OBJECTIVETo determine the interaction between miR-21 and DNA methylation in different breast cancer cells.

METHODSFluorescence tagged miR-21 inhibitor and its negative control (NC) were transient transfected into MCF-7 and MDA-MB-231 cell, the transfection efficiency was observed using fluorescence microscopy, and the miR-21 expression level and genome DNA methylation status before and after transfection were assessed by real-time PCR and bisulfite-qMSP respectively. To investigate the regulation effect of DNA methylation on miR-21, cells were treated with 5-AZA (2.5 µmol/L) for 72 h, with dimethyl sulfoxide (DMSO) treatment as its negative control (NC), and the expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and AKT(also known as Protein Kinase B), two downstream genes of miR-21 were detected by Western blot.

RESULTSThe expression of miR-21 in MCF-7 cell was significantly knocked down (P < 0.01) by miR-21 inhibitor, with the genome DNA methylation level (P < 0.05) and all the three Dnmts: Dnmt1, Dnmt3a, and Dnmt3b unregulated. In contrast, the miR-21 expression in MDA-MB-231 cell was elevated ( P < 0.01) by miR-21 inhibitor, meanwhile, down- regulated of genome DNA methylation (P < 0.05) and Dnmt3b expression, upregulation of Dnmt3a were also observed. In addition, treated with 5-AZA resulted in significant increases of miR-21 expression in both MCF-7 and MDA-MB-231 cells (P < 0.01), with the protein level of PTEN increased in MCF-7 cell, which was further involved in the downregulation of AKT.

CONCLUSIONThe regulation effects of DNA methylation by transient transfection of miR-21 in MCF-7 and MDA-MB-231 cells are almost opposite, whilst the expression of miR-21 in two cell lines were all upregulated by decreased DNA methylation level and our results may provide some experimental evidences for the future development of rational therapy for different breast cancer.

Azacitidine ; Breast Neoplasms ; genetics ; Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; MicroRNAs ; genetics ; PTEN Phosphohydrolase ; metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-akt ; metabolism ; Real-Time Polymerase Chain Reaction ; Up-Regulation

BACKGROUND: Advanced oxidation protein products (AOPPs) are a crucial pathogenic link to such long-term uremic complications in hemodialysis patients as immune system dysregulation, accelerated atherosclerosis, dialysis-related amyloldosis and so on. However, basic studies on AOPPs are relatively few, and one of the main reasons is the fact that it is difficult to obtain AOPPs with high pudty and biological activity.OBJECTIVE: To prepare, pudfy and indentify AOPPs, with the hope of searching for a method of preparing AOPPs with high purity and biological activity.DESIGN, TIME AND SETTING: A single sample observation was completed in the Clinical Biochemistry Section of Ecsomatics Department, Third Military Medical University of Chinese PLA from September to November in 2008. MATERIALS: Human serum albumin (HSA) was provided by Chengdu Rongsheng Company Ltd. Hitrap 26/60 sephacryl S-300 was purchased from GE Healthcare.METHODS: Hypochloric acid was used in the oxidation of purified HSA to prepare in vitro the AOPPs-modified HSA (AOPPs-HSA), which was then isolated by Hitrap 26/60 sephacryl S-300. Relative molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE), native polyacrylamide gel electrophoresis (PAGE) and molecular weight standards. Structural features and biological activities were identified in the experiment of tumor necrosis factor α (TNF-α) release from monocytes.MAIN OUTCOME MEASURES: ①The purification and gel electrophoresis results of HSA. ②The purification and gel electrophoresis results of AOPPs. ③The dose-effect relationship between AOPPs-HSA and TNF-α release from monocytes. RESULTS: The relative molecular mass of AOPPs-HSA was 670 000 according to SDS-PAGE, native PAGE and molecular weight standards. Moreover, AOPPs-HSA could encourage the release of TNF-α from monocytes. The time effects showed that TNF-α release volume significantly increased after 6 hours of stimulation by AOPPs-HSA (1 g/L) and reached a peak at hour 12. CONCLUSION: Highly purified and bioactive AOPPs can be successfully prepared by the above-mentioned method, which builds a basis for further study on AOPPs.

Objective Traditional nasolacrimal duct stenting possesses some shortcoming including difficulty of pulling ball head guide wire from the nasal cavity with turbinate hypertrophy and nasal septal deviation. The new method of nose-oral tube track establishment can overcome the forementioned and increase the successful rate. Methods 5 F catheter and arterial sheath were modified to be nasolacrimal duct stent delivery device respectively. Antegrade dacryocystography was taken firstly to display the obstructed site and followed by the modified protocol of inserting the guide wire through nasolacrimal duct and nasal cavity, and establishing the stent delivery track for retrograde stent placement. Results 5 epiphora patients with failure implantation by traditional method were all succeeded through the modified stenting(100%). During 6-mouth follow-up, no serious complications and reocclusion occurred. Conclusion The establishment of eye-nose-mouth-nose of external nasal guide wire track can improve the successful rate of nasolacrimal duct stenting.

