BACKGROUND AND AIM: To screen the marker genes related to oral leukoplakia,laying foundation for the research in the genetic mechanism of oral leukoplakia. MATERIALS AND METHODS: In order to identify marker gene candidates, differential gene expression between normal tissues and precancerous oral tissues were examined by Oligo Cancer Microarray(SuperArray,USA).We further validated several positively expressed genes by RT-PCR,searching for the related genes which participated in malignant transformation.Total RNAs were isolated from two tissues, Chemiluminescent Detection Kit (SuperArray Bioscience, catalog number D-01),X-ray film and GEArray Expression Analysis Suite(supplied on internet) were used to analyze the genechips. In order to quantitate the expression level of the chosen genes in RT-PCR,Kodak Gel image analysis system was used.Lastly,we made a correlation analysis on the results of these two technologies. RESULTS: RT-PCR showed that the expression Levels of CTNNB1,GDF15,FKBP8 and NF1 genes differed significantly between the precancerous tissues and the normal tissues(P<0.05).The genechip test confirmed these results(P<0.05):the genetic mechanisms of oral leukoplakia were very complicated,particularly associated with cell cycle regulation genes and cell growth and differentiation genes. CONCLUSION: This research formed the basis for further studies of the genetic mechanisms of oral leukoplakia and marker gene screening.

BACKGROUND AND AIM: To observe the ultrastructure of embryonic palate cells during cleft palate formation using the model of congenital cleft palate. MATERIALS AND METHODS: The model of congenital cleft palate was induced in NIH mice by intraperitoneal injection of dexamethasone on GD12.5. The control group was raised under the same condition except the injection of dexamethasone. The mice was sacrificed by cervical dislocation on GD13.5,GD14.5 and GD15.5. The fetal mice were removed and the heads were cut for light and electron microscopic studies.The embryonic palate and the palatal cells were examined by SEM and TEM during the process of the palate development and congenital cleft palate formation. RESULTS: In the control group, with the embryonic development, the cleft was diminished, basal epithelial membrane was broken down, pieces of nuclear chromatin became marginated, excretion appeared in the MEE cell, the matrix was abundant between EPM cells, palatine shelves were fused completely on GD15.5. In the experimental group, palatine shelf grew slowly, and was smaller than the control group at the same time point, MEE cells on the palatine shelf surface were connected tightly yet with the development of palatine shelves, basal membrane remained intact, epithelial cell became multiplayer, cilia appeared on the cell surface, the matrix was less between EPM cells, the cleft was formed on GD15.5. CONCLUSION: The development and differentiation of embryonic palatal cells were affected after being exposed to dexamethasone, which was associated with cleft palate formation.

BACKGROUND AND AIM: To evaluate the expression of the AY245441 gene in normal embyonic palatine process and the cleft palate of mice induced by retinoic acid and to explore the effects between the AY245441 gene function and the formation of cleft palate . MATERIALS AND METHODS: The expression of the AY245441 gene in embyonic palatine process tissues in the experimental group induced by retinoic acid and control groups were assessed by Real-time PCR from GD11-GD16 and in situ hybridization on GD13 and GD14． RESULTS: AY245441 was expressed in all normal and cleft palates at GD11-GD16. To the normal palates, the highest expressions were in GD11,GD13 and GD14, showing significant differences to the samples (P<0.05) taken at other gestational dates. The expression abundance of AY245441 in normal palates was stronger than that of cleft palates during GD11－GD16(P<0.05). AY245441 expression was detected in both the epithelium of the anterior region of normal developing palatal shelves at GD13 , in both the epithelium and mesenchyme of the anterior region of normal developing palatal shelves at GD14. AY245441 gene expression was detected in the epithelium , but was suppressed in the anterior region of cleft palates at GD13. AY245441 gene expression was absent in both the epithelium and mesenchyme of the anterior region of cleft palatal at GD14. AY245441 was absent in both the epithelium and mesenchyme of normal posterior palate and cleft palate at GD14. CONCLUSION: The AY245441 gene played an important role in the development of palate in mice .The suppressed expression of the AY245441 gene was related to the occurrence of cleft palate．

