OBJECTIVE: To study the effects of TGF-β1 shRNA in proliferation and adhesion of human salivary gland mucoepidermoid carcinoma cell Ms in vitro. METHODS: Ms cells, Ms cells transfected with empty vectors and Ms cells transfected with TGF-β1 shRNA were cultured in vitro. Cell count assay, clone formation assay were performed to measure the proliferation of Ms cells. Adhesive abilities were assessed by cell-to-cell adhesion and cell-to-FN adhesion. Coomassie brilliant blue staining assay was used to detect the changes in cytoskeleton and immunoflurescence was used to evaluate the expression of Ezrin protein on Ms cells. RESULTS: The population doubling times of TGF-β1 shRNA-transfected cells, empty vector-transfected cells and the control cells were 30.8，28.8 and 29.0, respectively. The rates of clone formation were 14.2％, 24.5％ and 25.1％, respectively. TGF-β1 shRNA transfection increased the cell-to-cell adhesion by 18.6％ in a 30-min adhesion assay, but inhibited the cell-to-FN adhesion by 35.3％ in a 1-h adhesion assay. Compared with control and Ms transfected with empty vectors, Ms cells stably transfected with TGF-β1 shRNA grew slowly. The ability of homogeneous adhesion was increased and that of heterogeneous adhesion was inhibited. TGF-β1 shRNA changed the cytoskeleton and down-regulated the expression of Ezrin protein. CONCLUSION: TGF-β1 shRNA could affect the proliferation and adhesion, which may be associated with the changes of cytoskeleton and Ezrin protein expression.

OBJECTIVE： To investigate the regulatory mechanism of oncogene BCR/ABL fusion gene on PTEN signaling pathway in proliferation and apoptosis of K562 cells in vitro. METHODS: To detect the mRNA levels of BCR/ABL, PTEN and mTOR in K562 cells treated with different concentrations of Imatinib by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and the protein levels of Akt, p-Akt by Western blotting technique. RESULTS: The expression level of PTEN mRNA was up-regulated and the mTOR mRNA was down-regulated with the reduction of BCR/ABL fusion gene in the initial 36 h after 1 μg/ml imatinib inhibiting K562 cells. Then the PTEN mRNA decreased and the mTOR mRNA expression restored, but the p-Akt was continuously down-regulated with the restoration of BCR/ABL fusion gene 48 h later. BCR/ABL and PTEN mRNA showed a positive correlation; whilst BCR/ABL had a negative correlation with mTOR mRNA and p-Akt protein. CONCLUSION: BCR/ABL fusion gene could regulate p-Akt, mTOR pathway by inhibiting PTEN expression in K562 cells and affected cell proliferation, apoptosis and cell cycle.

OBJECTIVE： To study the expressions of Nrf2 and NQO1 mRNA, Nrf2 protein by coal tar pitch (CTP) extract. METHODS： BEAS-2B cells were treated with different concentrations of CTP extract (0.00, 1.25, 2.50, 5.00, 10.00 μg/ml) for 24 h. Total cell RNA and protein were extracted. The expressions of Nrf2 and NQO1 mRNA and Nrf2 protein were measured by the reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS：The expressions of mRNA and protein of Nrf2 among test groups(1.25－5.00 μg/ml) were significantly lower than those in the control group. With increasing concentration of CTP extract, the expressions of Nrf2 and NQO1 mRNA, Nrf2 protein increased. However, the mRNA and protein of Nrf2 decreased in the CTP 10.00 μg/ml group. CONCLUSION： When the concentration of CTP extract was at a 1ow 1eve1，the expression of Nrf2 increased in parallel with the CTP extract concentration. So Nrf2 might induce the gene expression of NQO1, enhancing the antioxidant ability of BEAS-2B cells.

