Objective To construct a luciferase gene expression vector containing full-length 3' untranslated region(3'UTR)of mouse vascular endothelial growth factor-C(VEGF-C)gene,and to observe the effects of VEGF-C 3'UTR on luciferase gene expression by a double-fluorescence report system.Methods Polymerase chain reaction(PCR)was used to amplify VEGF-C 3'UTR and a 312bp VEGF-C coding region(CR)fragment from full-length VEGF-C cDNA in mouse Lewis lung cancer cells.The luciferase expression vectors containing VEGF-C 3'UTR or VEGF-C CR were constructed by subcloning the PCR products to luciferase reporter vector pGL3-Promoter using gene engineering technology,and then they were transfected to mouse Lewis lung carcinoma cells by LipofectamineTM 2000.The activities and mRNA expression of luciferase were detected by Dual-Luciferase Reporter System and quantitative RT-PCR,respectively.Results Mouse VEGF-C 3'UTR(429bp)and VEGF-C CR(312bp)were successfully amplified by PCR.The VEGF-C 3'UTR and VEGF-C CR fragments were successfully inserted into pTA2 vector,and then subcloned to pGL3-Promoter vector at Xba Ⅰ site by using restriction endonucleases analysis.The DNA sequences and insertion orientation of PCR products were all correct by sequencing analysis.The resulted luciferase expression plasmids were named pGL3-VEGF-C 3'UTR and pGL3-VEGF-C CR,respectively.Dual-Luciferase Reporter System detection and quantitative RT-PCR showed that in Lewis lung carcinoma cells,the activities of luciferase and expression of luciferase mRNA in the pGL3-Promoter group were higher than that in the pGL3-VEGF-C 3'UTR group,and there was no significant difference between pGL3-VEGF-C CR group and pGL3-Promoter group.Conclusion VEGF-C 3'UTR can inhibit luciferase gene expression.

Objective To explore whether alternatively activated macrophages (aaMphi) can transdifferentiate into lymphatic endothelial cells (LEC) under the inducement of VEGF-C, and to investigate the possible mechanisms involved in aaMphi-induced lymphangiogenesis. Methods An aaMphi model constructed by treating mouse macrophage cells RAW264.7 with mouse recombinant IL-4 for 24h was treated with different concentrations of recombinant mouse vascular endothelial growth factor-C (VEGF-C). After the clustering formed and the tube-like structures were detected in the matrigel, the aaMphi transdifferentiation system was finally decided to be constructed with the VEGF-C in the concentration of 100ng/ml. The mRNA expression of LEC specific markers, VEGFR-3 and Prox1, and aaMphi specific marker, Fizz1, were detected by real time quantitative RT-PCR on the 0, 7th, 14th, and 28th day, respectively, after VEGF-C stimulation. Formation of tube-like structure in the matrigel was observed with inverted phase contrast microscope in 28 consecutive days. Results The VEGF-C induced transdifferention system, incubated in EBM-2 medium and sustained by the matrigel, was successfully established. In this system, it was found that the mRNA expression of VEGFR-3 and Prox1 gradually increased, whilst that of Fizz1 decreased. The mRNA expression of VEGFR-3 and Prox1 reached the peak value, whilst that of the Fizz1 went down to the nadir, on the 14th day. No significant difference in values was found between the 14th day and the 28th day. During the period of the 7th day to the 28th day, distinct tube-like structures were gradually formed in the matrigel and the numbers increased in a time-dependant manner. Conclusion VEGF-C can induce the transdifferentiation of aaMphi into LEC by up-regulating the mRNA expression of VEGFR-3 and Prox1 in aaMphi, which is one of the possible mechanisms involved in aaMphi-induced lymphangiogenesis.

