Objective: To study the effect of breast cancer metastasis suppressor gene 1 (BRMS1) on the migration of human breast cancer cells. Methods: An eukaryotic expression vector containing BRMS1 was constructed and was transfected into human breast cancer MDA-MB-231-HM cells by using Lipofectin 2000. Expression of BRMS1 mRNA was detected by real-time PCR. The migration of the cells was assayed by Transwell test. Meanwhile, three pairs of siRNA targeting BRMS1 were designed. The most effective siRNA was screened by real-time PCR and transfected into MDA-MB-231 cells. The migration ability of the transfected cells was detected. Results: Compared with the cells transfected with empty vector, the number of MDA-MB-231-HM cells on the underlayer of transwell decreased by 51.9% after stably transfected with BRMS1. One pair of siRNA was selected which could notably down-regulated BRMS1 expression at mRNA level. After siRNA transfection, the number of MDA-MB-231 cells on the underlayer of transwell increased by 17.9%. Conclusion: BRMS1 suppresses the migration of breast cancer cells, which indicates that BRMS1 may suppress cancer distant metastasis by inhibiting migration of breast cancer cells.

Objective To assess the harvest method and application value of free-style anterolateral thigh perforator flap (ALTPF) in oral-maxillofacial reconstruction.Methods Fifty-three patients who suffered from oral and maxillofacial tumor underwent simultaneous reconstruction using free-style ALTPFs after radical resection from August,2013 to May,2014.Perforators of the ALTPF region were marked through hand-held Doppler probe preoperatively.Flaps were designed centered on perforators according to the defect size of the accepting site.Incisions were first made at the inner border of the designed flap.Perforators were exposed above the fascia lata femoris and then dissected retrogradely through the vastus lateralis muscle to harvest a vascular pedicle with desired caliber and length.Flap thinning was applied under microscope in some cases to compromise the need of the accepting site.Results All 53 flaps survived after transplantation while skin exfoliation occurred in 5 cases due to flap thinning.Four cases sustained partial necrosis and was cured by trimming and dressing changes.Five to 14 months' post-operative followup showed satisfactory accepting-site morphology with good speech function and swallowing recovery.All donor sites were closed primarily without skin-grafting,leaving no donor-site complications including incision disruption,scar hyperplasia and muscle strength degeneration of the lower limb.Conclusion Multiple perforators have been accu rately located preoperatively in free-style harvest approach of ALTPFs,thus optimal effects can be reached with decreased donor-site morbidity and improved aesthetic outcome to the uttermost,which accords with the refined,personalized and minimal invasive development concept of modem reconstructive surgery.

Objective To observe the effect of autophagy on paclitaxel-induced CaSki cell death through the regulation of the expression of autophagy gene Beclin1, and to explore the interaction and relationship between autophagy and apoptosis. Methods Eukaryotic expression vector pcDNA3.1-Beclin1 and RNA interference vector pSUPER-Beclin1 were transfected into human cervical cancer CaSki cells in vitro and screened for stable expression cell lines. The formation of autophagic vacuoles was observed with an electronic microscope. The expression of Beclin1 and LC3 was measured by Western blot. After being treated with paclitaxel, the change of cell proliferation was assessed by MTT assay, the percentage of apoptotic cells and autophagic cells were analyzed by flow cytometry. Results A lot of autophagic vacuoles were observed in pcDNA3.1-Beclin1 cells by electronic microscopy. Beclin1 and LC3 protein expression was up-regulated in CaSki cells transfected with pcDNA3.1-Beclin1, and was inhibited in cells transfected with pSUPER-Beclin1. MTT assay revealed the survival rate of CaSki cells was significantly decreased after being transfected with pcDNA3.1-Beclin1. After being treated with paclitaxel, the percentages of apoptotic cells and autophagic cells were both increased in pcDNA3.1-Beclin1 group compared with that of the blank control group especially the increase of apoptosis was particularly evident. Conclusion Autophagy and apoptosis have different roles in the process of paclitaxel-induced cervical cancer CaSki cell line death. Overexpression of Beclin1 in CaSki cells may enhance the apoptosis induced by paclitaxel.

