Objective: To investigate the effect of amiloride on in vitro invasive activity and uPA (urokinase-type plasminogen activator)system of human highly metastatic lung carcinoma cell line PGCL3. Methods: At 6 hours after treatment with amiloride at the concentrations of 25μmol/L，50μmol/L and 100μmol/L for PGCL3 cells，Transwell Chamber assay was performed to detect the effect of amiloride on the invasive and migratory capacity of PGCL3 cells.Effect of amiloride on the activity of uPA and PAI-1(plasminogen activator inhibitor-1)secreted by PGCL3 cells were measured by chromogenic substrate assay after PGCL3 cells were incubated with amiloride for 24 hours.RT-PCR was used to analyze the effect of amilorede on mRNA levels of uPA，uPAR(urokinase-type plasminogen activator receptor)and PAI-1.The expression levels of uPA，ERK2(extracellular regulated protein kinases 2)and ras protein were assessed by Western blot. Results: The number of cells through membrane was significantly decreased in invasion and migration test in vitro.The inhibitory rates of invasion and migration after treatment with amiloride of 100μmol/L were 37.7%±4.1%and 64.9%±4.9%.respectively,with a significant difference from those in the control group(P<0.01).At 24 hours after amiloride treatment，the chromogenic substrate assay showed direct inhibition of the activity of uPA and PAI-1 secreted by PGCL3 cells.No effect on the expression of uPAR in mRNA level was observed,but the expression of PAI-1 in mRNA level was significantly inhibited.Amiloride of 100μmol/L dramatically inhibited the expression of uPA mRNA.The expression level of uPA protein was decreased with the increase of the concentration of amiloride,but no effect was observed on the expression of ERK2 and ras in protein level.Conclusion: Amiloride can inhibit the invasion and migration of PGCL3 cells,through inhibiting the expression and activity of uPA and PAI-1.Amiloride is a potential agent to inhibit cancer invasion and metastasis.

Objective:To investigate the effect of dimethyl amiloride (DMA) on invasive activity of PGCL3 cells from a human highly-metastatic lung carcinoma cell line in vitro and elucidate its possible mechanism. Methods:The invasion and migration capacities of PGCL3 cells were measured by using Transwell chamber assay after pretreatment with DMA. The effects of DMA on the activity of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) secreted by PGCL3 cells were measured by chromogenic substrate assay. The effects of DMA on uPA, urokinase-type plasminogen activator receptor (uPAR), and PAI-1 mRNAs transcription were determined by RT-PCR. The expression levels of extracellular regulated protein kinases 2 (ERK2) and ras protein were assessed by Western blot. Results:DMA inhibited invasion and migration capabilities of PGCL3 cells in vitro, down-regulated the mRNA transcription of uPA, uPAR and PAI-1, as well as up-regulated the expression of ras protein. After 24-hour treatment, DMA reduced the activity of uPA at higher concentration, but DMA had no effects on the activity of secreted PAI-1 protein and expression of ERK2 protein. Conclusion:DMA inhibits the invasion and migration of highly-metastatic lung cancer PGCL3 cells. The mechanism might be associated with down-regulation of the expression of uPA system.

Objective Construct the finite element model of vertebral pedicle screw fixation in thoracolumbar fractures can obtain the intuitive systematic mechanical effects under different motion conditions, it has a very important significance to the patients' rehabilitation, evaluation of system stability and prognosis.In this study, the biomechanical effects of different motion states of thoracic and lumbar spine fracture models were analyzed by the finite element analysis method.Methods We selected 1 orthopedic health volunteers from Nanjing General Hospital of Nanjing Military Region in June 2014, established the finite element model by the CT scan data of the healthy volunteer, used the geometric cutting method for fracture simulation and replacement, assembled posterior orthopedic internal fixation system of vertebral pedicle screw, simulated the different motion states and obtained the stress nephogram and displacement graph of the nail-stick system..Results For the six-nail and two-rod internal fixation system of posterior nail-stick system which set nails through injured vertebrae, we used the finite element operation method to simulate the biomechanical characteristics of different modes of motion in space, it combined by the movement of six directions(flexion, dorsiflexion, left side bend, right side bend, left twist, right twist).The maximum stress(94.99±1.72MPa) and the maximum displacement(0.1854±0.0052mm) and the of the dorsiflexion were significantly lower than that of flexion[(126.82±5.56)MPa、(0.2502±0.0050)mm]、left side bend[(152.18±9.13)Mpa、(0.3172±0.0048)mm]、right side bend[(159.58±13.54)Mpa、(0.3315±0.0051)mm](P<0.05).Conclusion The method of finite element analysis can obtain clear and intuitive biomechanical data, which provides effective evidence for the evaluation of surgical efficacy, the development of postoperative rehabilitation methods and the evaluation of the stability of thoracolumbar spinal system.