Objective:To investigate the effects of antimalarial artemether on the proliferation of human lung adenocarcinoma cell line A549 in vitro and provide experimental data for the treatment of lung cancer with artemether.Methods:MTT assay was used to observe the inhibitory effects of artemether on the proliferation of A549 cells.Cell growth curve was draw according to the cell counts.The population doubling time was obtained in logarithmic growth phase.Cell cycle detection was observed by flow cytometry.H-E staining and transmission electron microscopy were used to observe the altered morphology of apoptotic cells. Results:Artemether has a significantly inhibitory effect on the proliferation of A549 cells in a dose-dependent manner in vitro,and the IC50 was 1.34 mg/L.The population doubling time in logarithmic growth phase in the artemether treatment group was(20.7?0.5) h compared to(32.2?0.3) h in the control group.The difference between two groups was statistically significant(P

Objective To explore the role of chemokine CXCL12 and its receptor CXCR4 in the directional migration of human prostate cancer(PCa).Methods The expression of CXCL12/CXCR4 in 18 human PCa samples and human PCa cell lines(PC3,DU145 and LNCap)was determined by immunohistochemistry and immunocytochemistry,respectively.Then the effect of CXCL12 on the migration and invasion of human PCa cell lines Was investigated by Matrigel invasion assay.Results Except 1 PCa sample,positive CXCR4 protein expression was detected in 17 clinical PCa samples.On the contrary,in 18 samples determined,only one sample expressed weak CXCL12 protein.CXCR4 rather than CXCL12 protein was exressed in PCa cell lines PC3,DU145 and LNCap.In addition,CXCL12 promoted the migration and invasion of PCa cell lines in a dose dependent manner in viiro,in which experiments PC3,LNCap cells were pretreated by antibody of CXCL12 or CXCR4 and then it was found the migrations of cells stimulated by CXCL12 were inhibited.Conclusion CXCR4 protein is expressed in human PCa and CXCL12/CXCR4 axis may play a significant role in the metastasis of prostate cancer.

The X-ray computed tomography (CT) appearance of 4 cases with pulmonary cryptococcosis (PC) diagnosed by pathological examination in our hospital was retrospectively analyzed. The appearances of PC on CT were various: solitary lesion in 1 case, multiple lesions in single lobe in 2, and multiple lesions in multiple lobes in 1. There were total 52 lesions in 4 cases; the diameter of nodules or masses was 3 - 75 mm. Cavitations were found in 1 case; lesions appeared obviously enhanced and one lesion showed central necrosis. Two cases underwent pulmonary lobectomy; and 2 cases received core cutting needle biopsies, after antifungal therapy for 3 months to 1 year the lesions showed being absorbed. In summary, the CT appearance of PC is non-specific with various modes and forms. PC should be considered when multiple nodules or masses scattered in subpleural zone, accompanied with ground-glass opacity and obviously enhanced. The examination of pathogen and pathology at the beginning is crucial for improving diagnostic accuracy.

AIM: To investigate the effect of glomerular intercellular interaction under high glucose concentration on the production of reactive oxygen species (ROS) and transforming growth factor ?_1 (TGF-?_1) in co-cultured human ECV304 cells, and to study the intervention with tea polyphenols (TPs). METHODS: The endothelial cells were cultured alone or co-cultured with mesangial cells in high glucose media with or without TPs for 0 h, 12 h and 36 h, respectively. The activity of SOD and the content of MDA in the media of the system were detected by spectrophotometry. The expression of TGF-?_1 mRNA in the endothelial cells was measured by using semi-quantitative reverse transcription PCR (RT-PCR). RESULTS: High glucose decreased the activity of SOD, increased the content of MDA and up-regulated the expression of TGF-?_1 mRNA in co-cultured ECV304 cells and the effect became more prominent than the single-cultured cells. TPs interrupted it more effectively. CONCLUSION: These data suggest that there is interaction between mesangial cells and ECV304 cells under high glucose concentration. The interaction may markedly up-regulate the production of ROS and the expression of TGF-?_1 in co-cultured ECV304 cells. TPs may protect ECV304 cells by intervening intercellular interaction.

