AIM: To detect the expression of the Calreticulin and HPV E2 Fusion Protein in B16, and study the effects on proliferation and apoptosis of B16 cell lines in vivo. METHODS: To construct eukaryotic fluoresce expression vector pEGFP-CRT-E2, pEGFP-CRT and pEGFP-E2. Then the recombinant plasmids were transfected into B16 cells by Lipofectamine 2000. The expression of proteins was detected by Western blot. The location of different GFP fusion proteins in B16 was tested by inverted fluoresce microscope. Flow cytometry was applied to detect the effects of fusion proteins on the growing of B16 and then the apoptosis effects of B16 induced by different proteins were observed. RESULTS: The correctly constructed recombinant plasmid pEGFP-CRT-E2 and the expression of CRT-E2 gene could be detected in B16 cells. Apoptosis of B16 cells could be detected after the transient transfection. Meanwhile, the apoptosis rate of B16 cells transfected by pEGFP-CRT-E2 was much higher than that of control cells. And cell cycle G1/G0 arrest was also found (P < 0.01). CONCLUSION: The eukaryotic expression plasmid pEGFP-CRT-E2 is successfully constructed and it could correctly express the fusion protein in B16 cells. And the B16 cells transfected by plasmid pEGFP-CRT-E2 could induce apoptosis.

Objective To investigate the appropriate incision for intact specimen extraction during retroperitoneoscopic radical nephrectomy. Methods One hundred and nineteen patients in need of retroperitoneoscopic radical nephrectomy were randomized into two groups. One group of 60 patients received intact specimen extraction through a muscle-splitting abdominal incision. The second group of 59 patients received intact specimen extraction through a muscle-cutting lumbar incision. All procedures were performed by the same team of surgeons, and the intact specimens were extracted by the same surgeon. Standard operative features were measured and recorded (operative time, the time of specimen extraction, incision length, specimen weight, the time to get out of bed, the recovery time of gastrointestinal function, postoperative hospital stay, analgesia requirement, and complication rate). Results The two groups were matched in regard to patient age, body mass index, the maximum diameter of the kidney, and the stage of TNM (each P>0.05). There were significant differences between the abdominal incision group and lumbar incision group in terms of operative time (99±14 min vs 115±12 min; P=0.000), incision length (4.9±0.3 cm vs 5.3±0.4 cm; P=0.000), the time of specimen extraction (14±2 min vs 24±6 min; P=0.000), analgesia requirement (35±27 mg vs 52±29 mg; P=0.002), the time to get out of bed (20±2 h vs 21±4 h; P=0.016). The differences were not significant between the 2 groups in terms of the recovery time of gastrointestinal function (21±3 h vs 20±4 h; P=0.457), hospital stay (6±1 d vs 6±1 d; P=0.476), and specimen weight (469±181 g vs 459±169 g; P=0.776). There was no complication of incision in the 2 groups at 12 months′ follow-up (rang, 6 to 18 months). Conclusion A muscle-splitting abdominal incision for intact specimen extraction is more appropriate than a lumbar incision during retroperitoneoscopic radical nephrectomy, with small incision, little injury, short operative time, quick recovery, and less pain.

Objective To investigate the clinical efficacy of adefovir dipivoxil in antiviral treatment of chronic hepatitis B and its effect on peripheral blood T lymphocyte subsets.Methods 40 cases of patients with chronic hepatitis B were selected from The Fourth People’s Hospital of Qinghai in October 2009 to June 2012,the patients were given the antiviral treatment of adefovir dipivoxil.Before treatment,3 months,6 months and 12 months after treatment,the changes of liver function indicators of alanine transaminase(ALT),aspartate transaminase(AST)and total bilirubin(TBiL),and hepatitis B virus-DNA(HBV-DNA)were recorded;the total effective rate was observed;the peripheral blood T lymphocyte subsets changes was tested and analyzed by flow cytometry.Results The ALT,AST and TBiL of the patients all improved good,and the indicators of ALT,AST and TBiL after 12 months decreased significantly than before treatment,the HBV-DNA testing of the patients recovered negative,the difference was statistically significant (P<0.05 );the total effective rate of hepatitis B patients is 92.5%;the peripheral blood T lymphocyte subsets has no significant improvement in patients after antiviral treatment for 3 months.After the treatment for 6 months and 12 months,the CD4 +T and CD4 +/CD8 +T were significantly higher than those before treatment,there was no significant changes in CD3 +T and CD8 +T before and after treatment.Conclusion Adefovir dipivoxil has a good clinical efficacy in antiviral treatment of chronic hepatitis B,and can significantly improve the patients’liver function,improve peripheral blood T lymphocyte subsets imbalance.

