Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients’ serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBV x gene and the I-SceI induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components—Patched1 (PTCH1) and Gli1 in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEP1), respectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines. Cyclopamine decreased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G₀/G₁ phase, while Shh induced the reverse effects and could partially prevent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic approach to NB.

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR)I and II, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both human population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 31 haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.

Clopidogrel was believed to be superior to aspirin by the well-known CAPRIE trial. However, no other large clinical trials demonstrated the same results, but all focused on the combination use of clopidogrel with aspirin, and combination therapy in CREDO was called the “Emperor’s New Clothes”. However, no one overturned the results of these clinical trials by quantitatively analyzing them. We reviewed ten large-scale clinical trials about clopidogrel. On the basis of results of CAPRIE, CREDO and CHARISMA trials, we re-estimated their minimal sample sizes and their powers by three well-established statistical methodologies. From the results of CAPRIE, we inferred that the minimal sample size should be 85 086 or 84 968 but its power was only 30.70%. A huge gap existed. The same was also true of CREDO and CHARISMA trials. Moreover, in CAPRIE trial, 0 was included in the 95% confidence interval and 1 was included in the 95% confidence interval for the relative risk. There were some paradoxical data in CAPRIE trial. We are led to conclude that the results in CAPRIE, CREDO, and from the subgroup analysis in CHARISMA trials were questionable. These results failed to demonstrate that clopidogrel was superior to aspirin or that clopidogrel used in combination with aspirin was better than aspirin alone. The cost-effectiveness analyses by some previous studies were not reliable.

The association between fasting plasma ghrelin levels and insulin resistance and blood pressure (BP) in octogenarians was investigated in this study. A total of 487 unrelated octogenarians (including 203 men and 284 women) were enrolled in this cross-sectional study at the Healthy Care Center of Shanghai East Hospital, Tongji University, China, from October 2008 to April 2009. Plasma ghrelin was determined by using the enzyme linked immunosorbent assay (ELISA). Insulin sensitivity was assessed using the homeostasis model of assessment-insulin resistance (HOMA-IR). The age of the participants ranged from 80 to 89 years (mean=83.9±4.8 years) with a body mass index (BMI) of 25.3±4.9 kg/m². Plasma ghrelin levels were 20.94±5.34 μg/L, being 20.89±5.53 μg/L in men and 21.38±3.73 μg/L in women respectively. Plasma ghrelin was not associated with systolic (P=0.981) or diastolic (P=0.724) BP, waist circumference (P=0.278), fasting insulin (P=0.246), fasting blood glucose (FBG) (P=0.693) and HOMA-IR (P=0.232). In the control cohort, no significant differences in plasma ghrelin were found between genders (P=0.489), and among subjects with hypertension (BP>140/90 mmHg) (P=0.284) and type 2 diabetes (P=0.776). In conclusion, fasting plasma ghrelin levels are not directly correlated with insulin resistance and BP among octogenarians.

This study examined the changes of activities of vitamin K-dependent clotting factors (VKDCF) under various pathological conditions and explored the relationship between acquired deficiency of VKDCFs and hemorrhage. Clinical data of 35 patients who were diagnosed as having acquired deficiency of VKDCF were retrospectively analyzed. Coagulation factors involved in the intrinsic and extrinsic pathways were detected in these patients and 41 control subjects. The results showed that the average activities of VKDCFs were decreased in the patients in comparison to the control subjects and significantly increased after treatment of these patients with vitamin K and blood products. Multivariate regression analysis indicated that decreased activity of VKDCF was not an independent risk factor for bleeding disorders owing to deficiency or metabolic disturbance of vitamin K. It was concluded that acquired deficiency of VKDCF occurs under a variety of pathologic conditions and is closely associated with hemorrhagic events. Administration of vitamin K and transfusion of blood products containing high concentrations of VKDCFs helps alleviate the hemorrhagic diseases.

