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.

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 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.

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 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 compare the difference in the efficacy on low back pain of cold-damp pattern between electric-thermal Bian-stone therapy and moxibustion box therapy.

METHODSForty-one cases of low back pain of cold-damp pattern were randomized into an electric-thermal Bian-stone therapy group (group A, 26 cases) and a box moxibustion therapy group (group B, 15 cases). In the group A, the electric-thermal Bian-stone was placed over Shenshu (BL 23) and Weizhong (BL 40). The temperature of stone was adjusted in accordance with patient's comfort. In the group B, moxibustion box was used over Shenshu (BL 23) and Weizhong (BL 40). The treatment was given once every day or every two days. Ten treatments made one session. The symptom and physical signs score of low back pain and the score of cold-damp syndrome were observed before and after treatment in the patients.

RESULTSThe symptom and physical signs score of low back pain and the score of cold-damp syndrome were all improved in the two groups (all P < 0.01). The changing rate of symptom and physical signs and syndrome were (37.04 +/- 32.68)% and (22.85 +/- 29.95)% in the group A, and were (47.29 +/- 22.08)% and (23.89 +/- 22.53)% in the group B, respectively, without significant difference in comparison between the two groups (all P > 0.05).

CONCLUSIONThe efficacy on low back pain of cold-damp pattern treated with the electric-thermal Bian-stone therapy is similar to that of moxibustion box therapy. This therapy is characterized as more convenient, safer operation and less pollution.

Acupuncture Points ; Adult ; Aged ; Aged, 80 and over ; Combined Modality Therapy ; Female ; Humans ; Low Back Pain ; therapy ; Male ; Medicine, Chinese Traditional ; methods ; Middle Aged ; Moxibustion

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

Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Fetal Blood ; Humans ; Infant, Newborn ; Lipopolysaccharides ; Lymphocytes ; metabolism ; Male ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Time Factors ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUND: There are few reports about vascularization in the repairing of bone defect by heterogeneous deproteinized bone.OBJECTIVE: To verify the vascularization characteristics of heterogeneous deproteinized bone, tissue engineering scaffold material, in the repairing of large segmental bone defect.DESIGN, TIME AND SETTING: The randomized controlled animal experiment was performed between March 2005 and February 2007 at the Third Military Medical University, Chongqing, China.MATERIALS: Twenty-four 10 to 12 months old goats, weighing (22.5±2.5)kg, were obtained from the Animal Center of the Third Military Medical University, Chongqing, China. Segmental bone defects of 20 percent right tibia middle and inferior diaphysis of the 24 goats were made.METHODS: The 24 goats were divided into test group (n=16) and control group (n=8) randomly. Goats in test group were implanted with deproteinized bone+autologous MSCs+recombinant human bone morphogenetic protein-2 (rhBMP-2), goats in control group were implanted with autograft bone, and all fixed with half-ring sulcated external fixator. Every 4 weeks, 3 goats were killed after ink perfusing through femoral artery. A thick slice of new bone tissue was made to observe the vascularization.MAIN OUTCOME MEASURES: Vascularization of new bone observed through gross anatomy and imaging; vascular network of new bone observed through thick section, blood vessel amount and area measured by Image-proplus really image analysis software.RESULTS: No goat was infected or dead. Animal soft tissue was dyed black, blood vessels'size, ditribution and network structure were observed in subcutaneous tissue, fascia and periosteum. At 4 weeks postoperation implant margin became crude in the defect area; at 8 weeks postoperation transparent bone absorbing area of different size and irregular shape appeared; after 12 weeks postoperation high-dense calcification shadow appeared at the ends of defect bone and new bone connected with the ends completely. On 4 to 24 weeks postoperation, the blood vessel amount became large, alignment became regular, and their size and distribution became uniform. It showed no significant difference in blood vessel amount and area between 2 groups (P>0.05).CONCLUSION: Composite of heterogeneous deproteinized bone+autologous MSCs+rhBMP-2 has the same vascularization degree. of autogeneous bone graft in repairing of large segmental tibia defect.