Background Hypoxia is a crucial factor of neovascularization.Many researches found that stromal cell-derived factor-1 (SDF-1) and integrin-linked kinase (ILK) play an important role in the neovascular disease.However,effect of SDF-1 and ILK in eye neovascular disease is below understood.Objective The aim of this study was to investigate the effect of hypoxia on the expressions of SDF-1 and ILK in cultured retinal pigment epithelium(RPE) cells in vitro.Methods RPE tissue was isolated from 4-week-old C57BL/6 mouse and was digested and cultured in DMEM/F12 with 10％ fetal bovine serum (FBS).The cells with 80％ confluence were collected and passaged.The third generation of cells were identified with cytokeratin 18 (CK18) antibody by immunochemistry.The cells were inoculated at the density of 5×104 cells/ml to free-serum DMEM/F12 for 24 hours and then were cultured in regular medium in the normoxic control group.RPE cells were cultured for 1 hour and 3,6,12,24,48,72 hours with 200 μmol/L CoCl2 in the hypoxia group.Reverse transcription-PCR(RT-PCR) was used to evaluate the expressing change of SDF-1 mRNA and ILK mRNA in RPE cells,and Western blot was used to assay the expressing change of SDF-1 protein and ILK protein in RPE cells in different time points.The detected outcomes were represented as the ratio of target gene A value/β-actin A value.Results Cultured cells showed the polygon in shape with the black pigment granules in cytoplasm.Over 90％ cells were positive response for CK18.Expressions of the SDF-1 mRNA and ILK mRNA were increased in different time points after CoCl2 co-cultured(SDF-1 mRNA:F=281.875,P=0.000 ;ILK mRNA: F=187.566,P=0.000),with the highest expressing value in hypoxia at 12 hours.No significant change in the expression of SDF-1 mRNA and protein was found 1 hour after CoCl2 co-cultured,but expressions of SDF-1 mRNA and ILK mRNA were significantly higher in 3,6,12,24,48 and 72 hours than the normoxic control group(P＜0.01).The expressions of SDF-1 protein and ILK protein were gradually ascended with the time increase of CoCl2 co-culture,showing a significant difference among different time points(SDF-1: F=44.719,P =0.000 ; ILK: F =144.481,P =0.000),and the up-regulation of SDF-1 protein and ILK protein expression was seen mainly in 3,6,12,24,48 and 72 hours after CoCl2 co-cultured in comparison with the normoxic control group (P＜0.01).Conclusions SDF-1 and ILK are involved in the hypoxic response of RPE cells and may play a potential role in ischemic/hypoxic retinopathy.

Objective To observe the effect of thumb pressing at Yangming Meridian on the function rehabilitation after total knee arthroplasty (TKA) for knee osteoarthritis (KOA).Method Sixty KOA patients after TKA were randomized into a treatment group (thumb pressing at Yangming Meridian plus conventional rehabilitation) and a control group (single conventional rehabilitation),30 cases each.The treatment group was intervened by thumb pressing and kneading manipulation at the acupoints of Foot Yangming Meridian and Foot Taiyin Meridian plus rehabilitation training of passive flexion and extension by CPM machine,while the control group was intervened by rehabilitation training of passive flexion and extension by CPM machine.The American Hospital for Special Surgery (HSS) for knee joint,the American Knee Society score (KSS) and the Western Ontario and the McMaster Universities Osteoarthritis Index (WOMAC) of KOA were used to evaluate the function.Result After the treatment,the HSS,KSS and WOMAC scores were changed significantly in both groups (P＜0.05).The improvements of HSS,KSS and WOMAC scores in the treatment group were superior to those in the control group,and the differences were statistically significant (P＜0.05).Conclusion The efficacy of thumb pressing at Yangming Meridian plus conventional rehabilitation for post-TKA KOA is superior to single conventional rehabilitation.

