Objective:To analyze the relationship between hepatitis B virus(HBV)gene mutation at 1896 in precore region with genotype and replication of HBV and the liver function of patients.Methods:HBV precore 1896 site mutation,the genotype of HBV and serum content of HBV DNA were determined by PCR in 60 patients positive of HBV DNA.Chemiluminescence miacropaticle immunoassay(CMIA)was used for detection of serum HBeAg and HBeAb.Liver function parameters were ob- tained by routine biochemistry method.Results:The alanine aminotransferase(ALT)level in HBV with 1896 site mutation was significantly higher than that in the wildtype virus.Site mutation at 1896 had no correlation with HBeAg,HBV genotype and HBV DNA content.HBV DNA content in patient with genotype C was significantly higher than that with genotype B(P

OBJECTIVETo study the effects of short hairpin RNA (shRNA) mediated gene silencing of β-catenin on the biological characteristics of esophageal carcinoma cells, and to provide theoretical and experimental evidence for the gene therapy of esophageal carcinoma through target inhibition of β-catenin gene.

METHODSSingle strand DNA was synthesized according to the hairpin RNA sequence, and then subcloned into eukaryotic expression vector pGenesil-3 to construct a shRNA-expression pDNAs driven by human U6 promoter of β-catenin (pGen-3-CTNNB1). One additional construct of random siRNA (pGen-3-con) without homologous to any human genes was constructed in a similar fashion as control.Positive clones were identified and verified by restriction cleavage and DNA sequencing analyses. pGen-3-CTNNB1 and pGen-3-con were then transfected into esophageal carcinoma cell line Eca-109 with liposome, respectively. Positive colonies were selected with G418. Expression of β-catenin protein and mRNA in the transfected and nontransfected Eca-109 cells were examined by Western blotting, immunofluorescence and RT-PCR, respectively. Xenograft tumor model was used to compare the tumorigenesis of three different cells.Expressions of β-catenin in all tumor tissues were examined by immunohistochemistry staining. The invasive abilities of three different cells were examined with transwell invasion filter and Matrigel.

RESULTSβ-catenin expression levels were found markedly decreased in Eca-109 cells transfected with pGen-3-CTNNB1. In vivo, transfection with β-catenin shRNA greatly impeded the tumor growth, pGen-3-con (1.18 ± 0.13) g, Eca-109 (1.38 ± 0.21) g, pGen-3-CTNNB1 (0.42 ± 0.09) g, P < 0.05. Immunohistochemistry staining showed a significantly decreased expression of β-catenin in β-catenin shRNA transfected cells than in random shRNA transfected and nontransfected cells (P < 0.05). The infiltration abilities of esophageal carcinoma cells were significantly suppressed, pGen-3-con (81 ± 5)/HPF, Eca-109 (77 ± 6)/HPF, pGen-3-CTNNB1 (41 ± 4)/HPF, P < 0.01; along with significantly decreased migration abilities, pGen-3-con (73 ± 5)/HPF, Eca-109 (69 ± 5)/HPF, pGen-3-CTNNB1 (38 ± 4)/HPF (P < 0.05).

CONCLUSIONSThere are abnormal expression of β-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109. RNA interference targeting β-catenin gene suppresses the growth of xenograft tumorigenesis in nude mouse and the invasiveness and metastatic capability of esophageal carcinoma cells.

Animals ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Gene Silencing ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Plasmids ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Random Allocation ; Signal Transduction ; Transfection ; Tumor Burden ; Wnt Proteins ; metabolism ; beta Catenin ; genetics ; metabolism ; physiology

OBJECTIVETo investigate the treatment efficiency of a new photodynamic therapeutic(PDT) drug synthesized by our laboratory toward MGC803 cells and related mechanisms.

METHODSBleaching method was used to evaluate the photostability of drug upon repetitive illumination. MTT assay was used to determine the ability of new drug killing MGC803 cells after PDT. Laser scanning confocal microscopy (LSCM) was applied to investigate the subcellular localization of drug in MGC803 cells (mitochondria and/or lysosomes). Hoechst staining and flow cytometry(Annexin V/PI double-staining) were performed to detect the death mode of MGC803 cells after PDT.

RESULTSThis new PDT drug had good stability to light irradiation after repetitive illumination. MTT assay showed no cytotoxicity towards MGC803 cells only by drug or only by irradiation(P>0.05), but intense lethal effect was observed with drug and light combination(P<0.05). The phototoxicity of medicine increased with the elevation of concentration, the LD50 was 1.74 μmol/L, and reaching plateau at the concentration of 3.12 μmol/L, even increasing the concentration. LSCM found that drug localized in lysosomes of MGC803 cells. Hoechst staining showed that the death mode of cells was mainly necrosis and Annexin V/PI double-staining proved this result further.

CONCLUSIONThis new PDT drug is an effective PDT sensitizer for MGC803 cells and the death mode of cells is mainly necrosis.

Apoptosis ; Cell Line, Tumor ; Humans ; Mitochondria ; Photochemotherapy ; Stomach Neoplasms ; drug therapy ; pathology

OBJECTIVETo investigate the early prognostic values of arterial lactate and base excess (BE) in patients with paraquat poisoning.