Objective To compare the effects of subgluteal(SG) and sub-subgluteal-fold(SSGF)approach for ultrasound-guided siatic nerve block. Methods One hundred forty-eight patients undergoing lower limb surgery were randomly divided into two groups to receive SG approaches and SSGF approaches to sciatic nerve block under real time ultrasound guidance. A combined posterior lumbar plexus block under ultrasound guidance was performed for sufficient surgery anesthesia. 20 ml of 0. 5% ropivacaine was used for sciatic nerve and lumbar plexus block separately. Measurements included skin-to-nerve distance,reorientation of the needle during block and execution time,rates of sensory and motor blockade after 15 min and 30 min of injection, quality of surgery blockade, duration of the sensory and motor block, and postoperative complications related to sciatic nerve block. Results In SSGF group, execution time and reorientation of needle for sciatic nerve block was significantly less than those of the SG group( P ＜0.01).But motor blockade in the SG group was quicker when compared with SSGF group ( P ＜0.01). There were no significant differences in the quality and duration of blockade between the two groups. Conclusions Both SG and SSGF approach can be used for sciatic nerve block with equal sensory and motor block rate,whereas sciatic nerve block via SSGF approach was faster and easy to perform than the SG one.

Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.

AIM:To explore relationship between the normal strehl ratio ( SR ) values of total aberrations/SR values of total higher-order aberrations and modulation transfer function ( MTF ) at total corneal at different pupil diameters in normal population.
METHODS: To exam the SR values of total aberrations and SR values of total higher-order aberrations of total corneals in 200 people ( 400 eyes ) using SIRIUS 3D topography system and analysis the corresponding root-mean-square ( RMS) .
RESULTS: The subjects with different pupil diameters (3. 0, 5. 0, 6. 0, 7. 0mm)'s exam results of total corneal were as following:SR value of total aberrations 100’(0. 45±0. 12), (0. 25±0. 06), (0. 17±0. 05), (0. 13±0. 04); SR value of total higher order ab cerrations 100’(0. 69±0. 14), (0. 34±0. 07), (0. 24±0. 05), (0. 16±0. 04);SR value of total aberrations 200’(0. 45±0. 12), (0. 24±0. 06), (0. 20±0. 04), (0. 16±0. 03); SR value of total higher order aberrations 200’(0.70±0. 13), (0. 35±0. 07), (0. 27±0. 06), (0. 20±0.04 ) . The SR values of each group decreases with the increases of pupil diameters. The SR values of total aberrations and SR values of total higher - order aberrations at total corneals are negatively correlated with corresponding RMS value. When the pupil diameter is small, the SR value of total aberrations is more related to higher frequency region of MTF. When the pupil diameter is big, the SR value of total aberrations is more related to lower frequency region of MTF.
CONCLUSION: The visual performance of normal people can be well reflected by SR values of total aberrations and SR values of total higher - order aberrations at total corneal.

Objective To study the clinical characteristics and prognosis of children aged 5 or above with stage 3/4 neuroblastoma ( NB).Method Among 180 children with NB admitted from March 2007 to June 2015, 54 were aged 5 or above with stage 3 or 4.The clinical characteristics , therapeutic efficacy and prognosis of above 54 cases were analyzed.Results There were 36 boys and 18 girls with a male to female ratio of 2∶1.The most common of primary site was retroperitoneum (41/54, 75.5%), followed by mediastinum (10/54,18.5%), spine and pelvic cavity (3/545,6%).After treated by average 9 cycles of chemotherapy , 34 cases ( 63%) achieved complete remission ( CR ) , 13 ( 24.1%) achieved partial remission (PR), 5 (9.3%) presented disease progression (DP), and 2 died (3.7%).Patients were followed up for median 30 months ( 8 -99 months ) , 24 cases died and 30 survived with a overall survival (OS) rate of 55.6%.In 30 survival cases, there were 23 cases (76.7%) of event-free survival (EFS) ,and 6 cases (20.0%) of PR and 1 case (3.3%) of DP.There were significant differences in prognosis among patients with different responses to first therapy (χ2 =8.963, P =0.003 ).Among 20 children with stage 4 NB treated by autologous peripheral blood stem cell transplantation ( APBSCT ) , 13 cases died and 7 survived with an average survival time of (73.55 ±8.89)months.Among 29 cases without APBSCT, 11 cases died and 18 survived with an average survival time of(40.19 ±5.52)months.There was no significant difference in survival between APBSCT and no-APBSCT.Conclusion Children aged 5 or above with NB tend to have advanced stage , relapse and long treatment cycle , but the survival still can be improved with the appropriate treatment .