Objective To develop europium (Ⅲ) [Eu (Ⅲ)] chelated microparticles for homogeneous immunoassay.Methods Anti-human PCT antibodies were labeled with Eu (Ⅲ) chelated nanoscale microparticles as the detection antibody,and another anti-human PCT antibody was labeled with biotin as the solid-phase antibody.Magnetic microspheres labeled with streptavidin were used to separate the complexes of Eu-IgM-PCT-IgM-Biotin.Results In the homogeneous immunoassay,the standard curve fit was not linear.The quadratic curve was Y =19170.12 + 75493.74X-26.00X2(r =0.9986).According to the standard curve,the limit of detection for PCT was 0.04 ng/ml.Conclusion The homogeneous immunoassay which uses Eu (Ⅲ) chelated microparticles is highly sensitive for detection of PCT recombinant antigens and may serve as a promising method to measure serum PCT levels in the future.

Objective To explore the distribution of arsenic speciafion and to estimate the effect of arsenic on glutathione(GSH)levels in the blood and liver of mice exposed to different concentrations of inorganic AsⅢ through drinking water.Methods Mice drank water containing arsenite at concentrations of iAsⅢ of 0(contr01),25,50,100 ms/L for 6 weeks.Blood and liver were sampled to asses$the levels of inorganic arsenic(iAs),monomethylarsenic acid(MMA),dimethylarsenic acid(DMA)by the method of hydride generation trapping and ultra-hypothermia coupled with atomic absorption spectrometry,and the level of GSH by the method of 5,5'-Dithio-bis (2-Nitrobenzoic acid).Results Leveh of iAs.MMA and DMA in blood and in liver increased along with the increase of iAs concentrations in drinking water.Primary methylated index(PMI)and secondary methylation index (SMI)of liver and blood were significantly higher in exposed groups than those in control group(P＜0.05).SMI of liver in 50 mg/L exposed group[(50.45±2.94)%]was significantly higher than those in 25 mg/L and 100 mg/Lgroups[(41.68±7.09)%and(41.19±8.87)%,respectively],the difference being statistically significant(P＜0.05).The ratio of iAs.MMA and DMA in blood and liver in exposed group were 2:3:5 and 4:3:3,the percentage of level of organic arsenic(MMA+DMA)were 80%and 60%.GSH in blood and liver in exposed group decreased along with iAs concentrations in drinking water and had significant differences compared with those in control group (P＜0.05).However,levels of GSH in liver and blood did not differ significantly between exposed groups and control group(P＞0.05).Conclusions Membolism of iAs in liver is maximized when the iAs concentrations in drinking water increases to a certain level.However,the percentage of arsenic speciation in blood is different from that in liver,suggesting that other organs and tissues may be capable of methylation of inorganic arsenic.The level of GSH in liver and blood in mice is a good mark tO reflect the toxicity of arsenic.

The value of tissue strain imaging (SI) in regional myocardial systolic anddiastolic function assessment was studied. In 18 patients with nonobstructive hypertrophic cardiomyopathy (HCM) and 20 age-matched healthy subjects, regional myocardial longitudinal peak systolic strain in eject time (represented by εet) was measured at basal, mid and apical segments of septal, lateral and posterior walls of the left ventricle (LV) and compared between groups. εet had no significant difference between segments in control group (P＞0.05), which displayed a decreasing trend from basal segments to apical ones. εet in the HCM group was significantly decreased (P＜0. 05) as compared with that in the healthy group. In the HCM group, εet in the midseptum was significantly less than at the basal and apical septum, and was also less than at the rest LV walls in the same group (P＜0.01). The systolic reversed εet was noticed in 35 % of the hypertrophic segments in HCM group. Significantly negative correlation existed between the absolute value of εet and wall thickness in the midseptum (r=- 0.83). The post-systolic strain(PSS) segment number the and amplitudes in healthy group were significantly less than those in HCM group (P＜0.05). Both regional myocardial systolic and diastolic functions were impaired in hypertrophic or non-hypertrophic segments in patients with the HCM, especially in hypertrophic segments. Strain imaging technique is a sensitive and accura tool in myocardial dysfunction assessment.