This study investigated the role of glucose in the biogenesis of high-density lipoprotein cholesterol (HDL-C). Mouse primary peritoneal macrophages were harvested and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose of various concentrations. The cells were divided into 3 groups in terms of different glucose concentrations in the cultures: Control group (5.6 mmol/L glucose), high glucose concentration groups (16.7 mmol/L and 30 mmol/L glucose). ATP-binding cassette transporter A1 (ABCA1) mRNA expression in the macrophages was detected by semi-quantitative RT-PCR 24, 48 and 72 h after glucose treatment. The results showed that ABCA1 mRNA expression in the 16.7 mmol/L glucose group was not significantly different from that in the control group at all testing time points (P>0.05 for each). In the 30 mmol/L glucose group, macrophage ABCA1 mRNA expression was not changed significantly at 24 h (P=0.14), but was substantially decreased by 40.4% at 48 h (P=0.009) and by 48.1% at 72 h (P=0.015) as compared with that in the control group. It was concluded that ABCA1 is of vital importance for HDL-C biogenesis. High glucose may hamper HDL-C biogenesis by decreasing ABCA1 expression, which contributes to low HDL-C level in diabetes.

FANCD2 is involved in DNA damage repair and maintenance of chromosome stability. The purpose of this study was to investigate the expression of FANCD2 in sporadic breast cancer tissues and its association with clinicopathological features. A total of 162 Chinese women with invasive breast carcinoma who had no family history in first-degree relatives and 12 normal breast tissues were examined. The expression of FANCD2 was detected by immunohistochemical staining based on a tissue microarray technique. SAS system was used to analyze the data. Twenty-one out of the 162 invasive breast cancers (13%) were negative for FANCD2. The mean percentage of FANCD2 positive cells was significantly lower in breast cancers than in controls (P<0.05). FANCD2 expression was significantly inversely associated with histological grade and TNM stage (P<0.05), but not with axillary lymph node status or other conventional prognostic markers such as ER, PR, Her-2 and PCNA (P>0.05). It was suggested that FANCD2 may play a critical role in breast carcinogenesis. It may become a valuable and independent marker for identifying women with sporadic breast cancer and evaluating the prognosis.

Over-expression of P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, represents one of the major mechanisms that contribute to multidrug resistance (MDR) in cancer cells. This study examined the effects of troglitazone, a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on P-gp-mediated MDR in SGC7901/VCR cells (a vincristine-resistant human gastric cancer cell line). The expression of P-gp was detected by RT-PCR and Western blotting, respectively. The SGC7901/VCR cells were treated with 0.1 mg/L vincristine (VCR) alone or in combination with 1, 5, 10 μmol/L troglitazone for 24 h. PPARγ was measured by electrophoretic mobility shift assay (EMSA). The intracellular concentration of Rhodamine123 (Rh123, a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp. The cell cycle and apoptosis were measured by flow cytometry. The results showed that the P-gp was increasingly expressed in SGC7901, BGC823 and SGC7901/VCR cells in turn, suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp. In the SGC7901/VCR cells, the expression level of total PPARγ was increased, however, the protein level and activity of PPARγ in the nuclei of cells decreased significantly. Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner. Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner. We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G₂/M phase and decreased the cell percentage in G₁ and S phase in a dose-dependent manner. Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner. It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity. Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells. It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

CD46 is not only identified as a complement regulatory protein which protects host cells from complement attack, but also a new co-stimulatory molecule for human T cells. CD3/CD46 co-stimulation can induce a T-regulatory 1 cell (Tr1)-specific cytokine phenotype in human CD4⁺ T cells. However, the role of CD46 as a co-stimulatory molecule in the modulation of the acquired immunity, such as transplant immunology, remains unclear. In this study, CD4⁺ T cells were isolated from human CD46-transgenic C57BL/6 mice by magnetic-activated cell sorting, and further induced by anti-CD3, anti-CD28 and anti-CD46 antibodies respectively, and anti-CD3/anti-CD28 antibodies, anti-CD3/anti-CD46 antibodies, or the monoclonal antibody panel against CD3/CD28/CD46. The levels of interleukin-2 (IL-2), γ-interferon (γ-IFN), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected in the supernatants of different groups. Suppression of allogeneic T cell proliferation were assessed by using mixed lymphocyte reaction (MLR) assay, in which monoclonal antibodies against CD46 were added to the culture. The results showed that CD3/CD28, CD3/CD46 and CD3/CD28/CD46 co-stimulation could significantly induce stronger proliferation of T cells than CD3 stimulation (P<0.05), and CD3/CD28/CD46 co-stimulation significantly increased the proliferation of T cells when compared with CD3/CD28 or CD3/CD46 co-stimulation (P<0.05 for each). IL-2 and γ-IFN levels were much higher in CD3/CD28 co-stimulation group than in CD3, CD28, CD46 and CD3/CD46 groups (P<0.05 for each). IL-10 and TGF-β levels were dramatically increased in CD3/CD46 co-stimulation group as compared with those in the CD3, CD28, CD46 and CD3/CD28 groups (P<0.05 for each). CD3/CD46 co-stimulation significantly inhibited the T cell proliferation and allogenic immune responses through the secretion of IL-10 and TGF-β in MLR (P<0.05). These results suggested that CD3/CD46 can induce Tr1 cells to modulate allogenic immune responses, and it may become a novel target for the development of new therapeutic approach for T-cell-mediated diseases. CD46 plays an important role in regulating the T cell-mediated immune responses by bridging innate and acquired immunity.

