Objective To establish a method of high resolution melting analysis (HRM) with unlabeled probe for detection of rt204 mutation in HBV P gene.Methods Plasmids with wild strain rt204M,mutant strains rt204I and rt204V were constructed,and the probes were designed and optimized.HRM plots were established by the constructed plasmids.A total of 185 samples were collected from patients with chronic hepatitis B in the Affiliated Hospital of School of Medicine of Ningbo University during May 2010 and May 2012.All samples were detected by HRM,and matched with characteristic melting pattern.rt204 mutations were screened,and then verified by DNA sequencing.Paired x2 test was used for the comparison of the detection of mutations.Results The melting temperatures for rt204I,rt204V and rt204M were 58.0℃,60.6℃ and 62.5℃,respectively.Among 185 samples,168 samples (90.8％) could be analyzed by HRM method,and 155 samples (83.8％) coule be successfully sequenced (P ＜0.01).In the 155 samples which were completely analyzed by HRM assay and sequencing,75 samples were rt204M,55samples were rt204I,and 25 samples were rt204V by using HRM method,with an overall mutation detection rate of 51.6％ (80/155) ; and by sequencing,110 samples were rt204M,30 samples were rt204I,and 15samples were rt204V,with an overall mutation detection rate of 29.0％ (45/155).The difference onmutation rates detected by the above two methods was of statistical significance (P ＜ 0.01).Conclusion HRM with unlabeled probe is simple,sensitive,rapid and specific for detection of rt204 mutation in HBV P gene.

Objective To study CEAmRNA、CK19mRNA and CK20mRNA in peripheral blood of lung cancer patients by Real-time RT-PCR,consequently discussing those result′s clinical significance.Methods 48 lung cancer patients,8 benign lung disease patients and 30 healthy volunteers of CEAmRNA,CK19mRNA and CK20mRNA in peripheral blood were detectd by Real-time RT-PCR.Results The levels of 48 lung cancer patients CEAmRNA in peripheral blood reach (16 264?28 765) copies/ml,of which 26 patients,results are positive; The levels of 48 lung cancer patients CK19mRNA in peripheral blood reach (14 891?27 244) copies/ml,of which 27 patients,results are positive; The levels of 48 lung cancer patients CK20mRNA in peripheral blood reach (10 924?21 678) copies/ml,of which 20 patients,results are positive. Among of 8 benign lung disease patients,there is just CK20mRNA was detected in one patient,both CEAmRNA and CK19mRNA were not detected in their peripheral blood. As for those 30 healthy volunteers,there is none of the mRNA was detected. Moreover,there is a marked positive correlation between the levels of CEAmRNA,CK19mRNA and CK20mRNA,the levels of CEAmRNA,CK19mRNA and CK20mRNA were correlated to the physiological cause or degree of lung cancer.Conclusion CEAmRNA,CK19mRNA and CK20mRNA in peripheral blood can be as auxiliary index for diagnosing lung cancer micrometastasis,also can monitor the lung cancer micrometastasis by quantity it.

Objective To investigate the expression of Tie-2 in rectal carcinoma and its relationship with invasion and metastasis in rectal carcinoma. Materials S-P immunohistochemical assay was used to detect the expression of Tie-2 in 40 cases of rectal carcinoma and 10 cases of normal rectal tissues. Results Tie-2 was mainly localized in the cytoplasm and nucleus of vascular endothelial cells in cancerous tissues and partly in the cytoplasm of some cancerous cells. The expression of Tie-2 in rectal carcinoma was significantly higher than that in normal rectal tissues (P<0.05); however, Tie-2 expression was not associated with differentiation, invasion depth and Dukes stage (P>0.05), but with lymphatic metastasis (P<0.05). Conclusion Tie-2 plays a key role in carcinogenesis and lymph node metastasis of rectal carcinoma.

