry and its positivity was compared among different clinicopathologic features. Results Fluorescent quantification PCR revealed gastric cancer tissues had significantly higher NEK-6 expression than para-cancer tissues ﹙0.002 80 ±0.001 36 vs. 0.001 91 ±0.001 48, P<0.05), NEK-6 expression was up-regulated in 31 gastric cancer tissues ﹙86.1%), and human gastric cancer cell lines had significantly higher NEK-6 expression than GES-1 cells, among whom BGC-823 and AGS cell lines were the highest. Invasion and migration tests showed that as compared to negative siRNA control group, ability of invasion and migration in BGC-823 and AGS cells after siRNA transfection was obviously decreased. In 94 paraffin samples, positive expression rate of NEK-6 was 60.6%﹙57/94),and NEK-6 expression was significantly associated with gastric cancer distant metastasis, lymph nodes metastasis and TNM staging ﹙all P<0.05). Conclusions NEK-6 expression is up-regulated in gastric cancer tissues, which is significantly associated with distant metastasis, lymph nodes metastasis and TNM staging. Down-regulation of NEK-6 expression can inhibit the ability of invasion and migration in gastric cancer cells.