Objective To study the angiogenic effects of adenovirus mediated human vascular endothelial growth factor 165 and human angiopoietin-1 (Ad 5-VEGF 165 , Ad 5-Ang-1) in rat models of hindlimb ischemia. Methods Rat models of hindlimb ischemia were established by ligation and peeling off rat′s femoral arteries. Ad-VEGF 165 and Ad-Ang-1 were intramuscularly transferred into the rat ischemic hindlimbs. The expression of VEGF 165 and Ang-1 were examined by Western blotting. Immunohistochemistry was performed to illustrate the effects on rat ischemic muscles after gene transferring. Results (1) Western blotting showed a high expression of VEGF 165 and Ang-1 in the ischemic hindlimb muscle transferred with Ad 5-VEGF 165 and Ad 5-Ang-1 VEGF 165 respectively. (2) There was no significant difference between groups on 7 days after the transfer. The capillary to muscle fiber ratio was significantly higher in the treating groups than that in control groups and were significantly higher in VEGF 165 +Ang-1 group than in VEGF 165 or in Ang-1 groups on day 14th and day 21th. (3) Many structured vessels surrounded by a layer of smooth muscle cells were found in Ad-VEGF 165 and Ad-VEGF 165 +Ad-Ang-1 groups, the number of SMA + vessel per muscle fiber was obviously higher than those in control groups. (4) Enormous cells positive for BrdU appeared in treated muscles in Ang-1, VEGF 165 , VEGF 165 +Ang-1 groups and many of them were positive for C-Kit, an antigen expressed by pluripotent marrow stem cells. Some C-Kit+ cells were incorporated in sites of neovascularization. Conclusion (1) Vascular endothelial growth factor 165 and angiopoietin-1 can promote neovascularization in rat models of hindlimb ischemia and the angiogenic effect is much more significant in Ad-VEGF+Ad-Ang-1 group. (2) VEGF 165 can increase the number of vessles that are coated with smooth muscle cells which shapes are similar to arterioles. (3) Not only angiogenic effect but perhaps vasculogenic effct also contribute to the neovascularization.

Clinical practice is an important part of cultivating the master of nursing specialist,this paper analyzed characteristics of clinical practice in domestic and foreign nursing students and revealed the problem in cultivation of master of nursing specialist in China,in order to provide references for the future construction and perfection of clinical practice of master of nursing specialist in China.

BACKGROUND:The type B synoviocytes induced by transforming growth factor β1(TGF-β1) have the potential to differentiate into chondrocyte,which can remain the phenotype in vitro. However,whether the transfected cell combined with scaffold can form cartilage tissues need further research. OBJECTIVE:Rabbit type B synoviocytes was transfected by liposome method in vitro,combined the cells with Pluronic-F127,and then implanted into nude mouse to construct tissue engineering cartilage. Additionally,to explore the feasibility of synoviocytes differentiate into chondrocytes.DESIGN,TIME AND SETTING:The randomized animal experiment of cytology observation. The experiment was performed at the Medical Research Center,the Second Affiliated Hospital of Sun Yat-sen University between April 2007 and May 2008.MATERIALS:Healthy New Zealand white rabbits,aged 3 months,and 12 BALB/c nude mice,aged 4 weeks,weighted 20 g.METHODS:The synovial membrane tissues were taken out from the rabbit knee,isolated by enzyme digestion,and then transfected. The positive cloning was screened by G418,and the expression of TGF-β1 and collagen Ⅱ was detected. The Pluronic-F127 was dissolved at 4℃ and prepared fluid with concentration of 30%. The fluid was mixed with cells. At the same time,there were 2 groups as the control group:chondrocytes with Pluronic-F127,and synoviocytes transfeced by pcDNA3.1(+)and Pluronic-F127. The cell density was 5x1010/L. Each compound (0.2 mL) was injected into the subcutaneous of the nude mouse back. Rats were sacrificed at weeks 4,6 and 8 to harvest samples.MAIN OUTCOME MEASURES:Cell growth curve;phenotype change of the cells after transfection;histological observation of the tissue in the subcutaneous of the nude mouse back. RESULTS:Cell growth curve demonstrated that the living activity of the transfected cells was temporary decreased,which returned into normal level at days 6,7 after transfection. At day 4 after transfection,TGF β1 were positive expressed,and at day 7,the collagen Ⅱ staining were positive. The compounds in the subcutaneous of the nude mouse back formed immature chondroid tissues at week 4,which turned to mature chondroid tissue at week 8,and the collagen Ⅱ staining were positive.CONCLUSION:The transfected synoviocytes can express the phenotype of chondrocytes in vitro,and form chondrocyte-likecells. The transfected synoviocytes with Pluronic-F127 can form chondroid tissues in nude mice.

