OBJECTIVE: To observe the inhibitory effect and strength of DVDMS-2-based-photodynamic therapy (PDT) on the growth of 4 kinds of cancer cells，S180 tumor in mice and human lung cancer cell H460 in nude mice. METHODS：The antiproliferative activity of DVDMS-2 was determined by MTT method. S180 tumor model was established in mice. All 35 mice were equally divided into 5 groups randomly：the negative control group，Photofrin positive control group and DVDMS-2 with low (38 J/cm2)，medium (76 J/cm2)，high (152 J/cm2) irradiation dose groups. DVDMS-2 at doses of 2.0 mg /kg was given intravenously to mice in low，medium and high irradiation dose groups on the day 2 after implantation of S180 cells. Negative control group was given saline in same volume，and positive control group was given intravenously. Photofrin at the dose of 10 mg/kg. PDT was given at 24 h after injection. All mice were killed at day 10 after PDT，and S180 tumors were taken out to measure the weight and calculate the tumor inhibition rate. Human lung cancer cells H460 transplant model was established in nude mice. DVDMS-2 at doses of 0.5，1.0 and 2.0 mg /kg was given intravenously on the day 7 after implantation of H460．Negative control group was given saline in same volume，and positive control group was given intravenously. Photofrin at the dose of 10 mg/kg. Then mice were treated with irradiation condition 76 J/cm2 (laser 630 nm) at 24 h after injection. All mice were killed at day 5 after PDT，and H460 tumors were taken out to measure the weight and size and calculate the tumor inhibition rate and the relative tumor-suppressing rate (T/C). Data were analyzed by SPSS software 13.0. RESULTS：Through the MTT assay，DVDMS-2-PDT could significantly inhibit the growth of cancer cells，including human hepatocellular carcinoma cells (HepG2)，human non-small lung cancer cells (H460)，human gastric cancer cells (BGC823) and human renal carcinoma cells (Ketr-3). MTT assay showed that the IC50 toward these tumor cells was 0.207-0.584 μg/mL. In the mouse model of S180 tumor，the tumor weight inhibition rates treated under irradiation condition (38，76，152 J/cm2) 24 h after DVDMS-2 (2 mg/kg) injection were 82.83%，88.56%，95.59% (P<0.05)，respectively. In human lung cancer cell H460 in nude mice，the tumor weight inhibition rates treated with DVDMS-2 (0.5，1，2 mg/kg) were 38.8%，47.9% and 53.9% (P<0.05)，respectively，under the irradiation condition of 76 J/cm2. CONCLUSION：DVDMS-2-PDT showed significant antitumor activities in vitro and in vivo.

OBJECTIVE: To investigate the effect of olaquindox on autophagy in human hepatoma G2 (HepG2) cells. METHODS：The HepG2 cells were treated with different concentrations (0，200，400，800 μg/mL) of olaquindox for 24 h. Then autophagy was analyzed by fluorescence microscope and flow cytometer following staining with monodansylcadaverine (MDC). HepG2 cells were treated with different concentrations (0，200，400，800 μg/mL) of olaquindox for 24 h or with 400 μg/mL olaquindox for different periods (0，1，3，6，12，24 h). Then the expressions of LC3-Ⅱ and Beclin 1 were determined by Western blot. RESULTS：Olaquindox-treated cells exhibited higher fluorescent density and more MDC-labeled particles in HepG2 cells compared with the control group. The percentage of MDC-positive cells was obviously increased after treatment with different concentrations (200，400，800 μg/mL) of olaquindox for 24 h. (P<0.05 or P<0.01). The expressions of LC3-Ⅱ and Beclin 1 increased with different concentrations and different time points of olaquindox treatment. Moreover，compared with the control group，the expression of LC3-Ⅱ and Beclin 1 were increased significantly with treatment of 400 μg/mL olaquindox for 24 h (P<0.01). CONCLUSION： Olaquindox enhanced the autophagic process in HepG2 cells.