BACKGROUND AND AIM: To explore the expression levels of pIgR/SC in lung cancer tissues and its clinical significance. MATERIALS AND METHODS: Tissue microarray (TMA) containing 484 sample of lung cancer, including 344 with squamous cell carcinoma (SCC), 93 with adenocarcinoma (ADC), 47 with small cell lung cancer (SCLC), and 30 normal controls from the same patients, were evaluated by immunohistochemistry (IHC). RESULTS: Immunohistochemical study showed that pIgR positive staining was found in 54.1％ lung cancers (especially in non-small cell lung cancer), significantly higher than in normal lung tissues(6.7％). The pIgR/SC expression was found in 66.3％ of SCC, 34.4％ and 4.3％ of ADC and SCLC specimens respectively (P<0.001). But pIgR/SC protein levels were not related to tumor differentiation and stage. CONCLUSION: Expression of pIgR/SC may reflect the histological difference of lung cancer, and over expression of pIgR/SC may happen at the early stage of non-small cell lung cancer.

BACKGROUND AND AIM: To study the protein expression of HBs and HBc genes brought into embryos via human spermatozoa. MATERIALS AND METHODS: Human spermatozoa were transfected with recombinant plasmid pIRES2-EGFP-HBV and were then fertilized with zona pellucida-free golden hamster ova. The 2-cell embryos with and without green fluorescence were collected by immunofluorescence assay and ELISA analysis, respectively. RESULTS: The immunofluorescence assay showed that no HBsAg- and HBcAg- positive signal was observed in 2-cell embryos without green fluorescence and the clear HBsAg- and HBcAg- positive signals were detected in ones with green fluorescence. ELISA analysis showed that the amount of HBsAg in a single 2-cell embryo was about 0.064 ng/ml and the positive result for HBcAg was detected. CONCLUSION: Human sperm-mediated HBs and HBc genes are able to express their proteins in early embryonic cells.

BACKGROUND AND AIM: To investigate the expression of Survivin protein and its relationship with cellular proliferation and apoptosis in differentiated thyroid carcinoma(DTC),and to identify the mechanism and pathway of Survivin in thyroid tumorigenesis. MATERIALS AND METHODS: The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique was used to measure the apoptosis index(AI). Expression of Survivin protein and proliferating cell nuclear antigen(PCNA) in 10 cases of nodular goiters (NG),10 thyroid adenomas (TA),30 papillary thyroid carcinoma(PTC) and 12 follicular thyroid carcinoma (FTC) were determined by immunohistochemistry staining. RESULTS: The positive staining rate of Survivin in thyroid carcinoma was higher than that in control group (P<0.01). The expression of Survivin in thyroid carcinoma was related to lymph node metastasis and clinical stage(P<0.05). In DTC group AI was negatively related with the PI in patients with positive survivin expression and in patients with negative Survivin expression(r=－0.72，r=－0.80，P<0.01),respectively.In DTC，the PI in patients with positive Survivin expression was higher than that in patients with negative Survivin expression. However,the AI in patients with positive Survivin expression was lower than that in patients with negative Survivin expression(P<0.01). CONCLUSION: Survivin demonstrated a closed relationship with cellular proliferation and apoptosis in differentiated thyroid carcinoma.It may play an important role in the development of thyroid carcinoma .

BACKGROUND AND AIM: To assess the growth-inhibiting effects of paclitaxel on p53-deficient human lung cancer H1299 cell line in vitro. MATERIALS AND METHODS: H1299 cells were exposed to paclitaxel at different concentrations or different treatment times, then the growth-inhibiting effects were determined by MTT assay, the change of the cell cycle and apoptosis were analyzed with flow cytometry. Fluorescence microscopy was used to observe the nucleolus change stained by Hoechst33342 and PI. RESULTS: The growth inhibition effect of paclitaxel on H1299 cells was time-dependent(P<0.05). With treatment concentration>1 nmol/L, the survival rate of H1299 cells in paclitaxel-treated groups decreased with increasing paclitaxel concentration, and showing significant difference with the survival rate in control group (P<0.05). The H1299 cells treated by 0.1－1 000 nmol/L paclitaxel were blocked at G2/M phase, and the percentage of G2/M phase increased in concentration-dependent manner. The proportion of apoptosis increased as concentration changed from 0 to 10 nmol/L(P<0.05). The induction of apoptosis was time dependent(P<0.05) and the percentage of G2/M phase was highest at 12 h with paclitaxel at 10 nmol/L. When treated with 1 000 nmol/L paclitaxel, H1299 showed the highest G2/M arrest at 24 h and some polyploidy cells appeared with prolonged treatment. Both concentrations could induce time-dependent apoptosis, but the higher concentration caused apoptosis later than the lower one. Apoptosis had no relationship with G2/M arrest(P>0.05). The coexistence of cell apoptosis and necrosis could be observed in paclitaxel-treated H1299 cells under fluorescence microscopy. CONCLUSION: Paclitaxel could induce apoptosis and necrosis of H1299 at the same time. It inhibited the growth of H1299 cells in vitro in time-and concentration-dependent manners.