Objective:To analyze risk factors that were associated with loss of correction curvature after short-segment restoration and fixation in cases who had single-segment thoracolumbar fracture.Methods:87 Cases who had experienced single-segment thoracolumbar fracture and had underwent short-segment restoration and fixation in our department from Jan 2008 to Jan 2011,and had complete follow-up imaging were included.Cobb angles were measured on lateral thoracolumbar X-ray preoperatively,postoperatively and before removal of internal fixation.And these included the angle formed by vertebras that located above and below injured vertebrae (α angle),superior endplate of injured vertebrae and its superior vertebrae (β angle),inferior endplate of injured vertebrae and its inferior vertebrae (γ angle),inferior and superior endplate of injured vertebrae (δ angle).T-test was used to analyze these angles and their changes.And correlation analysis was used to analyze relationships between α angle change and other risk factors.Results:When compared with preoperative angles,the mean α angle,β angle,γ angle and δ angle were all significantly increased (p＜0.05) after the operation.The mean α angle and δ angle before the removal of internal fixation were both significantly smaller than those after the operation (p＜0.05),and the mean change ofα angle was-2.85 degrees.After the correlation analysis,we found significant correlations between the change ofα angle and postoperative correction curvature(-0.342,p=0.026),injured region in endplate(0.374,p=0.015),and change of the δ angle(0.231,p=0.041).Conclusion:There was significant loss in the correction curvature before the removal of internal fixation.And the loss was significantly associated with postoperative correction curvature,injured region in endplate,and change of the δ angle.

Objective To investigate the clinical efficacy and safety of ultra pulse CO2 laser combined with collagen dressing in treatment of cutaneous amyloidosis.Methods A total of 68 cases of primary cutaneous amyloidosis patients according to the different treatment methods was divided into two groups:the control group (n =33 cases) that was given acitretin capsules oral and topical compound flumetasone ointment packet treatment,and observation group (n =35 cases) with ultra pulse CO2 laser combined with collagen dressing external treatment.Two groups were observed in patients with curative effect and adverse reaction.Results Disease of integral observation group after treatment was significantly lower than the control group(t =3.12,P ＜0.05).The observation group had the efficiency of [88.6％ (31/35)] that was significantly higher than that of control group[17/33(51.5％),x2 =11.23,P ＜.0.01].The observation group had significantly shorter healing time [(5.38 ± 1.81) d] compared to the control group [(10.75 ±2.06)d,t =11.39,P ＜ 0.01].The observation group did not have recurrence,which was significantly different from the control group (x2 =4.90,P ＜ 0.05).Conclusions Ultra pulse CO2 laser combined with collagen dressing in treatment of primary cutaneous amyloidosis is a kind of effective treatment methods with remarkable curative effect and less adverse reactions.It is worthy of clinical application.