METHODSSeventy-five patients with paraquat poisoning were divided into sudden death group (n = 10) who died within 24 h after admission, recent death group (n = 31) who died more than 24 h after admission, and survival group (n = 34). Arterial lactate and BE were measured on admission and at 24 h after admission. The prognostic values of arterial lactate and BE were analyzed.

RESULTSThe arterial lactate measured on admission was significantly higher in the sudden death group than in the recent death group and survival group (P < 0.01), but there was no significant difference in arterial lactate between the recent death group and survival group (P = 0.309). The BE measured on admission was significantly lower in the sudden death group than in the recent death group and survival group, and it was significantly lower in the recent death group than in the survival group (P < 0.01 or P < 0.05). At 24 h after admission, the recent death group had a significantly higher arterial lactate (P < 0.01) and a significantly lower BE (P < 0.01), as compared with the survival group. The logistic regression analysis showed that the two indices were significantly associated with prognosis (P < 0.01). On admission, the areas under the receiver operating characteristic (ROC) curve (AUCs) of arterial lactate and BE for predicting death were 0.692 and 0.787, respectively, and the cut-off values were 3.25 mmol/L and -1.75 mmol/L, respectively; the AUCs of arterial lactate and BE for predicting sudden death were 0.995 and 1, respectively, and the cut-off values were 7.1 mmol/L and -12.8 mmol/L, respectively. At 24 h after admission, the AUCs of arterial lactate and BE for predicting death were 0.743 and 0.822, respectively, and the cut-off values were 2.15 mmol/L and -5.55 mmol/L, respectively.

CONCLUSIONArterial lactate and BE have certain values in predicting the death, especially the sudden death, in patients with acute paraquat poisoning.

Adult ; Aged ; Arteries ; chemistry ; Female ; Humans ; Lactic Acid ; blood ; Male ; Middle Aged ; Paraquat ; poisoning ; Poisoning ; diagnosis ; Prognosis

OBJECTIVETo assess the effect of RNA interference-mediated gene silencing of plasma membrane-related Ca(2+)-ATPase-1 (PMR1) gene on the insulin secretion in islet beta cells NIT-1 in vitro.

METHODSA small interfering RNA duplex (siPMR1) corresponding to the nucleotides 337-357 of mouse PMR1 cDNA was introduced into NIT-1 cells via liposomes. The gene silencing effect was assessed by RT-PCR, and the total insulin level in the transfected cells was measured by radioimmunoassay.

RESULTSTransfection with siPMR1 resulted in obviously reduced PMR1 expression and increased insulin secretion in NIT-1 cells.

CONCLUSIONThe synthesized siPMR1 can significantly silence the expression of PMR1 and promote the secretion of insulin in the islet cells in vitro, which shed light on further studies of RNAi-based therapy of diabetes.

Animals ; Calcium-Transporting ATPases ; deficiency ; genetics ; Cell Line ; Gene Expression Regulation ; Insulin ; secretion ; Insulin-Secreting Cells ; metabolism ; secretion ; Mice ; RNA Interference ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo study the diagnostic value and pitfalls of ultrasound-guided core needle biopsy (CNB) of soft tissue tumors.

METHODSOne hundred and six cases of CNB specimens encountered during the period from 2007 to 2012 were enrolled into the study. The pathologic diagnosis using CNB was compared with that using surgical specimens. Diagnostic accuracy was analyzed using Chi-square test, with respect to the histologic pattern (such as spindle cell and myxoid), biologic behavior (benign versus malignant) and immunohistochemical results. The 59 cases of sarcoma were subdivided into three grades according to FNCLCC grading system.

RESULTSHistologic diagnosis could be made in 84.0% (89/106) cases. Thirteen cases were non-diagnostic on CNB. There were 4 cases on CNB showing diagnostic discrepancy with surgical specimens. Four cases of "benign lesions" on CNB found to be myxoid liposarcoma and lipoma-like liposarcoma upon resection. In general, myxoid pattern (9/17) seen on CNB showed less diagnostic correlation with surgical specimens, as compared to spindle cell and other histologic patterns (P < 0.01). The rate of diagnostic correlation was 79.7% (49/59) for the 59 cases of sarcoma studied, with grade 2 and grade 3 sarcoma showing better correlation (in contrast to 7/17 for grade 1 sarcoma) (P < 0.01). Comparative analysis showed no significant difference between benign/borderline tumors and sarcomas. The application of immunohistochemical study did not result in significant improvement in diagnostic accuracy on CNB.

