OBJECTIVE: To construct a series of cyclin-dependent kinase 2 (CDK2) mammalian cell expression vectors and to assess CDK2 expression in NIH3T3 cells. METHODS：RNA was isolated from esophageal cancer cells and the full coding sequence of CDK2 gene was obtained by RT-PCR. The PCR product was then cloned into T vector and subsequently subcloned into four eukaryotic expression vectors (pcDNA3，pcDNA4，pNTAP and pEGFP). The expressing plasmids were transfected into NIH3T3 cells and the expression of CDK2 was detected by western blot. RESULTS：The PCR product was about 900 bp and the sequence analysis showed that it was the full coding sequence of CDK2 gene. The product was subcloned into the eukaryotic expression vectors and four CDK2 expression vectors were constructed. Western blot showed that CDK2 could be expressed in the expression vector-transfected cells. CONCLUSION：Four CDK2 eukaryotic expression vectors were successfully constructed and CDK2 was effectively expressed in NIH3T3 cells.

OBJECTIVE: A chromosomal aberration (CA) screening test was established using eight-well chamber slides without adopting complicated procedures and chemical agents in the traditional chromosomal aberration test in vitro. The validation of the protocol was conducted using several genotoxic chemicals with various mechanisms of action and non-genotoxic compounds. METHODS：Optimized conditions for the test procedure in which cells were treated with cyclophosphamide (CP) for 6 h (for the +S9 condition) and mitomycin C (MMC) for 24 h (for the -S9 condition) were investigated. And then five known genotoxic chemicals including benzo(a)pyrene[B(a)P]，2-aminoanthracene(2-AA)，methyl methanesulphonate (MMS)，ethyl methansulfonate(EMS)，etoposide(Eto) and two non-genotoxic chemicals including ampicilin sodium salt(AS) and sodium chloride(SC) were used in the sensitive and specific validation research under these conditions. RESULTS：The CA percentage of CP (6 h，+S9) and MMC (24 h，-S9) showed dose-dependent accumulation. In the validation tests，B(a)P and 2-AA were positive only in (6 h，+S9) condition while MMS，EMS and Eto showed positive results in both +/-S9 conditions. AS and SC were negative in both +/-S9 conditions. CONCLUSION： The chromosomal aberration test using eight-well chamber slides could be considered as a simple，rapid and effective method，which could be used in genotoxicity screening in the early research phase of drug development.

OBJECTIVE: To investigate the effect of Skp2 in gastric carcinoma induced by Helicobacter pylori L-form(Hp-L) infection. METHODS ：The BGC-823 cells were co-incubated with different amount of Hp-L， and the expressions of Skp2 mRNA and protein were detected by in situ hybridization (ISH) and immunohistochemistry (IHC). Forty cases each of chronic atrophic gastritis(CAG)，gastric intestinal metaplasia(GIM)， gastric epithelial dysplasia(GED) and gastric carcinoma(GCa) were collected，and 40 mild chronic superficial gastritis (CSG) were also collected as control group. Hp-L was examined in each group by Gram’s staining. The expressions of Skp2 mRNA and protein were also evaluated. RESULTS ：By inversion microscope，infected BGC-823 cells showed increased division indicating growth acceleration. Expressions of Skp2 showed that the positive cells gradually increased，became more obvious with increasing concentration of Hp-L and the duration of treatment in a concentration-and-time-dependent manner . The positive rate of mRNA and protein of Skp2 in CAG，GIM，GED and GCa of the positive Hp-L groups were higher than the negative groups，but only the GCa group was significant (P<0.05). CONCLSION：Skp2 showed an obvious increase in gastric pre-cancerous lesions and gastric carcinoma induced by Hp-L infection，which indicated it may play an important role in the development of gastric carcinoma.

OBJECTIVE: To observe proliferative and morphological changes in human esophageal cancer cell differentiation induced by Hexamethylene Bisacetamide (HBMA). METHODS：EC9706 cells were treated by different concentration of HMBA(0，3，5，8，10 mmol/L) and the changes in proliferation and morphology before and after treatment were examined under light microscope ，while the effects were detected by growth curve，division index and HE staining. RESULTS：After treatment with HMBA the proliferation and growth of EC9706 cells were inhibited.，and the inhibitory rate rose with the increase of HBMA concentration. EC9706 cells tended to spread out flat，cell size was more consistent and regular under light microscope. CONCLUSION：Human esophageal cancer cells treated with HMBA appeared similar to normal epithelial cell morphology and structural features，displaying malignant phenotype reversal of tumor cells.

