OBJECTIVE: To study the effect of acid sphingomyelinase (ASMase) on endoplasmic reticulum stress (ERS) in vitamin E succinate (VES)-induced apoptosis in human gastric cancer cells. METHODS： SGC-7901 human gastric cancer cells were cultured in vitro. The cells were divided into four groups: desipramine (DES) group (cells were treated at 12.5 μmol/L for 2 h)，control group (containing 2% FBS RPMI-1640 culture medium)，VES+ DES group (cells were treated with DES at 12.5 μmol/L for 2 h，then with VES at 20 μg/ml) and VES group (20 μg/ml). ASMase activity detection kit was used to assess the ASMase activity of VES and VES + DES groups at 0，1.5，3 and 6 h. MTT assay was used to measure the growth inhibition at 12，24，48 h. Cell morphology was examined by the inverted microscope after treated for 24 h，and flow cytometry was used to evaluate the apoptosis，then we measured the ERS-related protein expressions of GRP78，GRP94，Perk，p-Perk，Chopand Caspase-4 by Western blot. RESULTS： ASMase activity continuously increased for VES (20 μg/mL) treatment，peaked at 3 h，then gradually decreased. ASMase activity of VES+DES group remained at a low level. There was no significant change in control group and DES group throughout the tests. Cell proliferation in VES group and VES+DES group was obviously inhibited after treatment for 12，24 and 48 h (all P<0.05) and showed a time-effect trend. Cell relative growth rates at different time points in VES+DES group were significantly higher than those in VES group(all P<0.05). The number of dead cells obviously increased in VES group，and the number of dead cells in VES+DES group decreased compared with VES group. Apoptosis rate evaluated by flow cytometry revealed that VES group (39.21%±1.90%) was significantly higher than the control group(3.91%±1.68%) and the DES group(4.07%±1.39%)(P<0.05)，VES+DES group (19.47%±4.46%) was significantly lower than the VES group(P<0.05). ERS-related protein GRP78，GRP94，p-Perk，Chop and c-Caspase-4 expression levels in VES+DES group were lower than those in VES group (all P<0.05). CONCLUSION：VES may induce apoptosis of human gastric cancer cell SGC-7901 cells though ERS by ASMase regulation pathways.

OBJECTIVE: To verify the lethal effects on cervical cancer cells by ultrasonic irradiation combined with lentivirus vector microbubble of double suicide gene，to lay the foundation of targeted gene therapy in cervical cancer. METHODS：After constructing lentivirus vector carrying the double suicide gene system (pLenti6-KDRP-CD/TK-EGFP)，transfecting it into the 293T cells，computing virus titer and mingling with microbubble (sonovue)，we then examined the encapsulating rate and drug loading capacity. The cervical cancer cells (HeLa) were divided to four groups：the blank group；the lentivirus vector group；the lentivirus vector microbubble group；ultrasonic irradiation+lentivirus vector microbubble group：(radiated 30 s by 0.25，0.5，1.0，2.0 W/cm2) 300 kHz. We examined the effect on proliferation of cervical cancer cells by MTT assay and apoptosis by flow cytometry. RESULTS：The virus titer，encapsulating rate and drug loadings capacity were assessed and the expression of EGFP were examined under the fluorescence microscope. MTT assay showed that proliferation of cervical cancer cells was obviously inhibited in the lentivirus vector group，the lentivirus vector microbubble group and 0.25 W/cm2+ the lentivirus vector microbubble group (P<0.05). When the intensity of ultrasound was higher than 0.5 W/cm2，the inhibition effect was more significant (P<0.01). With progressively lighter intensity of ultrasound，inhibition rates of cervical cancer cells were gradually increased until 2.0 W/cm2，when the inhibition rates tended to be steady. After 24 h，the inhibition effect decreased obviously (P<0.05). After 48 h，there was no significant change for this effect (P>0.05). The apoptosis rates of cervical cancer cells were obviously increased in the lentivirus vector group and lentivirus vector microbubble group (P<0.05). When combining with ultrasonic irradiation，the apoptosis rate of cervical cancer cells was more significant (P<0.01). CONCLUSION： Microbubble carrying lentivirus vector of double suicide gene could kill cervical cancer cells (HeLa). The lethal effect on cervical cancer cells was obviously increased by combining with ultrasonic irradiation.

