Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.

OBJECTIVETo establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress.

METHODSA model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim.

RESULTSTreatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too.

CONCLUSIONSThe lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Bcl-2-Like Protein 11 ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Endoplasmic Reticulum Stress ; drug effects ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; genetics ; metabolism ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Melanoma ; genetics ; metabolism ; pathology ; Membrane Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transcription Factor CHOP ; genetics ; metabolism ; Tunicamycin ; pharmacology

OBJECTIVE: To find the mutation of disease-causing genes of sudden unexplained death syndrome (SUDS) in the young by whole exome sequencing in one case. METHODS: One SUDS case was found no obvious fatal pathological changes after conventional autopsy and pathological examination. The whole exome sequencing was performed with the Ion Torrent PGM™ System with hg19 as reference sequence for sequencing data. The functions of mutations were analyzed by PhyloP, PolyPhen2 and SIFT. A three-step bioinformatics filtering procedure was carried out to identify possible significative single nucleotide variation (SNV), which was missense mutation with allele frequency < 1% of myocardial cell. RESULTS: Four rare suspicious pathogenic SNV were identified. Combined with the analysis of conventional autopsy and pathological examination, the mutation MYOM2 (8_2054058_G/A) was assessed as high-risk deleterious mutation by PolyPhen2 and SIFT, respectively. CONCLUSION Based on the second generation sequencing technology, analysis of whole exome sequencing can be a new method for the death cause investigation of SUDS. The gene MYOM2 is a new candidate SUDS pathogenic gene for mechanism research.
Autopsy ; Brugada Syndrome/genetics* ; Cause of Death ; DNA Mutational Analysis/methods* ; Death, Sudden/etiology* ; Exome ; Gene Frequency ; Genetic Testing/methods* ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Molecular Biology ; Molecular Diagnostic Techniques/methods* ; Molecular Sequence Data ; Mutation

BACKGROUNDThe mechanisms of pathological retinal neovascularization (RNV) remain unknown. Several microRNAs were reported to be involved in the process of RNV. Oxygen-induced retinopathy (OIR) is a useful model to investigate RNV. Our present work explored the expression and the role of microRNA-128 (miR-218) in oxygen-induced RNV.

METHODSOIR was used to establish RNV model. The expression level of miR-218 in the retina from OIR mice was assessed by quantitative real-time reverse transcriptase polymerase chain reaction. Fluorescein angiography was performed in retinae of OIR mice, and RNV was quantified by hematoxylin and eosin staining to evaluate the effect of pCDH-CMV-miR-218 intravitreal injection on RNV in OIR mice. Roundabout 1 (Robo1) expression was detected by Western blotting in mouse retinal vascular endothelial cells expressing a high or low level of miR-218 and retinal tissues from OIR mice. Cell migration was evaluated by scratch wound assay.

RESULTSIn OIR mice, the expression level of miR-218 was significantly down-regulated (P = 0.006). Retinal Robo1 expression was significantly increased at both mRNA and protein levels (P = 0.001, 0.008; respectively). miR-218 intravitreal injection inhibited retinal angiogenesis in OIR mice, and the restoration of miR-218 in retina led to down-regulation of Robo1.

CONCLUSIONSOur experiments showed that restoration of miR-218 inhibited retinal angiogenesis via targeting Robo1. MiR-218 contributed to the inhibition of retinal angiogenesis and miR-218 might be a new therapeutic target for preventing RNV.

Animals ; Cell Movement ; Cells, Cultured ; Mice ; Mice, Inbred C57BL ; MicroRNAs ; physiology ; Nerve Tissue Proteins ; physiology ; Oxygen ; pharmacology ; Receptors, Immunologic ; physiology ; Retinal Neovascularization ; prevention & control

OBJECTIVETo investigate the effects of poly-lactic-co-glycolic acid (PLGA) knitted mesh/collagen-chitosan hybrid scaffold (PCCS) on angiogenesis, and to explore the relative mechanisms.