The study investigated the effects of heat shock protein 70 (HSP70) antisense oligonucleotide (ASODN) on the proliferation and apoptosis of a human hepatocellular carcinoma cell line (SMMC-7721 cells) in vitro. HSP70 oligonucleotide was transfected into SMMC-7721 cells by the mediation of Sofast™ transfection reagent. Inhibition rate of SMMC-7721 cells was determined by using MTT method. Apoptosis rate and cell cycle distribution were measured by flow cytometry. Immunocytochemistry staining was used to observe the expression of HSP70, Bcl-2 and Bax. The results showed that HSP70 ASODN at various concentrations could significantly inhibit the growth of SMMC-7721 cells, and the inhibition effect peaked 48 h after transfection with 400-nmol/L HSP70 ASODN. Cytometric analysis showed the apoptotic rate was increased in a dose- and time-dependent manner in the HSP70 ASODN-treated cells. The percentage of cells in the G₂/M and S phases was significantly decreased and that in the G₀/G₁ phase increased as the HSP70 ASODN concentration was elevated and the exposure time prolonged. Immunocytochemistry showed that treatment of SMMC-7721 cells with HSP70 ASODN resulted in decreased expressions of HSP70 and Bcl-2 proteins, and an increased expression of Bax protein. It was concluded that the HSP70 ASODN can inhibit the growth of the SMMC-7721 cells and increase cell apoptosis by down-regulating the expression of HSP70. HSP70 ASODN holds promise for the treatment of hepatocellular carcinoma.

This study examined whether 1-methyl-tryptophan [1-MT, an indoleamine 2, 3-dioxygenase (IDO) inhibitor] could reduce CD4⁺CD25⁺ regulatory T cells (Tregs) proliferation and improve the anti-tumor efficacy of dendritic cells (DCs) pulsed with tumor cell lysate in the mice bearing pancreatic adenocarcinoma. The models of pancreatic adenocarcinoma were established in C57BL/6 mice by subcutaneous injection of Pan02 cells. Eight mice which were subcutaneously injected with PBS served as control. The expression of IDO was determined in tumor draining lymph nodes (TDLNs) and spleens of the murine pancreatic adenocarcinoma models. The prevalence of Tregs was measured in the TDLNs and spleens before and after 1-MT administration. The dendritic cells were pulsed with tumor cell lysate for preparing DC vaccine. The DC vaccine, as a single agent or in combination with 1-MT, was administered to pancreatic adenocarcinoma mice. The anti-tumor efficacy was determined after different treatments by regular observation of tumor size. The results showed that the levels of IDO mRNA and protein in tumor-bearing mice were significantly higher than those in the normal control mice. The percentage of Tregs in the spleen and TDLNs was also higer in tumor-bearing mice than in normal control mice (P<0.05). Foxp3 expression was significantly lower in the TDLNs and spleens of tumor-bearing mice administrated with 1-MT than that in normal control mice. Furthemore, in the mice that were administered 1-MT plus DC vaccine, the tumor was increased more slowly than in mice treated with DC vaccine or 1-MT alone, or PBS on day 36 (P<0.01). Our results indicated that 1-MT may enhance anti-tumor efficacy of dendritic cells pulsed with tumor cell lysate by downregulating the percentage of Tregs.

