China ; Coltivirus ; Encephalitis, Tick-Borne ; virology ; Humans

OBJECTIVETo explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA.

METHODSThree cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively.

RESULTSCompared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels.

CONCLUSIONHBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.

Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Down-Regulation ; Genes, Viral ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; MicroRNAs ; metabolism ; Trans-Activators ; genetics ; metabolism

Carcinoma, Hepatocellular ; diagnosis ; therapy ; Humans ; Liver Neoplasms ; diagnosis ; therapy ; United States

OBJECTIVEOur previous work indicated that overexpression of imprinting gene PEG10 is associated with malignant phenotype of hepatocellular carcinoma. The aim of this study is to explore whether disregulation of PEG10 leads to dysregulation of microRNAs.

METHODSIn silico analysis using TargetScan indicated that miR-122 could regulate the expression of PEG10. The expression of miR-122 in three hepatoma cell lines, Huh7, Hep3B and HepG2 and in primary human normal liver cell were compared using real time RT-PCR. After pre-miR-122 was transfected into HepG2 cell, the levels of PEG10 mRNA and protein were measured.

RESULTSIn silico analysis revealed that miR-122 could regulate the expression of PEG10. Real time RT-PCR indicated that miR-122 was not expressed in Hep3B and HepG2 cells, and only weakly expressed in Huh7 cells, but highly expressed in primary human normal liver cells. The expression of miR-122 was negatively correlated with the expression of PEG10. After pre-miR122 was transfected into HepG2, the mRNA level of PEG10 was not increased, whereas the protein level of PEG10 was increased.

CONCLUSIONmiR-122 may be involved in regulation of PEG10 expression.

Carcinoma, Hepatocellular ; genetics ; Gene Expression Regulation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; MicroRNAs ; genetics ; Proteins ; metabolism

Humans ; Liver Neoplasms ; therapy ; Molecular Targeted Therapy

OBJECTIVETo compare the efficacy difference in the treatment of insomnia between scraping technique of stuck needle and conventional acupuncture at Anmian (Extra).

METHODSOne hundred and thirty one cases were randomized into an Anmian group (68 cases) and a conventional acupuncture group (63 cases). In the Anmian group, Anmian (Extra) was selected. After arrival of qi, the stuck needling was adopted by rotating the needle gently in single direction, 2-3 rounds till the needle body was stuck tightly. Afterwards, the needle tail was touched gently with the index finger to fix the needle body and the needle handle was scraped gently with the thumbnail from bottom to up. The needle was retained for 30 min. In the conventional acupuncture group, Sanyinjiao (SP 6), Shenmen (HT 7) and Baihui (GV 20) were selected and stimulated with reducing technique by rotating the needles. The needles were retained for 30 min. The treatment was given once every day, continuously for 2 weeks in both groups. The score of each factor and the total score in Pittsburgh sleep quality index (PSQI) were assessed before and after treatment in the two groups. Additionally, the efficacies of two groups were evaluated.

RESULTSFor the patients in the conventional acupuncture group, the sleep quality and time of falling into spleen after treatment were improved as compared with those before treatment (all P < 0.01). The improvement of sleep time, sleep efficiency, sleep disturbance, hypnotic drug and daytime dysfunction, and PSQI total score did not present statistically significant difference as compared with those before treatment (all P > 0.05). After treatment, for the patients in the Anmian group, the factor score and total score in PSQI were apparently improved as compared with those before treatment (P < 0.01, P < 0.05). And the improvements of the above items were superior to the conventional acupuncture group (P < 0.01, P < 0.05). The total effective rate was 91.2% (62/68) in the Anmian group and was 74.6% (47/63) in the conventional acupuncture group. The clinical efficacy in the Anmian group was apparently superior to the conventional acupuncture group.

CONCLUSIONThe scraping technique of stuck needle at Anmian (Extra) achieves the superior effect on insomnia as compared with the conventional acupuncture.

Acupuncture Therapy ; instrumentation ; methods ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Needles ; Sleep ; Sleep Initiation and Maintenance Disorders ; physiopathology ; therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo evaluate the validity and reliability of Thread-tooth Arc-track Screw Plate System (TASPS) in the fixation fusion through expandable channels.

METHODSFrom August 2007 to August 2010, 108 patients with lumbar instability were treated with surgery, including 61 males and 47 females, ranging in age from 26 to 57 years, with an average of 41 years. All the patients were divided into two groups : minimally invasive fusion group and traditional operation group (54 patients in each group). The patients in the minimally invasive fusion group were treated with self-designed TASPS to conduct the fixation fusion through expandable channels by minimally invasive pedicle screw; and the patients in traditional operation group were treated with traditional interbody fixation fusion by pedicle screw. The data were collected and investigated at the 1st week, 3rd month and 1st year postoperatively. The comparative parameters of two groups contained the total operation time, the implanted time, the total amount of bleeding; the VAS score, ODI score and improvement rate at each investigated period; the intervertebral space height of preoperative and postoperative periods; the inefficiency rate of implantation and the fusion rate of postoperative period.