OBJECTIVE: To analyze the radiation-influence induced changes of the number and phenotype of the thymocytes and its regulatory subsets in mice. METHODS：Mice were exposed to 2 Gy 60Co γ-ray in a whole body manner，and thymocytes were separated and counted in 1，4 and 10 d after irradiation. The percentage of CD4+ T and reg in thymocytes and the expression of CD39，CD103 in Treg were analyzed using FACS. RESULTS：There were differences in time-response to radiation. The thymocyte and CD4SP T cell numbers were decreased significantly after whole body irradiation and reached a minimum in 4 days，but recovered clearly after 10 days. However the Treg cell decreased in a manner of non-statistical difference in 4 days，but decreased significantly in 10 days. The percentage of Treg increased first then decreased in a time-response manner. The percentage of CD39+ Treg cell and MFI of CD39 were increased after 4 days’ irradiation. The percentage of CD103+ Treg cell was also increased，however its MFI was deceased after 4 days’ irradiation. CONCLUSION：Treg cells were more resistant to radiation than other thymocytes，resulting in the immune suppression of whole body. The expressions of CD39 and CD103 were differentially regulated by radiation and may play an important role in mediating suppressive function of Treg.

OBJECTIVE: Exploring the environmental estrogenic effect and mechanism of cell proliferation induced by organic extracts from Songhua River. METHODS：1 000 L water sample was collected from upstream water (Sifangtai source water)of Songhua River in March 2011. Organic substances were extracted by GDX-102 resin. MCF-7 cells were cultured with different concentrations of organics extracts (the concentrations were equivalent to those from 25，100，400，1 600 and 3 200 mL of river water). We measured estrogenic effect by MTT assay and investigated invasion and migration of MCF-7 cells by wound healing assay and transwell assay. We also explored the influence of organic extracts on Bcl-2 and Bax protein expressions by immunocytochemical method. RESULTS：The estrogenic effect was gradually enhanced with increasing concentration of organic extracts，significantly different compared to negative control group (P<0.05). The estrogenic effect and promotion of cell invasion and migration were the strongest when the content of organic extracts was equivalent to 1 600 mL of river water. Both Bcl-2 and Bax proteins could be expressed in MCF-7 cells. Comparing to negative control，with increasing concentration of organic extracts， the expression of Bcl-2 protein was gradually increased while that of Bax protein decreased. CONCLUSION：Organic extracts from upstream water(Sifangtai source water) of Songhua River could promote the proliferation of MCF-7 cells by regulating the ratio of Bcl-2 and Bax proteins，and stimulate invasion and migration of MCF-7 cells.

OBJECTIVE: To study the different nuclear protein expression in primary focus of downward progressive type of nasopharyngeal carcinoma in patients with different clinical patterns according to TCM classification. METHODS：Nuclear proteins profiles in fire-toxin stagnation type，Qi-yin deficiency type and Qi-blood coagulation type were measured by two-dimensional gel electrophoresis and matrix assisted laser. RESULTS：Compared with the expression of nuclear protein spots from patients with Qi-yin deficiency type，there were 28 up-regulated protein spots and 12 down-regulated protein spots from patients with Qi-blood coagulation type. We also found 9 up-regulated protein spots and 19 down-regulated protein spots from patients with fire-toxin stagnation type. Compared with the expression of nuclear protein spots from patients with Qi-blood coagulation type，there were 25 up-regulated protein spots and 18 down-regulated protein spots from patients with fire-toxin stagnation type. Three differential proteins were identified successfully by MALDI-TOF-MS. Up-regulated proteins in one spot were heterogeneous nuclear ribonucleoprotein H (hnRNP H) and tubulin beta-1 chain (TUB1) from patients with Qi-blood coagulation type and the other down-regulated protein spot was type VI collagen alpha 2 chain (COLⅥA2) precursor. CONCLUSION：Heterogeneous nuclear ribonucleoprotein H (hnRNP H) and tubulin beta-1 chain (TUB1) in nasopharyngeal carcinoma tissues from patients with Qi-blood coagulation type were up-regulated，type VI collagen alpha 2 chain precursor in those tissues from patients with fire-toxin stagnation type was down-regulated.