OBJECTIVE: To observe the effects of arsenic trioxide (As2O3) on human bladder cancer T24 cell proliferation and apoptosis protease activating factor-1 (Apaf-1) gene expression. METHODS：T24 cells in the logarithmic growth phase，were digested with 0.25% trypsin into a cell density of 1×105/mL single cell suspension. Then cells were inculated with different concentrations (0，1，2，4，8 μmol/L) of As2O3，as well as blank control group for 24，48 and 72 h. Cell proliferation inhibition rate was evaluated by MTT. After cell treatment with As2O3 (0，1，2，4，8 μmol/L) for 72 h，deoxyribose situ terminal transferase labeling (TUNEL) method was used to measure apoptosis；RT-PCR and Western blotting to assess the impact of As2O3 on Apaf-1 mRNA and protein expression. RESULTS：Compared with the control group， the 2，4，8 μmol/L As2O3 dose groups could effectively inhibit the proliferation of T24 cells in a concentration-dependent manner (24 h，r =0.962，P=0.038；48 h，r =0.959，P=0.041；72 h，r =0.973，P=0.027). Cells treated with As2O3 at 1-8 μmol/L for 72 h， showed typical apoptotic changes in a concentration-dependent manner (r= 0.993，P=0.007); whilst Apaf-1 mRNA and protein expressions were also significantly increased in a concentration-dependent (mRNA，r =0.986，P=0.014；protein，r =0.989， P=0.000). CONCLUSION：As2O3 could effectively inhibit bladder cancer T24 cell growth，proliferation and induce apoptosis. The possible mechanism may be related to increased Apaf-1 gene expression.

 OBJECTIVE: To investigate whether vitamin E succinate (VES) could induce autophagy in SGC-7901，and to explore the role of autophagy in cell proliferation inhibited by the VES. METHODS：Human gastric cancer cells SGC-7901 were treated with VES. Electron microscopy was used to study autophagosome morphology，and fluorescence microscopy for intracellular punctuate fluorescence of green fluorescence protein-LC3 (GFP-LC3). Western blot evaluated the level of autophagy marker protein LC3，and the activities of Akt and mTOR，the critical targets of Akt/mTOR signaling pathway. SGC-7901 cells were pre-treated with autophagy inhibitor，chloroquine (CQ) and 3-methyladenine (3-MA)，then we used MTT to detect the suppression of cell growth. RESULTS：Typical autophagic vesicles and autolysosome in VES treatment groups were identified. With increasing doses of VES，the punctuate fluorescence began to gather and intensity. LC3-II protein levels increased in cells treated with VES，suggested that VES may probably induce autophagy in SGC-7901. VES down-regulated the activities of the phosphory-Akt and phosphory-mTOR，suggested that the activity of Akt/mTOR signaling pathway was inhibited by VES. Combined action of VES+CQ group and VES+3-MA group significately suppressed cell growth compared with VES alone (P<0.05). When autophagy was inhibited ，the rate of cell proliferation was greatly reduced，suggesting that VES- induced autophagy had a protective effect on cell proliferation. CONCLUSION：Vitamin E succinate induced protective autophagy in human gastric cancer cells SGC-7901 via the Akt/mTOR signaling pathway.

 OBJECTIVE: To find anti-tumor effective fractions of Carthamus tinctorius，and evaluate the cytotoxic activity of its different effective fractions observed. METHODS：Cytotoxicity of six samples of safflower extract isolated by macroporous resin were evaluated in vitro against K562 (human leukemia cancer)，SMMC7721 (human liver cancer)， HeLa (human cervical cancer)，H1299 and A549 (human lung cancer) by CCK-8 assay. Concentration-effect curves and IC50 values were calculated. RESULTS：Safflower samples showed better cytotoxicity against H1299 and A549 with IC50 values of 16.24 to 189.86 μg/mL，and moderate cytotoxic activity or no activity against K562，SMMC7721 and HeLa. Insoluble fractions of safflower extract had better cytotoxic activity than water-soluble fractions. 95% alcohol elution fraction of the safflower extract had obvious cytotoxicity against five kinds of tumor cell lines with IC50 values of 16.24 to 66.51 μg/mL. CONCLUSION：Safflower extract could inhibit a variety of cancer cells，especially human lung cancer cell. The effective fraction may be the fat soluble components.