BACKGROUND: In tissue engineering, three-dimensional biodegradable scaffolds are generally used as a basic structure for cell anchorage, proliferation. Currently, no perfect scaffold is available. OBJECTIVE: To observe the growth of rabbit bone marrow stromal cells (BMSCs) cultured in different-intensity three-dimensional fibrin glue in vitro, and to discuss the feasibility of fibrin glue used as a scaffold material of bone tissue engineering. DESIGN, TIME AND SETTING: The single sample observational study was performed at the China-Japan Union Hospital of Jilin University and School of Mechanical Engineering of Tianjin University of Technology from September 2007 to January 2008. MATERIALS: Fibrinogen and thrombin were mixed at various proportions, and prepared into different intensity fibrin glue. A month-old male New Zealand white rabbits, weighing 0.25 kg was used in this study. METHODS: Rabbit BMSCs were cultured and serial subcultivation in a CO2 incubator. And then the amplified BMSCs were collected and continue to be cultured in different intensity fibrin glue for 4 weeks. MAIN OUTCOME MEASURES：Observation of growing BMSCs is performed using the phase contrast microscope. The activity of BMSCs in fibrin glue at different stages was observed using hematoxylin-eosin staining. The ultrastructural changes of BMSCs were observed which had been cultivated in fibrin glue for 4 weeks. RESULTS: After growing in fibrin glue for 4 weeks, BMSCs showed strongly active status in low intensity fibrin glue and growing slowly or dying in high intensity fibrin glue. Under the electron microscope, BMSCs following 4 weeks culture in fibrin glue (proportation of fibrinogen and thrombin was 4:1) were found, with visible cellular organs, and BMSCs had good activities. CONCLUSION: BMSCs can spread and proliferate quickly in low intensity fibrin glue. The optimal proportion of fibrinogen and thrombin is 4: 1.

BACKGROUND: A superior composite scaffold hopes be constructed to resolve adhesion between seed cell and scaffoldmaterial.OBJECTIVE: To construct composite scaffolds with fibrin glue and xenogeneic inorganic bone and to explore the three-dimensional culture of rabbit marrow stromal cells (MSCs).DESIGN, TIME AND SETTING: Observational experiment was performed at the Department of Orthopedics of China-Japan Union Hospital and the Department of Mechanical Engineering, College of Mechanical Engineering of Jilin University from November 2007 to March 2008.MATERIALS: Fibrin glue was made by a certain ratio of fibrinogen and thrombin; bovine cancellous bone following defatting and deproteinization was mixed with fibrin glue to establish composite scaffold.METHODS: Rabbit MSCs were cultured in v#ro and transferred, and the MSCs were collected for three-dimensional culture withcombined scaffolds made of xenogeneic inorganic bone and fibrin glue.MAIN OUTCOME MEASURES: The growth and proliferation of MSCs were examined by phase-contrast microscope andtransmission electron microscopy.RESULTS: Phase contrast microscope showed that the MSCs could spread evenly in the combined scaffolds. After cultured 4 weeks, the MSCs formed into densely three-dimensional net. It could be observed under the transmission electron microscopethat there were micro-protrusions in local stromal cells at 4 weeks after culture, and the mitochondrion as well as ribosomes wasobserved in the cytoplasm with rough endoplasmic reticulum.CONCLUSION: The MSCs cultured in the combination of fibrin glue and xenogeneic inorganic bone scaffolds show a betteractivity, and they can proliferate rapidly.

