AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes.
METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289.
RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1.
CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.

In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells.
Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Coculture Techniques ; Culture Media, Conditioned ; Fluorouracil ; pharmacology ; Human Umbilical Vein Endothelial Cells ; chemistry ; Humans ; Liver Neoplasms ; Neoplastic Stem Cells ; cytology ; Phenotype

Objective To improve the teaching quality of medical genetics. Methods A medi-cal genetics questionnaire survey was designed and conducted among 2012 grade undergraduates of Kunming Medical University. The results of the questionnaire were statistically analyzed by Kruskal-wallis test. Results The students felt that, there were differences in the importance and difficulty of teaching sections (Z=395.818,Z=117.786,P<0.001). The practical abilities were different after learn-ing (Z=139.364,P<0.001). Most of the students had strong demand for clinical practice and genetic counseling, there were differences in the degree of demand in students of different majors (Z=8.432, P=0.038). Conclusion Method of adjusting teaching content and method was proposed and adding clinical practice in the future was recommend to improve the teaching quality .

Objective To summarize the clincical experience of transumbilical Laparoendoscopic Single-site (LESS) nephrectomy and to evaluate its safety and efficacy. Methods From December 2008 to August 2010, we have performed 20 cases of transumbilical LESS nephrectomy by Tri-Port system, of which 9 patients underwent LESS radical nephrectomy (left 8, right 1, stage T1 ), 1 patient underwent LESS radical resection of right ureteral carcinoma, 10 patients underwent LESS simple nephrectomy (left 5, right 5). The Tri-Port system was inserted transperitoneally through a 2 cm umbilical incision. A 5-mm 30° telescope was introduced through the port to visualize the operative field. Flexible equipment and standard laparoscopic equipment were used to perform the procedures.The incisions were extended to about 6cm in order to remove the specimens. Results Conversion to open surgery was necessary in one LESS radical resection of right ureteral carcinoma and one LESS simple nephrectomy, while the remaining 18 cases were successful (the addition of a single 5-mm port was necessary in 2 cases of LESS radical nephrectomy). The mean operative time was 197 min (85-510 min), mean estimated blood loss was 126 ml (50-400 ml), without blood transfusion in the perioperative period, mean postoperative hospital stay was 6.3 d (3-14 d), and mean duration of catheter drainage was 3.6 d (0- 14 d). Conclusions Transumbilical LESS nephrectomy is feasible, safe,minimally invasive and cosmetic. Long-term follow-up and a clinical control study are needed for evaluating clinical outcomes.

Enhancing medical students' humane quality education is an urgent requirement for modem medical mode transformation for medical education.The pharmacology teachers of Chongqing Medical University follow the modem education concepts and fully search the human spirit materials hidden in pharmacology,then actively explore how to integrate the humanity spirit education into the pharmacology teaching to achieve the changes of from exam-oriented education to quality education.

Objective To evaluate super-selective renal arterial embolization(SRAE)in treating severe renal hemorrhage when conservative treatment had failed. Methods SRAE was performed in 111 patients with severe renal hemorrhage who had failed to respond the conservative management.The clinical data,the way of embolization,the medication and the follow-up findings were retrospectively analyzed.Results Excellent results were obtained in all patients after SRAE and no serious complications occurred.The technical successful rate with single session was 95.5%(106/111).Gross hematuria disappeared within 1-4 days after the treatment.Two patients developed shock after renal embolization and had to receive surgery after the shock was controlled.Three patients had a recurrence of hematuria,the blood urine subsided after SRAE was employed again.A follow-up with a mean period of 37.4 months was carried out in 92 patients,and the follow-up checkups showed that the renal function was well preserved in all patients.Conclusion Super-selective renal artery catheterization and embolization is a safe and effective treatment for severe renal hemorrhage,it can maximally preserve the healthy renal parenchyma as well as the renal function.Therefore,this technique should be regarded as the treatment of first choice for patients with severe renal hemorrhage.

