OBJECTIVETo evaluate the specific killing effects of combination of recombinant adenovirus mediated double suicide gene driven by KDR promoter and survivin antisense oligonucleotide(ASODN) on colorectal cancer cells and vascular endothelial cells.

METHODSThe 293 packaging cells were transfected with the plasmids of pAdEasy-CDglyTK and the recombinant adenovirus were generated. The KDR expressive cells of SW620, ECV304 were infected with adenovirus, meanwhile survivinASODN was transferred into the same cells. The infection rate of adenovirus and transfection efficiency of survivinASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on SW620, ECV304 were examined through MTT method.

RESULTSThe cells which were infected with the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection efficiency. The high expression of CDglyTK gene was found in SW620, ECV304 cells infected with recombinant adenovirus and survivin ASODN decreased the survivin protein level. The survival rate of gene therapy group was significantly lower than that of negative group. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate of SW620 and ECV304 cells as compared with the AdKDR-CDglyTK or survivin ASODN used alone (P<0.05). The survival rate was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that AdKDR-CDglyTK used alone (P>0.05). The combined therapy of AdKDR-CDglyTK and survivin ASODN showed synergistic killing efficacy and more significant bystander effects.

CONCLUSIONThe combined gene therapy of AdKDR-CDglyTK system and survivin ASODN has stronger specific killing effects on colorectal cancer cells and vein endothelial cells.

Adenoviridae ; genetics ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; Endothelial Cells ; metabolism ; Genes, Transgenic, Suicide ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Oligonucleotides, Antisense ; genetics ; Receptors, Vascular Endothelial Growth Factor ; genetics ; Transcription Initiation Site

OBJECTIVETo compare the durability of quantum dots with that of green fluorescein for labeling survivin antisense oligonucleotide (ASODN) and investigate the difference in growth and apoptosis of cells transfected with the labeled survivin ASODN.

METHODSSurvivin ASODN labeled with quantum dots or green fluorescein was transfected into MCF-7 cells via Lipolifectmain(TM2000). The proliferation of MCF-7 cells was assessed with MTT assay, survivin mRNA expression determined by RT-PCR and its protein expression measured by Western blot analysis. The apoptosis rate of the transfected cells was estimated by flow cytometry, and the fluorescence distribution in the cells observed under fluorescent inverted microscope.

RESULTSThe mRNA and protein expressions of survivin were significantly decreased in the MCF-7 cells after cell transfection with survivin ASODN labeled with quantum dots or green fluorescein, and no significant difference was noted between the two labeling methods (P>0.05). Nor did survivin ASODN transfection with different labeling methods produced significant difference in cell proliferation and apoptotic rate (P>0.05). For green fiuorescein labeling, the fluorescence disappeared 4 days after transfection, whereas the fluorescence sustained for 1 week for quantum dots labeling.

CONCLUSIONSurvivin ASODNs labeled with quantum dots and green fiuorescein do not significantly differ in survivin expression or the transfected cell proliferation and apoptosis rate, but quantum dot labeling can be more stable with longer maintcnance of the labeling.

Apoptosis ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Fluorescein ; chemistry ; Gene Expression ; Humans ; Inhibitor of Apoptosis Proteins ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; genetics ; metabolism ; Oligonucleotides, Antisense ; chemistry ; genetics ; Quantum Dots ; Reverse Transcriptase Polymerase Chain Reaction ; Staining and Labeling ; methods ; Transfection

OBJECTIVETo study the loss of heterozygosity (LOH) on chromosome 3p in thyroid tumors.

METHODSLOH at 11 microsatellite loci was analyzed in 74 cases of thyroid tumors (including 20 follicular adenomas, 24 follicular thyroid carcinomas and 30 papillary thyroid carcinomas) by polymerase chain reaction and silver stain.

RESULTSLOH on chromosome 3p was detected in 71% of follicular thyroid carcinoma (17/24), 30% of the papillary thyroid carcinoma (9/30) and 10% of the follicular adenoma (2/20) case. Two minimal common deleted regions (CDR) (3p26-pter and 3p14.2-3p22) involving significant sites of LOH has identified in follicular thyroid carcinoma. There was also one CDR (3p25. 2-26.1) in papillary thyroid carcinoma.

CONCLUSIONSLOH is more frequently identified in follicular thyroid carcinoma than in papillary thyroid carcinoma and follicular adenoma. The 3 CDR on chromosome 3p may harbor tumor suppressor genes involved in the pathogenesis of follicular thyroid carcinoma and papillary thyroid carcinoma.