METHODSPLGA knitted mesh was integrated into collagen-chitosan scaffold (CCS) to construct PCCS with freeze-lyophilizing method, and CCS was made with the same method. The characteristics of morphology and water absorbing capacity among PCCS, PLGA knitted mesh, and CCS were compared in vitro. PCCS and CCS was respectively implanted into subcutaneous tissue of back on both sides in 24 SD rats, and the tissue specimens were harvested at post operation week (POW) 1, 2, and 4 according to the random number table to evaluate the level of angiogenesis by histopathological and immunohistochemical examinations. The expression levels of alpha smooth muscle actin (alpha-SMA) and vascular endothelial growth factor (VEGF) mRNA were examined by real-time quantitative RT-PCR. Data were processed with t test.

RESULTS(1) PLGA knitted mesh was closely integrated with sponge of collagen-chitosan in PCCS, and the porous structure of PCCS was similar to that of CCS. (2) Compared with that of PCCS [(506 +/- 15)%], the water absorbing rate of CCS and PLGA knitted mesh was respectively increased and decreased [(627 +/- 21)%, (195 +/- 15)%, with t value respectively 3.8, 11.9, P < 0.05 or P < 0.001]. (3) The scaffolds were filled with newly formed tissue in CCS at POW 4, while those in PCCS were observed at POW 2 with more homogeneous and abundant collagen. (4) Blood vessels could be induced, and they grew into scaffolds along with prolongation of implantation time in PCCS and CCS. The number of mature blood vessels in PCCS at POW 1, 2, 4 [(10.7 +/- 3.2), (18.6 +/- 2.1), and (30.3 +/- 4.5) branches per square centimeter] was respectively higher than that in CCS [(5.4 +/- 0.9), (10.8 +/- 4.2), and (23.6 +/- 1.7) branches per square centimeter, with t value respectively 4.6, 4.4, 4.5, P values all below 0.01]. (5) The expression levels of alpha-SMA and VEGF mRNA in PCCS at POW 1, 2, 4 were significantly higher than those in the CCS (with t(alpha-SMA) value respectively 1.26, 1.63, 2.17, with t(VEGF) value respectively 5.52, 2.07, 1.78, P values all below 0.01).

CONCLUSIONSPCCS is able to induce the ingrowth of blood vessels rapidly and promote their maturity. The mechanical properties and microstructures of scaffolds play synergistic role in the process of angiogenesis.

Animals ; Biocompatible Materials ; Chitosan ; pharmacology ; Collagen ; pharmacology ; Lactic Acid ; pharmacology ; Male ; Materials Testing ; Neovascularization, Physiologic ; drug effects ; Polyglycolic Acid ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo design and construct a kind of dermal regeneration template with mesh, and to preliminarily evaluate its biological characteristics.

METHODSPLGA mesh was integrated into CCS with freeze-drying method for constructing PLGA mesh/CCS composite (PCCS). The micromorphologies and mechanical properties among PLGA mesh, CCS, and PCCS were compared. PCCS and CCS was respectively implanted into subcutaneous tissue of SD rats (PCCS and CCS groups, 9 rats in each group). The tissue samples were collected at post operation week (POW) 1, 2, and 4 for histopathological and immunohistochemical observation. Protein levels of CD68, MPO, IL-1beta, IL-10 were examined by Western blot, with expression of gray value. Data were processed with one-way analysis of variance and t test.

RESULTSThree-dimensional porous structure of PCCS was similar to that of CCS. Mechanical property of PLGA mesh and PCCS was respectively (3.07 +/- 0.10), (3.26 +/- 0.15) MPa, and they were higher than that of CCS [(0.42 +/- 0.21) MPa, F = 592.3, P < 0.0001)]. The scaffolds were filled with newly formed tissue in PCCS group at POW 2, while those in CCS group were observed at POW 4. A large accumulation of macrophages was observed in both groups, especially at POW 2, and more macrophage infiltration was observed in CCS group. The protein level of IL-10 in PCCS group at POW 2 was obviously higher than that in CCS group, while the protein levels of CD68, MPO, IL-1beta were significantly decreased as compared with those in CCS group (with t value from -4.06 to 2.89, P < 0.05 or P < 0.01).