OBJECTIVE: To investigate the relationship between the expressions of osteopontin(OPN) and c-Met and its clinical significance in gastric cancer tissue. METHODS：Immunohistochemical method was used to detect the expression levels of OPN protein and c-Met protein in 69 tissue specimens from patients with gastric cancer，20 cases with nontypical hyperplasia，and 20 normal gastric mucosa specimens. We also investigated the relationships between their expressions and clinicopathological parameters；real-time quantitative reverse transcription-polymerase chain reaction(real-time PCR) was performed in 15 fresh gastric cancer samples and the corresponding normal tissue to detect mRNA expressions of OPN gene and c-Met gene. RESULTS：The positive rate of OPN was 69.6% in gastric cancer，which was significantly higher than 40.0% in nontypical hyperplasia tissues and 30.0% in normal gastric mucosa (P<0.05). The expression of OPN protein was closely correlated to the depth of invasion，TNM stage and lymph node metastasis. The expressions of OPN gene in gastric cancer tissues and normal gastric mucosa specimens were 2.08 and 0.46 times，respctively. The expression of OPN gene in gastric cancer tissues was significantly higher than normal gastric mucosa specimens (P<0.01). The positive rate of c-Met was 65.2% in gastric cancer，which was significantly higher than 30.0% in nontypical hyperplasia tissues and 20.0% in normal gastric mucosa (P<0.01). The expression of c-Met protein was closely correlated to the lymph node metastasis and TNM stage. The expressions of c-Met gene in gastric cancer tissues and normal gastric mucosa specimens were 0.21 and 0.03 times，respectively. The expression of c-Met gene in gastric cancer tissues was significantly higher than normal gastric mucosa specimens (P<0.01) . CONCLUSION：Increased expressions of OPN and c-Met may play important roles in the pathogenesis and progression of gastric cancer.

OBJECTIVE: To explore the relationship between the level of Methylation of the CpG island in the MT-3 gene in esophageal carcinoma in Kazaks and Hans ethnic groups. METHODS：The methylation of MT-3 gene was assessed by methylation specific polymerase chain reaction (MSP) in esophageal cancer tissues and adjacent tissues. RESULTS：The methylation rates of the MT-3 gene in esophageal cancer tissues and adjacent tissues were 66.7% (22/33) and 27.3% (9/33)，respectively in Kazaks，56.7% (17/30) and 26.7% (8/30)，respectively in Hans. Significant differences were seen in the methylation rates of MT-3 gene in esophageal cancer and adjacent tissues(χ2=10.280， P=0. 001； χ2=5.554，P=0.018). But there were no significant differences in the methylation rates in esophageal cancer and adjacent tissues between Kazaks and Hans. CONCLUSION：Aberrant promoter methylation of the CpG island in MT-3 gene may play an important role in the pathogenesis of esophageal squamous cell carcinoma，but it may not be a specific risk factor for esophageal cancer in the Kazaks.

 OBJECTIVE: To study the effect of doxorubicin on the development of cultured preantral follicles in female ICR mice and explore the preliminary mechanism of ovarian toxicity. METHODS：Cultured ovarian preantral follicles taken from day 12-14 female ICR mice were treated for 24 h with various concentrations of doxorubicin (0.4，0.8，1.6 and 3.2 μg/mL) on cultured day 2，6 or 11. Following the treatment，follicles were cultured to the endpoint. Follicle diameter measurement，follicle viability and cumulus-oocyte cell complexes (COCs) ovulation rate were used to confirm the effect on preantral follicle development. RESULTS：Compared to the control group， doxorubicin at the concentrations of 1.6 and 3.2 μg/mL could inhibit ovarian preantral follicle development (P<0.05)， decrease the viability (P<0.05) and inhibit COCs ovulation (P<0.05) in a concentration-dependent manner.  CONCLUSION： Doxorubicin could significantly inhibit the development of ovarian preantral follicles，decrease the viability and inhibit COCs ovulation，which could induce ovarian toxicity and dysfunction.

