Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.

Objective To explore the risk factors for clinical anastomotic leakage after resection of rectal cancer in China. Methods By meta-analysis we made a comprehensive analysis of the risk factors for clinical anastomotic leakage after resection of rectal cancer based on 19 articles published in China between January 1999 and January 2009. Results The anastomotic leakage rate was higher in the patients aged 60 years old and above than in those younger, with the combined odds ratio (OR) value being 0.50 (95% CI: 0.33-0.76) (P<0.01). The incidence rate was higher in the male patients than in the female ones, with the combined OR value being 2.17 (95% CI: 1.38-3.42) (P<0.01). The incidence rate in the patients with the distance of tumor from the lower margin to anal verge being 7cm and shorter was higher than that with longer distance, with the combined OR value being 1.79 (95% CI: 1.37-2.35) (P<0.01). The incidence rate in the patients who had received radiotherapy preoperatively was higher than that in those who had not, with the combined OR value of 3.66 (95% CI: 2.19-6.09) (P<0.01). The incidence rate in the patients who had received stapler anastomosis was higher than that in the patients who had received manual anastomosis, with the combined OR value being 0.70 (95% CI: 0.47-1.05), but there was no significant difference between them (P>0.05). The incidence rate was higher in the patients with diabetes mellitus than in the healthy ones, with the combined OR value being 3.16 (95% CI: 2.27-4.39) (P<0.01). The incidence rate was lower in the patients with Dukes A and B stages than in those with Dukes C and D stages, with the combined OR value being 0.61 (95% CI: 0.45-0.83) (P<0.01). The incidence rate in the patients with high malignance degree in clinicopathological types was higher than that with low malignance degree, with the combined OR value being 2.17 (95% CI: 1.38-3.42) (P<0.01). The incidence rate was lower in the patients who had received preventive colostomy than in those who had not, with the combined OR value being 0.39 (95% CI: 0.14-1.05), but there was no significant difference between them (P>0.05). The incidence rate was higher in the patients who had got selective operation than in those who had got emergency operation, with the combined OR value being 0.27 (95% CI: 0.13-0.56). Conclusion The risk factors of anastomotic leakage after resection of rectal cancer are as follows: 60 years old and above, male patients, diabetes mellitus, preoperative neo-adjuvant radiotherapy, the distance of tumor from the lower margin to the anal verge being shorter than 7cm, Dukes C and D stages, high malignance degree in clinicopathological types, and emergency operation.

Objective To study the effects of tetrandrine on apoptosis of HeLa cervical cancer cells qualitatively and quantitatively. Methods We measured tetrandrine-induced inhibition of HeLa cell proliferation at different concentrations and time points by MTT assay. The rate of Hela cell apoptosis induced by tetrandrine was detected by flow cytometer and confocal laser scanning microscope (CLSM). Results Tetrandrine inhibited the proliferation of HeLa cells in dosage- and time-dependent manners. Flow cytometry showed that the apoptosis rate was (51.8±0.97)% at the concentration of 15μmol/L, which was significantly higher than that in the control group (24.3±1.23)% (P<0.05). The cells treated with tetrandrine showed typical apoptotic morphology under CLSM. Conclusion Tetrandrine can inhibit proliferation and induce apoptosis of HeLa cervical cancer cells.

Objective To explore the expression of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-10 (MMP-10) in normal endometrium, atypical hyperplasia and endometrial adenocarcinoma. We aimed to study the relationship of MMP-3 and MMP-10 expressions with infiltration depth, histologic grade and clinical phase in order to explore the role of MMP-3 and MMP-10 in the occurrence, infiltration and metastasis of endometrial adenocarcinoma. Methods The expressions of MMP-3 and MMP-10 were measured by immunohistochemistry in 12 cases of normal endometrium, 12 cases of atypical hyperplasia and 42 cases of endometrial adenocarcinoma. Results The expression of MMP-3 was 73.8% (31/42), 25.0% (3/12) and 8.3% (1/12) in endometrial adenocarcinoma, atypical hyperplasia and normal endometrium, respectively, whereas the expression of MMP-10 was 76.2% (32/42), 33.3% (4/12) and 0(0/12), respectively. Both MMP-3 and MMP-10 expressions exhibited a pattern of decreased intensity in endometrial carcinoma, atypical hyperplasia and normal endometrium. Furthermore, the statistical analysis showed that the expression of both proteins was significantly greater in endometrial carcinoma than in atypical hyperplasia and normal tissue (P<0.01). Expressions of MMP-3 and MMP-10 had a significant correlation with such clinical parameters as histologic grade, depth of myometrial infiltration and clinical stage (P<0.01). Conclusion MMP-3 and MMP-10 can be used as tumor markers of endometrial carcinoma, and the combined detection of them can increase the detection rate of endometrial carcinoma.

