OBJECTIVETo study the influence of DNA polymerase beta (polbeta) gene silencing by small interfering RNA on biological behavior of human gastric cancer cell line BGC-823.

METHODSThe siRNA eukaryotic expression vectors targeting polbeta gene were constructed and transfected into BGC-823 cells by liposome. Stable cell lines were screened with G418. The expression levels of polbeta mRNA and protein were detected by real time PCR and Western blot in the cells of each group. The proliferation of each group was detected by flow cytometry and tumorigenicity was determined in nude mice.

RESULTSThe siRNA expression vector targeting polbeta gene was successfully constructed. The expression levels of polbeta mRNA and protein were significantly reduced in the experimental group transfected with siRNA expression vectors targeting polbeta, and the silencing effect of pRNAT-U6.1-sipolbeta2 (suppression degree was 83%) was stronger than that of pRNAT-U6.1-sipolbeta1 (depression degree is 56%). Compared with irrelevant siRNA control group, empty vector control group and untransfected group, the ratio of G0/G1 cells was increased, proportion of S phase cells and cell proliferation were decreased in the experimental group 1 cells transfected with pRNAT-U6.1-sipolbeta1 (P < 0.05). On the contrary, the ratio of G1/G0 was decreased, proportion of S phase cells and cell proliferation was increased in the experimental group 2 cells transfected with pRNAT-U6.1-sipolbeta2 (P < 0.05).

CONCLUSIONThe siRNA expression vectors targeting DNA polymerase beta gene can significantly inhibit the expression of polbeta mRNA. Neither high nor extremely low expression of polbeta is beneficial to maintain the cellular physiological functions. The expression of polbeta silenced to a proper level by siRNA may play an important role in inhibiting tumorigenesis.

Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Polymerase beta ; genetics ; metabolism ; Gene Silencing ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Random Allocation ; Stomach Neoplasms ; metabolism ; pathology ; Transfection ; Tumor Burden

OBJECTIVETo study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity.

METHODSStable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo.

RESULTSThe cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05).

CONCLUSIONSThe results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.

Adenoviridae ; physiology ; Animals ; Carcinoembryonic Antigen ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; metabolism ; pathology ; therapy ; Female ; Gene Silencing ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oncolytic Virotherapy ; Oncolytic Viruses ; physiology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tumor Burden

OBJECTIVETo construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells.

METHODSThe siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively.

RESULTSThe double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression.

CONCLUSIONThe siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.

Base Sequence ; Blotting, Western ; Cell Line, Tumor ; Cloning, Molecular ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Genetic Vectors ; genetics ; Histone Deacetylases ; biosynthesis ; genetics ; Humans ; Molecular Sequence Data ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Repressor Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo construct a lentiviral expression vector of human carcinoembryonic antigen (CEA), and identify its expression in dendritic cells (DCs).

METHODSHuman CEA-encoding sequence was amplified, purified, ligated with lentiviral vector plasmid pLentiGFP and verified by sequencing. The verified recombinant vector plasmid (pLentiGFP-CEA), the packaging plasmid p 8.2 and pVSV-G were transfected into 293T cells by Lipofectamine(TM) 2000 reagent. The supernatant of the cultured 293T cells was collected to infect the DCs. The expression of CEA in the transfected DCs was assayed by RT-PCR and Western blotting.

RESULTSCEA lentiviral vector was highly expressed in the transfected DCs as observed using fluorescence microscope 48 h after the the transfection. The human CEA gene was successfully amplified by RT-PCR with a length of about 2100 bp. Western blotting also showed CEA expression in the transfected DCs.

CONCLUSIONThe human CEA lentiviral expression vector has been successfully constructed and the functional CEA protein can be expression in the transfected DCs. This facilitates further studies of the function of CEA at the molecular level.

Carcinoembryonic Antigen ; biosynthesis ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection

BACKGROUNDOld pelvis fractures are among the most challenging fractures to treat because of their complex anatomy, difficult-to-access surgical sites, and the relatively low incidence of such cases. Proper evaluation and surgical planning are necessary to achieve the pelvic ring symmetry and stable fixation of the fracture. The goal of this study was to assess the use of three-dimensional (3D) printing techniques for surgical management of old pelvic fractures.

METHODSFirst, 16 dried human cadaveric pelvises were used to confirm the anatomical accuracy of the 3D models printed based on radiographic data. Next, nine clinical cases between January 2009 and April 2013 were used to evaluate the surgical reconstruction based on the 3D printed models. The pelvic injuries were all type C, and the average time from injury to reconstruction was 11 weeks (range: 8-17 weeks). The workflow consisted of: (1) Printing patient-specific bone models based on preoperative computed tomography (CT) scans, (2) virtual fracture reduction using the printed 3D anatomic template, (3) virtual fracture fixation using Kirschner wires, and (4) preoperatively measuring the osteotomy and implant position relative to landmarks using the virtually defined deformation. These models aided communication between surgical team members during the procedure. This technique was validated by comparing the preoperative planning to the intraoperative procedure.