Objective To evaluate the role of 5-HT1A receptors in distal cerebrospinal fluid (CSF)-contacting neurons in neuropathic pain (NP) in rats. Methods Forty male SD rats weighing 230-270 g were randomly divided into 4 groups (n = 8 each): sham operation group (group S); NP group; dimethyl sulfoxide (DMSO) group and 8-OH-DPAT (a specific 5-HT1A receptor agonist) group. NP was induced by chronic constrictive injury (CCI) in groups NP, DMSO and 8-OH-DPAT. Four silk ligatures were placed on the sciatic nerve at 1 mm intervals . In group S, the sciatic nerve was exposed but not ligated. 8-OH-DPAT and DMSO 1 μl were injected into the region where most of CSF-contacting neurons are present over 5 min on 7th day after CCI in groups 8-OH-DPAT and DMSO respectively. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured before CCI, on 7th day after CCI, and at 3 and 6 h after administration. The rats were sacrificed 6 h after administration, and the brain tissues removed for determination of the expression of 5-HT1A receptors in the distal CSF-contacting neurons by immunofluorescence. Results Compared with group S, PWL was significantly shorten and PWT decreased at T, in groups NP, DMSO and 8-OH- DPAT (P ＜ 0.01) . Compared with group DMSO, PWL was significantly prolonged and PWT increased at T2 and T3 in group 8-OH-DPAT ( P ＜ 0.01). The 5-HT1A receptor expression was significantly down-regulated in groups NP and DMSO compared with group S, while up-regulated in group 8-OH-DPAT compared with groups NP and DMSO ( P ＜ 0.01). There was no significant difference in 5-HT1A receptor expression between groups NP and DMSO ( P ＞ 0.05). Conclusion 5-HT1A receptors in distal CSF-contacting neurons are involved in the regulation of NP in rats.

Objective To investigate the effect of electroacupuncture at different points on auditory brainstem response (ABR) in a rat model of sodium salicylate-induced tinnitus.Method Forty-one male Wistar rats were randomly allocated to saline control (saline), sodium salicylate model (SA), electroacupuncture at Tinggong+ Yifeng (EA), electroacupuncture at Waiguan+ Zhongzhu (AA) and electroacupuncture at Zusanli+ Sanyinjiao (LA) groups. The saline group consisted of five rats and each of the other groups, nine rats. The model was made by intraperitoneal injection of sodium salicylate 275 mg/kg. The saline control group was injected with a corresponding volume of saline. Various acupoint electroacupuncture groups were given electroacupuncture at bilateral Tinggong+ Yifeng, Waiguan+ Zhongzhu and Zusanli+ Sanyinjiao, respectively, at 30 min after model making. Electroacupuncture lasted 30 min. The ABRs were recorded before model making and once every one hour for five consecutive hours after model making. The stimulus sounds were short clicks and tone bursts of frequencies of 4, 8, 16 and 32 kHz. The ABR threshold was used as an assessment index.Results Under the condition of clicks, there was a statistically significant difference in the ABR threshold at one to five hours after model making between the SA, EA, AA or LA group and the saline group (P<0.05), at two to five hours after model making between the EA or AA group and the SA group (P<0.05) and at one hour after model making between the LA and SA groups (P<0.05). Under the conditions of 4, 8 and 16 kHz, there was a statistically significant difference in the ABR threshold at one to five hours after model making between the SA, EA, AA or LA group and the saline group (P<0.05). Under the condition of 32 kHz, there was a statistically significant difference in the ABR threshold at one to five hours after model making between the SA, AA or LA group and the saline group (P<0.05). Under the condition of 4 kHz, there was a statistically significant difference in the ABR threshold at two to five hours after model making between the EA and SA groups (P<0.05) and at four to five hours after model making between the AA and SA groups (P<0.05). Under the condition of 8 kHz, there was a statistically significant difference in the ABR threshold at two to five hours after model making between the EA or AA group and the SA group (P<0.05). Under the condition of 16 kHz, there was a statistically significant difference in the ABR threshold at two, four and five hours after model making between the EA and SA groups (P<0.05). Under the condition of 32 kHz, there was a statistically significant difference in the ABR threshold at one to five hours after model making between the EA and SA groups (P<0.05).Conclusion Electroacupuncture at both periauricular and forelimb points can improve the ABR threshold in sodium salicylate-treated rats. The effect of electroacupuncture at periauricular points is superior to that at forelimb points.