OBJECTIVE: We investigated the promoter methylation of secreted frizzled-related protein 4(SFRP4) and SFRP5 gene in gastric cardia adenocarcinoma (GCA). METHODS: Methylation specific PCR (MSP) method was used to examine the methylation status of the 5' CpG island of SFRP4 and SFRP5 genes in 94 tumors and 47 corresponding normal tissues. RESULTS: Methylation frequencies of SFRP4 and SFRP5 genes in tumors were 68.1％(64/94)and 79.8％(75/94), respectively, significantly higher than that in corresponding normal tissues (8.5％ and 12.8％, respectively)(P<0.01). Methylation frequencies of SFRP4 in poor differentiation group (92.6％,50/54)was significantly higher than that in moderate and poor-moderate differentiation groups (35％,14/40). Methylation frequencies of SFRP5 in lymph node metastasis group (89.3％,50/56) was significantly higher than that in no lymph node metastasis group (65.8％,25/38). Methylation frequencies of SFRP5 in poor differentiation group was higher than that in moderate and poor-moderate differentiation groups, and SFRP4 in lymph node metastasis group was higher than that in no lymph node metastasis group but without significant difference(P>0.05). 57 cases of GCA showed simultaneous methylation of SFRP4 and SFRP5 genes, including 18 moderate and poor-moderate differentiation tumors, and 39 moderate differentiation tumors. Simultaneous methylation frequencies of SFRP4 and SFRP5 genes in moderate andpoor-moderate differentiation groups was significantly lower than that in moderate group. CONCLUSION: SFRP4 and SFRP5 genes might be associated with oncogenanesis of GCA and hypermethylation of these genes might be related to the malignant behavior of GCA.

OBJECTIVE: The anti-tumor activity of extract from cochinchina momordica seed (CMS) and its probable mechanism were investigated. METHODS: The growth-inhibitory effect of ethanol extract and water extract of Cochinchina Momordica Seed (CMSEE and CMSWE) on A549, MDA-MB-231, TE-13, B16 tumor cell lines and PBMC were examined by MTT assay. Morphological changes of tumor cells were analyzed by light microscope and Wight-Giemsa staining. The ratio of cell cycle and apoptosis rate were analyzed with Flow Cytometry (FCM). RESULTS: CMSEE could significantly inhibit the proliferation of tumor cell lines in a dose-dependent manner(P<0.01), but CMSWE had no such effect . Both CMSEE and CMSWE showed no obvious influence on growth of PBMC. After treatment with CMSEE, B16 cells showed apoptosis morphology and the apoptotic rate was increased as compared to control group, meanwhile the proportion of B16 cells in the G0/G1 phase was elevated significantly(P<0.01). CONCLUSION: CMSEE significantly inhibited tumor cell growth in vitro by arresting cell cycle and inducing apoptosis of tumor cells.

OBJECTIVE: To study the effects of selective cyclooxygenase-2 inhibitor NS-398 combined with octreotide on growth and apoptosis of human colon carcinoma cell. METHODS: Lovo cells were treated with NS-398, octreotide or both. The inhibitory effect on the proliferation of Lovo cells was measured by MTT assay. Morphologic changes were examined by electron microscopy, apoptotic percentage and cell cycle of Lovo cells were measured by flow cytometry. The expression of COX-2 mRNA was detected by RT-PCR. RESULTS: NS-398 and octreotide markedly inhibited cells growth in a time-dependent manner, combined treatment could significantly enhance the inhibitory effect than either alone (P<0.05). Apoptotic cells were studied with electron microscope. The apoptotic ratio induced by combined treatment was hihger than other groups. Furthermore, cell cycle analysis showed that S phase cells were decreased and quiescent G0/G1 phase cells accumulated (P<0.05). Compared with control group, the levels of COX-2 mRNA was down-regulated in three experimental groups (P<0.05). CONCLUSION: NS-398 combined with octreotide could synergistically inhibit growth and induce apoptosis of colon carcinoma cell, which may be attributed to cell cycle block and decrease in COX-2 mRNA expression.

OBJECTIVE: Three sesquiterpene lactones 1-O-acetylbritannilactone, isoalantolactone and britannilactone were isolated from the roots of Inula helenium and the flowers of Inula japonica. The antitumor activities of the three sesquiterpenoids in the cell lines of HeLa, HEC-1, SHIN3, HOC-21 and HAC-2 were measured, and the relationship between structure and activity and the possible mechanisms were explored. METHODS: The effect of three sesquiterpene lactones on five cell lines was measured by MTT assay in vitro. The apoptosis induced by isoalantolactone was examined by flow cytometry. RESULTS:Isoalantolactone exhibited excellent anti-proliferative activities on HeLa, SHIN3 and HOC-21 and HAC-2 cell lines, while 1-O-acetylbritannilactone and britannilactone demonstrated weak anti-proliferative activities on HeLa, SHIN3, HOAC-21 and HAC-2 cell lines even at the concentration of 100 μmol/L. The apoptotic rate in isoalantolactone group with the concentration of 12.5 μmol/L was higher than that in PBS group. CONCLUSION: MTT assay indicated that anti-proliferative activities in HeLa, SHIN3, HOAC-21 and HAC-2 cell lines were closely related to structure, 1,10-seco of eudesmane sesquiterpene lactone resulted in reduced anti-proliferative activity. The anti-tumor activity of isoalantolactone was probably achieved by inducing HeLa cell apoptosis.