OBJECTIVE: Multidrug resistance (MDR) is a major obstacle for effective treatment of tumors. Previous studies of MDR tended to concentrate in a single or a few related genes and their protein products. We focused on the total protein with the method of proteomics. The aim of this study was to investigate differently expressed protein in B-MD-C1 and B-MD-C1 (ADR+/+) cells by proteomic analysis. METHODS：The holoproteins of human carcinoma of endometrium cell lines B-MD-C1 and B-MD-C1 (ADR+/+) were measured by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS：Thirty-five differential proteins were analyzed by peptide mass fingerprinting. The differentially expressed proteins could be divided into six groups based on their functions：molecular chaperones，cystoskeleton proteins(actin-related protein 3，lamin-B1 and tubulin beta chain)，metabolic enzymes (78 kDa glucose-regulated protein，retinal dehydrogenase)，proteins associated with cell cycle (eukaryotic initiation factor 4A-III)，proteins associated with cell proliferation，differentiation and apoptosis (endoplasmic reticulum protein，endoplasmic reticulum protein)，and proteins associated with carbohydrate metabolism (60S acidic ribosomal protein，alpha-enolase). CONCLUSION：These differentially expressed proteins provided some clues to the mechanism of tumor cell resistant to cisplatin，providing the basis of searching for potential target of carcinoma.

OBJECTIVE: We investigated the promoter and exon1 methylation of mothers against decapentaplegic homolog 4 (Smad4) and its correlation with protein expression of TGF-β1 in gastric cardia adenocarcinoma (GCA). METHODS：Methylation-specific PCR approach，RT-PCR and immunohistochemistry methods were used to examine the methylation status of the 5' CpG island，mRNA and protein expression of Smad4. Immunohistochemistry method was used to detect the protein expression of TGF-β1 in tumors and corresponding normal tissues. RESULTS：For the promoter site，Smad4 was methylated in 6/110 (5.5%) tumor specimens. For the 5' UTR of exon1，Smad4 was methylated in 24.5% (27/110) tumor specimens. Both were significantly higher than that in corresponding normal tissues (P<0.05). Methylation frequency of Smad4 in the 5' UTR of exon1 was significantly higher than that in the promoter site (P<0.01). Smad4 mRNA and protein expression of tumor tissues were significantly lower than that in corresponding normal tissues and was inversely correlated with its methylation status. Seventy-two of 110 (65.5%) tumor tisssues demonstrated positive immunostaining for TGF-β1 and the frequency of positive TGF-β1 protein expression was significantly higher than that in corresponding normal tissues (P<0.01). The protein expressions of TGF-β1 were significantly different among various stages and differentiations of GCA (P<0.05). The protein expression of Smad4 was inversely correlated with TGF-β1 in GCA. CONCLUSION：Hypermethylation of Smad4 in 5' UTR of exon1 and the higher protein expression of TGF-β1 may play important roles in the development of gastric cardia adenocarcinoma.

OBJECTIVE: To elucidate the molecular mechanism of low concentration of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) that interfered with the function of EGFR- mediated cellular signal transduction pathway，by inducing the epidermal growth factor receptor(EGFR)clustering，which was similar to that of epidermal growth factor treatment. METHODS： Eukaryotic expression vector of EGFR cytoplasmic domain gene was constructed and transfected into Lec-1 cells. Then the recombinant EGFR cytoplasmic domain protein was purified，and treated with MNNG for 1 h，at concentrations of 0.25，0.5，and 1.0 μmol/L. Enzyme linked immunoassay was used to measure the protein tyrosine kinase activity of recombinant EGFR cytoplasmic domain. Untreated recombinant cytoplasmic domain protein tyrosine kinase was used as control. RESULTS：Compared with normal control group，tyrosine kinase activity of EGFR was significantly inhibited by 0.5 μmol/L MNNG (P＜0.01). CONCLUSION：Low concentration of MNNG interfered with the signal transduction pathway of EGFR by supressing its tyrosine kinase activity.