CONCLUSIONSUltrasound-guided CNB is a reliable tool in pathologic diagnosis of soft tissue tumors and shows a high accuracy rate especially for high-grade sarcoma. Tumors with myxoid pattern, lipomatous tumors and grade 1 sarcomas are associated with lower diagnostic accuracy on CNB. Correlation with clinicoradiologic findings would also be helpful in diagnostic evaluation and surgical planning.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biopsy, Large-Core Needle ; methods ; Diagnostic Errors ; Extremities ; Female ; Humans ; Liposarcoma, Myxoid ; diagnosis ; diagnostic imaging ; pathology ; Male ; Middle Aged ; Neoplasm Grading ; Sarcoma ; diagnosis ; diagnostic imaging ; pathology ; Soft Tissue Neoplasms ; diagnosis ; diagnostic imaging ; pathology ; Ultrasonography, Interventional ; methods ; Young Adult

To analyze the genetic characteristics of echovirus 6 (E6) isolated from meningitis and encephalitis cases in Shandong Province, China, we collected cerebrospinal fluid samples from meningitis and encephalitis cases in Shandong Province from 2007 to 2012 for virus isolation. Viral RNAs were extracted from positive isolates, and complete VP1 coding regions were amplified by RT-PCR and sequenced. Homology comparison and phylogenetic analysis were performed. Six isolates were identified as E6 by microneutralization assay and molecular typing. The homology analysis showed that the six isolates had 78. 6%-99. 8% nucleotide and 95. 5%-100. 0% amino acid identities with each other, as well as 76. 9%-78. 4% nucleotide and 92. 3%-95. 1% amino acid identities with the prototype strain (D' Amori). The phylogenetic analysis based on the integrated VP1 sequences indicated that all Shandong E6 isolates could be separated into four clusters, designated as A, B, C, and D. The six E6 isolates belonged to clusters A, B, and D. Our study reveals high genetic differences between Shandong E6 isolates and suggests different transmission lineages of E6 co-circulated in Shandong Province.
Amino Acid Sequence ; Child ; Child, Preschool ; China ; epidemiology ; Echovirus 6, Human ; classification ; genetics ; isolation & purification ; Encephalitis ; epidemiology ; virology ; Female ; Genetic Variation ; Humans ; Infant ; Male ; Meningitis ; epidemiology ; virology ; Molecular Sequence Data ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

Objective To construct the recombinant varicella zoster virus (VZV)carrying 3xflag gene,3xflag was added to the VZV open reading frame 7 (ORF7)using GalK-based homologous recombination.Methods GalK and 3xflag gene fragments with 50 bp VZV ORF7 homologous arms were amplified by PCR.The obtained fragments were purified and transferred to the competent cells of VZV bacterial artificial chromosome (SW102-VZVWTBAC). The clones of VZV ORF7 with 3xflag (SW102-VZV ORF7-3xflag-BAC)were obtained by homologous recombination and selection from medium containing GalK and replaced GalK. The recombinant plasmids were extracted and transfected into ARPE-19 cells.The effect of VZV ORF7-3xflag on ARPE-19 cells was observed.Results The clones of VZV ORF7 with 3xflag (SW102-VZV ORF7-3xflag-BAC)were obtained.The virus patches with green fluorescence were observed three days after SW102-VZVWTBAC and SW102-VZV ORF7-3xflag-BAC were transfected into ARPE-19 cells.Western blot showed that ORF7 expression was effectively enhanced with 3xflag.Conclusion The recombinant VZV carrying 3xflag gene was obtained,which suggests that GalK-based homologous recombination is convenient,efficient and accurate in manipulating the gene virus of interest.

OBJECTIVETo investigate the clinicopathological features, treatment, and prognosis of gastric neuroendocrine carcinoma and gastric carcinoma with neuroendocrine cell differentiation.

METHODSA total of 19 patients were treated for gastric neuroendocrine cancer or gastric cancer with neuroendocrine differentiation in the Beijing Cancer Hospital from January 1997 to December 2008. Clinical data were retrospectively analyzed.

RESULTSFourteen patients had neuroendocrine carcinoma in the gastric cardia (n=9) or gastric body(n=5), and 5 patients had gastric cancer with neuroendocrine differentiation in the gastric cardia(n=2), the antrum(n=2), and the entire stomach(n=1). According to the International Classification of Disease for Oncology(2000), patients were divided into gastric carcinoid type I((n=2, 10.5%), type III( sporadic gastric carcinoid (n=9, 47.4%), small cell carcinoma of the stomach(n=3,15.8%), and gastric cancer with neuroendocrine cell differentiation(n=5, 26.3%). Clinical manifestations were mostly non-specific. Diagnosis was based on pathological and immunohistochemical examination. Eighteen patients underwent surgery including radical subtotal gastrectomy and total gastrectomy, of whom 3 underwent simultaneous resection of the liver metastasis. The remaining one patient with small cell carcinoma of the gastric body received chemotherapy alone because of unresectable liver metastasis. The survival rate was 73.7% at 1 year and 38.6% at 3 years.

CONCLUSIONSGastric neuroendocrine carcinoma usually develops in the cardia and body of the stomach. Gastric carcinoma with neuroendocrine cell differentiation can occur in any locations of the stomach. Immunohistochemistry is important to the diagnosis. Radical resection is the main treatment.

Aged ; Carcinoid Tumor ; diagnosis ; pathology ; therapy ; Carcinoma, Neuroendocrine ; diagnosis ; pathology ; therapy ; Carcinoma, Small Cell ; diagnosis ; pathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Stomach Neoplasms ; diagnosis ; pathology ; therapy

Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.
Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Histocompatibility Antigens Class I ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; beta 2-Microglobulin ; biosynthesis ; genetics