OBJECTIVE: Pyrosequencing was applied to study the effect of whole genome amplification (WGA) on the fidelity of DNA methylation. METHODS：EpiTect Whole Bisulfitome Kit was used to perform DNA amplification after whole genome bisulfite modification of L02，A549 and HCT116 cells. The DNA methylation of three specific genes (MGMT，GSTP1，P16) and LINE 1 sequence were analyzed by pyrosequencing. RESULTS：The DNA amplification obtained was 246 folds higher in DNA content and > 1 000 bp fragment length which met the basic requirements of DNA methylation detection. The DNA methylation level of LINE1 did not change after WGA，indicating that WGA had no effect on the whole genome methylation. However，the change of specific DNA methylation after WGA correlated with their original methylation level. When the original methylation rate was higher than 60%，the methylation condition of three genes remained stable after WGA. There was significant change with irregular trend of DNA methylation level after WGA when the initial DNA methylation ranged between ≥20%-60%. DNA methylation rate of three genes declined significantly in all cells when the initial DNA methylation level was lower than 20%. In addition，pyrosequencing result showed that the effect of WGA on genes CpG sites methylation level was similar to that on its mean methylation rate. CONCLUSION：WGA technology was suitable for the study of whole genome methylation and specific gene with higher methylation rate (≥60%). However，we must take caution for using the WGA technology when the rate of DNA methylation was relative low (< 60%).

OBJECTIVE: The aim of this study was to identify the expressions of E2F5 and MYC gene located in chromosomal region 8q21–24 in prostate cancer (PCa)，adjacent benign prostate tissues and PCa cell lines，to analyze their expression levels and to explore theirs significances in PCa. METHODS：DNA qPCR analysis was carried out to evaluate the copy number changes of E2F5 and MYC genes in PCa tissues and PCa cell lines. Western blot and immunohistochemical staining were used to assess expressions of E2F5 and MYC in PCa and BPH tissues，and analyzed together with clinical pathological parameters. RESULTS：DNA qPCR revealed a significant copy number gain of E2F5 and MYC in PCa tissues but not in PCa cell lines (P<0.05 or P<0.01). In addition，Western blot analysis and immunohistochemical staining both found the significantly higher expression of E2F5 and MYC proteins in PCa tissues than those in adjacent benign specimens (all P<0.01). Moreover，the overexpression of E2F5 protein was significantly associated with a high Gleason score (P<0.01)，an advanced clinical stage (P=0.01)，a positive metastasis (P<0.01) and biochemical recurrence (P<0.01). The overexpression of MYC was more frequently found in PCa tissues with positive metastasis (P=0.02) and biochemical recurrence (P=0.02). Interestingly，there was a close correlation in the expression level of MYC in PCa tissues with that of E2F5 (rs=0.5，P<0.01). CONCLUSION：Overexpressions of E2F5 and MYC may play an important role in the development and progression of PCa. E2F5 may be a novel potential candidate marker for malignant PCa.

OBJECTIVE: To explore the effect and mechanism of different doses of apigenin on in vitro maturation of Xenopus oocytes. METHODS：Healthy Xenopus oocytes were cultured in different doses (20，80，160 and 500 μmol/L) of apigenin. Meanwhile negative and positive control groups were set. Then we observed and recorded the rate of germinal vesicle breakdown (GVBD) per hour，confirmed the transformation of cell nucleus and chromosome by means of trichloroacetic acid fixation and fluorescence staining. Finally oocytes were collected at 8 h in each group，and the expressions of p-Erk1，Mos and CyclinB2 were detected by Western blot. RESULTS：Compared with negative control group，GVBD rates were all highly increased when apigenin-treated dose were 80，160 and 500 μmol/L，and time were 4-8 h (P<0.05). The expressions of p-Erk1，Mos and CyclinB2 were all markedly increased when the dose of apigenin were between 80 and 500 μmol/L (P<0.05). CONCLUSION：Apigenin may play an important role in promoting the maturation of Xenopus oocytes in vitro through regulation of mature promoting factor (MPF) and mitogen-activated protein kinase (MAKP).