OBJECTIVE: To investigate the expression of c-IAP2，Bcl-2，STAT3，cyclinD1，PTEN， VEGF associated with apoptosis -ralated gene Survivin in Kazak’s esophageal carcinoma tissues and its adjacent non-carcinoma tissues and to find relationships between Survivin and the above six genes. METHODS：RT-PCR was used to detect the expressions of these six genes in carcinoma tissues and its adjacent non-carcinoma tissues in 32 Kazaks esophageal cases，Western-bolt performed to evaluate the levels of proteins. RESULTS：In Xinjiang Kazak's esophageal cancer，The expression rates of these six genes in 32 cases were over 50.0％，with c-IAP2 at 93.75%，cyclinD1 at 87.5%，VEGF at 84.38%. The expression levels of c-IAP2 STAT3 PTEN and VEGF were significantly increased in carcinoma compared to the adjacent non-carcinoma tissues(P<0.05). There were differences in the expressions of c-IAP2，STAT3，PTEN and VEGF mRNA between carcinoma and its adjacent non-carcinoma tissues (P<0.05). PTEN and VEGF showed similar expression status in the level of transcription and translation. There were correlations between Survivin and c-IAP2，STAT3，PTEN and VEGF expressions in carcinoma tissues (P<0.05). We found that the expression of cyclinD1 correlated with c-IAP2，Bcl-2 and PTEN mRNA；and the expression of PTEN correlated with c-IAP2， TAT3， cyclinD1 and VEGF mRNA (P<0.05). Expression of cyclinD1 correlated with differentiated degree and stage；the expression of Bcl-2 and STAT3 correlated with lymph node metastasis whilst the expression of VEGF correlated with sex，lymph node metastasis (P<0.05). CONCLUSION：Survivin played an important role in promoting carcinogenesis and development of esophageal cancer in Kazak's，through regulating apoptosis，cell cycle and angiogenesis. These six genes could be identified as molecular markers for early diagnosis，and prognosis prediction in Xinjiang Kazak's esophageal cancer.

OBJECTIVE: To study treatment effects of Integripetal rhodiola herb，Salvia miltiorrhiza and lotus flower pollen on injuries induced by phosgene in mice. METHODS：50 mice were randomly divided into 5 groups including Integripetal rhodiola herb group，Salvia miltiorrhiza group，lotus flower pollen group，positive control group and negative control group. The mice in negative control group were exposed to room air and the mice in other groups were exposed to 11.9 mg/L phosgene for 1 minute. After 20 minutes，mice in treatment groups were treated with integripetal rhodiola herb，Salvia miltiorrhiza and lotus flower pollen by 0.1ml/10g intragastric administration. Mice were sacrificed after 4 hour. The protein content in bronchoalveolar lavage fluid (BLAF)，the content of malondialdehyde (MDA) and reduced glutathione (GSH) and blood cell count were determined. RESULTS：The protein content of BLAF in integripetal rhodiola herb group decreased significantly compared with that of positive control (P<0.05). Peripheral blood platelet count increased significantly in mice treated with integripetal rhodiola herb and Salvia miltiorrhiza compared with positive control without treatment (P<0.05). MDA content decreased in three treatment groups and was significantly decreased in Integripetal rhodiola herb compared with positive control group (P<0.05). GSH content increased in three treatment groups and was increased significantly in Integripetal rhodiola herb group compared with positive control (P<0.05). CONCLUSION：Integripetal rhodiola herb， Salvia miltiorrhiza and lotus flower pollen exerted a therapeutic role on lung damage induced by phosgene in mice. Integripetal rhodiola herb efficacy was better than that of Salvia miltiorrhiza and lotus flower pollen.

OBJECTIVE: To investigate the association of calcitonin-related polypeptide alpha (CALCA) protein expression level with cervical lesion pathogenesis. METHODS：We collected 104 cases of paraffin-embedded cervical tissue specimens of Uighur women with cervicitis，low grade squamous intraepithelial lesion (LSIL)，high grade squamous intraepithelial lesion ( HSIL) and cervical squamous cell carcinoma (CSCC)，and evaluated the CALCA protein expression by immunohistochemistry. RESULTS：CALCA protein was strongly expressed by cervical epithelial cells in most cases of cervicitis，but the expression level changed from strong to weak or loss of expressions with the development of cervical intraepithelial neoplasia and cervical carcinoma. The rates of CALCA protein expression loss in the groups of cervicitis，LSIL，HSIL and CSCC were 45.5% (15/33)，75% (12/16)，80.8% (21/26)and 82.8% (24/29)，respectively. The expression level was negatively correlated with cervical lesion pathogenesis (r = -0.361，P<0.01)，and the difference was statistically significant for HSIL and CSCC compared to cervicitis (P＜0.01)，but not for LSIL (P＞0.05). CONCLUSION：This study showed that the loss of CALCA protein expression was to some extent，associated with the development of cervical pre-cancerous lesions and cancer.

OBJECTIVE: To assess the DNA damage by comet assay in cervical cancer patients undergoing radiotherapy. METHODS：Blood samples were collected from 15 cervical cancer patients exposed to radiation at the accumulated dose of 0，10，20，30，40，50，60 Gy. DNA damage was measured by comet assay. RESULTS： Rate of lymphocyte DNA tailing in radiation groups increased significantly compared with to pre-radiation (P<0.01)，showing a linear relationship from 0 to 60 Gy. The regression equation was y =12.133+0.230x，r =0.964 (P<0.05). The tail length showed significant increase in radiation groups compared with pre-radiation (P<0.01). At the accumulated dose of 30 Gy，the tail length reached the peak value. The tail length decreased a little，but remained at a higher level still when dose was higher than 30 Gy, compared to pre-radiation (0 Gy)(P<0.01). There was no dose-effect relationship between tail length and accumulated dose. CONCLUSION：Radiotherapy may induce DNA damage in cervical cancer patients. Evaluation of the radiation- damage by comet assay has a high sensitivity.