This study examined whether insulin-stimulated hypoxia-inducible factor 1α (HIF-1α) expression plays a crucial role in promoting the proliferative vitality and invasive capability in human pancreatic cancer cells. PANC-1 cells were divided into three groups: Control group, insulin group and insulin+YC-1 (a pharmacological inhibitor of HIF-1α) group in terms of different treatments. Cells in the insulin group or insulin+YC-1 group were treated with insulin (0.1, 1, 10 and 100 nmol/L) alone or combined with 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1, 0.1, 1, 10 and 100 μmol/L). HIF-1α mRNA and protein expression in PANC-1 cells was determined by real-time RT-PCR and Western blotting respectively. Cell proliferation and invasion were measured by using growth curve and invasion assay, respectively. Western blot analysis demonstrated that insulin dose-dependently increased the HIF-1α protein expression, and YC-1 could dose-dependently block this effect. However, neither insulin nor YC-1 altered HIF-1α mRNA levels in PANC-1 cells. Moreover, insulin could enhance the proliferation and invasion of PANC-1 cells, while YC-1 could weaken this effect. It was concluded that the malignant proliferation and local invasion of pancreatic cancer cells may be related to high-insulin microenvironment. The tumor biological behavior change resulting from high-insulin microenvironment may be associated with the increased expression of HIF-1α protein.

The purpose of this study was to pool information in epithelial ovarian cancer by combining studies using Affymetrix expression microarray datasets made at different laboratories to identify novel biomarkers. Epithelial microarray expression information across laboratories was screened and combined after preprocessing raw microarray data, then ANOVA and unpaired T test statistical analysis was performed for identifying differentially expressed genes (DEGs), followed by clustering and pathway analysis for these DEGs. In this work, we performed a combination analysis on microarrays from three different laboratories using gene expression data on ovarian cancer and obtained a list of differential expression profiles identified as potential candidate in aggressiveness of ovarian cancer. The clustering and pathway analysis explored the different molecular basis of different ovarian cancer stages and potential important regulatory pathways in ovarian cancer development. Our results showed that combination of microarray data from different laboratories in the same platforms may overcome biases derived from probe design and technical features, thereby accelerating the identification of trustworthy DEGs, and demonstrating the advantage of integrative analysis in gene expression studies on epithelial ovarian cancer research.

This study examined the role of PI3K/Akt pathway in radiosensitization of DNA damage of cervical carcinoma cells. The 50% inhibition concentration (IC₅₀) of cisplatin and docetaxel in HeLa cells was detected by Mono-nuclear cell direct cytotoxicity assay (MTT) in vitro. HeLa cells were treated by cisplatin/docetaxel of 10 percent of IC₂₀ alone or combined with LY294002 for 24 h, and then radiated by different doses of X-ray. The cell survival ratio was obtained by means of clone formation. One-hit multi-target model was fitted to the cell survival curve to calculate dose quasithreshold (Dq), mean lethal dose (D₀), 2Gy survival fraction (SF₂) and sensitization enhancement ratio (SER). The pAkt and total Akt expression was detected by Western blotting and DNA damage by neutro-comet electrophoresis. The HeLa cells were randomly divided into 7 groups in terms of different treatments: Control; radiation treatment (RT) group; LY294002+RT group; cisplatin+RT group; docetaxel+RT group; LY294002+cisplatin+RT group; LY294002+docetaxel+RT group. The apoptosis ratio of each group was measured by flow cytometry. The results showed that docetaxel and cisplatin significantly enhanced the phosphorylation of Akt in radiation-treated HeLa cells. The Dq, D₀ and SF2 in LY294002-contained groups were lower than those in docetaxel or cisplatin+RT group. The SER in the LY294002+docetaxel+RT group was 1.35 times that of the docetaxel+RT group, and that in the LY294002+cisplatin+RT group 1.26 times that of the cisplatin+RT group. The Comet electrophoresis showed that tail distance in the LY294002+cisplatin+RT group or LY294002+docetaxel+ RT group was longer than in the cisplatin+RT group or docetaxel+RT group. The apoptosis ratio in the LY294002+cisplatin+RT group or LY294002+docetaxel +RT group was higher than in the cisplatin+RT group or docetaxel+RT group. It was concluded that inhibiting PI3K/Akt pathway can increase the effect of docetaxel and cisplatin on the radiosensitivity of HeLa cells and DNA damage resulted from radiation.

