Reverse phase high-performance liquid chromatography (RP-HPLC) system for analysis of vitamin D3 soft capsules with soybean oil matrix / 复旦学报（医学版）

Objective To develop a method for the determination and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in reverse phase high performance liquid chromatography (RP-HPLC) system.Methods RP-HPLC system had Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm,5 ttm) with detection wavelength of 265 nm,column temperature of 25 ℃ and flow rate of 1 mL/min.Retention behaviors of vitamin D3 and its 3 isomers were studied by altering the mobile phase.Firstly,acetonitrile was mixed with different proportions of methanol,water and ethanol as the mobile phase to investigate the effects of these 4 mobile phase components on the retention behavior of vitamin D3 and its 3 main related substances (isomers) on a C18 column.Then,a suitable mobile phase was selected for content determination and impurity analysis according to the retention behavior study.Results The recovery was only 80.55％-84.37％ with 100％ acetonitrile as the mobile phase.The addition of ethanol in acetonitrile was found to make remarkably significant improvement.Recovery rate was achieved between 98.07 ％ and 103.23 ％ with V (acetonitrile) ∶V (ethanol) =90∶10 as the mobile phase,while improving pealk shape.The method showed good linearity [(0.52-5.2) x 10-4 t mol/L,R2＞0.999] and fine density (RSD＜2.32％) which can be used for determination.For impurities profile,it could be achieved using V (acetonitrile):V (water) =95:5 as the mobile phase which can obviate interference from soybean oil matrix.Conclusions The method established in this experiment can easily and accurately determine the content and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in a RP-HPLC system.

Mechanism of photodynamic therapy against human esophageal carcinoma xenografts in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism of photodynamic therapy (PDT) in nude mice bearing human esophageal cancer cell line Eca-109 xenografts.</p><p><b>METHODS</b>A nude mouse model bearing human esophageal carcinoma was established by subcutaneous transplantation of Eca-109 cells. The mice were then randomized into 4 groups, namely hematoporphyrin derivative (HpD)-PDT group (given HpD and laser irradiation), exclusive laser irradiation group, exclusive HpD group and blank control group. In HpD-PDT group, the mice were exposed to irradiation at the light energy density of 120 Jsol;cm(2) delivered via a DIOMED 630 PDT system 24 h after intraperitoneal HpD injection, and the mice in exclusive laser irradiation group received only laser irradiation. Three days later, all the nude mice were sacrificed for determination of malondialdehyde (MDA) production, immunohistochemistry for caspase-3 protein and HE staining of the tumor tissue.</p><p><b>RESULTS</b>The MDA level was significantly higher in HpD-PDT group than in the other 3 groups (P<0.01), and comparable between the latter 3 groups. Expression of caspase-3 protein was similar between HpD-PDT group and the blank control group (P>0.05). Under light microscope, HE staining visualized massive tissue necrosis in HpD-PDT group with homogeneous red staining.</p><p><b>CONCLUSION</b>In human esophageal carcinoma xenografts in nude mice, HpD-PDT generates singlet oxygen to result in direct tumor cell damage and cause MDA production. Caspase-3 may not be activated in the apoptotic pathway, suggesting that this pathway may not be caspase-3-dependent.</p>

Radioimmunotherapy with 131 I-labeled humanized anti-HBsAg Fab and anti-nucleus antigen monoclonal antibody chTNT in nude mice bearing human hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the feasibility and efficacy of radioimmunotherapy with 131 I-labeled humanized anti-HBsAg Fab (131 I-anti-HBsAg Fab) combined with 131 I-labeled anti-nucleus antigen monoclonal antibody chTNT (131 I-chTNT) in nude mice bearing human hepatocellular carcinoma.</p><p><b>METHODS</b>Nude mice bearing subcutaneous human hepatocellular carcinoma xenografts were treated by intratumoral injection of 131 I-anti-HBsAg Fab and/or 131 I-chTNT, and the changes in the tumor size and alterations in the radioactivity concentration in the tumor and non-tumor tissues were observed.</p><p><b>RESULTS</b>The tumor inhibition rate in mice treated with 131 I-anti-HBsAg Fab combined with 131 I-chTNT (73.09%) was significantly higher than that in mice treated with 131 I-anti-HBsAg Fab (47.8%) or 131 I-chTNT (54.26%) alone. Combined treatment also resulted in significantly higher tumor-to-normal radioactivity concentration ratios than the treatment with the single agents.</p><p><b>CONCLUSION</b>Intratumoral injection with 131 I-labeled monoclonal antibodies can increase the radioactivity concentration in the tumor and enhance the efficacy of the radioimmunotherapy in nude mice bearing human hepatocellular carcinoma.</p>