OBJECTIVE: To identify the key transcriptional regulatory regions in oral squamous cell carcinoma (OSCC) cell lines，and lays the groundwork for investigating the mechanism of ITGB6 transcriptional regulation in OSCC. METHODS：The recombinant pGL2 luciferase reporter constructs containing different lengths of 5' -flanking DNA fragments upstream of transcription initiation site of ITGB6 gene were constructed，and were transfected into two OSCC cell lines TCA8113 and SAS. The promoter activities were detected using dual-luciferase reporter assay system and the potential transcription factor binding sites at key transcriptional regulatory region of ITGB6 gene were predicted by bioinformatics method. RESULTS：The recombinant reporter constructs containing different lengths of 5' -flanking region of ITGB6 gene were obtained. When the length of ITGB6 5' -flanking region was reduced from -187 to -35 and -35 to +27，the transcriptional activity decreased significantly. Two potential Ets-1 binding sites and one potential IRF-4 binding site were identified in the region of -187 to +27 of ITGB6 gene. CONCLUSION：The -187/-35 and -35/+27 regions are the two key transcriptional regulatory regions of ITGB6 gene in OSCC cell lines.

OBJECTIVE: To investigate the effects of tributyltin(TBT) on the activity of protein phosphatase 2A(PP2A) in the mouse spleens，livers and brains. METHODS：Mice were orally dosed with 0，10，20 and 60 mg/kg of body weight TBT for 24 h and 96 h，and the activity of PP2A was assessed in the mouse spleens，livers，and brains. RESULTS：PP2A activity in the spleens was obviously inhibited in 20 and 60 mg/kg groups treated for 24 h and 96 h compared to the control group (P< 0.01). PP2A activity in the livers was significantly inhibited in the highest dose of TBT (60 mg/kg) for 96 h compared to the control group (P < 0.05). With regard to the PP2A activity in the brains，there were no statistical significance differences between the control and treatment groups (P>0.05). CONCLUSION：This study suggests a critical role of PP2A in the TBT immunologic and hepatic toxicity mechanisms.

OBJECTIVE: To study time-course and dose-response relationship of ENU-induced rat phosphatidylinositol glycan class-A(Pig-a) gene mutation and explore the potential of this assay to be integrated into repeat dose study，and to establish a rat Pig-a gene mutation assay. MEHTODS：Fifteen rats were randomly assigned to be treated with PBS and ENU (20 mg/kg and 40 mg/kg) for 3 consecutive days by oral gavage. Jugular blood samples were collected on days -1 (the day before administration)，15，30 and 45. Erythrocytes were enriched and incubated with Anti-CD59-PE and nucleic acid dye solution，and then analyzed with flow cytometer. RESULTS：On days 15，30 and 45，the Pig-a gene mutation frequencies in RBCs and RETs in low and high dosage groups were elevated significantly compared with that in control group (all P values <0.01)，and the mean frequencies in high dosage group were about 2-3 times of that in low dosage group. The Pig-a gene mutation frequencies were maintained at high levels during days 15-45. CONCLUSION：The in vivo Pig-a gene mutation assay was established successfully in rats. Time-course results suggested that this assay can be integrated into repeated-dose study.

OBJECTIVE: Based on the published literature of heavy metal toxicity on Caenorhabditis elegans，and after controlling the effects of other factors，we analyzed and compared the toxicity of heavy metals on Caenorhabditis elegans. METHODS：We used Scan It software to extract data from 20 related literature which contained figures expressing different indices of toxicity of various heavy metals on Caenorhabditis elegans，we then built a Meta regression model using toxicity indices as the dependent variables，and different metals，concentration，exposure time，etc. as independent variables. RESULTS：The order of heavy metal on the mortality of Caenorhabditis elegans was：Mercury (Hg)＞Copper (Cu)＞Lead (Pb)＞Chromium (Cr)＞Aluminum (Al)＞Cadmiun (Cd)＞Cobalt (Co)＞Nickel (Ni)＞Zinc (Zn)；affecting body size was：Pb＞Silver (Ag)＞Hg＞Co＞Al＞ Cr＞Zn＞Calcium (Ca)＞Ni；the effect on general time was：Ni＞Al＞Co＞Ag＞Cr＞ Zn＞Cu＞Pb； with head thrashes was：Hg＞Pb＞Ni＞Ag＞Cu＞Zn＞Cr＞ Co＞Cd＞Al. CONCLUSION：Meta regression model could be used to integrate information on the toxicity of different heavy metals，and to sort them in order. However，more experimental data on sorting of toxicity is necessary to validate the toxicity of some metals.