Objective To investigate the clillicoradiologic characteristics of MarchiafavaBignami disease (MBD). Methods The clinical and neuroimaging findings, including lesion morphorogy, distribution, signal intensity/density in 7 MBD patients were retmspectiVely analyzed.Diffusion-weighted imaging(DWI)was performed in 5 cases.Of the 7 cases,4 underwent both CT and MRI,2 only CT and 1 MRI. Results with extensive iso-or slightly hypo-intensity on T1-weighted images and hyper-intensity on T2-weighted images, 5 patients were manifested as acute onset, in which white matter (WM) was symmetrically involved in bilateral periventricular and frontal subconical regions. Punctate or linear hypo-intensity on FLAIR images was found in the atrophied corpus callosum in 1 case presented as chronic type, with scattered patchy hyper-intensity in periventricular WM and frontal subcortical WM. DWI showed markedly hyper-intensity with diffusion restriction in 2 cases in acute phase with apparent diffusion coefficient(ADC)values(0.52～0.55)×10-3 mm2/s.Brain atrophy was found in all the 7 patients. Conclusions The MRI features of MBD are characteristic and may be associated with the clinical spectrum and prognosis.

Objective To investigate the effect of exogenous kallikrein on apoptosis of the neurons aroundthe cerebralinfarctareain rats. Methods Thirty rats wjth cerebral infarction induced by middle cerebral artery occlusion(MCAO)were assigned randomly into 3 groups(n=10),namely the blank control group,saline group,and pAdCMV-HTK group.In the pAdCMV-HTK group,kallikrein gene was delivered into the cerebral ischemie lesion via a replication-defective adenovims using stereotaetic injection technique, and the expression of exogenous kallikrein was detected immunohistoehemically.TUNEL staining was performed to evaluate the neuronal apoptosis around the infarct area,and RT-PCR used to detect the mRNA expressions ofbcl-2,bax and caspase-3 in the brain tissues. Results At 24 h aftertreatment there were some HTK expressed cells found in group C and peal(at 72 h after treatment.While compare with group B and group C,there existed significant difference(112±6.1,68±4.2,59±3.9,P<0.05).At 72 h after treatment,the NSS of group C was significantly lower than that ofgruop B and A(6.70±0.16,8.13±0.16,7.93±0.20,P<0.05);7 days after the treatment,the difference was more significant(5.14±0.18,7.82±0.14,7.91±0.10,P<0.01).Apoptotic cells were mostly seen around the infarct area.The ratsinpAdCMV-HTK group showed significantly reduced number of cells positive for TUNEL staining as compared to those in the saline and blank control groups at 3 days(10.1±0.9,16.7±1.1,and 20.4±0.8,respectively)and 7 days after the treatment(15.2±1.2,33.6±1.3,and 28.8±1.7,respectively)(P<0.05).The mRNA levels ofbc1-2.bax and caspasc-3 were elevated in all the groups at 24 h,peaked at 72 h,and decreased gradually till 7 days alter the treatment.Compared with those in the other two groups，bcl-2 mRNA level in the pAdCMV-HTK group increased slightly P>0.05) while bax and caspase-3 mRNA levels decreased markedly(P<0.05) 72 h and 7 days after the treatment.Conclusion Kallikrein can inhibit neuronal apoptosis around the cerebral infarct and improve the neurological fimction of rats following cerebral infarction probably by reducing the expressions of such apoptotic factors as bax and caspase-3.

Objective To investigate the effects ofkallikrein gene transfer on microvascularproliferation around the cerebral infarct and on the recovery of regional cerebral blood flow (rCBF)following ischemia/reperfusion injury in rats. Methods The rats with cerebral ischemia/reperfusioninjury induced by middle cerebral artery occlusion (MCAO) were randomly assigned into blank controlgroup, saline group, and pAdCMV-HTK treatment group and received corresponding injections into thetissues around the infarct area. Each group was divided into 3 subgroups (n=10) for observation at 12, 24and 72 h after the treatment. The neurological deficits of the rats before and after the treatment wereevaluated using neurological severity scores (NSS), and the expressions of exogenous human tissuekallikrein (HTK) and vascular endothelial growth factor (VEGF) in the brain tissues were detectedimmunohistochemically. TIC staining was performed to measure the changes in the infarct size.14C-iodoantipyrine tracing technique was used to define the rCBF in the rats. Results Compared tothe blank control group, the cerebral infarct size was significantly reduced in pAdCMV-HTK group 24 hafter the treatment, and was further reduced at 72 h (P<0.05). At 24 h after the treatment, the NSS inpAdCMV-HTK group was significantly lower than that in the blank euntrol and saline groups (P<0.05),and was further reduced at 72 h (P<0.01). After MCAO, the VEGF-positive cells were found mostly inthe cortex and the white matter around the infarct area. The expression of VEGF in pAdCMV-HTK groupwas markedly higher than that in the other two groups at 12, 24, and 72 h after the treatment (P<0.05). Inall the 3 groups, the rCBF around the infarct was slightly decreased as compared to that in thecontralateral hemisphere, pAdCMV-HTK slightly increased the rCBF 12 h after the injection (P>0.05),and significant increase in the rCBF occurred 24 h and 72 h after the injection (P<0.05). ConclusionKallikrein gene transfer following cerebral ischemia/reperfusion injury promotes vascular proliferationaround the infarct and increases the rCBF to reduce the infarct volume and attenuate neurological deficitsin rats.