RESULTSThe accuracy of the 3D printed models was within specification. Production of a model from standard CT DICOM data took 7 hours (range: 6-9 hours). Preoperative planning using the 3D printed models was feasible in all cases. Good correlation was found between the preoperative planning and postoperative follow-up X-ray in all nine cases. The patients were followed for 3-29 months (median: 5 months). The fracture healing time was 9-17 weeks (mean: 10 weeks). No delayed incision healing, wound infection, or nonunions occurred. The results were excellent in two cases, good in five, and poor in two based on the Majeed score.

CONCLUSIONSThe 3D printing planning technique for pelvic surgery was successfully integrated into a clinical workflow to improve patient-specific preoperative planning by providing a visual and haptic model of the injury and allowing patient-specific adaptation of each osteosynthesis implant to the virtually reduced pelvis.

Adolescent ; Adult ; Female ; Fractures, Bone ; diagnosis ; pathology ; Humans ; Imaging, Three-Dimensional ; methods ; Male ; Middle Aged ; Pelvic Bones ; surgery ; Reconstructive Surgical Procedures ; Young Adult

Laggera pterodonta is commonly used for treating influenza in Southwest China, especially in Yunnnan province. The main clinical effects of L. pterodonta include anti-influenza, anti-microbial, anti-inflammatory. To investigate the anti-influenza A (H1N1) virus effect of L. pterodonta, neutralization inhibition and proliferation inhibition tests were performed. MDCK culture method was used to observe the cytopathic effect (CPE) of extracts from L. pterodonta in inhibiting influenza A (H1N1) virus and haemagglutination titre of H1N1 virus in vitro. The culture medium were collected at 24 h, 48 h, 72 h, 96 h, and detected by Real time RT-PCR, in order to compare the effect of different extracts from L. pterodonta on in vitro proliferation of H1N1, virus. The result of neutralization inhibition test showed that hemagglutination titer of ethyl acetate extract were 8 times lower at 72 h; in proliferation inhibition test, hemagglutination titer of ethyl acetate extracts reduced by 2 and 4 times. According to the results of Real time RT-PCR test, the H1N1 inhibition ratio of ethyl acetate extract was 72.5%, while the proliferation inhibition ratio of ethyl acetate extract was 25.3%; as for petroleum ether extracts, the H1N1 inhibition ratio was 60.2%, while the proliferation inhibition ratio was 81.4%. In conclusion, both ethyl acetate extract and petroleum ether extract of L. pterodonta have significant neutralization and direct proliferation inhibition effects on influenza A virus.
Asteraceae ; chemistry ; China ; ethnology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Influenza, Human ; drug therapy ; virology ; Medicine, Chinese Traditional

OBJECTIVETo prospectively investigate the effectiveness of suction drainage for correction of maxillofacial deformities caused by cystic lesions of the mandible.

METHODSSuction drainage was performed in 21 cases with large cystic lesions of the mandible which had expanded facial contour. Clinical and radiological examinations of these patients were carried out regularly. The volume of the cavity was measured during treatment. The expansion indexes on axial CT image and area of the cyst on a panoramic radiograph were measured preoperatively and postoperatively. The curettage via intraoral incision was completed until the extent of disease significantly shrunk.

RESULTSAfter a mean duration of suction drainage of 5.1 months, the volume and the size on the panoramic radiographic of the cystic lesions were reduced by an average of 84% and 63% respectively. The expansion indexes were reduced notably during treatment. Computerized tomography of the mandible showed that the contour of expanded mandible was restored greatly and notable new bone was formed in the area of cortex perforation.

CONCLUSIONSSuction drainage is a useful treatment modality for the primary management of giant cystic lesion of the mandible, and can fast correct maxillofacial deformities caused by bony expansion and perforation in the area of cystic lesions.

Adolescent ; Adult ; Female ; Humans ; Male ; Mandibular Diseases ; complications ; therapy ; Maxillofacial Abnormalities ; etiology ; therapy ; Middle Aged ; Prospective Studies ; Suction ; Young Adult

OBJECTIVETo investigate the correlation of pathology, bone morphogentic protein (BMP) expression, CT value with the ossification of thoracic ligamentum flavum (TOLF) to afford the evidence to choose appropriate treatment methods.