Objective To investigate expression levels of epithelial mucin 1 (MUC1) and epitbelial mucin15(MUC15) in elderly patients with papillary thyroid carcinoma and assess the role of MUC1 and MUC15 in the pathogenesis of thyroid papillary carcinoma.Methods Protein expression of MUC1 and MUC15 was detected by immunohistochemistry in 10 samples from normal thyroid tissue adjacent to thyroid adenoma,57 samples from papillary thyroid carcinoma (PTC),and 14 samples from PTC in neck lymph node metastasis.Results Expression rates of MUC1 in normal thyroid tissues,thyroid papillary carcinoma,and lymph node metastatic carcinoma were 40.0％,75.4％,64.3,respectively,and the rates for MUC15 were 0,73.7％,71.4％,respectively.The positive expression rate of MUC1 was higher in PTC tissues than in normal thyroid tissues (x2 =5.10,P=0.02) and,compared with normal thyroid tissues,the positive expression rate of MUC15 increased in PTC tissues and lymph node metastatic carcinoma (x2 =12.25 and 19.75,both P＜0.05)MUC15 protein expression was higher in micro-PTC (less than or equal to 1 cm in diameter) than in carcinoma larger than 1 cm in diameter (90.9％ vs.62.9,x2 =5.48,P=0.02).MUC15 expression was higher in PTC without lymph node metastasis than in PTC with lymph node metastasis (83.8％vs.55.0％,x2 =5.55,P=0.02).MUC1 expression was positively correlated with MUC15 expression in thyroid papillary carcinoma (r=0.35,P=0.01).Conclusions MUC1 and MUC15 may have synergistic effects in the initiation and progression of PTC.MUC15 may play a role in regulating tumorigenesis of thyroid papillary carcinoma in early stages and can potentially serve as a supplementary marker in the screening of micro-thyroid papillary carcinoma.

Objective To construct a lentiviral vector-based short hairpin RNA (shRNA) targeting the gene encoding tryptophan-aspartate containing coat protein ( TACO) and to evaluate its inhibitory effect on the expression of TACO , and to further elucidate its effects on the phagocytosing and intracellular killing of My-cobacterium tuberculosis (M.tb) by macrophages and the possible mechanisms.Methods Three shRNA frag-ments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentivi -ral vector pSicoR .The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells.Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW 264.7 cells transfected with the concentrated lentivirus .The most effective lentivirus was screened out to transfect the RAW 264.7 cells for 48 hours, followed by infection those cells with M.tb strains.The entry and intracellular survival of M .tb strains in RAW264.7 cells were de-termined by bacterial culture at indicated time points .The colocalization of M .tb and lysosomes was detected by immunofluorescence staining .The cyto-ID autophagy kit was used to detect the cellular autophagy and the auto-phagy-associated protein LC 3 was determined by Western blot assay .Results The recombinant lentiviral vec-tors were successfully constructed and confirmed by sequencing analysis .Decreased expression of TACO in RAW264 .7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours.The most effective lentivirus , LV-pSRT1, decreased the expression of TACO by 85.24%and 69.00%at the mRNA and protein levels, respectively.The bacterial loads in LV-pSRT1 trans-fected RAW264.7 cells were significantly decreased at the time point of 0 h after M.tb infection as compared with those in the control lentivirus treated cells (5.50×104 vs 8.1 ×104, P<0.05).Compared with the RAW264 .7 cells transfected with control lentivirus , the survival rate of intracellular M .tb strains in LV-pSRT1 transfected cells was significantly decreased at the time point of 48 h (134.54% vs 213.58%, P<0.05) and 72 h (148.18%vs 262.96%, P<0.05) considering the bacterial load at the time point of 0 h as the standard. The immunofluorescence staining demonstrated that the colocalization of M .tb strains with lysosomes was signifi-cantly enhanced in LV-pSRT1 transfected cells as compared with that in control lentivirus treated cells (75.67%vs 10.66%, P<0.05).Moreover, significantly enhanced autophagy and relative expression of LC 3Ⅱ protein were observed in RAW264.7 cells with TACO gene knockdown (16.20%vs 8.50%, P<0.05;0.51 vs 0.34, P<0.05).Conclusion The lentiviral vector-based shRNA targeting TACO gene could effectively knockdown the expression of TACO protein , decrease the entry and increase the intracellular killing of M .tb strains in mac-rophages.The enhanced intracellular killing of M .tb strains by macrophages was associated with the increased fusion of M.tb-containing phagosome and lysosome .