CONCLUSIONSPCCS has excellent mechanical property with appropriate three-dimensional porous structure. Meanwhile, it can rapidly induce formation of new tissue and vascularization, and it has a prospect of serving as a dermal substitute.

Animals ; Cells, Cultured ; Chitosan ; chemistry ; Collagen ; chemistry ; Extracellular Matrix ; chemistry ; Lactic Acid ; chemistry ; Male ; Materials Testing ; Polyglycolic Acid ; chemistry ; Prosthesis Design ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Skin, Artificial ; Tissue Engineering ; methods ; Tissue Scaffolds

BACKGROUND AND OBJECTIVERadiotherapy is effective in treating nasopharyngeal carcinoma (NPC). This study evaluated the treatment efficacy, toxicity, and prognostic factors of intensity-modulated radiotherapy (IMRT) in the treatment NPC.

METHODSBetween September 2003 and September 2006, 305 patients with NPC were treated with IMRT in Fujian Provincial Cancer Hospital. IMRT was delivered as follows: gross tumor volume (GTV) received 66.0-69.8 Gy in 30-33 fractions, high-risk clinical target volume (CTV-1) received 60.0-66.65 Gy, low-risk clinical target volume (CTV-2) and clinical target volume of cervical lymph node regions (CTV-N) received 54.0-55.8 Gy. Patients with stages III or IV disease also received cisplatin-based chemotherapy. All patients were assessed for local-regional control, survival, and toxicity.

RESULTSWith a median follow-up of 35 months (range, 5-61 months), there were 16, 8, and 39 patients who had developed local, regional, and distant recurrence, respectively. The 3-year rates of local control, regional control, metastasis-free survival, disease-free survival, and overall survival were 94.3%, 97.7%, 86.1%, 80.3%, and 89.1%, respectively. Multivariate analyses revealed that T-classification had no predictive value for local control and survival, whereas N-classification was a significant prognostic factor for overall survival (P < 0.001), metastasis-free survival (P < 0.001), and disease-free survival (P = 0.003). For stages III-IV disease, concurrent and adjuvant chemotherapy did not influence prognosis. The most severe acute toxicities included Grade III mucositis in 14 patients (4.6%), Grade III skin desquamation in 90 (29.5%), and Grades III-IV leucocytopenia in 20 (6.5%). There were 7% patients with Grade II xerostomia after 2 years of IMRT, no Grades 3 or 4 xerostomia was detected.

CONCLUSIONSIMRT provided favorable locoregional control and survival rates for patients with NPC, even in those with locally advanced disease. The acute and late toxicities were acceptable. N-classification was the main factor of prognosis. Further study is needed on chemotherapy for patients with NPC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Cisplatin ; administration & dosage ; Combined Modality Therapy ; Disease-Free Survival ; Female ; Follow-Up Studies ; Humans ; Leukopenia ; etiology ; Lymphatic Metastasis ; Male ; Middle Aged ; Mucositis ; etiology ; Nasopharyngeal Neoplasms ; drug therapy ; pathology ; radiotherapy ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Radiotherapy Dosage ; Radiotherapy, Intensity-Modulated ; adverse effects ; methods ; Retrospective Studies ; Survival Rate ; Xerostomia ; etiology ; Young Adult

OBJECTIVETo investigate the therapeutic effect of temporal-zygomatic expanded flaps pedicled with orbicularis oculi muscle (00M) for sub-orbital defects.

METHODS16 cases with sub-orbital defects were treated. The expanders were implanted at temporal-zygomatic region at the first stage. At the second stage, temporal-zygomatic expanded flaps pedicled with OOM were designed and transferred to repair the suborbital defects.