OBJECTIVE: To establish mouse preantral follicles 3-dimensions in vitro growth (3D-IVG) system and an efficient in vitro fertilization system. METHODS：Preantral follicles (PFs) of the 12 d old Kunming mice were isolated. 150 μm PFs were selected from each ovary. Then，PFs were cultured in 3D-IVG system for about 6 d. Ovulation of surviving follicles was induced by HCG and EGF. 36 h later，the mucificated cumulus-oocyte-complexes (COCs) and the matured oocytes were collected. Germinal vesicle breakdown (GVBD)，germinal vesicle (GV) oocytes and the matured oocytes were calculated under the stereomicroscope. Chromosomal abnormalities of matured oocytes including numerical and structural aberrations were examined and recorded by C-band strains compared with that of matured oocytes in vitro convention (IVC)．We put sperms and mature COCs together for 24-48 h，then， observed the zygote formation. RESULTS：In 3D-IVG system，the survival rate of the follicles was 82.5% and the mean diameter of follicles increased rapidly. After 6 d of culture ，the survival rate was 91.7％，the ovulation rate induced by hormone was 82.6％，6.1％ oocytes arrested at GV stage，45.4％ oocytes showed GVBD and 48.5％ oocytes emitted the first polar body．After 6 d 3D-IVG culture，the development rate (48.5%) of PFs was strikingly lower than in vivo oocytes superovulation (IVC，82.9％) (P<0.05). However，the chromosomal abnormalities of 3D-IVG have no significant increase comparing with that of oocytes in vitro maturation (IVM) and oocytes superovulation in vivo. We observed that those matured oocytes cultured from 3D-IVG system could get fertilized，from which we can draw a conclusion that they obtained the ability of developing to embryos. CONLUSION：We established and optimized mouse preantral follicles 3D-IVG system，which may provide a new method for reproductive toxicology and embryo engineering．

OBJECTIVE: To evaluate the in vitro genotoxicity of PBS [poly(butylene succinate)] in human peripheral blood mononuclear cells. METHODS：The cytotoxicity in PBMCs of PBS including different concentrations (200，100，50，25，12.5，6.25，3.125 mg/mL) was assessed by MTT and the genotoxicity of extracts was evaluated by chromosomal aberration test and micronucleus test. Meanwhile RPMI-1640 and medical biodegradable L-PLA (L-polylactide) were used as control groups. RESULTS：There was little toxicity in the extracts of PBS and no IC50 was valued. There were no cells with chromosomal aberration after co-culture with 200 mg/mL PBS for 48 h under metabolic and non-metabolic activation. The results of micronucleus test compared with the RPMI-1640 and L-PLA groups were not significantly different (P＞0.05). CONCLUSION：PBS showed less genotoxicity in peripheral blood mononuclear cells under this experimental condition. It may be a promising medical biodegradable polymer because of excellent biocompatibility.

OBJECTIVE: To detect the plasma protein levels of tissue inhibitor of metalloproteinases 2 (TIMP2) in lung cancer patients and healthy controls，and analyze its clinical significance. METHODS：Plasma TIMP2 level in 114 patients with primary lung cancer and 100 healthy subjects were measured by an enzyme immunoassay (ELISA). RESULTS：The plasma level of TIMP2 in patients with primary lung cancer (74.56±16.70) ng/mL was significantly lower than the healthy controls (170.98±48.61) ng/mL (P<0.01). The TIMP2 level in stage Ⅲ+Ⅳ(81.57±15.62) ng/mL were significantly higher than those in stage Ⅰ+Ⅱ(69.16±15.63) ng/mL (P<0.01). The plasma TIMP2 levels were elevated in the patients with tumor >3 cm (P<0.01)，and correlated with distant metastasis (P<0.01). When the TIMP2 cutoff value was set to 103.29 ng/mL，sensitivity of lung cancer detection was 97.4%， and specificity was 82.8%. CONCLUSION：Measurement of plasma level of TIMP2 protein may contribute to the diagnosis of lung cancer.