Objective To evaluate the significance of carbohydrate antigen 50 (CA50) expression in colorectal carcinoma. Methods Immunohistochemical staining was used to detect CA50 expression in 10 cases of normal colorectal mucosa and 40 cases of cancer mucosa. Results The expression of CA50 increased in normal colorectal mucosa, cancer distant mucosa, cancer adjacent mucosa and cancer mucosa, and there were significant differences among them (P<0.05). The expression of CA50 in colorectal carcinoma was correlated with the degree of differentiation, Duke's stage and lymphatic metastases (P<0.05). Conclusion The expression of CA50 can be used as a valuable index in evaluating the biological characteristics and prognosis of colorectal carcinoma.

Objective To evaluate the clinical effects and experiences of short stem ceramic-on-ceramic total hip arthroplasty in the treatment of young and middle-aged patients with femoral head necrosis.Methods From December 2009 to March 2011,40 patients with femoral head necrosis were treated primarily with short stem ceramic-on-ceramic hip arthroplasty (43 hips) through posterior-lateral or anterior-lateral approach.There were 23 males,17 females； with the mean age of 40.5 years (range,27-55 years).All cases were stage Ⅳ or more according Steinberg stage.The patients were followed up at the 3,6,12 months after operation,and then once each year.Standard X-ray films were taken at the follow-ups.Femoral prosthesis subsidence was assessed through X-ray films.Harris scores system and WOMAC Osteoarthritis Index were recorded before and after operation to evaluate clinical effects of surgery.Results Thirty-eight patients (41 hips) were followed up for 21-36 months,with an average of 31.3 months.The femoral stem subsided 0.68±2.90 mm at the last follow-up.After operation,hip pain of patients was relieved obviously.The Harris score improved from 38.7±12.1 (15-66) preoperatively to 95.2±4.49 (83-100) postoperatively,WOMAC Index from 66.7±12.0 (49-94) to 12.8±5.6 (0-24).During follow-up period,there was no serious complication such as femoral neck fracture,infection of wound,pulmonary embolism,et al.No dislocation,implant prosthesis failure and infection were observed.Conclusion The short stem hip arthroplasty preserves bone stock to a great extent.The short stem implant showed a good stability and osseous integration.The wear rate of ceramic-on-ceramic bearing surface is extremely low.Short stem hip arthroplasty plus ceramic-on-ceramic bearing surface might be a good option for the young and middle-aged patients with femoral head necrosis.

Objective To investigate the correlation between acquired drug resistance-related genes and mobile genetic elements from pandrug-resistant Acinetobacter baumannii. Methods Fifty-three horizontal transfer drug resistance-related genes ( β-lactamases,aminoglycoside and quinolones resistance related) and 12 mobile genetic elements (including zygosity plasmid,transposon,insertion sequence and integron) were detected by polymerase chain reaction (PCR) in 20 clinical isolates of pandrug-resistant Acinetobacter baumannii.Index cluster analysis was performed to explore the correlation.Results In 20 strains of pandrug-resistant Acinetobacter baumannii,there were 3 types of β-lactamases related genes (TEM-1,ADC-30,OXA-23 ),4 types of aminoglycoside modifying enzyme genes [ aac (3)-Ⅰ,aac(6′)-Ⅰ b,ant( 3″)-Ⅰ and aph( 3′)-Ⅰ ],and 5 kinds of mobile genetic elements ( int Ⅰ 1,tnpU,tnp513,IS26 and ISAba1 ). Index cluster analysis showed high correlations between resistance genes [TEM-1,ADC-30,aac( 6′)-Ⅰ b,ant( 3″)-Ⅰ,abeB,qacE Δ1] and mobile genetic elements ( int Ⅰ 1,tnpU,tnp513,IS26,ISAba1 ).Conclusion Clinical isolated pandrug-resistant Acinetobacter baumannii carries several acquired drug resistance-related genes and mobile genetic elements,and there may be a close association between them.