BACKGROUND AND AIM: To evaluate the expression of cell proliferation and apoptosis in congenital pulmonary cyst and investigate their effects in the development of congenital pulmonary cyst. MATERIALS AND METHODS: Immunohistochemical method was used to measure the expressions of Ki-67、Bcl-2 and Bax in normal lung tissue from 5 children(control group 1)，relative normal lung tissue around the cyst from 5 patients with congenital pulmonary cyst(control group 2)and abnormal lung tissue from 30 with congenital pulmonary cyst. RESULTS: The expressions of Ki-67,Bax and the ratio of Bax/Bcl-2 in congenital pulmonary cyst were significantly higher than those in the two control groups(P<0.05)while the expression of Bcl-2 was markedly lower(P<0.01).In those with congenital pulmonary cyst, the expressions of Bax and Bcl-2 in ciliated epithelial cells(CEC) were much lower than that in non-ciliated epithelial cells (P<0.01) while the ratios of Bax/Bcl-2 was much higher (P<0.01). CONCLUSION: Cell proliferation and apoptosis in congenital pulmonary cyst were significantly higher than those in control groups. This indicated that the imbalance of both could play important roles in the development of congenital pulmonary cyst.

BACKGROUND AND AIM: To investigate the mechanisms for cytotoxicity of furazolidoze (FZD) to human hepatocarcinoma cell line (Hep G2). MATERIALS AND METHODS: After Hep G2 cells were exposed to FZD at the different concentrations(0、0.78、1.56、3.12、6.25、12.50、25.00、50.00 μg/ml)for 24 h, the proliferation effect was measured by MTT assay whilst the cell cycle distribution was assessed by flow cytometry. The mRNA of S stage checkpoint was evaluated by RT_PCR. RESULTS: MTT assay showed that Hep G2 survival rate decreased with increasing FZD concentration, whereas the survival rate was significantly higher with concentration above 6.25 μg/ml(P<0.05). Cell growth assay showed that the growth of Hep G2 was significantly slower than control. Treated with FZD (0－3.12 μg/ml), cells in S stage increased, while those in G1 stage decreased. The expression level of p21 increased and the expression of cyclinE, cyclinA ,Cdk2 decreased after treatment with FZD. CONCLUSION: FZD exhibited cytotoxicity to Hep G2 in vitro and induced cell arrest at S stage, and it may exert antiproliferative effect through activation of the S stage damage checkpoint.

BACKGROUND AND AIM: To evaluate the toxic reactions and severity in dogs fed continuously with selenium-enriched garlic. MATERIALS AND METHODS: Wistar dogs were divided into four groups (3.00 g/kg、1.50 g/kg、0.75 g/kg and a solvent control), fed continually for 12 weeks and observed for 2 weeks after stopping the medicine. General condition, haematology and biochemical tests, electrocardiogram, urine test, visceral dissection and coefficient and histopathology were performed. RESULTS: Dogs in the high dose group vomited intermittently, and appetite was decreasd. Serum total bilirubin was obviously increased, the albumin lowered, the coefficients of thymus gland and spleen went up. No other toxics reactions were observed. CONCLUSION: The selenium-enriched garlic was a mild alimentary tract stimulant with obvious increase in the serum total bilirubin, at 30 times the clinical dose. The dose of 0.75 g/kg in dogs had no obvious toxicity reaction.

BACKGROUND AND AIM: Coculture of 60Co-γ irradiated and nonirradiated osteogenic sarcoma U2OS cells was examined for the bystander effects and the interfering effects of Trolox. MATERIALS AND METHODS:groups with 5 samples each:control group,irradiated group,effect group and intervention group,the Trolox control group. In the irradiated group, U2OS cells were treated with 60Co -γ at the dose of 3 Gy and then cultured. In the effect group, after the medium of the irradiated cells was changed, and kept for 1 h, cells with digested with trypsin and collected. After that, the irradiated and nonirradiated cells were co-cultured in the ratio of 1∶1. The intervention group was treated the same as the effect group except the addition of Trolox (10 μg/ml final concentration) . At different time points, cells from the five groups were collected. The cell activity was determined by MTT; the colony formation efficiency and micronucleus frequency were assessed by counting method; the survival rates were estimated by Trypan Blue Staining; and the MDA content in the culture medium was determined by chemical fluorometric method. RESULTS: Compared with the control group, cells in the irradiated group exhibited decreased survival rate (P<0.05), suppressed cell activity (P<0.05), decrease in colony formation efficiency (P<0.05), increase in micronucleus frequency (P<0.05) and increase in MDA content in the culture medium(P<0.05). With the intervention of Trolox, exhibitions of the above indexes were greatly improved. CONCLUSION: Trolox could suppress the bystander and other effects as an antioxidant.