OBJECTIVE: To explore the effects of subchronic exposure to formaldehyde on ovarian reserve function in female rats. METHODS: Forty female Sprague-Dawley (SD) rats were randomly subdivided into a control group and three formaldehyde groups. The rats in formaldehyde groups were intraperitoneally treated with formaldehyde at 0.2, 2.0 and 20.0 mg/(kg•d)for 14 days. Radioimmunoassay was used to detect estradiol(E2), luteinizing hormone (LH), follicle stimulating hormone (FSH) and Inhibin B. Ovary and body weights of female rats were measured and the organic coefficient of ovary was counted. RESULTS: Compared with the control group, the levels of serum estradiol (E2) and Inhibin B in the groups treated with formaldehyde decreased significantly (P<0.05). The level of serum FSH increased and the organic coefficient of ovary decreased significantly(P<0.05), while the levels of LH didn't change obviously(P>0.05). CONCLUSION:The ovarian reserve function of female rats can be impaired after subchronically exposed to formaldehyde for 14 days.

OBJECTIVE: To study the time-course and dose-response of chromosome aberrations in human blood lymphocyte induced by carbon ion irradiation. METHODS: Peripheral blood samples from volunteers were irradiated with carbon ions with a mean LET of 46 keV/μm. For the time-course study, the irradiation doses were 2 Gy and 4 Gy. First division metaphase cells were collected after different culture times of 48,72 and 84 h, and chromosome aberrations were determined by using sister chromatid differential staining method. For dose-response study, the irradiation doses were 0，0.5，1，2，3and 4 Gy. First division metaphase cells were collected after a culture time of 48 h, and chromosome aberrations were determined by using conventional chromosome technology. RESULTS: There was no significant difference between the frequencies of “dic＋ring” obtained in these three culture times. The frequency of “dic＋ring” increased linearly with doses, with the equation of Y=0.0005＋0.689D. CONCLUSION: In these irradiation conditions, there was no time-course effect in PBL chromosome damage induced by 12C ions. A culture time of 48 h is acceptable for studing the biological effectiveness of carbon ions, and the number of chromosome aberrations increased linearly with dose.

OBJECTIVE： To establish model of HIV-1 gene transmission and experimental basis for true vertical transmission of HIV via female germ cells. METHODS： PIRES2-EGFP-gag plasmid containing HIV-1 gag gene was injected into mouse ovaries to transfect germ cells. Induction of superovulation was then used to produce oocytes. PCR and fluorescence in situ hybridization (FISH) were performed to detect the intergration of HIV-1 gag DNA in chromosomes of oocyte. RESULTS：It was possible to identify oocytes which carried the HIV-1 gag gene by the presence of green fluorescence. PCR demonstrated the positive bands for HIV-1 gag gene in the tested oocytes with green fluorescence. FISH revealed the positive signals for HIV-1 gag DNA in chromosomes of the oocytes. CONCLUSION： HIV-1 gag gene was able to pass through zona pellucida and plasma membrane of oocyte and became integrated into the host genome. Our results provided solid evidence that HIV-1 gag gene could be transmitted vertically to the next generation via female germ line.

OBJECTIVE: To explore the value of Zona-free Hamster Oocyte Sperm Penetration Assay (SPA) and DNA integrity monitoring in in vitro fertilization-embryo transfer (IVF-ET). METHODS: 41 men from infertile couples opting for IVF-ET were divided into two groups of pure male sterility or female infertility depending on the cause of infertility.They were assessed by comparing male ages,fertility index,rates of penetration,DNA fragmentation,IVF fertility, ovum cleavage,good quality embryo,implantation and clinical pregnancy. RESULTS: Two groups showed significant differences in penetration rates,fertility index,DNA fragment rates(P<0.05)，but no differences in male ages,the rates of IVF fertility, ovum cleavage,good quality embryo, implantation and clinical pregnancy(P>0.05). In groups of male sterility and female infertility,obvious correlations were observed in penetration rates with the rates of IVF fertility,ovum cleavage and good quality embryo,and DNA fragment rates with the rates of IVF fertility, ovum cleavage and good quality embryo, respectively(P<0.05). CONCLUSION: SPA and DNA fragmentation monitoring may be valuable in assisted reproductive technology.