OBJECTIVE: To study on the immunotherapy effects of HSP70 polypeptide complex on colon cancer in mice，and to explore its antitumor mechanism. METHODS：The Colon26 cells were cultured at 42 ℃ for 1 h， then incubated at 37 ℃ for 8 and 12 h，and tumor cell lysates containing high expression of HSP70 polypeptide complex were prepared by repeated freezing and thawing methods. Tumor-bearing mice model was established with Colon26 cells. Tumor cell lysate containing high expression of HSP70 polypeptide complex (test group)，tumor cell lysate containing HSP70 polypeptide complex at 37 ℃ routine culture (control groupⅠ) and PBS (control groupⅡ) were repeatedly injected subcutaneously，in vivo tumorigenicity，growth and survival were monitored.　Expressions of CD3+， CD4+ and CD8+ T cells on the mice splenic monocuclear surface were analyzed by flow cytometry. ELISA was used to assess the production of TGF- β1 and IFN-γ in plasma of the tumor-bearing mice. RESULTS：After tumor-bearing mice were treated with tumor cell lysate containing high expression of HSP70 polypeptide complex，tumor growth，tumor volume and weight were significantly lower than control groupsⅠ and Ⅱ，the survival time was longer (P<0.05). Splenic mononuclear surface molecules of CD8+ T cells was significantly increased in the experimental group compared with the control groupsⅠand Ⅱ (P<0.05). CD3+ and CD4+/CD8+ T cells as well as TGF-β1 significantly reduced (P<0.05) in the plasma in mice of the experimental group compared with the control groupsⅠand Ⅱ，while IFN-γ level increased significantly (P<0.05). CONCLUSION：HSP70 polypeptide complex inhibited the tumor growth significantly，prolonged survival period，indicating that the peptide complex has obvious anti-tumor effects in mice by modulating immunology functions.

OBJECTIVE: To observe the inhibitory effect of FOLFOX4 combined with 1-MT on subcutaneous xenografts of gastric cancer in mice，and its influence on the expression of indoleamine-2，3-dioxygenase (IDO) in gastric cancer tissue. METHODS：Transfect the IDO eukaryotic expression plasmids (pcDNA3.1-IDO) into mice gastric cancer cell line MFC. The cell line expressing IDO stably was established. Expression of IDO was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Suspension of the MFC cells transfected with pcDNA3.1-IDO plasmid were inoculated subcutaneously in mice. Mice gastric carcinoma subcutaneous xenografts model expressing IDO highly was established (n=32). Blank control (n=8，untransfected MFC cells) and negative control (n=8，transfected MFC cells) were set up. The 32 model mice were randomly divided into 1-MT treatment group， FOLFOX4 treatment group，FOLFOX4+1-MT combined treatment(saline) group and non-treatment group (positive control group). There were eight mice in each group. The general condition of the mice and the differences in tumor quality were observed. Twelve day after cell suspension injection，all of the mice were sacrificed. The tumors were excised and weighed. Average tumor size and the tumor inhibitory rates of each group were calculated. The expression of IDO in gastric carcinoma tissues was evaluated by immunohistochemistry. RESULTS：Tumor of mice gastric carcinoma subcutaneous xenografts model expressing IDO highly was greater than blank control and negative control groups (P<0.05) and the expression of IDO in gastric carcinoma tissue was also increased significantly (P<0.05). After given 1-MT，FOLFOX4 and FOLFOX4+1-MT treatments，the food intake of model mice increased to varying degrees，more agile than before，and lethargy was also improved to different extents. The symptoms of FOLFOX4+1-MT combined treatment group were basically resolved. The tumors of 1-MT treatment，FOLFOX4 treatment and FOLFOX4+1-MT combined treatment group were smaller than no treatment group (P<0.05). The tumour inhibitory rates were 8.91%，80.20%，86.13%，respectively. FOLFOX4+1-MT combined treatment group tumor was less than 1-MT treatment group and FOLFOX4 treatment groups (P<0.05). The expressions of IDO in gastric carcinoma tissue of 1-MT treatment，FOLFOX4 treatment and FOLFOX+1-MT combined groups were lower than non-treatment group (P<0.05). The expression of IDO in gastric cancer tissue of FOLFOX4+1-MT combined treatment group was lower than 1-MT treatment group and FOLFOX4 treatment groups (P<0.05). CONCLUSION： 1-MT could inhibit the growth of subcutaneous xenografts of gastric carcinoma and the expression of IDO in gastric carcinoma tissue in mice. It could play synergistic antitumor role together with FOLFOX4.