Objective To put forward the prevention advice on foreign body suction,and to discuss the effica-cy,safety and application experience of flexible bronchoscopy in the diagnosis and treatment of tracheobronchial foreign bodies in children. Methods The treatment experience of 38 cases for airway foreign body removal with flexible bron-choscopy and granulation tissue proliferation in Argon plasma coagulation ( APC ) ( argon knife ) combining carbon dioxide( CO2 ) cryotherapy in Bayi Children′s Hospital Affiliated to General Hospital of Beijing Military Command from January 2013 to December 2014 were reviewed,and the clinical data including age,gender,treatment time for inhaled foreign body,clinical and X-ray manifestations,location of the foreign body,treatment with bronchoscopy with APC and cryotherapy,complications and outcomes were analyzed. Results There were 38 cases of patients including 31 male (81. 6%) and 7 female(18. 4%),aged from 10 months to 14 years old,with mean age 28. 5 months;among them there were 30 cases with definite history of foreign body,accounting for 78. 9%;the most common clinical symptom was cough among the cases,accounting for 84. 3%;X ray showed 15 cases with ipsilateral lung atelectasis,accounting for 39. 5%, emphysema in 17 cases,accounting for 44. 7%,pneumonia change in 6 cases,accounting for 15. 8%,there were 11 ca-ses who had lung computerized tomography examination when coming to the hospital,and only 1 case could be seen to have foreign body shadow;microscopic examination found that inhaled foreign body in the right bronchus accounted for 57. 9%,and peanut was the main foreign body inhalation in this group;only 4 cases(10. 5%) had definite diagnosis and foreign body removal within 24 h after foreign body aspiration,moreover,34 cases(89. 5%) with foreign body aspi-ration got the diagnosis and treatment after 24 h;crying was the primary inducement for inhaled foreign body. All the 38 cases of children with inhaled foreign body experienced removal under flexible bronchoscopy. There were 19 cases (50. 0%) who had granulation tissue proliferation around the foreign body,among which 5 cases of foreign body was wrapped by the proliferation of granulation tissue,with APC dealing with the granulation tissue of foreign body surface to remove foreign body after exposure,then giving CO2 cryotherapy. Nevertheless,there were 9 cases of foreign body who had granulation tissue but was not wrapped,receiving CO2 cryotherapy directly after the foreign body removal. One case of this group had bradycardia during the surgery,and 2 cases had postoperative bleeding,but there was no death cases with foreign bodies removal. Conclusions Education is the key to prevent foreign body aspiration in infants under 3 years old. Flexible bronchoscopy is safe to remove foreign bodies from the respiratory tract and has fewer complications, so it is one of the alternative methods in diagnosis and treatment of foreign body inhalation.

Objective To explore the operating methods,the effectiveness and safety for 2 cases of tracheal in-tubation related severe subglottic stenosis under laryngeal mask by using bronchoscopy in the diagnosis and interven-tional treatment. Methods Two male patients ( age at 4 and 11 months) both had difficulty breathing and weaning failure after endotracheal intubation,who were diagnosed with severe subglottic stenosis through CT and bronchoscopy examination. All parents signed their consent after being fully informed of treatment risks. Two cases were treated under bronchoscopic holmium laser combined with argon plasma coagulation and freezing treatment by using the laryngeal mask. The clinical data, complications and postoperative outcome and follow-up were analyzed. Results Two cases were operated successfully,the subglottic granulation tissue of 1 case was completely removed,2 cases of patients had subglottic narrow annular completely removed, and there was no problem for 4. 0 mm bronchoscope to get through,postoperative tracheal catheter was successfully extracted,dyspnea was significantly improved. Two cases imme-diately showed well open after treatment,7 d,30 d and 90 d through endoscopic examination. During the operation,oxy-gen saturation decreased and heart rate increased fast or slowed down,but after operation and oxygen got resumed,they immediately recovered,and there were no abnormal hemodynamic changes during and after operation. The two cases were still under follow-up. Conclusions The laryngeal mask holmium laser,argon plasma coagulation and cryotherapy can be used to remove acquired granulation tissue hyperplasia caused by subglottic stenosis,which is safe and effective, and the short-term effect was remarkable. Long-term effect still needs to be further assessed through follow-up.