Objective To investigate the feasibility of using a novel docetaxel-loaded polycaprolactoneTween 80 (PCL-Tween 80) nanoparticles for glioma therapy.Methods Two types of docetaxel-loaded nanoparticles were made from commercial polycaprolactone (PCL) and a self-synthesized PCL-Tween 80 copolymer using a modified solvent extraction/evaporation method.A C6 glioma cell line was used to investigate the uptake of polymeric nanoparticles by brain cancer cells.In vitro cancer cell viability was assessed using MTT toxic assay.Results The nanoparticles were found by FESEM to have a spherical shape and be around 200 nm in diameter.The copolymers could encapsulate 10% of the drug in the nanoparticles and release 34.9% of the encapsulated drug over 28 days.The drug-loaded PCL-Tween 80 nanoparticles showed better in vitro cytotoxicity towards C6 cancer cells than Taxotere at the same drug concentration.Conclusion Nanoparticles using PCL-Tween 80 copolymer as drug delivery vehicles may have a promising outcome for glioma patients.

Objective:To observe the effects of acupuncture on the characteristics of neuro-electrophysiological activity in hippocampal CA1 and CA3 areas of rats with post-traumatic stress disorder (PTSD).Methods:Fifty Sprague-Dawley (SD) rats were randomly divided into a blank group,a model group,a grasping group,a Western medicine group and an acupuncture group,with 10 rats in each group.Except for the blank group,rats in the other 4 groups all received the combined stress modeling method.Rats in the Western medicine group were intragastrically administrated with paroxetine hydrochloride,those in the acupuncture group received acupuncture intervention,those in the grasping group received grasping fixation,and those in the model group and the blank group did not receive any interventions.After 14 d of intervention,the interspike interval (ISI) and power spectral densities (PSD) were analyzed and mapped by in vivo multiple channels to record the neuron clusters discharge in the hippocampal CA1 and CA3 areas.Results:Compared with the blank group,ISI was prolonged in the CA1 and CA3 areas of the model group and the grasping group,and the concentrated PSD distribution area moved down (P＜0.05 or P＜0.01).Compared with the grasping group,the ISI of the CA1 and CA3 areas in the Western medicine group and the acupuncture group was shortened,and the concentrated PSD distribution area moved up (P＜0.05 or P＜0.01).The ISI and PSD distributions in the CA1 and CA3 areas of the acupuncture group were not statistically different from those in the Western medicine group (both P＞0.05).Conclusion:Both acupuncture and paroxetine hydrochloride can significantly regulate the neuro-electrophysiology activity of hippocampal CA1 and CA3 areas in PTSD rats,which may be one of the mechanisms of acupuncture intervention to promote PTSD recovery.

OBJECTIVETo reveal the sequence variations of the full coding region of the human alpha (1,beta/1,4) fucosyltransferase 5 gene (FUT5) in a Chinese Han population.

METHODSThe whole coding region of the FUT5 gene was amplified and sequenced in a total of 30 unrelated Chinese Han individuals. The PCR products containing the nucleotide variants observed in the study were subcloned into plasmid pcDNA to determine all potential haplotypes in the investigated population. Genetic polymorphisms of C560T (rs778970) and C484A loci were further analyzed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RLFP) method.

RESULTSIn addition to seven previously reported base substitutions, two novel polymorphisms, namely C484A (Leu162Met) and T684C, were found in the coding region of the FUT5 gene in the 30 individuals. Seven haplotypes were identified by subcloning the variants into plasmid and subsequent DNA sequencing. The allele frequencies in the rs778970 locus in 160 Chinese Han individuals was 0.3031 for 560C and 0.6969 for 560T, while no polymorphism was detected in the C484A locus.

CONCLUSIONThe sequence of the coding region in the human FUT5 gene demonstrated high genetic diversity, and the allelic distribution of the rs778970 locus in the Chinese populations is polymorphic.

Alleles ; Asian Continental Ancestry Group ; ethnology ; genetics ; Base Sequence ; Fucosyltransferases ; genetics ; Gene Frequency ; Genetic Variation ; genetics ; Haplotypes ; Humans ; Open Reading Frames ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Population Groups ; genetics ; Sequence Analysis, DNA