OBJECTIVE: To investigate whether combining 1-D-MT with FOLFOX4 could be useful to improve the immune tolerance of gastric cancer- bearing mice. METHODS：By using the lipofectamineTM 2000，the eukaryotic expression plasmid pcDNA3.1-IDO and empty vector pcDNA3.1 (+) were transfected in a MFC cell line，setting a control group. The expression of IDO was detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot. Animal model of gastric cancer- bearing mice were established to receive normal saline (NS)，FOLFOX4，1-D-MT and 1-D-MT+FOLFOX4 therapy. By using flow cytometry，Treg cell ratio change was analyzed and FOXP3 mRNA expression change was assessed by RT-PCR. RESULTS：The expression of IDO mRNA and protein in the group trancfected with pcDNA3.1-IDO was obviousely ligher than the MFC group and the empty vector group (P<0.05). Treg cell ratio and FOXP3 mRNA expression in the transfected IDO negative control group with NS therapy was higher than the MFC and the empty vector groups (P<0.05). The Treg ratio and FOXP3 mRNA in 1-D-MT+FOLFOX4 and 1-D-MT groups were less than in FOLFOX4 group (P<0.05)，with 1-D-MT+FOLFOX4 group more markedly than 1-D-MT group (P<0.05). CONCLUSION：Combining 1-D-MT with FOLFOX4 could reduce Treg cell ratio and FOXP3 expression，thus reducing the immune escape.

OBJECTIVE: To investigate the effect of (-) epigallocatechin gallate(EGCG) on mRNA expressions of hMLH1 and hMSH2 in human lymphoblast cell lines. METHODS: Total RNA was isolated from GM12593 and GM13705 cells treated with EGCG(0，5，10，20 μmol/L) for 6 days. Real-time fluorecence quantification PCR(FQ-PCR) was conducted to measure the mRNA expression levels of hMLH1 and hMSH2. RESULTS: The mRNA expressions of hMLH1 and hMSH2 significantly increased at 20 μmol/L EGCG in GM12593 cells，and were significantly higher than equal concentrations in GM13705 cells. The effect of EGCG on expressions of target genes were insignificant in GM13705 cells. CONCLUSION: EGCG had the potential to up-regulate the expressions of hMLH1 and hMSH2， contributed to the promotion of mismatch repair and maintaining genomic stability in normal human lymphoblast cell line GM12593.

OBJECTIVE: To study the effect and mechanism of ginseng polysaccharide (GPS) on tamoxifen (TAM)-induced apoptosis in human breast cancer cells. METHODS： Human breast cancer cells MCF-7 were treated with different doses of GPS for 48 h. MTT assay was used to evaluate the minimal effective GPS dose. Then cells were treated with GPS at minimal effective dose combined with different doses of TAM. Bürgi formula was used to assess the synergy effect between GPS and TAM. Cellular mitosis was detected by Giemsa staining assay. Apoptosis was studied by DAPI staining analysis and flow cytometer. The expression of Fas，Caspase-9 and Parp was measured by Western blot. RESULTS：GPS at 40 μg/mL was the minimal effective dose on MCF-7 cells. Combined treatment of GPS at 40 μg/mL and TAM at 1 μg/mL showed the best effect in cell growth inhibition (q=1.82). Combination of GPS and TAM synergistically enhanced apoptosis of MCF-7 in comparison with GPS or TAM alone (q=2.19，P<0.05). Combinated treatment of GPS and TAM significantly inhibited cellular mitosis compared with GPS or TAM alone (P<0.05). Treatment of GPS+TAM induced increased level of Fas as well as cleavage of Caspases-9 and Parp. CONCLUSION：Combined treatment of GPS and TAM induced apoptosis in MCF-7 cell through Fas signal in a synergetic manner.