OBJECTIVE: To study the protective effect and mechanisms of ethyl pyruvate(EP) on pulmonary inflammation induced by chlorine gas. METHODS：Adult male SD rats were exposed to chlorine gas of 2 536 mg/m3 or normal air for 20 min in a whole-body dynamic exposure chamber. EP (40 mg/kg) or vehicle(saline) was given by intraperitoneal injections immediately after chlorine gas exposure. Rats were killed at 6 hours and 24 hours after chlorine gas exposure. At 6 hours，levels of TNF-α，IL-1β and IL-8 in bronchoalveolar lavage fluid (BALF) and the nuclear translocation of nuclear factor κB (NF-κB) in the lung were evaluated. At 24 hours，the expression of HMGB1 and the activity of type-ⅡA secretary phospholipase A2 (sPLA2-ⅡA)were measured. RESULTS：At 6 hours after chlorine gas exposure，levels of TNF-α，IL-1β and IL-8 in BALF as well as NF-κB nuclear translocation increased significantly(P<0.05). Furthermore，at 24 hours，HMGB1 expression and sPLA2-ⅡA activity too were significantly increased(P<0.05). EP treatment attenuated these changes(P<0.05). CONCLUSION： Chlorine gas induced inflammation in the lung via activation of inflammatory mediators at early as well as late stage. EP treatment attenuated inflammation in the lung induced by chlorine gas.

OBJECTIVE: To study the combined effects of formaldehyde and benzene on the development of mice embryo after implantation. METHODS：Pregnant mice were randomly divided into four groups and exposed to different concentrations of formaldehyde and benzene starting from embryo implantation for 15 days. The concentrations of formaldehyde and benzene were 0 mg/m3 of formaldehyde and 0 mg/m3 of benzene for control group，(0.10±0.01) mg/m3 of formaldehyde and (0.11±0.01) mg/m3 of benzene for low dose group，(5.00±0.30) mg/m3 of formaldehyde and (5.50±0.40) mg/m3 of benzene for medium dose group，(10.00±0.50) mg/m3 of formaldehyde and (11.00±0.50) mg/m3 of benzene for high dose group. The general condition of pregnant mice，number of newborns as well as 24-hour survival of newborns were recorded. The body weight of newborns along with liver weight were also recorded to calculate liver coefficient. Total RNA was extracted from liver to detect the expression of cell cycle regulatory genes，Cdk2， CyclinE，Cdk7，CyclinH，by RT-PCR. RESULTS：The average weight of pregnant mice in high dose group was lower than the remaining groups (P<0.05) while the number of abortion was higher (P<0.05). In addition，the number of newborns and 24-hour survival of newborns in the high dose group were also lower than other dose groups，and the difference was statistically significant (P<0.05).The expression of Cdk2 in the medium and high dose groups were stronger than that in control group and low dose group. Besides，the expression of CyclinE in high dose groups was stronger than any other groups. The difference was statistically significant (P<0.05). The expressions of Cdk7 and CyclinH were not statistically significant (P>0.05) among the groups. CONCLUSION：Exposure of pregnant mice to formaldehyde and benzene after implantation could lead to abortion，the reduction of newborns and their survival. It could also cause hepatocyte cell cycle regulation abnormalities in newborn mice

 OBJECTIVE: To explore the cytotoxic effects of 9 different solvents with different volume fractions on CHL cells in vitro. METHODS：MTT was used to study the inhibition rates of different solvents on CHL cells at 0.1%，0.3%，1%，3%，10% volume fractions，and thus to calculate the volume fraction of each solvent causing cell inhibition rate of 20%. RESULTS：When the inhibition rates of DMSO，diethylene dioxide，acetonitrile， acetone， tetrahydrofuran，glycerine，acetic acid，methanol and ethanol on CHL cells reached 20%，the volume fractions were 1.0%，0.2%，1.5%，5.4%，0.2%，3.2%，0.000 07%，1.7% and 2.1%，respectively. CONCLUSION：When performing chromosome aberration test，one should first fully understand the characteristics of the test substance，on this basis， to select the appropriate solvent according to their characteristics and toxicities.

OBJECTIVE: To explore the teratogenic toxicity of konjac mannan oligosaccharide，hydrolyzed by mannase，in SD rats during sensitive period of teratogenesis. METHODS：Pregnant rats were divided randomly into four groups (12 in each group). Three groups were fed with konjac mannan oligosaccharide solution (625，1 250，2 500 mg/kg) while the negative control group was given deionized water. During the sensitive period of teratogenesis (7th to 16th day of gestation)，pregnant rats of each group were given test sample orally once on day 20 and then sacrificed. Nidation sites，living and dead embryos，embryonic and fetal development were evaluated. Meanwhile the appearance and skeletal abnormalities were examined. RESULTS：In konjac mannan oligosaccharide treated groups，the indexes including the weight of pregnant rats and uteri including fetuses，living and dead fetus rates，body weight and length and tail of fetuses were not significantly different from negative control (P>0.05). No abnormality of appearance，skeleton or organ was identified in fetuses of treated groups. CONCLUTION：Under our exprimental conditions，konjac mannan oligosaccharide showed neither maternal toxicity nor teratogenicity in pregnant SD rats.