Objective To analyze the immunological characteristics of Trichinella spiralis secretory antigen P53 and to evaluate its value in diagnosis of trichinellosis. Methods An open read frame of secretory antigen P53 was cloned from Trichinella spiralis by reverse transcriptasepolymerase chain reaction (RT-PCR) and then sequenced. Bioinformatics analysis was performed to search for its homologues in other helminths and predict its potential linear B cell epitopes and T cell epitopes. The sequence coding mature peptide was inserted into prokaryotic expression vector pET28a(+) and the purified recombinant product was identified by Western blot using serum samples of patients infected with Trichinella spiralis or other helminth. Results Bioinformaties analysis results showed that there was no P53 homologue in other helminths, which indicated that there were many linear B cell epitopes and T cell epitopes in TsP53. The recombinant P53 antigen only reacted with the serum samples of patients infected with Trichinella spiralis without any cross-reaction with the serum of patients infected with other helminths. Conclusion P53 has strong immunogenicity and immunoreactivity, which may be a promising candidate for developing Trichinella spiralis specific diagnostic method.

Objective To explore the prevalence of 11 efflux pumps in isolates of multidrugresistant Escherichia coli(MDR-ECO). Methods Efflux pumps emrB, emrD , emrE, mdfA, sugE, mdtl,tehA, oqxA, qacE△1, qacE and smr-2 were detected by polymerase chain reaction(PCR)in 20 MDR-ECOs isolated from clinical samples. Results Efflux pumps emrB, emrD, emrE, mdfA, sugE, mdtI, qacE △1, tehA and oqxA were detected, and 8 efflux pumps were found in the same strain. Conclusion Multidrug- resistance in Escherichia coli may be related with efflux pumps.

OBJECTIVE:To observe the clinical efficacy and safety of Xiaozhen zhiyang spray in the treatment of EGFRI-asso-ciated rash. METHODS:A total of 60 malignant tumor patients suffering from rash induced by EGFRI were divided into trial group (40 cases)and control group(20 cases)according to the patient's willingness. Control group didn't received any therapy for rash. Trial group received Xiaozhen zhiyang spray for several times a day according to the degree of rash as 1-2 times/d for first degree, 2-3 times/d for second degree,3-5 times/d for third degree,and the treatment course lasted for 2 weeks. Rash degree and improve-ment,itching degree and improvement,daily life quality index(DLQI)score before and after treatment as well as the occurrence of ADR were compared between 2 groups. RESULTS:before treatment,there was no statistical significance in rash and itching de-gree,or DLQI score between 2 groups(P>0.05). After treatment,rash and itching degree of trial group were improved significant-ly compared to before treatment and control group,with statistical significance(P<0.05). The total response rates of rash and itch-ing therapy in trial group were significantly higher than control group(67.50% vs. 20.00%,70.00% vs. 15.00%),with statistical significance(P<0.05). DLQI score of trial group was significantly lower than before treatment and control group,with statistical significance(P<0.05). There was no statistical significance in DLQI score of control group before and after treatment(P>0.05). No obvious ADR was found in trial group. CONCLUSIONS:Xiaozhen zhiyang spray can effectively relieve EGFRI-associated rash and itching as well as improve the quality of life for patients.

The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells. The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α, and the expression of β-catenin protein was detected by Western blotting. The cultured rabbit nucleus pulposus cells were divided into 4 groups. In group A, the cells were cultured with normal medium and served as control group. In group B, the cells were cultured with TNF-α and acted as degeneration group. In group C, the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group. In group D, the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP, serving as intervention group. The expression of type II collagen, proteoglycan, β-catenin, and MMP-13 in each group was detected by immunocytochemistry and RT-PCR. The result showed that TNF-α increased the expression of β-catenin and MMP-13, and significantly inhibited the synthesis of type II collagen and proteoglycan, which resulted in the degeneration of nucleus pulposus cells. This effect could be obviously reversed by DKK1. We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway, and increase the expression of MMP-13, thereby resulting in disc degeneration. Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue. The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.