 OBJECTIVE: To evaluate the antioxidative effect of saponin from Aralia taibaiensis，and provide some basic data for its further application in the prevention and treatment for oxidative stress-related diseases. METHODS： The total antioxidant capacity of saponin was determined by ferric-reducing ability of plasma (FRAP) method. In the cellular study，tert-butyl hydroperoxide (tBHP) was employed as an oxidant to induce oxidative stress，and saponin (5 μg/mL) was pre-incubated with the cells 12 h before challenged by tBHP in the protective group. Then cellular ROS was labeled with DCFH-DA and determined by flow cytometry. The protein levels of nuclear respiratory factor 2 (Nrf2)，heme oxygenase-1 (HO-1)，γ-glutamate-cysteine ligase (GCL) were evaluated by Western blot. RESULTS：The saponin showed marked antioxidative effect. tBHP induced massive reactive oxygen species (ROS) production，for which saponin pre-treatment showed significant inhibition. Saponin pre-treatment also significantly inhibited the tBHP-stimulated protein expressions of Nrf2，HO-1 and GCL. CONCLUSION：Saponin from Aralia taibaiensis has potent antioxidative activity，indicating its potential clinical application in oxidative stress-related diseases.

OBJECTIVE: To study the apoptotic effects of human bile duct carcinoma MZ-Cha-1 cells induced by curcumin. METHODS：After the MZ-Cha-1 cells induced by 0，10，20 and 40 μg/mL curcumin for 12，24，36 h，the apoptotic effects of curcumin were studied using the methods of MTT to detect the curcumin on MZ-Cha-1 cell suppression，and selected 10 μg/mL of curcumin treatment MZ-Cha-1 cells after 24 h，using Giemsa staining and Hoechst33258 dye to assess MZ-Cha-1 cell apoptosis，using flow cytometry to detect the cell cycle distribution. RESULTS：Compared with control group，the curcumin on MZ-Cha-1 bile duct carcinoma cell had obvious inhibitory effect，and in dose- and time- dependent mamer (P<0.05). Proliferation of MZ-Cha-1 cells could be inhibited by 10 μg/mL curcumin after 24 h. Light microscope showed that the morphology of the cells treated with curcumin appeared shrunken. Concentrated cell nucleus appeared granular and fluorescent by Hoechst33258 staining. Flow cytometry analysis showed that G0/G1 phase was increased and G2/M phase decreased (P<0.05). CONCLUSION：This study suggested that curcumin could induce apoptosis of the human MZ-Cha-1 cells effectively.

OBJECTIVE: To determine the association of Prohibitin (PHB) 3'- untranslated region (UTR) polymorphism with the risk of breast cancer in eastern Guangdong of southern China．METHODS：A case-control study of 231 breast cancer patients and 169 healthy controls were enrolled into this study. Genomic DNA was extracted from whole blood cells. Single nucleotide polymorphisms of rs1049620，rs9893420 and rs4987083 were determined by the high resolution melting (HRM) method．Data were analyzed via Logistic regression analysis and Chi-square test. RESULTS：PHB gene at rs9893420 and rs4987083 sites showed single genotype in breast cancer group and control. There were no significant differences in frequencies of G/G，A/G and A/A at rs1049620 between patients and controls(P>0.05). Meanwhile，no significant correlation could be found between rs1049620 SNP and clinico-pathologic characteristics of breast cancer such as histological classification and expression of hormone receptor. CONCLUSION： Our results do not lend support to the hypothesis that PHB polymorphism contributes to susceptibility of breast cancer.