RESULTSAll the expanded flaps survived completely. 11 cases were followed up for 3 months to 3 years. The flaps had no contracture with a good match of color and texture with surrounding tissue. The scar was also inconspicious.

CONCLUSIONSThe temporal-zygomatic expanded flaps can be used for large size defects below orbit. It is very flexible, leaving minimal morbidity for the whole face.

Adolescent ; Adult ; Child ; Facial Injuries ; surgery ; Facial Muscles ; surgery ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Skin ; injuries ; Surgical Flaps ; Tissue Expansion ; Treatment Outcome ; Young Adult

BACKGROUNDThe mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).

METHODSIn vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified.

RESULTSIn vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals.

CONCLUSIONSVEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

Animals ; Eye Proteins ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factors ; analysis ; RNA, Small Interfering ; genetics ; Retinal Neovascularization ; therapy ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; physiology

OBJECTIVETo investigate the efficacy and safety of percutaneous radiofrequency perforation and valvuloplasty in infants with pulmonary atresia with intact ventricular septum (PA/IVS).

METHODSFour infants (body weight 4 - 10 kg) aged 11 months, 9 months, 12 days and 9 months old, respectively, were hospitalized for dyspnea and cyanosis. All patients had a continuous murmur in the left second intercostal space. Doppler echocardiogram showed membranous pulmonary atresia with intact ventricular septum. Right ventriculogram showed a tripartite right ventricle, vasiform infundibulum, and membranous pulmonary valve atresia without ventriculocoronary connections. Descending thoracic aortogram showed good-sized confluent pulmonary arteries being filled from a ductus arteriosus. All the patients were taken up for radiofrequency perforation followed by a balloon dilatation. A 6F Judkins right coronary guiding catheter was positioned in the right ventricular outflow tract and under the atretic pulmonary valve membrane. The radiofrequency perforation catheter along with coaxial injectable catheter was then passed through the right coronary guiding catheter, using it as the guide to the imperforate membrane. The proximal end of the radiofrequency perforation catheter was then connected to radiofrequency generator. After the cusps of pulmonary valve were perforated, the coaxial injectable catheter was moved into the main pulmonary artery. A tiny floppy-tipped coronary guidewire was then passed through the coaxial injectable catheter into the main pulmonary artery and directed through the patent ductus arteriosus into the descending thoracic aorta or directed into pulmonary arteriola. Thereafter, serial balloon dilation catheters were introduced across the pulmonary valve, and dilations were sequentially performed with increasing balloon diameters. The balloon was dilated until the concave of the balloons disappeared. The radiofrequency energy (5 to 8 W) was delivered for 2 to 5 seconds once, but commonly twice, to perforate the valves. After a predilation with a 3 mm x 20 mm to 5 mm x 20 mm balloon at 6 - 14 atm pressure, the valve was subsequently dilated with 10 mm x 30 mm to 14 mm x 30 mm balloon once or twice. The duration of procedures was 120 to 150 min and exposure time was 25.4 to 43.9 min.

RESULTSThe primary procedure was successful in all the infants except one who died early of cardiac perforation with tamponade. After a follow-up period ranging from 2 to 8 months (mean 4.3 m), the remaining 3 survivors achieved complete biventricular circulation. Two of them were awaiting occlusion of the patent ductus arteriosus and 1 needed right ventricular outflow tract reconstruction because of infundibular obstruction.

CONCLUSIONPA/IVS consists of 0.7% to 3.1% of congenital heart defects. 85% of the untreated patients die within half a year. Surgical repair for the infants with PA/IVS is associated with a high mortality. In carefully selected patients with PA/IVS, radiofrequency perforation and balloon dilatation of the pulmonary valve is feasible and may represent a new alternative to surgery due to its low mortality and avoidance of cardiopulmonary bypass.

Balloon Occlusion ; Catheter Ablation ; methods ; Catheterization ; methods ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Pulmonary Atresia ; physiopathology ; therapy ; Pulmonary Valve ; surgery ; Ventricular Septum