Objective To investigate the status of clinical practice of master of nursing specialist (MNS) and provide references for completing the clinical practice patterns of master of nursing specialist in intensive care units.Methods Totally 56 clinical nursing postgraduate students were investigated from 10 nursing colleges by questionnaire survey.Results 46.43％ students were not satisfied with effect of clinical practice;69.64％ students though there was little difference between postgraduate and undergraduate students in clinical teaching method; 82.14％ students considered clinical teachers couldn't meet their leaming needs; the main reasons affecting the effect of clinical practice included inadequate teaching method (73.21％),impertinent teaching contents (69.64％) and unreasonable time arrangement (51.79％);55.36％ students wanted to be nursing educators; 50.00％ students wanted to be nursing specialist.Conclusions The effect of clinical practice of clinical nursing postgraduate students did not achieve the target.Clinical teaching couldn't meet the learning needs.Students are lack of recognition to capability and quality of master of nursing specialist.We should highlight the special characteristics of intensive care nursing,regulate the clinical teaching method and strengthen the publicity of master of nursing specialist to optimize structure of nursing talent.

PurposeTo develop a novel RGD microbubbles (RGD-MBs) and to evaluate the targeted binding effect with endothelial cells in vitro.Materials and MethodsThe RGD peptide was coated onto the microbubbles through biotin-avidin linkage including 10 μg/ml and 30 μg/ml groups. The microbubbles not carrying RGD peptide were obtained as negative control. Blocking studies were performed with pre-incubation of the cells with RGD peptide for 2 hours. The microbubbleswere characterized by Accusizer 780 and optical microscope. The binding specificity of RGD-MBs withανβ3-expressing mouse endothelial cells was determined with competitive inhibition experiments in vitro. The attachment study was performed using parallel plate flow chamber to investigate the dynamic adhesion on endothelial cells at various shear stresses.ResultsThe RGD-MBs had an average diameter of (4.09±0.07) μm. The binding RGD-MBs per cell were 2.98±0.35 for 10 μg/ml RGD and 1.78±0.23 for 30 μg/ml RGD. RGD-MBs binding to mouse endothelial cells decreased 54.64% and 67.00% in the presence of RGD peptide at a concentration of 10 μg/ml and 30 μg/ml respectively. When the shear stress was under 1.5 dyne/cm2, the accumulation rate was increased with the increase of shear stress (P<0.05). Accumulation rate reached the maximum (48.72±4.26) RGD-MBs/min at wall share stress of 1.5 dyne/cm2, and decreased as sheer stress >1.5 dyne/cm2 (P<0.05). Conclusion The RGD-MBs can specifically bind to endothelial cells, indicating its usefulness as ultrasonic molecular probe in monitoring integrinανβ3 expression during tumor angiogenesis, and is potentially valuable for in tumor early-staging and prognosis.

Many clinical studies showed that the traditional Chinese medicine (TCM) syndromes in stroke have been dynamically changing since the onset of the disease. The changing of TCM syndromes can be attributed to multiple correlative factors such as age, sex, area distribution, underlying diseases, and constitutional factor. Data-driven methods involving multivariate statistical methods and descriptive approach have been used to analyze the regularity of dynamically changed TCM syndromes of stroke. However, expressing non-linear relationship between symptom or correlative factors and syndrome patterns by data-driven models is challenging. Model-driven methods involving artificial neural networks and Bayesian networks are new methods for studying the changes in TCM syndromes in patients with stroke. In this review, the authors summarized the studies of dynamically changed patterns of stroke syndromes based on data-driven methods and some clinical trials on TCM syndromes based on model-driven methods. Further studies are needed to improve the understanding of the dynamically changing regularity of TCM syndromes for stroke by using model-driven methods so as to develop appropriate and timely TCM treatments.