 OBJECTIVE: To study the subchronic dermal toxicity of sodium lactate in rats，and analyze the target organ damage and its reversibility. METHODS：Sodium lactate was given to rats by transdermal administration at dose of 2 000，1 000 and 500 mg/kg for 90 days. Some rats from each group were sacrificed at 7th，13th week and 4 weeks after recovery，blood count and biochemistry were tested，ophthalmic and urinalysis tests were performed，wet weight of individual organs and the coefficient values were determined，and anatomic pathology were observed. RESULTS：With all treatment doses，K+，Cl- decreased and Na+，Ca2+ increased，protein precipitation was found in the bladder lumen. High dose group showed urinary protein and bilirubin concentration increased，specific gravity reduced. The above had dose-response relationship. Na+ values in high dose group remained elevated in the recovery stage. CONCLUSION： Sodium lactate showed significant toxic effects in rats as determined by mild disturbance of electrolyte and abnormal urinary protein.

OBJECTIVE: To investigate whether GFP transgenic mice can be used as a new animal model for hprt gene mutation test. METHODS：ENU was used to induce the gene mutation of GFP transgenic and normal mice. The dynamic of frequencies of micronuclei in peripheral blood PEC was compared between GFP transgenic and normal mice after ENU induction. DNA and mRNA sequences of hprt were tested in GFP transgenic mice. PCR-sequencing was used to detect six exons 1 and three exons 9. RESULTS：The dynamic of frequencies of micronuclei was consistent between GFP transgenic and normal mice after ENU induction. The materials of GFP transgenic mice were easier to confirm than those of normal mice. It reduced the false positive rate and false negative rate as well. The hprt gene sequences of GFP transgenic mice were complete. There were no significant differences in hprt gene sequences between GFP transgenic and normal mice. DNA sequences of exons 1 were consistent with the wild-type mouse. Two A∶T→T∶A substitutions occurred in exons 9 of hprt gene from mutation clone. CONCLUSION：GFP transgenic mice can be used as animal models for mutation test. GFP transgenic mice can be used as the animal model with high accuracy for hprt gene mutation test，which may reduce the difficulty of the test.

OBJECTIVE: To establish male rat fertility and early embryo developmental toxicity test (I section) positive control model，cyclophosphamide was given by i.p. before mating. METHODS：Male Sprague-Dawley rats were divided into control group，cyclophosphamide I and cyclophosphamide II groups. Cyclophosphamide groups were given intraperitoneally on day 1 of 9 weeks (100 mg/kg) and the first 5 days of 9 weeks (20 mg/kg). Male rats were sacrificed after mating，the parameters were observed and tested．RESULTS：Positive groups male animals showed reversible depilatory. Animal body weight and then food consumption were decreased significantly when compared with the control group，although no significant differences in the hormone level were found．Weights of auxiliary reproductive organ were significantly decreased，time for penis erection was significantly longer，sperm count and sperm activity ratio were decreased，and sperm malformation rate was increased significantly. CONCLUSION：Cyclophosphamide exeited significant effects on male fertility，and both treatment methods could be used to establish positive fertility and early embryo developmental toxicity test model.

OBJECTIVE: To establish a new technique of multiple immunoenzyme histochemistry staining by using computer image processing technology. METHODS：We stained Hepatitis B virus surface protein (HBs)， asiologlycoprotein receptor (ASGPR) and proliferating cell nuclear antigen (PCNA) on the same hepatocellular carcinoma slice using the improved streptavidin-peroxidase method and combined these three figures into one that displayed the expression of these proteins together with the assistance of computer image processing technology. RESULTS：The positive signals of three proteins in the hepatocellular carcinoma slice were clear and strong without antibody cross reactivity. The precessed image could objectively display the exact site of positive signals in the original pictures and show the expression level of these different proteins together in one picture. CONCLUSION：This established method could mark multiple proteins in one slice together.