Inhibitory effects of recombinant human endostatin on the metastatic potential and invasiveness of hepatocellular carcinoma HCCLM6 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of recombinant human endostatin (rh-ES) on cell adhesion, metastatic potential and invasiveness of hepatocellular carcinoma HCCLM6 cells in vitro.</p><p><b>METHODS</b>The changes in the cell proliferation status of HCCLM6 cells treated with different concentrations of rh-Endostatin in vitro was measured with MTT assay, and their invasiveness and migration were assayed using transwell cell culture chamber. The cell adhesion assay was carried out on 96-well plate precoated with matrigel.</p><p><b>RESULTS</b>Rh-ES showed inhibitory effect against the proliferation of HCCLM6 cells after a 72-h treatment. The adhesion, metastatic potential and invasiveness of the cells were obviously inhibited by rh-Endostatin in a dose-dependent manner.</p><p><b>CONCLUSION</b>Rh-ES inhibits the adhesion, invasiveness and migration of hepatocellular carcinoma cells in vitro, and the mechanism needs further investigation.</p>

Effects of bevacizumab and cisplatin on human lung adenocarcinoma A549/DDP xenografts in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of bevacizumab with or without cisplatin (DDP) on the growth of lung adenocarcinoma A549/DDP cell xenografts in mice.</p><p><b>METHODS</b>Human lung cancer A549/DDP cells was subcutaneously transplanted in to 25 nude mice, which were randomly divided into control group (group A), bevacizumab group (group B), DDP group (group C), combined treatment group (group D) and half-dose combined treatment group (group E). After corresponding treatments for 4 consecutive weeks, the tumor inhibition rate was evaluated, tumor microvessel density (MVD) measured with immunohistochemistry, and the mRNA expression of apoptosis-associated gene (bcl-2) and multidrug resistance genes (LRP and GST-pi) assessed by RT-PCR.</p><p><b>RESULTS</b>The tumor growth inhibition rates in groups B, D, and E with bevacizumab treatment were 20.96%, 51.67% and 50.95%, respectively, and the two combined treatment groups showed better effects. MVD in these 3 groups were 18.6-/+1.14, 13.6-/+1.14, and 14.4-/+0.55, respectively, and no significant difference was found in MVD between DDP group and the control group. Compared with the control group, the 3 bevacizumab-treated groups showed decreased expression of bcl-2 genes in A549/DDP tumors at a comparable amplitude, and LRP and GST-pi mRNA expression showed no significant differences between the 5 groups.</p><p><b>CONCLUSION</b>Bevacizumab has synergetic inhibitory effect with conventional chemotherapy against lung adenocarcinoma A549/DDP cell xenografts in mice by inhibiting angiogenesis of the tumor, and may enhance the sensitivity of A549/DDP cells to DDP by inducing cell apoptosis.</p>

Procyanidins inhibit proliferation and promote apoptosis of the prostate cancer cell line LNCaP / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of procyanidins on proliferation and apoptosis of a hormone-dependent prostate cancer cell line LNCaP in vitro.</p><p><b>METHODS</b>LNCaP cells were cultured in the presence of procyanidin (at 100, 200 and 300 microg/ml, respectively). After 24, 48 and 72 h of culture, the morphological changes of LNCaP cells were examined, and the inhibition of cell proliferation was measured by MTT assay and cell apoptosis evaluated by flow cytometry.</p><p><b>RESULTS</b>After procyanidins treatment, some LNCaP cells presented characteristic morphological changes. Procyanidins inhibited the growth of LNCaP cells in a concentration- and time-dependent manner. Flow cytometry analysis showed that procyanidins induced apoptosis of LNCaP cells and the percentage of apoptotic cells increased with the concentration of procyanidins administered and also the elongation of treatment time.</p><p><b>CONCLUSION</b>Procyanidins inhibit the proliferation and promote the apoptosis of prostate cancer cells.</p>