Objective To investigate the effect of endoplasmic reticulum stress in renal damage caused by hyperlipidemia. Methods Forty male Wistar rats were randomly divided into two groups: normal control group (NC, n=20) and high-fat group (HF, n=20). Rats in NC group were fed with normal diet while those in HF group were fed with high-fat diet. Five rats in each group were randomly chosen in week 4, 8, 12 and 18. Serum lipid, urine protein in 24 hours and the pathological changes of renal tissues were observed; the apoptosis of renal cells was detected by TUNEL staining; the expression of GRP78 protein in the kidney was examined by immunohistochemistry. The expression of GRP78 mRNA and CHOP mRNA was examined by RT-PCR. Results Compared with those in NC group, serum lipid as well as the expression of GRP78 mRNA and protein in the kidney were increased in week 4, 8, 12 and 18 in HF group(P<0.05). In contrast, urine protein in 24 hours, the apoptosis index of renal cells and the expression of CHOP mRNA were increased in week 8, 12 and 18 (P<0.05). Conclusion CHOP pathway of endoplasmic reticulum stress is involved in renal damage caused by hyperlipidemia.

Objective To study the effects of sesamin on lipid metabolism disorders in hyperlipidemia rats. Methods The hyperlipidemia SD rat models were established with high-fat diet. These rats had been interfered by sesamin for seven weeks, and then had their levels of serum lipids, antioxidant enzymes and peroxidation products determined. The morphological changes in their livers were observed. Results Sesamin could reduce the hyperlipidemia rats' serum total cholesterol, triglyceride, low-density lipoprotein cholesterol and apolipoprotein B levels, and properly increase high-density lipoprotein cholesterol and apolipoprotein A contents (P<0.05). It also reduced the rats' free radical metabolites in serum MDA contents, raised the activities of superoxide dismutase and glutathione peroxidase, and enhanced the body's capacity of inhibiting hydroxyl free radicals (P<0.05). Sesamin of high, medium and low dosage had different effects in relieving liver steatosis of the hyperlipidemia rats. Conclusion Sesamin can regulate lipid metabolism of hyperlipidemia rats, alleviate oxidative stress of the body, and improve liver steatosis.

Objective To evaluate the role of EP_2 in synaptic transmission in dentate gyrus of hippocampus. Methods Field excitatory post-synaptic potentials (fEPSP) were recorded at the perforated path-granule neurons in dentate gyrus in vitro. Results ① Butaprost, an agonist of EP_2, enhanced the synaptic transmission in dentate gyrus and decreased the paired-pulse ratio, and these effects were reversed by bath application of AH6809 (EP_2 antagonist). ② Application of Forskolin alone or with AH6809 elevated the slope of fEPSP. ③ The Butaprost-induced responses were mediated via PKA, ERK and IP_3 signal pathways. Conclusion Multiple signal pathways were involved in the EP_2 activation-mediated enhancement of synaptic transmission.

Objective To construct wtp53/junB fusion gene and its eukaryotic expression vector in order to provide the basis for further application of polygene union therapy in hepatocellular carcinoma. Methods Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and gene recombination techniques were used to construct the eukaryotic vector of pEGFP-C1-wtp53/junB fusion gene, which carries the enhanced green fluorescent protein (EGFP). The transfection of pEGFP-C1-wtp53/junB in hepatoma HepG2 cells was detected by the location of green fluorescence. Results The DNA sequence of wtp53/junB fusion gene was successfully cloned into the pEGFP-C1 plasmid and the sequence was the same as what we expected. Green fluorescence located on cell nucleus proved that pEGFP-C1-wtp53/junB was transfected into HepG2 cell line successfully. Conclusion We successfully constructed the eukaryotic vector of pEGFP-C1-wtp53/junB fusion gene, which carries the EGFP, and transfects it into human hepatoma cell nucleus. It may lay the basis for studying the synergetic effect of wtp53 and junB in hepatocellular carcinoma.

Objective To examine matrix proteoglycan metabolic markers and probe into the turnover of matrix proteoglycan and enzyme-mediated role of matrix metalloproteinases (MMPs) and aggrecanases in reparative tissues with tissue engineering cartilage. Methods Tissue-engineered cartilage was constructed by cancellous bone matrix gelatin (BMG) with allogeneic chondrocytes in vitro for 2 weeks, then implanted to repair osteochondral defects of rabbit knee joint. Samples were obtained 6 months later to explore the expressions of 3-B-3(-) epitope, MMPs, MMP-generated epitope BC-4 and aggrecanases-generated epitope BC-13. Results In repaired tissues, the expression of 3-B-3(-) epitope increased, but that of MMPs and MMP-generated epitope BC-4 reduced. There was no expression of aggrecanases-generated epitope BC-13. Conclusion Expressions of 3-B-3(-), MMPs, BC-4 and BC-13 can help probe into the matrix proteoglycan turnover in reparative cartilage tissues. Anabolism exceeds catabolism in the repaired tissues. MMPs play an important role in the conservative baseline turnover of proteoglycan and remodeling of the graft tissues.

Objective To evaluate the expression and function of mammalian target of rapamycin (mTOR) and its substrate kinase P70S6K in mTOR/P70S6K signal pathway in cervical carcinogenesis. Methods The expression of mTOR and P70S6K in normal cervix uteri or cervical cancer was detected by the immunohistochemistry and RT-PCR methods. Results Compared to those in normal cervix uteri, both mRNA and protein of mTOR/P70S6K in cervical carcinomas were significantly increased (P<0.05). There was a positive correlation between mTOR gene and P70S6K gene expression in cervical cancer (r=0.746, P<0.001). Conclusion High expression of mTOR and P7OS6K may play an important role in the pathogenesis and development of cervical cancer.