RESULTSAll incisions were healed by first stage without any complications such as dural tear, injury of nerve root or cauda equine, intervertebral space infection. The patients in the minimally invasive fusion group needed longer operative time than that of the traditional operation group, but had less total amount of intraoperative and postoperative bleeding and shorter implanted time of pedicle screw than those of the traditional operation group. The VAS scores of two groups at the 1st week after operation significantly decreased compared with that of the 1st day before the operation, and the difference was of high statistical significance. And the VAS scores of two groups at the 3rd month and 1st year after operation also significantly decreased compared with that of the 1st day before the operation, and the difference was highly statistically significant. By comparing the VAS score and ODI score at the 1st week, 3rd month and 1st year postoperatively, the results showed that the VAS score and ODI score of the minimally invasive fusion group were all lower than those of the traditional operation group, and the differences were of high statistical meaningfulness. After 1 year, the improvement rate of the minimally invasive fusion group was (77.46 +/- 6.34)%, while that of the traditional operation group was (72.73 +/- 4.49)%, and the difference was highly statistically significant (P < 0.01). The intervertebral space heights of two groups remarkably increased. At the 3rd month after operation, the difference of intervertebral space heights of the two groups was of no statistical significance. At the 1st year after operation, intervertebral space heights of the two groups were lost to some extent, but the height of the minimally invasive fusion group was higher than that of the traditional operation group, and the difference was statistically significant. The fusion rates of the two groups after 1 year were 100%.

CONCLUSIONThe fixation fusion through expandable channels by minimally invasive pedicle screw possesses the characteristics of fewer traumas, less intraoperative blood loss, less postoperative pain and rapid recovery. Since its intervertebral fusion rate is similar to the open surgery, it can be viewed as one effective approach for the treatment of lumbar instability diseases. And TASPS is reasonably designed, easy to install and reliably fixed with good reduction effect, which can be applied through expandable channels.

Adult ; Bone Plates ; Bone Screws ; Female ; Humans ; Joint Instability ; surgery ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; Spinal Fusion ; methods

OBJECTIVETo compare the efficacy of endoluminal ultrasonography (EUS) and spiral computed tomography (SCT) in preoperative staging of rectal carcinoma.

METHODSBoth EUS and SCT were performed prior to surgery in 68 patients with rectal carcinoma. After radical surgery, the preoperative findings were compared with histologic findings of the operative specimens, and the efficacy of EUS and SCT in staging the rectal carcinoma were evaluated.

RESULTSFor T stage, EUS accuracy was 86.8%, while SCT was 70.6%. The difference was significant (P<0.05). For N stage, EUS accuracy was 67.6% and SCT was 63.2%. The difference was not significant (P>0.05).

CONCLUSIONEUS is superior to SCT for the judgement of tumor infiltration depth, but neither is able to provide satisfactory assessment of lymph node metastases.

Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; methods ; Rectal Neoplasms ; diagnostic imaging ; pathology ; Tomography, Spiral Computed ; Ultrasonography, Interventional

OBJECTIVETo extend the use of vector-derived siRNA by generating multiple shRNAs in the same plasmid.

METHODSConstruct a vector that expresses shRNAs targeting on Ku70 and Ku80 in tandem. The gene silencing efficiency of each shRNA was verified previously. After identification by restriction digestion and DNA sequencing, the reconstructed plasmid, named psiRNAKus, was transfected into the human hepatoma cell line HepG2. The tandem-shRNA-induced silencing of targeted genes was determined by RT-PCR at RNA level and Western blot at protein level.

RESULTSThe shRNAs encoded by psiRNAKus down-regulated both the expression of Ku70 and Ku80.

CONCLUSIONThe vector-derived siRNA delivery system that allows multiple shRNA species to be expressed from the same vector may be of value in experimental and therapeutic applications.

Antigens, Nuclear ; genetics ; DNA-Binding Proteins ; genetics ; Gene Knockdown Techniques ; Hep G2 Cells ; Humans ; Ku Autoantigen ; Plasmids ; RNA ; genetics ; RNA, Small Interfering

OBJECTIVETo construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair.

METHODSThe eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis.

RESULTSRestriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.

CONCLUSIONSGenomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.

Carcinoma, Hepatocellular ; genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; Flap Endonucleases ; genetics ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; Plasmids