ObjectiveTo explore the relationship of trefoil factor family 3 (TFF3),vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α in gastric cancer SGC-7901 cells under hypoxic condition and try to investigate the mechanism of TFF3 in the genesis and development of gastric cancer. Methods The hypoxic model of gastric cancer SGC-7901 cell was induced by CoCl2 Gastric cancer cell line SGC-7901 cells were transfccted with pU6-siTFF3 plasmid which carrying RNAi targeted to human TFF3 and pU6-mock.Puromycin was selected as screening medicine.The stable and specific TFF3 inhibited gastric cancer cell line was established. Gastric cancer cell line SGC-7901 and TFF3 RNAi targeted gastric cancer cell line SGC 7901 were cultured under hypoxic condition and normoxic condition. The expression of TFF3,VEGF and HIF-1a at protein and mRNA level were detected by RT-PCR,Western blot and ELISA assay.The distribution and expression of TFF3 and HIF-1α in gastric cancer cell line SGC-7901 cells uuder normoxia and hypoxic condition were determined with immunofluorescence staining.Results The expressions of HIF-1a,TFF3 and VEGF in gastric cancer SGC-7901 cell increased under CoCl2 induced hypoxic condition (33.4 =1.8,14.8 ± 1.1 and 15.1 ± 1.2,respectively). Under hypoxie condition,the expression of VEGF and HIF-1α protein reduced in stable TFF3 RNAi SGC-7901cells.Conclusion TFF3 mediated the regulation of VEGF and HIF-1α expression under hypoxic condition.TFF3might be a potential anti-angiogenic target in gastric cancer treatment.

Objective To explore the influence of pelvis obliquity in lateral position to acetabular component orientation during total hip arthroplasty (THA),and the method to correct.Methods Fifty patients (62 hips) were performed THA with posterolateral incision in lateral position by the same team.The patients were randomized and divided into experimental group (EX,with 25 cases,34 hips) and control group (CON,with 25 cases,28 hips).In EX group,the acetabular components were placed by means of the gradienter and plumb correcting technique during THA.While in CON group,the acetabular components were placed by traditional method during THA.The acetabular abduction angles were measured postoperatively,and compared between the two groups.Results The average obliquity of pelvis was-1.647°±4.512°in EX group when putting the patient in lateral position before correcting.Through the application of gradienter and plumb,the average abduction angle of acetabular component was 42.685°±3.355° postoperatively,with the difference of 1.962°±1.515° compared with the preoperative angles.And in CON group,the average abduction angle of acetabular component was 44.534°±4.844° postoperatively,with the difference of 4.244°±3.042°.The difference of abduction angle in CON group was much higher than that in EX group (P＜0.05).Conclusion The pelvic obliquity when putting the patient under lateral position will affect the surgeons'judgments of placing acetabular component during THA,furthermore,lead to inconsistency among the abduction angles obtained preoperatively,intraoperatively and postoperatively.By applying the correcting method with gradienter and plumb,the discrepancy can reduce obviously between the abduction angle measured postoperatively and that of measured during operation comparing with traditional method.

Objective:To analyze, at cellular level, whether the mouse B16-F1 melanoma cells with OAZI-1 overexpression could activate antigen-presenting cells and promote the phagocytotic and antigen-presenting efficiencies of mouse peritoneal macrophage and bone marrow derived DC on tumor cells. Methods:The plasmid pcDNA3. 1(+)/OAZI-1 was transfected into B16-F1 cells by Li-pofectamine2000 reagent. The positive clones with OAZI-1 overexpression ( B16/OAZI-1 ) were identified by Western blot assay and RT-PCR. Macrophages from abdominal cavity and DC from bone marrow were collected from BALB/c mouse. The B16-F1 cells transfected with the pcDNA3. 1(+) (B16/3. 1) were used as the control cells in this experiment. B16-F1 cells and macrophages were co-cultured for 4 h at a 1∶5 ratio and DC were co-cultured with B16-F1 cells at 1∶1 ratio for 4 h. And then the phagocytotic efficiencies were assayed by flow cytometry. DC were co-cultured with B16-F1 cells at 1∶1 ratio for 24 h and then the expression of mature DC surface marker molecules CD40,CD80,CD86 were determined by flow cytometry. The DC activated by the tumor cells were co-cultured with mouse spleen lymphocytes for 24 h, and then IFN-γ content in culture medium was analyzed by ELISA. Results: Phagocytotic assay showed that,compared to the control cells,the OAZI-1 overexpression in B16-F1 cells significantly enhanced the engulfment of B16-F1 cells by macrophages ( 24. 7% vs 53. 9% ) and DC ( 8. 2% vs 13. 8%) . When DC were co-incubated with OAZI-1 overexpressed B16-F1 for 24 h,the expression levels of CD40,CD80,CD86 on the DC surface,which were the molecular markers for matured DC,increased from 24. 2%,20. 8% and 16. 4% to 46. 8%,32. 5% and 36. 1% respectively. Co-culture of tumor-activated DC with the spleen lymphocytes resulted in an increased IFN-γcontent in the culture medium(32. 9 pg/ml vs 15. 1 pg/ml). Conclusion:The tumor cells with OAZI-1 overexpression can be engulfed more efficiently by macrophages and DC. And this process can induce the maturation and activation of DCs. Matured DC could induce T cell activation and then activate the anti-tumor immune response.