Aim To establish a hepatic carcinoma cell line with stable voltage-gated chloride channel 3 ( ClC-3 ) gene silencing through the lentivirus-mediated short-hairpin RNA ( shRNA ) method and investigate the effects of gene silencing on invasion and migration. Methods Three lentiviral vectors coding shRNA tar-geting ClC-3 gene were constructed, the recombinant plasmids were packaged into mature lentivirus by 293FT cells, and then the lentiviruses were harvested, concentrated and titrated. MHCC97H cells were infec-ted with the recombinant lentiviruses and then were se-lected to obtain cell lines stably expressing ClC-3 shR-NA. The efficiency of ClC-3 mRNA and protein ex-pression interference were determined by real-time flu-orescence quantitative PCR and Western blot, respec-tively. The effects of ClC-3 gene interference on inva-sion and migration of MHCC97 H cells were performed by Transwell chamber assays with or without Matrigel and cell scratch assay. Results The recombinant lentiviral vectors were successfully constructed and four lentiviruses were acquired after packaged by 293 FT cells. One negative control cell line and three cell lines with ClC-3 gene interference ( MHCC97 H/shClC-3-1 , shClC-3-2 and shClC-3-3 ) were successfully construc-ted after MHCC97 H cells were infected with lentivirus-es. The expression level of ClC-3 mRNA and protein in three ClC-3-silenced cells were obviously lower than the negative control cells ( P <0. 01 ) , MHCC97 H/shClC-3-2 cells showed the greatest inhibition of ClC-3 mRNA and protein expressions. As compared with the negative control cells, the ClC-3 gene interference sig-nificantly decreased invasion and migration of MH-CC97 H cells in vitro ( P <0. 01 ) . Conclusion The hepatic carcinoma cell lines with stable ClC-3 gene si-lencing were successfully established and the ClC-3 gene interference could significantly inhibit invasion and migration of MHCC97H cells.

Objective To investigate the relationship of TCM constitution and syndrome elements based on patients with hyperhomocysteinemia. Methods A survey was conducted among 1316 patients with diabetes, coronary disease, hypertension and cerebral infarction. Totally 326 patients with hyperhomocysteinemia were incorporated in diagnostic data and their constitutions were determined. At the same time, syndrome elements identification method for syndrome elements was applied for judgment, and consistency of the constitution and syndrome elements was analyzed. Results The survey showed that prevalence rate of population with hyperhomocysteinemia was 24.77%;there were 6 cases with mild nature, and 320 cases with biased constitution;phlegm dampness, blood stasis, qi deficiency of constitutions 110 cases, 73 cases and 59 cases, respectively;blood stasis, phlegm, qi deficiency, and dampness were the main syndrome elements of hyperhomocysteinemia, of 100 cases, 98 cases, 73 cases and 59 cases, respectively;the consistency of phlegm dampness constitution and syndromes of phlegm was the most obvious (Kappa=0.89, P<0.05). Conclusion Phlegm dampness, blood stasis, and qi deficiency were the most common constitutions of hyperhomocysteinemia;blood stasis, phlegm, qi deficiency and dampness were the main syndrome elements of hyperhomocysteinemia patients. The two results were consistent, and the consistency of phlegm syndrome and phlegm dampness constitution was the most obvious.

Objective To analyze the clinical effects of individualized comprehensive physiotherapy interventions for temporomandibular disorders (TMD).Methods Data of 307 patients with TMD treated between April 2011 and March 2012 in the authors' department were collected and reviewed.All the patients were treated with individualized comprehensive physiotherapy approach based on the patient's category in research diagnostic criteria for TMD (RDC/TMD),such as patient education,ultrashort-wave diathermy,ultrasound therapy,soft tissue massage,joint mobilization and stabilization exercise.The treatment was administered for 3 weeks.The baseline and endpoint outcome assessment measures were maximum active mouth opening (mm),visual analogue scale (VAS) score and joint clicking (100％ before treatment).Results At the end of treatment,the patient's maximum active mouth opening [(36.95 ± 6.59) mm],VAS score (1.21 ± 0.62) and joint clicking [(29 ± 17) ％] improved significantly (P ＜ 0.05) compared to baseline.Conclusions Individualized comprehensive physiotherapy interventions can improve symptoms of TMD,such as joint clicking,pain,limited mouth opening.