OBJECTIVE: To investigate the toxicity on target organ after continuous intramuscular injection of ethyl pyruvate injection. METHODS：The high，medium and low dose groups and solvent control group were set up，according to the age，weight and gender. Each group had 15 animals. EP injections were given intramuscularly at 2 400，1 600，50 mg/kg，and equal volume of solvent in hindlimbs or buttocks for 14 days. We measured blood indicators and documented the extent of EP toxicity in rats. RESULTS：14 days after EP injection all animals survived. But there was overt toxicity within 5 minutes after high dose injection such as convulsion，pronation，hindlimb rigor and tachypnea. But these signs subsided gradually. No such serious toxicity were observeol in the medium and low dose groups. No flaccid toxicity was found in any group after drug withdrawal. CONCLUSION：EP injection at high dosage (2 400 mg/kg) exerted overt toxicity in the central nervous system，but reversible for 30 minutes after injection. Thus，as a single dose，EP injection had no long-term toxicity in rats.

OBJECTIVE: To explore the genetic toxicity of cetirizine amidine sodium and to evaluate whether cetirizine amidine sodium would change the genetic material in the body. Providing necessary experimental data for its development and application as a new drug. METHODS：Three tests were used to study the genetic toxicity of cetirizine amidine sodium： bone marrow cell micronucleus test，in vitro mammalian cells chromosome aberration test and salmonella typhimurium typhimurium mammals microsomal enzyme test (Ames test). RESULTS：The rates of micronucleus in each dose group and negative control group showed no significant difference (P>0.05). Cell chromosome aberration frequency in each group and in negative control group revealed no significant difference. Comparing with the solvent control group，the number of revertant colonies of TA97，TA98，TA100，TA102 strains did not increase twice or more times. CONCLUSION： Under this experimental condition，cetirizine amidine sodium did not demonstrate any genetic toxicity.

 OBJECTIVE: We used the D21S11 loci in the IdentifilerTM system analysis to predict Down’s syndrome. METHODS：Eleven samples of 5 confirmed and 4 suspected cases of Down’s syndrome family were amplified by the common IdentifilerTM kit in forensic DNA paternity identification，and their products were analyzed by ABI3130 DNA automatic sequencer. After the family cumulative paternity indexes were calculated to confirm their phylogenetic relationship，the three body loci of peak number and peak area ratio were analyzed. When there were three STR loci alleles fluorescence peak ( peak area ratio of 1∶1∶1) or two alleles fluorescence peak (peak area ratio of 2∶1 or 1∶2 or peak area ratio had not reached 2∶1/1：2，but <0.65 or > 1.8)，Down’s syndrome could be predicted . RESULTS： D21S11 locus contained highly polymorphic information. The detection results were consistent with the results of chromosome karyotype in five children diagnosed with Down's syndrome. The detection results of 10 samples in the four measured families with 11 samples were consistent with the results of chromosome karyotype and 1 sample being false negative. CONCLUSION：The IdentifilerTM kit D21S11 loci in the detection of Down’s syndrome had the characteristics of being simple fast，direct and the test effect was good，but there were false negative results. Future research will expand samples size and the number of loci detected on chromosome 21，to improve the accuracy.

OBJECTIVE: To evaluate the reproducibility and transferability of the erythrocyte(RBC) based Pig-a mutation assay and explore the potential of combining this assay and micronucleus(MN) analyses into one study. METHODS：20 rats were randomly assigned to be treated with PBS and different doses of (10，20 and 40 mg/kg) N-ethyl-N-nitrosourea (ENU) for 3 consecutive days by oral gavage. Jugular blood samples were collected on days -1 (the day before administration)，14，and 30 and evaluated for Pig-a mutantion frequencies in whole peripheral blood RBCs and reticulocytes (RETs). Day 4 samples were scored for micronucleated reticulocyte frequencies. RESULTS： While ENU-induced Pig-a mutation frequencies were consistent with previously reported results，analysis rate and number of cells evaluated were dramatically increased. ENU also induced significant increases in MN-RET frequencies. CONCLUSION： The in vivo Pig-a mutation high-throughput assay was established successfully in rat. Results with ENU indicated that the new Pig-a scoring methodology was reproducible Pig-a mutation and MN analyses could be readily combined into one study.