BACKGROUND AND AIM: The suppressive effects of cortex periplocae on human breast cancer BT_549 cells and the expressions of p16 and p27 were analyzed to demonstrate the possible mechanisms. MATERIALS AND METHODS: The suppressive effects of Cortex periplocae on proliferation of BT_549 cells was analyzed with MTT method. The cell cycle and apoptosis rate of BT_549 cells treated with Cortex periplocae were examined by flow cytometry. Expressions of p16 and p27 mRNA and proteins were assessed by semiquauntitative RT_PCR and flow cytometry. RESULTS: Cortex periplocae could obviously inhibit proliferation of BT_549 cells (P<0.01). Compared to control group, after treatment with Cortex periplocae, the cell number in G0/G1 phase of BT_549 cells was increased(P<0.01), the apoptosis rate was also increased significantly(P<0.01) as well as the expression of p16 and p27(P<0.05). CONCLUSION: Cortex periplocae exerted significant inhibitory effects on BT_549 cells in vitro which was probably related to improving the expressions of p16 and p27.

BACKGROUND AND AIM:To investigate the effects of lycium barvarum polysaccharides(LBP) on the reproductive system in male rats under chronic psychologic stress. MATERIALS AND METHODS: Rats were randomly divided into 6 groups:control group, stress group,high dose LBP group(hLBP),low dose LBP group(lLBP) , hLBP＋stress group and lLBP＋stress group.Sexual homone levels and sexual organ indexes were measured after four weeks. RESULTS: The sperm count and activity were increased significantly, and abnormality percentage was reduced in stress group. LBP protected the spermatogenic cells and promoted the normal development of testicular germ cells. CONCLUSION: LBP could enhance the reproductive function of chronic mentally stressed rats. The mechanism might be through adjustment of hypothalamus_pituitary_gonadal axis and target receptors.

BACKGROUND AND AIM: To study the effects of polychlorinated biphenyl(PCB) on rat liver structure and function. MATERIALS AND METHODS: 40 male Wistar rats were divided into four groups , A group was control group , B group (fed with 10－8 mol/L PCB)， C group(10－7 mol/L PCB), D group(10－6 mol/L PCB) as experimental groups.After three months, all rats were killed . Immunohistochemical and biochemical methods were used to observe the effects of PCB on liver structure and function in rats. RESULTS: The strutural damage of the liver was related to the dose of PCB, with D group showing more severe damage than that of others. PCB could induce c-fos expression in the liver, which was higher in C and D groups compared with other groups (P<0.05). PCB could promote the expression of bcl-2 in liver,with higher expression in C and D groups than that of A and B groups. PCB also could affect biochemical function of liver, such as URIC,TBLL,DBLL,AST,ALT,TP3,LDH to increase in C and D groups compared with A and B groups. CONCLUSION: PCB could lead to liver injuries in the rats.

BACKGROUND AND AIM: To explore the glyphosate-induced mutagenesis in mice. MATERIALS AND METHODS: In micronucleus test(MNT), glyphosate was given twice via intragastric administration to different groups of mice at different concentrations(2 320 mg/kg, 1 160 mg/kg, 580 mg/kg) at a 24-hour interval. Mice were killed 6 hours after last treatment to determine the micronucleus rates of polychromatic erythrocytes(PCE) in the bone marrow. For sperm shape abnormality test, all mice were treated with glyphosate for 5 consecutive days at concentrations of 1 160 mg/kg, 290 mg/kg and 580 mg/kg. In the 1st and 4th week after the first treatment, half of mice were killed each time to regularly examine the sperm aberration rates, to count the total number of sperm, to weigh the testis and the epididymis，and to observe the change of organ coefficient. RESULTS: The micronucleus rate of 2 320 mg/kg group was significantly higher than that of negative control(P<0.01).A dose-dependent response was observed between the treatment dose and the micronucleus rate(r=0.588,P<0.01). One week after the first treatment, compared with negative control, the 580 mg/kg, 1 160 mg/kg groups both showed increasing rate of sperm aberration and decreased total number of sperm(P<0.05，P<0.01)，while the 1 160 mg/kg group showed significantly decrease in epididymis weight and its coefficient(P<0.05). Four weeks after the first treatment, by comparing to negative control, 1 160 mg/kg group showed increasing rate of sperm aberration and decreased total number of sperm(P<0.05)，as well as a decline in the weights of testis and epididymis. CONCLUSION: In this study, glyphosate showed reproductive toxicity in mice and certain mutagenesis.

