Objecfive To investigate the expression of erbB4 and PTEN in cutaneous squarnous cell carcinoma(SCC)tissue and their significance.Methods Immunohistochemical Elivision method was used to measure the protein expression of erbB4 and PTEN in tissue specimens from 52 patients with SCC (11 cases with and 41 cases without lymph node metastasis)and 10 normal human controls.Results The positivity rate of erbB4 was significantly higher in the patients with SCC than in the normal human controls (75%vs.10%,x2=12.77,P<0.01),and in the patients with lymph node metastasis than in those without (100%vs.68.29%,P<0.05).Significant differences were also observed in the expression rate of PTEN between the patients with SCC and the normal controls (48.08%vs.100%,x2=9.20,P<0.01),between the patients with well-differentiated SCC and those with poody differentiated SCC(78.57%vs.36.84%,P<0.05),and between the patients with lymph node metastasis and those without (9.09%vs.58.54%,P<0.01).The expression of erbB4 was negatively correlated with that of PTEN (r=-0.42.P<0.01).Conclusion The disturbalice in mutual regulation between erbB4 and FIEN seems to be involved in the initiation.progression and metastasis of SCC.

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

Objective To compare the differences of cardiac function and interstitial remodeling between diastolic heart failure(DHF) and systolic heart failure(SHF) rabbit models. Methods To establish DHF model with abdo-mial aorta constriction and SHF model with abdomial aorta constriction plus aortic insufficiency. The cardiac func-tion was examined by UCG parameters and homodynamic parameters. The collagen content was measured through hydroxyproline colorimetric assay and shown as collagen area(CA), collagen volume fraction(CVF) and area ratio of Ⅰ to Ⅲ type collagen with PSR. Results Compared with control group, there were significantly increased thick-ness and stiffness of myocardium, impaired diastolic function but normal ejection fraction (EF), and significantly increased collagen content, CA, CVF and area ratio of Ⅰ to Ⅲ type collagen in DHF group; heart chamber was sig-nificantly enlarged, systolic function decreased, and collagen content, CA, CVF significantly increased, but ratio of Ⅰ to Ⅲ type collagen decreased in SHF group(P <0.05 or P <0.01). Conclusion DHF and SHF rabbit mod-els were established successfully, which can simulate clinical profiles and provide technical support to future re-search.

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Objective To investigate the protective immunity of the recombinant thioredoxin of Schistosoma japonicum(reSjcTrx)in mice. Methods Thirty 6-week old female C57BL/6 mice were randomly divided into 3 groups with 10 each: reSjcTrx with Montanide ISA720 adjuvant, adjuvant control, and infection control. Mice were vaccinated subcutaneously at week 0, 2, 4 with reSjcTrx emulsified in Montanide ISA720 adjuvant. The mice in adjuvant group was injected three times with Montanide ISA720 and saline only. Mice in infection control group were given no injection. Three weeks after final injection, each mouse was challenged with 30?1 cercariae of S. japonicum (Chinese strain). At the week six after challenge, all mice were sacrificed and perfused. The number of recovered worms and eggs from liver tissue of mice were counted. Sera were collected from mice before immunization, before challenge and before killing. The anti-SjcTrx antibodies in sera were detected with ELISA. Results ELISA showed a high level of specific IgG antibodies in mice immunized with the reSjcTrx. The worm reduction rate and egg reduction rate of reSjcTrx immunization group were 22.8% and 29.5% respectively, significantly higher than those of the control groups (P﹤0.05). SDS-PAGE and Western blotting revealed that the molecular weight of expressed protein was around Mr 14 000 and could be recognized by sera from rabbit infected with S.japonicum and from mice immunized with reSjcTrx. Conclusion The reSjcTrx induces certain protective immunity against schistosomiasis japonica in mice.

Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C.parvum gene (L16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 method was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C.parvum oocysts, while Chelex-100 method is simpler, quicker and more reliable for DNA extraction of the parasite.

Objective To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. Methods Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheather sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. Results The results suggested that the size of oocysts was (7.4?0.32)?m by(5.4?0.21)?m and the ratio of length and width was 1.37?0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of C.andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C.andersoni clade based on the sequence of SSU rRNA and COWP gene. Conclusion What isolated from naturally infected cow feces has been identified as C. andersoni.

Objective To investigate the heterogeneity of chemosensitivity in colorectal cancer using an ATP-tumor chemosensitivity assay (ATP-TCA) and the feasibility of individual chemotherapy.Methods An ATP-TCA were used to determine the effect of 16 single or combined cytotoxic drugs in surgical specimens from 50 patients with colorectal cancer.Results There were considerable differences in chemosensitivity between individuals.The most active single drugs in the assay was identified as Navelbine, Hydroxycamptothecin, 5-Fluorouracil and Paclitaxel; 34.1%, 31.6%, 27.6% and 24.3% of specimens showed sensitivity to them, respectively.5-Fluorouracil+Mitomycin+Aytarabine was found to be the most effective combination, for 100% (11/11)specimens were sensitive to this regimen.5-Fluorouracil+Cisplatin+Adriamycin and Gemcitabine+Cisplatin were moderately active regimens.Conclusions There was the heterogeneity of the in vitro chemosensitivity in colorectal cancer.The use of the ATP-TCA provides a method of selecting appropriate anti-cancer drugs in colorectal cancer.

Objective To evaluate the efficacy and safety of CT-guided percutaneous argon-helium cryoablation in treating hepatic metastases from nasopharyngeal carcinoma. Methods The clinical data of 16 patients with hepatic metastases from nasopharyngeal carcinoma, who had received percutaneous argonhelium cryoablation therapy, were retrospectively analyzed. The quality of life before and after therapy, and the postoperative complications were recorded. After argon-helium cryoablation therapy, the progression-free survival (PFS), the overall survival (OS), and the one-month, 3-month, 6-month, 12-month postoperative tumor local control rates were calculated. Results In all 16 patients, no severe complications occurred and the quality of life was significantly improved after argon-helium cryoablation therapy. The one-month, 3-month, 6-month, 12-month postoperative tumor local control rates were 100％, 87.5％, 80.0％ and 58.3％respectively. The median PFS was 11 months (95％CI: 8.4-13.6 months), and the median survival time was19 months (95％CI: 9.2-28.8 months) . After argon-helium cryoablation therapy, the half-year, one-year and2-year survival rates were 93.8％, 75％ and 43.8％ respectively. Conclusion For the treatment of hepatic metastases from nasopharyngeal carcinoma, CT-guided percutaneous argon-helium cryoablation is minimally-invasive, safe and effective with reliable curative effect.

Objective: To establish a method for determination the S-phenylmercapturic acid in urine by dispersive solid-phase extraction using Humic Acid/Fe₃O₄ magnetic nanocomposite as adsorbent. Methods: The 5 ml of urine samples were adjusted to pH 1.0 and extracted by Fe3O4@HA. Then the analytes were separated on EC-C₁₈ capillary column and detected by HPLC-VWD. The S-phenylmercapturic acid was characterized by the retention time and quantified by peak area and external standard method. Results: The standard curves of SPMA showed significant linearity between 0.04~1.00 mg/L (r=0.999 7) . The average recovery was 94.2%~102.4%. The intra-day and inter-day precisions (RSD) were 2.9~6.7% (n=6) and 3.1~7.5% (n=6) respectively. The detect limit of SPMA was 0.012 g/L (S/N=3) . Conclusion This method is simple, rapid, sensitive and accurate. It is applicable for determination of SPMA in the urine of works who were exposed to benzene.