Adenocarcinoma, Follicular ; genetics ; Adenoma ; genetics ; Adult ; Aged ; Carcinoma, Papillary ; genetics ; Chromosome Mapping ; Chromosomes ; Chromosomes, Human, Pair 3 ; genetics ; Female ; Genes, Tumor Suppressor ; physiology ; Heterozygote ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; Middle Aged ; Thyroid Neoplasms ; genetics ; Young Adult

Objective To establish the rabbit VX2 tumor as a model for pyriform sinus carcinoma and to observe its biological features. Methods VX2 tumor was implanted into the pyriform sinus of 15 rabbits by direct laryngoscope. Fifteen rabbits were randomized into 3 groups (average of 5 rabbits per group). Observation of the tumor growth and evaluation of the histopathological characterization were taken on one group each at the of time 14, 21 and 28 days after transplantation respectively. Results Tumors were found grown in the pyriform sinus of all 15 rabbits with a success implantation rate of 100%. Dysphagia, body weight loss, rhinorrhea and short of breath appeared in the rabbits 28 days after transplantation. The metastasis rates of deep cervical were 100% in all three groups. The metastasis rates of submandibular lymph nodes were 3/5, 4/5 and 5/5 in 14-day, 21-day and 28-day group respectively. The metastasis rates of paratracheal lymph nodes were 0,4/5 and 5/5 in 14-day,21-day and 28-day group respectively. There were opposite side lymph node metastasis of deep cervical, submandibular and paratracheal in 4,3 and 5 rabbits on 14, 21 and 28 days after transplantation respectively. The median diameter for deep cervical, submandibular and paratracheal neck lymph nodes were 1.50, 0.60 and 0.0 cm in 14 days; 1.60, 0.80 and 0.50 cm in 21 days; 1.80,0.8 and 0.65 cm in 28 days (P>0.05). Conclusions The animal model for pyriform sinus carcinoma is established successfully. The metastasis of deep cervical lymph node could be induced from day 14 after VX2 transplantation.

OBJECTIVETo study the clinicopathologic features of granulocytic sarcoma.

METHODSThe clinical and pathologic findings of 38 cases of granulocytic sarcoma were retrospectively analyzed. Immunohistochemical study was performed and the literature was reviewed.

RESULTSThe age of patients ranged from 2 to 77 years (mean = 43.3 years). The male-to-female ratio was 1.5:1. Major clinical presentations included superficial lymph node enlargement and painful soft tissue mass. Follow-up data were available in 18 patients; and 14 of them died of tumor-related diseases. The average duration of survival of the patients was 16.9 months. Histologically, the tumor cells were relatively uniform in appearance and small to medium in size. The cytoplasm was scanty and pale in color. The nuclei were round or focally irregular, with fine chromatin and inconspicuous nucleoli. Mitosis figures were readily identified. Scattered immature eosinophilic myelocytes were seen. Immunohistochemical study showed that the tumor cells in all cases expressed MPO and CD43. Most cases were also positive for CD68, lysozyme, CD99 and TdT. The staining for CD3, CD20, CD79a, pan-cytokeratin and PLAP were negative.

CONCLUSIONSGranulocytic sarcoma is a known histologic mimicker of non-Hodgkin lymphoma, Ewing sarcoma/PNET and embryonal rhabdomyosarcoma. Detailed morphologic examination, when coupled with immunohistochemical study, is useful in arriving at a correct diagnosis.

Adolescent ; Adult ; Aged ; Burkitt Lymphoma ; metabolism ; pathology ; Child ; Child, Preschool ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Humans ; Leukosialin ; metabolism ; Lymph Nodes ; pathology ; Male ; Middle Aged ; Muscle Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Ovarian Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Peroxidase ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Retrospective Studies ; Sarcoma, Ewing ; metabolism ; pathology ; Sarcoma, Myeloid ; drug therapy ; metabolism ; pathology ; surgery ; Skin Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Survival Rate ; Young Adult

OBJECTIVETo evaluate the selectively killing effects of combination of adenovirus mediated double suicide gene driven by kinase-domain insert containing receptor (KDR) promoter and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells.

METHODSHuman embryonal kidney cells 293 were transfected with the plasmids of pAdEasy-KDR-CDglyTK and the adenovirus was generated. The KDR expressive cells of MCF-7, ECV304 were infected by adenovirus and survivin ASODN was transferred into the same cells meanwhile. The infection rates of adenovirus and transfection efficiency of survivin ASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on cells were assessed by MTT assay.

RESULTSThe cells infected by the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection rate. The high expression of CDglyTK gene was found in the two kinds of cells and survivin ASODN decreased the survivin protein level. When survivin ASODN was transferred into MCF-7, ECV304 cells, the survival rates were 56.4% +/- 3.8% and 55.9% +/- 3.6% respectively. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate comparing with using each treatment alone (P < 0.05) and the survival rate decreased gradually with the increasing of the concentration of GCV and 5-FC. But the survival rate for combined gene therapy group was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that of AdKDR-CDglyTK group (P > 0.05). The combination of survivin ASODN and AdKDR-CDglyTK therapy showed synergistic killing efficacy and more significant bystander effects.