OBJECTIVE: To explore the suppression mechanism of MC-LR on lymphocyte immune function in mice. METHODS: Mice splenic lymphocytes were treated with MC-LR at the dose of 0, 1, 3, 5, 10 μg/ml in vitro. The T and B lymphocyte proliferations were examined by MTT assay. The production of IL-2 in cells was measured by ELISA and the apoptosis rate of lymphocytes was deteceted by FCM. RESULTS: The lymphoproliferation was suppressed by MC-LR at all doses. The production of IL-2 was increased at 1 μg/ml but decreased after treatment with MC-LR at 5 and 10 μg/ml. The rate of lymphocyte apoptosis was increased at 10 μg/ml in vitro. CONCLUSION: MC-LR could decrease the production of IL-2 of mouse lymphocyte but increase its apoptosis in vitro. MC-LR could markedly inhibit mice lymphocyte proliferation in vitro.

OBJECTIVE: To explore the protective effect of Schisandrins extract on liver ultrastructure in rats with CCl4 poisoning. METHODS: Twenty five Wistar rats were randomly divided into the control group, the CCl4 poisoned 24 h and 10 d groups, the Schisandrins 24 h and 10 d groups, five groups with 5 rats in each group. The rats of the CCl4 poisoned and the Schisandrins groups were given one peritoneal injection of CCl4(200 ml/L in vegetable oil) at a dosage of 0.2 ml/100 g body weight, the rats of the control group were given the same volume of vegetable oil. Then Schisandrins extract(0.5 ml/100 g body weight)were given orally to rats of the Schisandrins group daily，while saline was given to rats of the poisoned and the control groups. The rats were sacrificed at different time points. The morphologic changes of the hepatocytes were examined by transmission electron microscopy. RESULTS: The ultrastructure of hepatocytes of rats were changed in the CCl4 poisoned 24 h group, the cytoplasm of hepatocytes was loosed and vacuolated, mitochondria were swollen, with cristae breakdown or loss, and the endoplasmic reticulum appeared lamellar. The nuclear membrane was shrunken and ruptured, and the nuclear chromatin condensed and marginated. The hepatocytic nuclei showed obvious atypia in rats of the CCl4 10 d group. By comparing with that of the CCl4 group,the ultrastructure of hepatocytes in rats of the schisandrins group was less severely damaged. CONCLUSION: The use of Schisandrins extract in the treatment of CCl4 poisoned rats exerted a protective effect on liver ultrastructure.

OBJECTIVE： To examine the association between the degree of pollution of the water around residential Kunming and mutagenicity. METHODS：The micronucleus test of Vicia faba root tip cell was used to detect the water quality，and to survey the chemical oxygen demand(CODcr).The micronuclear rates and Pollution Index(PI) were determined and the F-test was used to evaluate the difference in micronuclear rates among different samples. RESULTS: There was significant difference in micronuclear rates of every site(P<0.01). PI values of 7 among 9 sampling sites were over 2.00. The micronuclear rates were higher in the sites with higher PI. There was no obvious pattern in the results of CODcr. CONCLUSION：The degree of pollution was directly related to the level of mutagenicity.But the water mutagenic activity was not directly related to the chemical oxygen demand.

OBJECTIVE: To investigate the anti-mutation effect of the extracts of pinecones. METHODS：The anti-mutation effect of extracts of pinecones was measured by micronucleus test, with different doses of pinecones alkaline extract solution(50、250 and 500 mg/kg) as samples, cyclophosphamide as the positive group. RESULTS: The mice marrow cells MN rate of high, middle and low doses of extract solution plus cyclophosphamide treatment groups：16‰±0.58‰, 34.75‰±0.85‰ and 40.25‰±0.48‰,respectively. Compared with physiological saline group the difference in MN rate was statistically significant (P<0.01). For extract solution in low dose group compared with CP group, the difference was not statistically significant (P>0.05). In the high and middle dose groups, the difference was statistically significant (P<0.05),inhibition rates were 22.35％ and 65.35％, respectively. Between male and female mices the difference was not statistically significant (P>0.05). CONCLUSION: The extracts of pinecones could suppress mice marrow cells induced by cyclophoshomide, with an obvious dose-response relation. It also showed anti-mutation effects. The extracts of pinecones may have positive effect on tumor prevention.

OBJECTIVE: To study the mutagenicity of Fufang Xiaojingtong capsules (FFXC). METHODS: The mouse sperm morphology test , the mouse sperm chromosomal aberration test and the mouse bone marrow micronucleus test were used to evaluate mutagenicity of FFXC. RESULTS: The results of sperm morphology rates, the sperm chromosomal aberration test and the bone marrow micronucleus rates were negative. CONCLUSION: FFXC had no mutagenic effects in our study.