Objective To construct the eukaryotic plasmid expression vector mediated short hairpin RNA(shRNA) interference targeting TLR4 gene,and transfect it into pancreatic adenocarcinoma cell line PANC1,then screen stably transfected clonal cell line.Methods Three shRNA interference expression plasmid vectors targeting the TLR4 gene were constructed,named TLR4-1,TLR4-2,TLR4-3.The shRNA plasmid with highest inhibitory efficiency was selected and transfected into PANC1 cells with liposome.The silencing efficiency and transfection efficiency of TLR4-shRNA was assayed with real-time quantitative PCR and flow cytometry analysis.Monoclonal cell with stable transfection of TLR4-shRNA were selected by geneticin 418 (C418) and limiting dilution analysis.Results Transient transfection efficiency of PANC1 was (46.72 ±5.06) ％.TLR4 mRNA expressions were 0.025 ± 0.004,0.027 ± 0.003,0.019 ± 0.006in cells transfected with TLR4-1,TLR4-2,TLR4-3,respectively,which were significantly lower than that in untransfected group (0.061 ±0.018) and negative control group (0.057 ±0.015,P ＜0.05).The transfection efficiency of TLR4-3 vector in stably transfected clones [(82.79 ±8.16)％] was significantly higher than that of transient transfection (P =0.001 ).The expression of TLR4 mRNA was decreased to 0.010 ± 0.002,which was significantly lower than that of transient transfection ( P =0.001 ).The expression of TLR4 protein was (0.54±0.32) ％,which was significantly lower than that of untransfected cells [( 87.42 ± 5.00 ) ％] and that of negative control [(82.9±5.00)％,P =0.000].Conclusions Stable transfection PANC1 cell lines with TLR4 gene silencing are successfully identified.

ObjectiveTo evaluate and compare long-term clinical outcomes of arthroscopic anterior cruciate ligament (ACL) reconstruction with remnant preservation versus remnant resection.MethodsFrom October 1999 to May 2005,standard ACL reconstruction with autogenous quadrupled hamstring tendons under arthroscopy using remnant resection with transtibial techniques was conducted in 87 patients.From June 2005 to May 2010,ACL reconstruction with autogenous quadrupled hamstring tendons using outside-in bone tunnel establishment and remnant preservation was performed in 221 patients.Only the patients who underwent simple ACL reconstruction together with more than three years follow-up records were enrolled in the study,including 66 patients in remnant preservation group and 39in remnant resection group.ResultsPatients of both groups were all followed up for 36-60 months,which showed insignificant difference between groups ( P ＞ 0.05).Compared with the preoperative data,the latest follow up data demonstrated satisfactory outcomes in both groups.The two groups showed no significant differences regarding the preoperative data including age and gender distribution,average time from injury to surgery,joint instability extent and functional score of the affected knee ( P ＞ 0.05 ).There was no statistical difference in regards of the thigh muscle atrophy and Lachman test between groups ( P ＞0.05).However,the remnant preservation group was superior to the remnant resection group in aspects of the range of motion of the affected knee,Lysholm score,stability in anterior drawer test,stability in pivot shift test and international knee documentation committee (IKDC) grade ( all P ＜ 0.05 ). ConclusionCompared with standard ACL reconstruction using remnant resection with transtibial techniques,the ACL reconstruction using outside-in bone tunnel establishment and remnant preservation has better longterm clinical outcomes including stability and function of the affected knee.

Objective To evaluate the clinical application of multi-slice spiral CT(MSCT)for foreign body of trachea or bronchus in children.Methods Twenty cases suspected foreign body were examined with MSCT.The post-processing reconstruction included multiple planar reconstruction(MPR),CT virtual bronchoscopy(CTVB),shaded surface display(SSD),minimum intensity projection(MinIP),and volume rendering(VR).Results The foreign bodies located at trachea in 1 case and at bronchia in 19 cases.The foreign bodies included 18 fragments of peanut and melon seeds,one shrimp and one plastic-tube.All cases were confirmed by fiberoptic bronchoscopy or course of diseases.The position and form of foreign bodies,as well as the relationship between foreign bodies and bronchial muous membrane could be showed by MPR,CTVB,SSD,MinIP and VR.Conclusion MSCT scanning is a significant noninvasive and sensitive diagnostic technique in detecting the foreign body in trachea or bronchus.