Objective To evaluate the effects of pregabalin combined with hydrochloric oxycodone on patients with ma-lignant neuropathic pain (MNP). Methods A total of 66 patients with MNP was divided into control group or treatment group randomly. The patients in control group received only hydrochloric oxycodone, and treatment group were treated with the combina-tion of pregabalin and hydrochloric oxycodone. Numeric rating scale (NRS) score was used to evaluate the analgesic effects. Med-ical outcomes study sleep scale (MOS-SS,Chinese version) was used to evaluate the improvement of sleep disorder. The changes of depression or anxiety were investigated by 17-item Hamilton Depression Rating Scale (HAMD-17) or Hamilton Anxiety Scale (HAMA), respectively. Side effects were accessed by Acute and Subacute Toxicity Grading Criteria of Anticancer Drugs (WHO). Results The pain control rate of treatment group was 87. 1% , which was superior to that of control group (58. 6% ) (P<0. 05). The improvement of sleep interference, and the quality and quantity of sleep in treatment group were also superior to that in control group (P<0. 05). After the treatment, depression and anxiety was attenuated in both groups, and the improvement degree in treatment group was higher than that in control group (P<0. 05). No obvious side effects were found in either groups. Conclusion The combination therapy of pregabalin and hydrochloric oxycodone is the better way to treat MNP.

Aim To explore clinicalpathological features and the pathogenesis of lymphocytic mastitis, an uncommon breast benign disease. Methods The clinical pathological characteristics of 7 patients with lymphocytic mastitis were studied retrospectively. All cases were evaluated by using paraffin-embedded sections and by immunohistochemical staining with mouse anti-CD20, -CD45RO, and -CD68 antibodies. T and B lymphocytes infiltrated in the lobules of mammary gland were quantitatively analyzed according to stereoscope. The glucose regulation protein 94 (grp 94)/glycoprotein 96(gp 96), a member of heat shock protein family was also investigated in these cases by using immunohistochemical staining. Results It was showed that 4 cases were women suffering from insulin dependent diabetic mellitus (IDDM). One case was a woman without diabetic history. The history of the other two cases was not clear. The histopathological features of all 7 cases were lobulitis, perilobulitis and catheter ductitis with infiltration of lymphocytes accompanied with atrophy of lobule in mammary glands and homologous fibrosis of stroma. The result of immunohistochemical staining showed that most of infiltrated lymphocytes were B lymphocytes, while the small proportion of the cells were T lymphocytes, and the difference was significant(P〈 0.01). There was the expression of grp 94 in the cytoplasm of epithelium cells of lobules and ducts in normal mammary glands. A proportion of lymphocytes infiltrated in lobules and perilobules expressed grp 94. Some infiltrated cells expressed CD68. Conclusion A portion of lymphocytic mastitis is related closely to insulin dependent diabetic mellitus. Both humoral immunity and cellular immunity are probably involved in the pathogenesis of lymphocytic mastitis. Because of its unique pathologic and clinical features, lymphocytic mastitis should be defined as an independent mastitis and distinguished from other chronic inflammatory and fibrosing conditions in breast.

Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.

Objective To investigate treatment and prognosis of patients with osteosarcoma of the extremities.Methods A total of 311 patients with osteosarcoma of the extremities,who had undergone treatment in our institute from 1998 to 2008,were enrolled in this retrospective study.Kaplan-Meier survival curve and Cox regression model were used to analyze the correlation between survival rate and variables including patients' demographics,chemotherapy,surgery,complications,and tumor metastasis.Results Among 311 patients,there were 206 males and 105 females,aged from 5 to 56 years (average,18.6 years).A total of 282 patients underwent aggressive or radical surgery,including 149 cases of limb salvage surgery and 133 cases of amputation surgery.One hundred and five patients underwent standard chemotherapy and 206 patients underwent non-standard chemotherapy.The 5-year survival rate was 57.4％ in patients treated with standard chemotherapy,36.3％ in patients treated with non-standard chemotherapy,16.8％ in patients with lung metastasis,50.7％ in patients without lung metastasis,56.6％ in patients who underwent limb salvage surgery,31.8％ in patients who underwent amputation surgery,44.6％ in patients with Enneking stage Ⅱ B and 33.1％ in patients with Enneking stage Ⅲ.For patients treated by amputation surgery,because non-standard chemotherapy which was performed in most of them and other confounding factors,the 5-year survival rate of them was lower.The Cox regression analysis showed that lung metastasis and non-standard chemotherapy were associated with inferior outcomes.Conclusion Neoadjuvant chemotherapy combined with aggressive or radical surgery could cure about 60％ of patients with osteosarcoma of the extremities.Lung metastasis and non-standard chemotherapy are risk factors that severely affect prognosis.