Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3. 1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3. 1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5% and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3. 1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.     

Preoperative Neutrophil Lymphocyte Ratio and Platelet Lymphocyte Ratio Cannot Predict Lymph Node Metastasis and Prognosis in Patients with Early Gastric Cancer: a Single Institution Investigation in China

In the present study, we aimed at exploring the applied value of preoperative neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) in the prediction of lymph node metastasis (LNM) and prognosis in patients with early gastric cancer (EGC). We retrospectively analyzed a total of248 consecutive patients who underwent curative gastrectomy to be identified T1 stage gastric adenocarcinoma between January 1, 2010 and May 1, 2016 in a single institution. According to median preoperative NLR and PLR value, we divided the patients into four groups: high NLR >1.73 and low NLR <1.73, high PLR >117.78 and low PLR <117.78. Furthermore, to evaluate the relationship between preoperative NLR and PLR values, we categorized patients according to cutoff preoperative NLR-PLR score of 2 [high NLR (>1.73) and high PLR (>117.78)], 1 [either high NLR or high PLR], and 0 [neither high NLR nor high PLR], Statistical analyses were conducted using SPSS 20.0 software. The results showed that the preoperative NLR or PLR values, lower or higher, could not predict the LNM in patients with EGC (both P=0.544>0.05). The invasive depth of tumor was significantly correlated with LNM of EGC (P0.001). Kaplan-Meier plots illustrated that preoperative NLR and PLR values were not associated with overall survival (OS) in patients with EGC. It was concluded that the preoperative NLR and PLR may be the predictors for LNM and prognosis in patients with advanced gastric cancer; nevertheless, they cannot predict LNM and prognosis in patients with EGC.     

Value of Quantitative Tissue Velocity Imaging in the Detection of Regional Myocardial Function in Dogs with Acute Subendocardial Ischemia

This study evaluated the application of quantitative tissue velocity imaging （QTVI） in assessing regional myocardial systolic and diastolic functions in dogs with acute subendocardial ischemia. Animal models of subendocardial ischemia were established by injecting microspheres （about 300 μm in diameter） into the proximal end of left circumflex coronary artery in 11 hybrid dogs through cannulation. Before and after embolization, two-dimensional echocardiography, QTVI and real-time myocardial contrast echocardiography （RT-MCE） via intravenous infusion of self-made microbubbles,were performed, respectively. The systolic segmental wall thickening and subendocardial myocardial longitudinal velocities of risk segments before and after embolization were compared by using paired t analysis. The regional myocardial video intensity versus contrast time could be fitted to an exponential function： y=A·（1-exp-β·t）, in which the product of A and β provides a measure of myocardial blood flow. RT-MCE showed that subendocardial normalized A·β was decreased markedly from 0.99±0.19 to 0.35±0.11 （P〈0.05） in 28 left ventricular （LV） myocardial segments after embolization, including 6 basal and 9 middle segments of lateral wall （LW）, 8 middle segments of posterior wall （PW） and 5 middle segments of inferior wall （IW）. However, there was no statistically significant difference in subepicardial layer before and after embolization. Accordingly, the ratio of A·β of subendocardial myocardium to subepicardial myocardium in these segments was significantly decreased from 1.10±0.10 to 0.31±0.07 （P〈0.05）. Although the systolic wall thickening did not change 5 min after the embolization in these ischemic segments （29%±3% vs 31%±5%, P〉0.05）, the longitudinal peak systolic velocities （Vs） and early-diastolic peak velocities （Ve） recorded by QTVI were declined significantly （P〈0.05）. Moreover, the subendocardial velocity curves during isovolumic relaxation predominant     

Construction of Eukaryotic Expression Plasmid of Human PRX3 and Its Expression in HEK-293FT Cells

To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could he expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.     