METHODSTwenty-three patients aged 35 - 65 years old had TOLF in my hospital as case. Their courses of disease were 2 months to 9 years. The values of blood calcium, blood phosphorus and AKP in them were normal. The 5 peoples aged 21 - 35 years old who presented fracture of thoracic but not the ligamentum flavum ossification were selected as control. We excluded those who have DISH, ankylosing spondylitis, fluorosis and other disease related with TOLF. The lesion locus were scanned and mensurated by CT. The pathology characteristics were classified into immature ossification and mature ossification by general observation, histology examination. BMP were measured by the immunohistochemical (IHC) staining techniques.

RESULTSThe CT value was significantly higher in the case group (547.2 +/- 131.4) than controlled group (137.7 +/- 10.6) (t = 6.922, P = 0.000). Further, the CT value in the mature ossification (702.9 +/- 17.7) was significantly higher than the immature (480.5 +/- 180.2) (t = 5.623, P = 0.000). In addition, BMP both expressed negative in the mature ossification and the controlled group, but positive in the immature ossification. BMP expression was significantly different between the immature ossification and the mature (chi2 = 70.000, P = 0.000).

CONCLUSIONSThe CT values, pathological types and BMP expression results are similar to evaluate the ossification degrees of ligamentum flavum, and then could be indirectly judged the maturation degrees of TOLF by CT to confirm the treatment methods before operation.

Adult ; Bone Morphogenetic Proteins ; metabolism ; Case-Control Studies ; Female ; Humans ; Immunohistochemistry ; Ligamentum Flavum ; diagnostic imaging ; metabolism ; pathology ; Male ; Middle Aged ; Ossification, Heterotopic ; diagnostic imaging ; metabolism ; pathology ; Thoracic Vertebrae ; Tomography, X-Ray Computed ; Young Adult

OBJECTIVETo assess the different causes of thoracic ossification of the ligamentum flavum (TOLF).

METHODSFrom July 1989 to November 2005, 142 cases were diagnosed the TOLF, in which 121 were operated. The lesions were classified into three types on the basis of the clinical result: (1) In such primary group (Group 1, 90 cases), without incorporation disease and Ca, P and AKP was all normal; (2) In systemic ossified TOLF group (Group 2, 30 cases), 6 cases ankylosing spondylitis, 3 cases DISH, 10 cases fluorosis, 11 cases OPLL; (3) In local spine disease group (Group 3, 22 cases), 5 cases fracture in spine, 4 cases spine TB, 13 cases posterior marginal intraosseous cartilaginous node. Such clinical feature was analysed, moreover surveyed the thoracic kyphosis angle, upper thoracic kyphosis angle, lower thoracic kyphosis angle and the vertebra body wedge change. The effect was assessed using Epstein Scale.

RESULTS(1) In Group 1, the mainly type was connected type (67/90, 74%). The ossified ligamentum flavum was mainly located at the lower thoracic and thoracic-lumber levels. The local type was less. In Group 2, the mainly type was connected type (21/30, 70%). The local type was none. The lesions figure was the most. In Group 3, the local type was the most (18/22, 82%). (2) In Group 1, the ossified ligamentum flavum was mainly located at the upper and lower thoracic levels (225/486, 47%). In Group 2, mainly located at the whole thoracic, some include cervix and lumber. In Group 3, mainly location was related with the location of primary disease. (3) In group 1, the curve was normal in 81% (73/90) of cases. In Group 2, the curve was abnormal in 87% (26/30) of cases. In Group 3, the curve was normal in the 82% (18/22) of cases.

CONCLUSIONSThe TOLF relates with systemic ossify disease, the change of load on the spine, aging and so on. It should be classified according to its causes.

Adult ; Aged ; Female ; Humans ; Ligamentum Flavum ; pathology ; Male ; Middle Aged ; Ossification, Heterotopic ; classification ; etiology ; pathology ; Retrospective Studies ; Thoracic Vertebrae

The changes of microRNA expression in rat hippocampus after traumatic brain injury (TBI) were explored. Adult SD rats received a single controlled cortical impact injury, and the ipsilateral hippocampus was harvested for the subsequent microarray assay at three time points after TBI: 1st day, 3rd day and 5th day, respectively. We characterized the microRNA expression profile in rat hippocampus using the microRNA microarray analysis, and further verified microarray results of miR-142-3p and miR-221 using quantitative real-time PCR. Totally 205 microRNAs were identified and up-/down-regulated more than 1.5 times. There were significant changes in 17 microRNAs at all three time points post-TBI. The quantitative real-time PCR results of miR-142-3p and miR-221 indicated good consistency with the results of the microarray method. MicroRNAs altered at different time points post-TBI. MiR-142-3p and miR-221 may be used as potentially biological markers for TBI assessment in forensic practice.