BACKGROUND: Presently, it is a certain preventive effect of local emplacement of various isolated materials during operation and postoperatively drug-controlled inflammatory reaction on adhesion between epidural and nerve root after laminectomy.OBJECTIVE: To study the preventive effect of chitosan and titanium net on epidural cicatricle adhesion after laminectomy.DESIGN, TIME AND SETTING: Randomized controlled animal study, which was performed in Guiyang Medical College from June to December 2005.MATERIALS: Eighty adult rabbits, weighing (2.0±0.2) kg, were used to establish vertebral plate resection models. Chitosan was provided by Shanghai Qisheng Biological Agent Industry Company, and titanium net by Guizhou Kelun Pharmacology Company Limited.METHODS: Eighty adult rabbits were randomly divided into four groups, including control group (0.5 mL saline), titanium net group (titanium net in the size of 1.0 cm × 0.8 cm was covered on defect region of vertebral plate and fixed on processus spinosus of upper and lower cone, bilateral muscles and soft tissues), chitosan group (2 mL chitosan), and chitosan + titanium net group (combination of chitosan and titanium net). Six rabbits were sacrificed in the 2nd and 4th weeks postoperatively, and eight rabbits were chosen as the samples in the 8th week.MAIN OUTCOME MEASURES: Morphological changes of cicatricle.RESULTS: Cicatricle was clearer in the control group. Titanium net could relieve peripheral cicatricle of dura mater and effectively isolate peripheral cicatricial tissue and spinal dura meter. Chitosan could remarkably prevent from cicatricle adhesion and inhibit inflammatory reaction. The combination of chitosan and titanium net could effectively inhibit proliferation of cicatricle surrounding dura meter.CONCLUSION: Association of chitosan and titanium net can effectively inhibit cicatricle adhesion surrounding dura meter after laminectomy.

The genetically modified mice , as a helpful model , have been widely used in life scientific research . However, several new issues appeared subsequently with the wide application of the genetically modified mice .Here, we mainly discussed and analyzed the problems in the management and usage of genetically modified mice , which underlies the foundation for establishing management practice of the genetically modified mice .

Objective To investigate the inhibitory effects of trichostatin A(TSA)on bladder cancer cell lines and its synergetic effect with anticancer drugs in treating bladder cancer in vitro and in vivo.Methods The inhibitory effects of TSA on human bladder cancer cell lines in vitro were detected by MTT assay.Hoechst staining was used to observe morphology for apoptotic cells after TSA treatment.Western blot was used to detect expression of acetyl-histone H3 and survivin.In vivo synergetic effects of TSA with anticancer drugs were detected in bladder cancer model rats.Results TSA significantly inhibited growth of bladder cancer cell lines in concentration and time dependent manner.Better results of tumor inhibition have been achieved when it was combined with DDP,MMC and ADM than used alone.After TSA treatment,the survivin expression in bladder cancer cells decreased and acetyl-histone H3 expression increased.Intravesical application of TSA combined with MMC can significantly inhibited tumor growth and progression.Conclusion TSA has direct anti-cancer effect and can enhance the action of several chemotherapy agents markedly.TSA may be an excellent candidate agent for intravesical application to treat bladder cancer.

OBJECTIVETo observe the clinical effect of chronic fatigue syndrome (CFS) treated with moxibustion at Gaohuang (BL 43).

METHODSWith stratified block randomization, 72 patients accorded with inclusive criteria were divided into a moxibustion at Gaohuang (BL 43) group (moxibustion group) and an acupuncture group, 36 cases in each one. In the moxibustion group, Gaohuang (BL 43) was treated with big moxa cones as the main acupoint, 10 cones a time; Qihai (CV 6) and Zusanli (ST 36) were added with big moxa cones, 7 cones a time. In the acupuncture group, acupoints were the same as those in the moxibustion group, and twirling reinforcing method was used after qi arriving, 60 times one minute and 360° with range. In the two groups, 10-day treatment was made into one course and there were two days between courses. The treatment was given once a day for 3 courses. Changes of fatigue assessment index (FAI) before and after treatment and clinical effects were observed.

RESULTSThe total effective rate was 88.9% (32/36) in the moxibustion group, which was better than 72.2% (26/36) in the acupuncture group apparently (P < 0.05). After treatment in the two groups, FAI scores were obviously declined compared with those before treatment (both P < 0.01) and FAI score in the moxibustion group was apparently lower than that in the acupuncture group (P < 0.05).

CONCLUSIONMoxibustion at Gaohuang (BL 43) can improve the FAI score of patients with CFS and the clinical efficacy is definite.