OBJECTIVE: Two procedures were chosen to study cyclophosphamide (CP) as a positive control in teratogenicity test of rats. One procedure was dosing CP 4.8 mg/kg from gestation day 6 (GD6) to GD15 continuously (group A). The other procedure was single dosing of CP 12 mg/kg on gestation day 12 (GD12) (group B). METHODS：The successfully mated SD rats were divided into three groups randomly，namely group A (dosing CP 4.8 mg/kg from GD6 to GD15 continuously)，group B (single dosing CP 12 mg/kg on GD12) and group C (the saline negative control group). The general condition of the treated rats was observed and the body weight was recorded every three days. Pregnant rats were euthanized by CO2 on GD20 for autopsy and reproductive organs were weighed. Fetal appearance， internal organs and skeletal malformations were checked and recorded. RESULTS：General conditions in group A and B were normal when compared with group C . Weights of uterus with placentas，placentas and uterus in group B were statistically lower than group C (P<0.05). Fetal appearance in group A showed no significant difference when compared with group C ，while in group B there were obvious deformities，including meningocele，microcephaly， brain exposed，club-foot，etc. Fetal internal organs examination in group A showed only the lateral cerebral ventricle enlarged or congested. However in group B internal organs malformations were found including the third and lateral cerebral ventricles enlarged or congested，and abnormal cardiac atrium or cardiac ventricle. Fetal skeletal examination showed little malformation in group A，with no significant difference when compared with group C. In group B，skeletal deformities incidence was higher in most bones than group C，mainly in the head and thorax，such as basisphenoid abnormalities，abnormal number of ribs，etc. CONCLUSION：In this experiment，dosing CP 12 mg/kg on GD12 induced more severe and more types of fetal malformation than dosing CP 4.8 mg/kg from GD6 to GD15 continuously. Therefore，dosing CP 12 mg/kg on GD12 was recommended as an effective positive control procedure in teratogenicity test of rats.

OBJECTIVE: To investigate the in vitro exposure and the genetic toxicity of native whole cigarette smoke，and to provide the basis for mutagenic evaluation of cigarette smoke. METHODS：Using VITROCELL VC10 smoke machine，dilution system and Ames exposure module，whole smoke was examined by Ames assay by adjusting the rate of air and diluted smoke，and the number of cigarette. RESULTS：Under the optimum condition， 5 mL/min diluted smoke and 4 cigarettes，whole smoke showed clear genetic toxicity with 30% S9 mixture and without the overlay agar. CONCLUSION：It was possible to analyse the effects of native whole smoke directly and achieve a dose-dependent induction of revertants. The procedure of exposing bacteria directly to gases and complex mixtures offers new possibilities in the field of inhalation genotoxicology for its evaluation in the Ames assay.

OBJECTIVE: To explore the sources and control methods of non-sample bands in the protein SDS-polyacrylamide gel electrophoresis. METHODS：The sample loading buffer，and its different component parts without protein sample were directly added to the gel and electrophoresed to identify the non-sample bands. Impact on non-sample bands of gradient concentrations of reducing agent and glycerol in the buffer were also assessed. The effective methods for removing the non-sample bands were further verified by pretreatment comb for preparation gel. Finally，method for reducing non-sample bands was tested by SDS-PAGE electrophoresis of protein of Vibrio parahaemolyticus. RESULTS：Non-sample bands appeared as long as the reducing agent and glycerol were both present in the buffer solution with comb pretreated by ordinary commercially available detergents，but the intensity of the bands showed no significant dose relationship with the concentration of reducing agent or glycerol. Treating the comb with 8 mol/L urea liquid，the non-sample bands showed weakening or even disappeared. CONCLUSION：Non-sample bands were caused by pollution of the comb. Treating the comb with a strong protein dissolving agent，such as 8 mol/L urea may be useful to reduce as eliminate non-sample bands.