BACKGROUND:The ankle is one of the most important joints of human body. Medial maleolar fractures are very common, and there are lots of surgical methods to treat it. A traditional approach is open reduction and internal fixation. As views changed, percutaneous cannulated screw internal fixation become increasingly popular, but various clinical studies are stil needed to analyze the efficacy of these two methods. OBJECTIVE:To compare the recovery of joint function after medial maleolus fracture repaired by open reduction and internal fixation and percutaneous cannulated screw fixation. METHODS:A total of 63 cases of medial maleolus fracture, who were treated in the Department of Orthopedics, People’s Hospital of Xinyi City from March 2009 to March 2013, were enroled. According to repair plan, they were divided into two groups: open reduction and internal fixation group (n=29) and percutaneous cannulated screw fixation group (n=34). Ankle function was assessed in accordance with Kofoed ankle score standard on admission and at 3 months after repair. RESULTS AND CONCLUSION:The patients were folowed up for 3 to 12 months. Among 29 cases in the open reduction and internal fixation group, the wound was healed at grade A in 27 cases and at grade B in 2 cases; 29 cases were reset, and no poor reduction was found. In the percutaneous cannulated screw fixation group, 34 cases had healing at grade A, with the presence of good reduction. Al patients experienced bone union, and no infection appeared. In accordance with Kofoed score, at 3 months of folow-up, the satisfaction rate was 97% in the open reduction and internal fixation group, and 100% in the percutaneous cannulated screw fixation group. These data suggest that both open reduction and internal fixation and percutaneous cannulated screw fixation for medial maleolus fracture obtained positive effects, but percutaneous cannulated screw fixation showed smal surgical trauma, which could reduce the rate of infection and contributed to early functional recovery of ankle joint.

This study was aimed to establish a method for assay of 4-terpineol, α-terpineol, bornyl acetate and methyl-n-nonylketone in Houttuynia cordata with gas chromatography-mass spectrometry (GS/MS) for the first time in order to provide experimental evidences for its quality control. Undecylen was analyzed quantitatively. GC/MS method was used in the content determination of 4 kinds of components in Houttuynia cordata. GC conditions were that the temperature was firstly increased to 70oC for 5 min, and then 5oC·min-1 to 140oC for 5 min. Then, the temperature was increased by 10oC·min-1 to 250oC for 10 min. The temperature of the injection port was 230oC. The carrier gas was high purity helium. The flow rate was 1.0 mL·min-1. The split ratio was 10:1. The sample quantity was 1 μl. MS conditions were that the detector was MS, EI was 70ev, the temperature of four poles was 150oC, the temperature of ion source was 230oC. The results showed that there was good linearity of four components, which were 4-terpineol, α-terpineol, bornyl acetate and methyl-n-nonylketone obtained within the ranges of 6.492~129.84μg·min-1, 1.097~21.944 μg·min-1, 12.128~242.56 μg·min-1, and 84.76~169.52 μg·min-1, respectively. The average recoveries of the four components were 81.2%, 80.6%, 88.3%, and 84.6%, respectively. The RSDs were 1.5%, 2.3%, 1.1%, and 0.7%, respectively. It was concluded that the method was simple, sensitive, and easy to operate. The simultaneous measurement of multiple indexes on the component for quality evaluation of Houttuynia cordata can effectively reflect the quality of Houttuynia cordata. It can also be used in the quality assessment of fresh Houttuynia c ordate .