Effect of Antisense Oligodeoxynucleotide of Vascular Endothelial Growth Factor C on Lymphangiogenesis and Angiogenesis of Pancreatic Cancer

In order to investigate the effect of antisense oligonucleotide (ASODN) of vascular en-dothelial growth factor C (VEGF-C) on lymphangiogenesis and angiogenesis of pancreatic cancer, antisense and scamble-sense oligonucleotide of VEGF-C were constructed, and the model of nude mice with orthotopically xenografted human pancreatic cancer cells (Panc-1) was established. Thirty nude mice were randomly divided into 3 groups: PBS control group (group A), scramble-sense con-trol group (group B) and antisense group (group C). All nude mice were treated once every 2 days as 3 times per week, for 3 weeks (oligonucleotide 10 mg/kg every time). After treatments were com-pleted, ELISA method was used to examine the concentration of VEGF-C in plasma and immunohis-tochemical method to examine microvessel density (MVD), lymphtic vessel density (LVD) of pan-creatic cancer. The results showed that the expression of VEGF-C was inhibited significantly in group C. The concentrations were 237.5±41.5, 221.5±52.3 and 108.6±14.9 pg/mL in groups A, B and C re-spectively (P<0.01). LVD in groups A, B and C was 13.8±2.1, 12.4±1.9 and 4.2±1.6 respectively (P<0.01). MVD in groups A, B and C was 27.5±8.7, 25.9±4.2 and 19.4±5.6 respectively with no sig-nificant difference among the groups (P>0.05). It was suggested that VEGF-C ASODN decreased the expression levels of VEGF-C in nude mice with orthotopically xenografted human pancreatic cancer, and it could inhibit lymphangiogenesis, but had no significant effect on angiogenesis.     

Increased Expression of PI-3K in Asthmatic Rat T Lymphocytes

In order to explore the expression of PI-3K in T lymphocytes of asthmatic rats and the relationship between PI-3K and activation of T lymphocytes, 24 Wistar rats were randomly divided into 4 groups: normal control group, asthmatic one-week group, asthmatic two-week group and asth-matic four-week group. T cells were purified from blood of each rat and the expression of PI-3K was observed by immunocytochemical fluorescence staining, the semiquantitative fluorescence intensity was measured by HPIAS-2000 analytic software, and the expression of IL-4 in supernatants was de-tected by ELISA. The results showed that the fluorescence intensity of T lymphocytes in asthmatic groups was significantly higher than that in normal control (P<0.001), indicating that the expression of PI-3K in T lymphocytes of asthmatic rats was significantly higher than that in those of normal controls, and the difference between acute and chronic stage asthmatic groups was significant (P<0.05). The expression levels of IL-4 protein in supernatants of asthmatic T lymphocytes were sig-nificantly higher than those in the normal controls (P<0.05). There was a significant positive correla-tion between the expression of PI-3K in T lymphocytes and the IL-4 protein expression in super-natants (r=0.583, P<0.01). It was suggested that PI-3K signal pathway may participate in the proc-esses of activation and other cytological effects of asthmatic T lymphocytes, thus may play an impor-tant roles in the pathogenesis of asthma.     

Enhanced Response of Acute Monocytic Leukemia Cells to Low-dose Cytarabine by 1,25-dihydroxyvitamin D3

Low-dose cytarabine combined with differentiating or DNA hypomethylating agents, such as vitamin D compounds, is a potential regimen to treat acute myeloid leukemia (AML) patients who are unfit for high-intensity chemotherapy. The present study aimed to determine which subset of AML would be most responsive to low-dose cytarabine with the differentiating agent 1,25-dihydroxyvitamin D3 (1,25-D3). Here, firstly, cBioPortal database was used and we found out that vitamin D receptor (VDR) was highly expressed in acute monocytic leukemia (M5) and high VDR expression was associated with a poor survival of AML patients. Then, we confirmed that 1,25-D3 at clinical available concentration could induce more significant differentiation in acute monocytic leukemia cell lines (U937, MOLM-13, THP-1) and blasts from M5 patients than in non-monocytic cell lines (KGla and K562) and blasts from M2 patient. Finally, it was shown that the combination of 1,25-D3 and low-dose cytarabine further increased the differentiating rate, growth inhibition and G0/G1 arrest, while mild changes were found in the apoptosis in acute monocytic leukemia cell lines. Our study demonstrates that the enhanced response of acute monocytic leukemia cells to low-dose cytarabine by 1,25-D3 might indicate a novel therapeutic direction for patients with acute monocytic leukemia, especially for elderly and frail ones.