OBJECTIVE: To identify the relationship between the pathogenesis of leukemia and Flt3 gene by detecting the mutations in juxtamembrane domain coded by Flt3. METHODS：We tested exons14，15 of Flt3 gene using PCR，PCR-SSCP and gene sequence in 60 leukeamia patients’ bone marrow samples. RESULTS：8 Flt3-ITD mutations，another 3 Flt3-L576P point mutations were found in juxtamembrane domain in 60 patients，including 1 patient with a deletion of intron 14. CONCLUSION：Flt3-L576P is a new point mutation site，which might be associated with the pathogenesis and development of leukemia.

OBJECTIVE: To study the inhibition effect of LY255283，a BLT2 receptor antagonist，on different human cancer cell lines. METHODS：The expressions of BLT2 receptor in SMMC7721、A549、Bel7402、BGC823、Eca109、HeLa、HL60、MCF7 and Pc3 cell were evaluated by RT-PCR. The inhibition effect of LY255283 on the nine tumor cell lines was analyzed by MTT assay. Apoptotic cells were evaluated by flow cytometry and fluorescence microscopy. RESULTS：All nine tumor cell lines expressed BLT2 receptors. LY255283 inhibited the proliferation of all nine tumor cell lines，the IC50 values were 0.21，11.17，1.28，5.9，7.9，1.59，0.14，1.25 and 31.28 μmol/L， respectively. The apoptotic rates of MCF-7 cells induced by LY255283 at 1 and 10 μmol/L were 20.47% and 31.28%，respectively，which were significantly increased compared with blank control group (8.66%) and DMSO control group (11.71%) (P<0.01). CONCLUSION：BLT2 receptor was expressed in nine human tumor cell lines，and the specific antagonist LY255283 could inhibit the proliferation of tumor cells probably by inducing apoptosis.

OBJECTIVE: To develop a method for detecting the mutations of BMP4 gene by matrix assisted laser desorption ionisation-time of flight mass spectrometry method (MALDI-TOF-MS). METHODS：BMP4 gene fragments were amplified by PCR using DNA extracted from blood samples of 100 non-syndromic cleft lip with or without cleft palate patients and 91 healthy unrelated individuals in Guangdong province. The mutations of BMP4 gene were analysed by MALDI-TOF-MS and identified by sequencing. RESULTS：The method for fast and high throughput genotyping BMP4 gene has been set up. The mutations of R162Q，R198X and T102A in BMP4 coding region were not found in 191 samples in our study. CONCLUSION：MALDI-TOF-MS is a fast，high throughput and accurate method for BMP4 gene mutation detection.

OBJECTIVE: To assess the mutagenic effects of Henna，which is a special hair dye in Xinjiang. METHODS：Micronucleus test and sperm malformation test were performed to analyze the mutagenicity of Henna. RESULTS：The rates of micronuclei in three Henna groups (with concentrations of 5 000，2 500，1 250 mg/kg，respectively) and control group were 0.8‰，1.0‰，0.9‰ and 0.8‰，respectively. There was no significant difference between Henna groups and control group (P>0. 05). The rates of abnormal sperm in three Henna groups and control group were 1.4%，1.6%，1.4% and 1.3%，respectively. There was no significant difference between Henna groups and control group (P>0. 05). CONCLUSION：Henna showed no mutagenicity under this experimental condition.

OBJECTIVE: To investigate the effect of Duyiwei ( lamiophlomis rotate) capsule on the expression of proliferating cell nuclear antigen (PCNA) in Wistar rats. METHODS：40 Wistar rats，half male half female，were randomly divided into 5 groups：model group，PTCA group，Duyiwei (Lamiophlomis rotate) capsule 0.50，1.25，2.50 g/kg，positive control group (salvia miltrorrhiza tablet 1.0 g/kg) . Oral administration of drugs was performed in each group，continuously 5 days. Then all rats were treated by percutaneous transluminal coronary angioplasty (PTCA) for the establishment of carotid balloon injury model. Then drug treatment continued after PTCA for 28 days. Carotid artery segment underwent routine biopsy immunohistochemistry and PCNA expression determination. RESULTS：In the normal artery，these was low level or no expression of PCNA. In the arterial intima of the model groups，33.71% was PCNA-positive with each dose of Duyiwei capsule group showing significant differences (P <0.05). CONCLUSION： Duyiwei capsule significantly inhibited the expression of PCNA， which may be one of its mechanisms in preventing restenosis after carotid artery injury in rats.