Objective To study the relationship between the CT value and injury of ultrastructure in posttraumatic acute diffuse brain swelling(PADBS). Methods The change of CT value of brain tissue was analyzed at posttrauma and preoperation in 9 patients, in combination with the ultrastructure in brain parenchyma in 36 specimen taken from operations. The relationship between the descend of CT value and ultrastructure injury was analysed.Results The CT value of brain in preoperation was lower than it in posttrauma first scanning(2.5～4.3 HU).The capillary distention and stenosis and the diffuse edema in pericapillary and intercellular were observed under transmission electron microscopy(TEM). The nucleolus of neuronal cells displaced to membrane or disappeard. Chromation agglutionation, nuclear membrane circuity, perinuclear diffuse lipid drops and blankspace were detected. The mitochondrion swelling, mitochondrial crest blurring or effacement, rough endoplasmic reticulum distension and its’ granules detachmen were also seen under TEM. Axolemma edema, microfilaments and microtubules derangement in axis-cylinder were found too. The similar phenomena existed in astrocyte.Conclusion The descent of CT value in PADBS was relevant to the aggravation of vasogenic cerebral edema, cytotoxic cerebral edema and ultrastructure injury in brain parenchyma.

Objective To explore the clinical significance of integrated screening of RET in a Chinese multiple endocrine neoplasia type 2A(MEN 2A)family and to evaluate the feasibility and effectiveness of prophy-lactic total thyroidectomy to MEN 2A-related medullary thyroid carcinoma ( MTC).Methods Medical history was obtained from 10 family members in a 3-generation south China family .Systemic investigations including bio-chemical tests, imaging examinations and germline RET screening were performed .3 asymptomatic mutation car-riers underwent prophylactic total thyroidectomy .Results RET screening showed a heterozygous missense muta-tion of TGC to CGC at codon 634 on exon 11 in 6 members(p.C634R), which was completely consistent with the clinical manifestations.There were 4 males and 2 females.The initial mean diagnostic age of 33.5 years(ranging from 19 years to 65 years) and the mean maximum diameter of MTC was 2.3 cm(ranging from 0.7 cm to 5.2 cm). Among them 3 members had palpable neck masses (1 case with diarrhea).Right total thyroidectomy +right level Ⅵlymph-node dissection with modified right neck dissection in one case , and bilateral total thyroidectomy +bilat-eral level Ⅵlymph-node dissection in 2 were performed .In other 3 asymptomatic mutation carriers , prophylactic total thyroidectomy +bilateral level Ⅵ lymph-node dissection were also performed .Among them, 1 case of a-symptomatic pheochromocytom ( PHEO) underwent cortical-sparing adrenalectomy before MTC .After the first op-eration, 4 patients still presented a high value of calcitonin , among whom 1 patient( T3N 1bM 0-1) underwent re-operation for 3 times after the initial operation and presented metastasis to bone after 130 months, taking vandet-anib orally up to now;2 patients underwent reoperation at 6 and 7 months after initial operation respectively (T1N 1bM0 and T2N 1bM0), and the other one patient was closely monitored and followed up for 22 months(T2N 1b M0).Moreover, The calcitonin levels dropped to normal in the other 2 asymptomatic cases(T1N0M0) who were followed up for 20 months.Conclusions Pedigree screening can work up an early diagnosis and improve the prognosis of MEN 2A.Integrated screening of RET and pre-operative calcitonin level measurement and prophylac-tic thyroidectomy for asymptomatic RET mutation carriers are reasonable and effective .