Avastin combined with cisplan inhibits malignant ascites production in nude mice bearing transplanted ovary carcinoma with high VEGF expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a nude mouse model of malignant ascites with human ovarian carcinoma cell line OVCAR3 which highly expresses VEGF and evaluate the therapeutic of Avastin combined with cisplan.</p><p><b>METHODS</b>Forty-eight nude mice with malignant ascites resulting from intraperitoneal transplantation of human ovarian carcinoma cell line OVCAR3 were treated with intraperitoneal injection of Avastin, cisplan, their combination, and PBS, respectively, to observe the effect on ascites development, VEGF content in the ascites, peritoneal permeability, development of new vessels and number of tumor cells in the ascites.</p><p><b>RESULTS</b>Avastin obviously inhibited ascites accumulation and peritoneal capillary permeability, reduced VEGF protein level and microvascular density in the tumor tissues and the number of red cells and tumor cells in the malignant ascites, and prolonged the survival of the mice. The combination of Avastin and cisplan further enhanced the therapeutic efficacy of Avastin.</p><p><b>CONCLUSION</b>The bio-chemotherapeutic strategy with Avastin combined with cisplan can be a promising method for treatment of malignant ascites.</p>

Percutaneous radiofrequency ablation therapy with left single lung ventilation for liver carcinoma in the hepatic dome / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the advantages of percutaneous radiofrequency ablation (PRFA) therapy with contralateral single lung ventilation (SLV) for liver carcinoma in the hepatic dome (LCHD).</p><p><b>METHODS</b>The clinical data of 10 patients (the SLV group) with LCHD consecutively treated from January to December 2006 were retrospectively analyzed. And another 10 cases (the control group) with LCHD treated from January 2004 to December 2005 were selected with a strict inclusion criterion for compared test according to rules of same diagnosis, similar tumor bulk and site, same sex, similar age and liver function. The patients' ages and tumor diameters of the 2 groups were compared with t-test and the rates of complications and incomplete tumor ablation were compared with chi2-test.</p><p><b>RESULTS</b>There was no statistical difference in ages and tumor diameters between the 2 groups (P > 0.05). The average number of radiofrequency ablation needle punctures in the SLV group was significantly less than the control group (3.4 +/- 0.4 vs. 6.1 +/- 0.8, P < 0.01). There was no bronchial intubation related complications like hypoxemia, atelectasis, lung infection and no puncture related complications like pneumothorax, hemothorax, hemoperitoneum and bile leakage in the SLV group. Two cases in the control group had complications including pneumothorax (n = 1) and pleural effusion (n = 1). There was no mortality in the 2 groups. Though the rate of incomplete tumor necrosis in the SLV group was not statistically lower than that in the control group (10% vs. 40%), the occurrence rate of the undesirable event (complication and incomplete tumor necrosis) of the SLV group was significantly lower than that of the control group (10% vs. 60%, P < 0.05). The durations and costs of operating procedure were not significantly different between the 2 groups.</p><p><b>CONCLUSION</b>Left SLV makes PRFA for LCHD more efficient, effective and safe.</p>

Main factors affecting the effect of photodynamic therapy against human esophageal cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the tumor cell-killing effect of photodynamic therapy against human esophageal cancer cells in vitro and identify the main factors affecting the effect.</p><p><b>METHODS</b>Human esophageal cancer Eca-109 cells were incubated for 24 h in vitro with hematoporphyrin derivative (HpD) and Photofrin at different concentrations prior to exposure to a light energy density of 15 J/cm(2) delivered from a DIOMED 630 PDT system. The cell killing effect was also evaluated for different HpD concentrations combined with 3 light energy densities (10, 30, and 50 J/cm(2)), respectively. The cell survival rate was measured using MTT assay, and fluorescence spectrometry was used to detect the intracellular photosensitizer fluorescence of the tumor cells after incubation with HpD for 4 h.</p><p><b>RESULTS</b>The cell survival rate after incubation with the two photosensitizers at different concentrations were significantly different, and under the 3 different light energy densities, incubation of the cells with different HpD concentrations also resulted in significantly different cell survival rates (P<0.05). At the 4 low photosensitizer concentrations and with different light energy densities, the cell survival rates were similar (P>0.05), but the 4 higher photosensitizer concentrations resulted in significant difference in the cells survival (P<0.05). Correlation analysis showed that the intracellular photosensitizer concentration was positively correlated to the photosensitizer concentrations in cell incubation (r=0.997).</p><p><b>CONCLUSION</b>When the light source remains constant, the light energy density, the kinds of photosensitizers and their concentrations are the main factors affecting the Eca-109 cell-killing effect of PDT.</p>