CONCLUSIONThe combined therapy with AdKDR-CDglyTK system and survivin ASODN shows obvious killing effects on breast tumor cells and vein endothelial cells.

Adenoviridae ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Cell Survival ; Endothelial Cells ; metabolism ; pathology ; Female ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Oligonucleotides, Antisense ; genetics ; Plasmids ; Promoter Regions, Genetic ; Receptors, Vascular Endothelial Growth Factor ; genetics ; Transfection

OBJECTIVETo construct a cDNA library from human liver tissue of cirrhosis.

METHODSThe total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.

RESULTSThe titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb).

CONCLUSIONA high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

DNA, Complementary ; biosynthesis ; Gene Library ; Hepatitis B, Chronic ; complications ; genetics ; virology ; Humans ; Liver Cirrhosis ; genetics ; virology ; Polymerase Chain Reaction ; Recombination, Genetic ; Transcription, Genetic ; genetics

OBJECTIVETo retrospectively analyze the clinical value of diffusion-weighted MR imaging in the detection of prostate cancer in suspected patients.

METHODSBetween January 2009 and December 2010, the 551 patients suspected as prostate cancer underwent prostate biopsy. Patients in group A were accepted to a transrectal ultrasound (TRUS) guided transrectal prostate biopsy (n = 410), while patients in group B were accepted to a diffusion weighted imaging (DWI) and TRUS jointly guided transrectal prostate biopsy (n = 141). The two groups were divided into 4 subgroups by prostate specific antigen (PSA) < 10 µg/L, 10 µg/L ≤ PSA < 20 µg/L, 20 µg/L ≤ PSA < 50 µg/L and PSA ≥ 50 µg/L. Then, the diagnostic rates of prostate biopsy guided by combination of DWI and TRUS with only TRUS were compared.

RESULTSThe diagnostic rate of patients with PSA < 10 µg/L, 10 µg/L ≤ PSA < 20 µg/L, 20 µg/L ≤ PSA < 50 µg/L and PSA ≥ 50 µg/L were 12.1%, 31.1%, 48.0%, 91.2% in group A, and 23.7%, 35.5%, 66.7%, 96.3% in group B, respectively. In the patients with PSA less than 10 µg/L, there were significant differences in diagnostic rate between the two biopsy techniques (χ(2) = 4.405, P < 0.05).

CONCLUSIONThe combination of DWI and TRUS showed the potential to guide biopsy to cancer foci in patients suspected as prostate cancer. For patients with PSA < 10 µg/L, a DWI and TRUS jointly guided transrectal prostate biopsy was recommended.

Biopsy, Needle ; methods ; Endosonography ; Humans ; Magnetic Resonance Imaging ; Male ; Prostate ; diagnostic imaging ; pathology ; Prostatic Neoplasms ; diagnosis ; pathology ; Retrospective Studies

OBJECTIVETo construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B.

METHODSThe total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.

RESULTSThe titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%.

CONCLUSIONA high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

DNA, Complementary ; genetics ; Gene Library ; Hepatitis B, Chronic ; genetics ; Humans ; Liver ; metabolism ; pathology ; Polymerase Chain Reaction ; RNA ; analysis ; genetics

OBJECTIVETo evaluate the inhibition effects of DNAzymes specific to Hepatitis B Virus(HBV) s gene and e gene on the expressions of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg).

METHODSDNAzymes DrzBS and DrzBC specific to HBV s gene ORF A157UG and e gene ORF A1816UG, respectively, were designed and synthesized. The inhibition effects of DrzBS or DrzBC on the expressions of HBV s and e genes were observed in 2.2.15 cells.

RESULTSThe expression of HBV s or e genes was dramatically depressed after 2.2.15 cells treated by DrzBS or DrzBC. The concentration for effective inhibition was within 0.1-2.5 micromol/L and the inhibition showed a dose dependence within that concentration range. The maximum inhibition was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The inhibition was maintained for 72 hours. The efficiency of inhibiting HbsAg, HbeAg in 2.2.15 cells by DrzBS, DrzBC was higher than that by antisense oligonucleotides for the same target genes. The concentrations for effective inhibition of the DNAzymes were at least 10-fold lower compared with antisense oligonucleotides. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed.

CONCLUSIONDrzBS and DrzBC can highly block the expressions of HBV s gene and e gene in 2.2.15 HBV cell model and are proved a specific and effective anti-HBV gene therapeutic means.

DNA, Catalytic ; pharmacology ; therapeutic use ; DNA, Viral ; analysis ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Genetic Therapy ; Hepatitis B ; therapy ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B e Antigens ; genetics