Objective To verify the dose delivery accuracy of volumetric-modulated arc therapy plan by log-file analysis of linear accelerator that can be created when a dynamic delivery occurs.Methods Accelerator log file in binary format recorded the accelerator execution plan for each control point corresponding to the gantry angle,multi-leaf collimator leave position,cumulative machine monitor units ( MU).These information were read from the accelerator log file with Matlab7.1,then the original control points in the plan file replaced the corresponding information for the log,which generated a new plan.New plan was exported into the planning system to reculculate the dose.The volume dose histogram (DVH) and dose distribution was contrasted to determine the accuracy of the accelerator plan of implementation between two plans.Results Compared with the original plan,antry angle difference over ± 1° accounted for about 35％ of the entire arc of control points in 4 of 12 arcs and the percentage of the leave error of ±0.5 mm was about 95％.MU error of a single control point was larger,but the cumulative MU for each are was small which was located between-0.09％ to 0.11％ in the selected 12 arcs.Between the targets,the maximum dose,minimum dose,the mean dose differences were from-0.07％ to 0.42％,-0.38％ to 0.40％,0.03％ to 0.08％,respectively.The maximum dose and mean dose differences of organs at risks were located from-1.16％ to 2.51％,-1.21％ to 3.12％,respectively.Conclusions Accelerator log-file analysis to verify the VMAT plan nan be supplyed to the experimental method supplement.

Objective To investigate the experimental methods of transferring the synoviocytes with the reconstructed pcDNA3.1-TGF-β1 gene by the liposomes and study the feasibility of self-induction of synoviocytes to the chondrocytes in vitro so as to provide a scientific and experimental basis for the further gene enhanced tissue engineering research in articular cartilage repair.Methods Synoviocytes were cultivate in vitro and purified to construct the eucaryotic expression plasmid carrying the recombinant rabbit TGF-β1 gene.By means of Lipofectamine 2000,the synoviocytes were transfected with pcDNA3.1-TGF-β1 (experimental group) and with pcDNA3.1 ( + ) blank plasmid (control group).The synoviocytes free from transfection was set as blank group.After 48 hours of transfection,the cells were screened by G418.Representative sections from among the positive clones were used for RT-PCR assay of instant expression of TGF-β1.The other sections were used for immunohistochemical analysis with antibodies to TGF β1.Screening of cells by G418 was continued for 12 days of cell counting and drawing the growth curve.The antigens of TGF-β1 and collagen Ⅱ were examined every week three weeks after transfection.All images were processed by using analysis instrument (Image-Pro Plus V6.0).Statistical analysis was conducted with SPSS 13.0 software package.The difference between groups was tested by using variance ( ANOVA) analysis.Results The transfection efficiency in experimental group was 18% ,with temporary decreased living activity of the transfected cells shown by growth curve.The cell population decreased to 3.6 × 104/ml four days after transfection.After 72 hours of transfection,the positive fragment of TGF-β1 was detected by RT-PCR assay,and immunohistochemical staining of antibiotics to TGF-β1 showed positive particles only in the experimental group.After three weeks of tranfection,the immunohistochemical analysis with antibodies to TGF-β1 and type Ⅱ collagen showed that there were positive particles in the transfected cells in the experimental group,with no positive particle in the control and blank groups.According to the results of Image-Pro Plus V6.0,PU value of anti-TGF-β1 was (19.04±1.26) seven days after transfection,while that of control and blank groups were (4.07 ± 0.65)and (3.23 ±0.56) respectively,with statistical difference (P<0.05).PU value of anti-type Ⅱ collagen in experimental group,control group and blank group was (13.74 ± 1.27),(4.62 ±0.56) and (3.93 ± 0.38) 14 days after transfection,with statistical difference ( P < 0.05).Conclusions Transfection of the rabbit articular synoviocytes with the reconstructed pcDNA3.1-TGF-β1 gene can be successfully accomplished by using Lipofectamine 2000 method.The transfected synoviocytes can express TGF-β1 and excrete type Ⅱ collagen,have chondrogenic potential when transfected by pcDNA3.1-TGF-β1 gene and can be used as candidate cells for repair of articular cartilage.