Objective To explore the correlations of biochemical factors as gamma-aminobutyric acid (GABA) level with amino acid metabolism level in the blood, levels of intestinal immunoglobulin A (IgA)+complement factor 3 (C3), feeding intolerance and fatiguability in children with hypotonia cerebral palsy. Methods Ninety-six children with hypotonia cerebral palsy,admitted to our hospital from January 2009 to January 2012,were chosen in our study; questionnaire was used to understand the clinical symptoms of the children; the blood ammonia level,hepatic function and IgA+C3 level were obtained from routine blood chemistry testing, and amino acid metabolism was detected by blood tandem mass spectrometry; the correlations of GABA level with amino acid metabolism level in the blood, levels of IgA+C3, feeding intolerance and fatiguability were statistically analyzed. Results In all the 96 children with hypotonia cerebral palsy,63 (65.63％) had low arginine; 52 had both decreased arginine and elevated blood ammonia levels, enjoying negative correlation (r=-0.776,P=0.000); 42 had decreased arginine and reduced levels of IgA+C3 enjoying positive correlation (r=0.351,P=0.000); both decreased arginine level and feeding intolerance were noted in 47 with positive correlation (r=0.372,P=0.000).In these 96 children,30 (31.25％) had carnitine metabolism abnormality,including decreased propionyl carnitine/free carnitine or propionyl carnitine/acetylcarnitine levels in 21 (21.88％),increased hydroxyl palmitoyl carnitine/hydroxyl Palm enoyl carnitine level in 9 (9.37％),and decreased cysteine content in 3 (3.12％). Conclusion Metabolic abnormalities of arginine,carnitine and cysteine are noted in children with hypotonia cerebral palsy; a lot of exercise will consume arginine,carnitine and cysteine,which causes fatigue; children with low blood arginine content might also have increased blood ammonia level,reduced IgA+C3 level,trends of vomiting,susceptibility to infection and feeding difficulties,and therefore,children's mental state,immune function and exercise tolerance ability are affected.

To assess the normal value of left ventricular twist (LVtw) and examine the changeswith normal aging by 2-dimensional ultrasound speckle-tracking imaging (STI), 121 healthy volunteers were divided into three age groups: a youth group (19-45 y old), a middle-age group (46-64 y old ) and an old-age group (≥65 y old). Basal and apical short-axis images of left ventricular were ac- quired to analyse LV rotation (LVrot) and LVrot velocity. LVtw and LVtw velocity was defined as apical LVrot and LVrot velocity relative to the base. Peak twist (Ptw), twist at aortic valve closure (AVCtw), twist at mitral valve opening (MVOtw), untwisting rate (UntwR), half time of untwisting (HTU), peak twist velocity (PTV), time to peak twist velocity (TPTV), peak untwisting velocity (PUV), time to peak untwisting velocity (TPUV) were separately measured. The results showed that the normal LV performs a wringing motion with a clockwise rotation at the base and a counterclock- wise rotation at the apex (as seen from the apex). The LVtw velocity showed a systolic counterclock- wise twist followed by a diastolic clockwise twist. Peak twist develops near the end of systole (96%±4.2% of systole). With aging, Ptw, AVCtw, MVOtw, HTU and PUV increased significantly (P<0.05) and UntwR decreased significantly (P<0.05). However, no significant differences in TPUV, PTV and TPTV were noted among the 3 groups (P0.05). It is concluded that LV twist can be meas- ured non-invasively by 2-dimensional ultrasound STI imaging. The age-related changes of LVtw should be fully taken into consideration in the assessment of LV function.