Hematoporphyrin derivative-mediated photodynamic therapy for human nasopharyngeal carcinoma: a comparative study with CNE2 and C666-1 cell lines in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate biological effect of hematoporphyrin derivative (HpD) photodynamic therapy (PDT) on in vitro cultured nasopharyngeal carcinoma (NPC) cell lines CNE2 and C666-1.</p><p><b>METHODS</b>CNE2 and C666-1 cells cultured in vitro were incubated in a medium containing HpD at different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 microg/ml) for 4 h followed by exposure to different light doses (2, 5, 10, and 20 J/cm2) using a diode laser at 630 nm with power density of 20 mW/cm2. After 24 h of incubation with HpD-PDT, the survival rate of CNE2 and C666-1 cells were analyzed by MTT assay.</p><p><b>RESULTS</b>HpD-PDT produced effective killing of CNE2 and C666-1 cells cultured in vitro, and the killing effects were positively correlated with HpD concentration and the irradiation dose. Exposure of CNE2 and C666-1 cells to irradiation dose of 20 J/cm2 resulted in the IC50 of 0.7 and 1.2 microg/ml, respectively (P<0.01). With the same HpD concentration and irradiation dose, the survival rate of C666-1 cells, however, was significantly higher than that of CNE2 cells (P<0.05).</p><p><b>CONCLUSION</b>HpD-PDT may result in effective killing of CNE2 and C666-1 cells cultured in vitro, although C666-1 cells are less sensitive to HpD-PDT than CNE2 cells.</p>

Construction and identification of recombinant adenoviruses containing the gene fragments encoding Her2/neu extracellular and transmembrane domains / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct eukaryotic expression vectors using recombinant adenovirus containing the gene fragments encoding Her2/neu extracellular first ligand-binding domain (Her2-ECD), full-length extracellular domain (Her2-ECD), and extracellular and transmembrane domain (Her2-TM).</p><p><b>METHODS</b>The cDNAs were amplified by RT-PCR and inserted into shuttle pAdTrack-CMV plasmids. Viral plasmids were obtained from homologous recombination in E. coli BJ5183, and transfected into 293 cells via liposome. Formation of viral plaque and expression of green fluorescent protein were observed by fluorescence microscopy, and the target proteins were detected by Western blotting.</p><p><b>RESULTS</b>The target cDNA fragments were amplified by PCR with expected lengths and the DNA sequences were confirmed against Genbank. Formation of viral plaque, expression of green fluorescent protein and the target proteins were detected in 293 cells transfected by the viral plasmids, which showed elevated expression of Her2/neu protein with the increase of multiplicity of infection (MOI).</p><p><b>CONCLUSION</b>The eukaryotic expression vectors using recombinant adenovirus have been successfully constructed for expression of Her2/neu extracellular and transmembrane domains.</p>

Preparation of hematoporphyrin-herceptin photoimmunoconjugate for photoimmunotherapy / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare photoimmunoconjugate of hematoporphyrin (HP) and herceptin, and study its killing and apoptosis-inducing effect on tumor cells BT-474.</p><p><b>METHODS</b>HP-herceptin photoimmunoconjugate was synthesized with EDCI as the condensator. After exposure of the cells to 630 nm laser, the killing effect of the conjugate and cell apoptosis were evaluated by MTT assay and flow cytometry.</p><p><b>RESULTS</b>Compared with free HP at equivalent dose, the immune reactivity, killing effect and the apoptosis-inducing effect of HP-herceptin immunoconjugate on BT-474 cells was enhanced (P<0.05).</p><p><b>CONCLUSION</b>The killing effect of HP-herceptin immunoconjugate is stronger than free HP on BT-474 cells.</p>

Adsorption condition optimization for anti-HBsAg Fab fragment separation and purification from E. coli using Streamline SP / 南方医科大学学报

<p><b>OBJECTIVE</b>To optimize the adsorption condition of cation-exchange chromatographic media Streamline SP for separation and purification of anti-HBsAg Fab fragment from E. coli.</p><p><b>METHODS</b>The adsorption of the target protein for separation and purification by the cation-exchange chromatographic media Streamline SP was tested using test tube method in balanced buffer solution with different pH values and ion concentrations. The adsorption effect was then verified by cation-exchange chromatography using 1-ml Streamline SP prepacked column and 28-ml Streamline SP self-assembly column.</p><p><b>RESULTS</b>According to the experiment results of test tube method, the loading buffer with pH of 4.4 and ionic concentration of 100 to 600 mmol/L could achieve optimal target protein adsorption effect by cation-exchange chromatographic media Streamline SP, as verified by cation-exchange chromatography with 1-ml SP prepacked column and 28-ml Streamline SP self-assembly column.</p><p><b>CONCLUSION</b>The optimal condition of cation-exchange chromatography selected by test tube method can be applied for separation and purification of anti-HBsAg Fab fragment from E. coli.</p>