BACKGROUND AND AIM: Study on toxicity effects of aconitine on rat Leydig cells. MATERIALS AND METHODS: Primary cultured cells of rat Leydig cells was used in this study. Leydig cells were isolated from testis of adult male SD rats. To purify Leydig cells, testicular cells were centrifuged with discontinuous Percoll gradients, and the Leydig cells were identified by 3β-HSD staining. Leydig cells were exposed to aconitine with concentrations of 5×10, 5×102, 5×103 and 5×104 ng/ml. Cell viability was determined by the MTT method. Concentration of MDA and activity of SOD were determined by assay kits, and concentration of testosterone in the media were measured by specific radioimmunoassay. RESULTS: Cell viability showed no significant difference in all aconitine dose groups compared to DMSO control group at 24 h and 48 h culture period(P>0.05).Concentration of MDA and activity of SOD revealed no significant difference in all aconitine dose groups compared to DMSO control group at 24 h culture period (P>0.05). The concentration of testosterone also showed no significant difference in all dose groups compared to DMSO control group at 4 h culture period(P>0.05). CONCLUSION:This study demonstrated that aconitine at 5×10, 5×102, 5×103 and 5×104 ng/ml demonstrated no obvious toxicity on Leydig cells.

BACKGROUND AND AIM: To prepare specific monoclonal antibody (mAb) against rabbit NADP(H)-dependent retinol dehydrogenase／reductase (NRDR) for further exploration of its structure and function. MATERIALS AND METHODS: The BALB/c mice were immunized with the recombinant rabbit NRDR expressed by genetic engineering. Splenocytes of immunized mice were collected and fused with the mouse myeloma cell line NS-1 cells. Hybridomas that secreted rabbit NRDR mAb were cloned with limited dilution method. Characteristics of mAb(Ig subclasses，titers and specificities) were identified and determined by indirect ELISA and Western blot. RESULTS: From over 80 positive hybridomas which secreted anti-rabbit-NRDR mAbs，three clones of hybridoma were obtained, and designated as NR1、NR2 and NR5. They were all of IgG1 subclass. The cell culture supernatant titers of NR1,NR2 and NR5 were1∶20,1∶40 and 1∶20,respectively. Ascite titers of NR1,NR2 and NR5 mAb were 1∶106, 1∶107 and 1∶106, respectively. Western blot analysis showed that mAbs had specific binding abilities with NRDR in rabbit liver and recombinant rabbit NRDR. CONCLUSION:The hybridomas secreting mAbs to rabbit NRDR were established successfully and primarily identified，which could lay the basis for further research on the biological structure and function of NRDR.

BACKGROUND AND AIM: Multi-color fluorescence in situ hybridization(MFISH) was employed to evaluate the frequency of aneuploid sperm in patients with asthenospermia. MATERIALS AND METHODS:Patients were divided into 2 groups: 6 with asthenospermia and 3 normal healthy men used as control. MFISH and probes for chromosomes including X,Y and 18 were used to determine the frequency of aneuploidy in sperms. RESULTS: Analysis was performed on 30 305 sperms. The rate of hybridization was 99.98％. Nullisomy kinds included XX18, YY18, XY18, X1818, Y1818. Nullisomy frequencies were 0.133％％±0.095％,0.135％±0.231％,0.438％±0.415％,0.507％±0.973％,0.432％±0.705％,respectively, and were 0.011％±0.020％,0.006％±0.010％,0.051％±0.075％,0.023％±0.040％,0.034％±0.034％ in control group.Aneuploidy in the sperms of test group and control group were 2.02％ and 0.25％, respectively, significantly higher in the test group(P<0.01).CONCLUSION: Sperm aneuploidy can be reliably determined by sperm MFISH analysis in men with asthenospermia.