Acupuncture Points ; Adult ; Fatigue Syndrome, Chronic ; therapy ; Female ; Humans ; Male ; Middle Aged ; Moxibustion ; Treatment Outcome ; Young Adult

Background and purpose: MicroRNAs (miRNAs) play an important role in tumor biological behavior. miRNAs are down-regulated or up-regulated in various cancer types, triggering abnormal cell differentiation, proliferation and apoptosis. This study was designed to investigate the expression and clinical signiifcance of miR-187*in colorectal cancer (CRC), and further to investigate its roles in promoting cell apoptosis. Methods:The expressions of miR-187* in 40 CRC cases were examined by real-time quantitative reverse transcription-PCR (qRT-PCR). The relationship between miR-187*expression and clinical features of CRC was analyzed. HCT116 cells were transfected with a miR-187*mimic and the apoptosis of the transfected cells were examined by lfow cytometry (FCM). Results:The expression of miR-187*was down-regulated in CRC tissues 0.165 (0.106, 0.428) compared with those in normal tissues 0.334 (0.211, 0.712) (P<0.05), especially in mucinous carcinoma and older age CRC (P<0.05). Transfection of HCT116 cells with a miR-187*mimic up-regulated the expression of miR-187*and increased cell early apoptosis (P<0.05). Conclusion: The expression level of miR-187* was lower in CRC. miR-187* expression correlates with histological type and age. Transfection of HCT116 cells with a miR-187*mimic accelerates apoptosis of tumor cells, suggesting that miR-187*is a potent tumor suppressor.

BACKGROUND:The bone morphogenetic protein 2 (BMP2)/vascular endothelial growth factor (VEGF) dual gene activated nanobone putty has been constructed in the previous experiments.
OBJECTIVE:To investigate the effects of osteogenesis and osteogenic gene expression in mice by implanting BMP2/VEGF dual gene activated nanobone putty.
METHODS:Twenty-four Kunming mice (48 sides) were randomly divided into four groups. Animals in each group (12 samples) were injected different materials into the right thigh muscle pouches:nanobone putty+hBMP2/VEGF plasmid;nanobone putty+hBMP2 plasmid;blank plasmid+nanobone putty;nanobone putty only. The effects of osteogenesis were evaluated by radiography, histology and molecular biology analysis in 2, 4 weeks after operation.
RESULTS AND CONCLUSION:Bone-like tissues were observed in groups of nanobone putty+hBMP2/VEGF plasmid and nanobone putty+hBMP2 plasmid after operation. There was apparent BMP2 and VEGF mRNA expression in group of nanobone putty+hBMP2/VEGF plasmid. Group of nanobone putty+hBMP2/VEGF plasmid was significantly better than group of nanobone putty+hBMP2 plasmid in the alkaline phosphatase levels, the speed of osteogenesisas and amount of new bone (P<0.05). Groups of blank plasmid+nanobone putty and nanobone putty had no obvious osteogenesis performance. Either BMP2/VEGF dual gene activated nanobone putty or BMP2 gene activated nanobone putty had the osteogenic ability in vivo. And the former was significantly enhanced in the speed and quality of osteogenesis.

Objective To investigate the therapeutic effects and mechanism of Artemisia argyi ferment substance on systemic Candida albicans infection. Methods The model of systemic Candida albicans infection was established in immunosuppressed mice. The model mice were randomly divided into the model control,Artemisia argyi ferment substance( AAFS) at different doses(100,200,and 400 mg·kg-1 )and fluconazole group(20 mg·kg-1 ),30 mice in each. Mice in each treatment group were given therapeutic drugs by gavage for 5 consecutive days,twice daily. The survival of mice was determined 21 days after the model was set up. The serum levels of IFN-γand IL-2 were determined by ELISA. The proliferation activity of T lymphocyte in the spleen was detected by MTT assay. The number of living fungi in liver and kidney tissues was counted. Results Compared with the model control,AAFS at middle and high doses and fluconazole significantly increased the survival rate of mice,the serum levels of IFN-γand IL-2,and the proliferation activity of T lymphocyte in the spleen,but decreased the number of living fungi in tissues(P〈0. 01). Compared with low dose AAFS,middle and high doses of AAFS and fluconazole showed significantly different effect on each index(P〈0. 05 or P〈0. 01),but there was no difference among these groups(P〉0. 05). Conclusion AAFS at 200-400 mg·kg-1 has inhibitory effects on systemic Candida albicans infection in mice,the mechanism of which is related to increasing the proliferation of T lymphocyte in spleen and the levels of IFN-γand IL-2 in serum.