AIM:To investigate the effect of angiotensin II type 1 receptor (AT1R)-calcineurin (CaN) signa-ling pathway on the expression of sodium current channel Nav 1.5 at mRNA and protein levels in the hypertrophic ventricu-lar myocytes from neonatal rats .METHODS:The ventricular myocytes were isolated from the ventricles of 1-day-old neo-natal Sprague-Dawley rats and were divided into 4 groups according to different drug intervention as control group , pheny-lephrine (PE) group, losartan (Los)+PE group and cyclosporin A (CsA)+PE group.The method of RNA interference mediated by adenovirus carrying short hairpin RNA ( shRNA) was used to knock down the gene which encodes the beta subtype of CaN A subunit (CnAβ) and the cells were divided into 4 groups as Ad-Null group, Ad-Null+PE group, Ad-CnAβshRNA1 group and Ad-CnAβshRNA1+PE group.The mRNA expression of brain natriuretic peptide ( BNP) ,β-my-osin heavy chain (β-MHC) and Nav1.5 was detected by RT-qPCR.The protein levels of CnAβand Nav1.5 in the whole-cell extracts were determined by Western blot analysis .RESULTS:Treatment of the neonatal rat ventricular myocytes with PE for 24 h increased the protein-to-DNA ratio and the mRNA expression of BNP and β-MHC.The size of the cell surface was also increased after PE treatment .Treatment of the cells with PE increased the protein expression of CnAβ, and re-duced the protein expression of Nav 1.5.Both Los and CsA prevented those effects of PE .The mRNA expression of Nav1.5 was reduced by PE , and no significant difference of Nav 1.5 mRNA expression among PE group , Los+PE group and CsA+PE group was observed .Silencing of CnAβin the neonatal rat ventricular myocytes using Ad-CnAβshRNA1 inhibited the ability of PE to increase the mRNA expression of BNP , and diminished the ability of PE to reduce the protein expression of Nav1.5.CONCLUSION:AT1 R-CaN signaling pathway participates in regulating protein expression of Nav 1.5 in the hy-pertrophic ventricular myocytes from neonatal rats .

OBJECTIVETo explore effects of color Doppler ultrasound and magnetic resonance imaging (MRI) in diagnosis intramuscular hemangioma of skeletal muscle.

METHODSFrom December 2000 to January 2013, 54 patients treated by operation confirmed as intramuscular hemangioma of skeletal muscle by pathology postoperatively, there were 19 males and 35 females aged from 11 to 59 years old (averaged 33.6); the courses of disease ranged from 2.5 to 15 years with an average of 5.2 years. Thirty-eight patients were checked by color Doppler ultrasound, and 14 patients were inspected by MRI. All patients were treated by operation. Postoperative operative time, blood loss in operation, and complications and pathology postoperatively were observed, and IMH clinical effective evaluating standard were used to evaluate clinical outcomes.

RESULTSForty-three patients were followed up from 7 to 49 months with an average of 28.4 months. Operative time was (53 to 187) min with average of 76.3 min, blood loss was (70 to 350) ml with an average of 223.6 ml. No infections and death occurred. Thirty-five patients were diagnosed by color Doppler ultrasound and 13 patients were confirmed by MRI. Twenty patients were capillary type, 22 patients were spongy vascular type and 12 patients were mixed type according to Brown pathological type. In accordance with IMH clinical effective evaluating standard, 29 cases obtained excellent results, 8 moderate and 4 dissatisfaction and 2 poor.

CONCLUSIONColor doppler ultrasound and MRI get a high rate diagnosing patients with intramuscular hemangioma and have an significant valuable in clinical application, and surgical operation which has advantages of relieve symptoms obviously, improve life quality and reduce recurrence rate, could receive good curative effect.

Adolescent ; Adult ; Child ; Female ; Hemangioma ; diagnosis ; pathology ; surgery ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Muscle, Skeletal ; pathology ; Ultrasonography, Doppler, Color

BACKGROUND:Human umbilical vein endothelial cells transfected with lentivirus containing enhanced green fluorescent protein can be easily traced. The optimal multiplicity of infection and time for producing strong fluorescence intensity can lay the foundation of tracing human umbilical vein endothelial cells in animal models.
OBJECTIVE:To observe expression of lentivirus containing enhanced green fluorescent protein in human umbilical vein endothelial cells, and thereby to find a stable method to label human umbilical vein endothelial cells.
METHODS:Using 0.1%col agenase perfusion digestion, we isolated human umbilical vein endothelial cells, which then were placed into a culture medium containing 20%fetal bovine serum and endothelial cellgrowth factor and observed under an inverted microscope. Fol owing digestion, centrifugation and suspension, the cells were counted and divided into four groups, 5.0×105 cells in each group. After cells were seededonto 24-wel plates, 10μL serum-free Dulbecco’s modified Eagle’s medium was added into the blank group, and lentiviruses containing enhanced green fluorescent protein were added into another three groups for celltransfection respectively at multiplicities of infection of 2, 3, 4. There were three dishes in each group.
RESULTS AND CONCLUSION:After cultured for 5-7 days, isolated cells grew into a single layer and exhibited a cobblestone-like arrangement under a light microscope. In addition, factor VIII related antigen test was positive. A green fluorescence was visible at 24 hours of transfection, and peaked at 72 hours. Transfection efficiency was in a linear growth with the multiplicity of infection. Up to the 21st day of transfection, the green fluorescence was stil visible. After 0, 7, 14, 21 days of transfection, the number of human umbilical vein endothelial cells showed no difference between the transfection group with the multiplicity of infection=3 and blank group, suggesting the proliferative ability of cells has no changes after transfection with lentivirus containing enhanced green fluorescent protein. These findings indicate that the lentivirus containing enhanced green fluorescent protein can highly transfect human umbilical vein endothelial cells, and green fluorescent protein can sustainably express for 21 days but cannot impact the cellproliferation.

Objective o evaluate the characteristics and treatments of the medial-extension type of posterior malleolar fractures.Methods Data of 75 patients with posterior malleolar fractures from May 2007 to December 2010were retrospectively analyzed.13 patients whose X-ray showed Cotton fracture while CT scan showed medial-extension type of posterior malleolar fractures were involved in this study.There were 8 males and 5 females,with an average age of 40.3 years old (range,15-75 years).The mechanisms of injuries were as follow:6 patients with falling injury,4 patients from motor vehicle accidents and 3 patients from severe sprain.All the patients combined with distal fibular fracture.The preoperative clinical manifestations included foot and ankle swelling,deformity and restricted movement.The fracture line could be found on coronary X-ray.10 of those patients had double lines sign in medial malleolus.According to Haraguchi CT scan classification system,8 patients were Type Ⅰ fractures (61.6％,8/13),3 with Type Ⅱ fractures (23％,3/13),and 2 with Type Ⅲ fractures (15.4％,2/13).Posterior medial incision,cannulared screws after reduction were conducted.Wound and fracture healing were recorded postoperatively.Function was evaluated according to Baird-Jackson criterion.Results All 13 cases had been followed up for 8-45 months (mean 16 months).Post-operation X-ray showed articular surface displacement was less than 1mm; widening of the medial ankle mortise was no more than lmm; anatomy reduction was achieved or approximately achieved.All cases got union and the union period was 12-20 weeks with an average of 15.1 weeks.The incisions were primary healed in all patients.According to Baird-Jackson criterion,10 cases were excellent and 3 were good.76.9％(10/13) patients got excellent results.No instrument failure,fracture displacement,and infection were found.All patients could walk without accessory appliance.Conclusion Most of medial-extension type of posterior malleolar fractures have articular cartilage damage.It may be caused by rotational force combined with axial load.It needs open reduction and internal fixation early.The posterior medial incision has certain superiority.

Objective: To explore the role of angiotensin receptor type I (AT1)-calcineurin (CaN) signaling pathway in transient outward potassium ion channel (Ito) remolding in hypertrophic atrial myocytes of neonatal rats. Methods: 1 day old neonatal rats’ atrial myocytes were isolated and the cells were divided into 4 groups:①Control group, normal cells were cultured for 24 h,②Stretching group, the cells were cultured for 24 h with mechanical stretching to induce hypertrophy,③Telmisartan group, the cells were treated by telmisartan at 1 μmol/L for 1 h, then cultured for 24 h and ④Cyclosporin-A (CsA) group, the cells were treated by CsA at 0.25 μg/ml for 1 h, then cultured for 24 h. The ratios of protein/DNA in myocytes were compared between Control group and Stretching group, cell hypertrophy was deifned by mRNA expression of atrial natriuretic peptide (ANP). Ito changes were detected by whole-cell patch clamping technique, proteins expressions of Kv4.3 and CaN A subunit were examined by Western blot analysis. Results: Compared with Control group, Stretching group showed obviously decreased Ito density and Kv4.3 protein expression, while increased CaN A protein expression; Compared with Stretching group, the above effects were reduced in Telmisartan group and CsA group. Conclusion: AT1-CaN signaling pathway was involved in the regulation of Ito channel remodeling in hypertrophic atrial myocytes of neonatal rats.

Objective:To establish an HPLC method for the determination of patulin in health foods containing hawthorn to im-prove the quality control of patulin in related health foods. Methods:An Agilent TC-C18(2)(250 mm ×4.6 mm, 5 μm) column was used with the temperature of 30℃. The mobile phase consisted of 0. 8% tetrahydrofuran and the flow rate was 1. 0 ml·min-1 . The detection wavelength was set at 276nm. Results:The calibration curve of patulin was linear within the range of 1-20ng(r=1. 000 0), the average recovery was 92. 1% and RSD was 2. 2%(n=6). Conclusion:The method is simple, reliable and accurate, which can be used in the content determination of patulin in health foods containing hawthorn.

A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.