The growth-inhibiting and apoptosis-inducing effects of WW domain-containing oxidoreductase (WWOX) gene on ovarian cancer cell line A2780 were investigated. The full length cDNA of human WWOX gene was amplified from normal human ovary tissues. The correct cDNA of full length WWOX was subcloned into eukaryocytic expression vector pCMV. After introduction of WWOX gene into cancer cells with liposome, the WWOX mRNA and protein level in the cancer cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. The growth activities of cancer cells were detected by Trypan blue staining. The clone formation assay in soft agar was employed to observe the proliferation of the cancer cells. Apoptosis was examined by DNA ladder and acridine orange-ethidium bromide fluorescent staining. The results showed that 72 h after WWOX gene transfection, the WWOX expression was increased significantly (P<0.01). The growth of ovarian cancer cells was decreased by 16.41% to 38.49% (P<0.01). The clone formation abilities were reduced (P<0.01). Some cancer cells presented the characteristic morphological changes of apoptosis with obvious ladder bands on electrophoresis. The apoptosis rate was (20.7±6.0)% (P<0.01). It was concluded that over-expression of WWOX gene could induce apoptosis and inhibit the growth of ovarian cancer cells, which might be potentially useful in the gene therapy of ovarian cancers.

This study examined the association of expression of vascular endothelial growth factor (VEGF), a promoter of angiogenesis, with endothelium dysfunction in preeclampsia. The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. VEGF expression in the human umbilical vein endothelial cells (HUVECs) was blocked by small interfering RNAs (siRNAs). The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers. The cell proliferation and cell-secreted nitric oxide (NO) level were detected by MTT method and nitrate reductase assay, respectively. The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue. The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects, although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high. Moreover, VEGF deficit could lead to endothelium cell dysfunction, and the administration of VEGF could protect endothelium cells from injury. It was concluded that lack of VEGF contributes to endothelium dysfunction, which may lead to the occurrence and development of preeclampsia.

To investigate the efficacy and the clinical value of total laparoscopic radical hysterectomy (TLRH) for the treatment of uterine malignancies, we performed a retrospective review of 87 patients with cervical cancer and 23 patients with endometrial carcinoma who underwent TLRH at Union hospital between June 2008 and September 2009. Data collected included operative time, estimated blood loss, lymph node count, time for the recovery of normal temperature and time to resumption of normal bladder function, intraoperative and postoperative complications. The procedure was completed laparoscopically in 108 patients. Two patients were converted to laparotomy due to common iliac vein injury. The mean overall operative time was 200.6±38.6 min; the mean operative blood loss was 280.5±128.3 mL; he mean number of pelvic lymph nodes that were resected wa26. 0±5.8. The time for recovery of normal temperature and the normal bladder function after the operation was 5.8±2.9 d and 15.2±4.3 d. There were 2 (1.8%) common iliac vein injuries during the operation and 10 (9.1%) bladder retentions post operation. It was concluded that TLRH is feasible, minimally invasive and provides promise for the treatment of uterine malignancies.

In order to investigate the apoptotic pathway of rabbit annulus fibrosus (AF) cells induced by mechanical overload, an experimental air-pressure model was established in this study to pressurize the rabbit AF cells in vitro. Cells were randomly divided into five groups in which the cells were exposed to a continuous pressure of 1.1 MPa for different lengths of time (0, 5, 12, 24 and 36 h). The cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) assay and flow cytometry; the alterations in mitochondrial membrane potential were measured by fluorescence microscopy and fluorescence spectrophotometer; the activities of caspase-8 and 9 were determined by spectrophotometry. The results showed that after the cells were subjected to the pressure for 24 or 36 h, the cell proliferation was inhibited; the ratio of cell apoptosis was increased; the mitochondrial membrane potential was decreased; the activity of caspase-9 was enhanced; no activity changes were observed in caspase-8. The results suggested that treatment with a pressure of 1.1 MPa for more than 24 h can lead to the proliferation inhibition and the apoptosis of rabbit AF cells in vitro, and the mitochondrial-dependent pathway is implicated in the pressure-induced AF cell apoptosis.

This study aimed to determine whether aldosterone could induce vascular cell apoptosis in vivo. Thirty-two male rats were randomly divided into 4 groups: vehicle (control), aldosterone, aldosterone plus eplerenone or hydralazine. They were then implanted with an osmotic mini-pump that infused either aldosterone or the vehicle. Systolic blood pressure (SBP) was measured weekly by the tail-cuff method. After 8 weeks, plasma aldosterone concentration (PAC) and renin activity (PRA) were determined by radioimmunoassay. Aortic apoptosis was examined by TUNEL assay. The levels of cytochrome c and caspase-3 were determined by Western blotting and the expression of Bax and Bcl-2 was detected by immnuohistochemistry and Western blotting. The results showed that as compared with control group, aldosterone-infused rats exhibited: (1) an increase in SBP; (2) significantly elevated PAC with depressed PRA; (3) elevated aortic vascular cell apoptosis accompanied with higher levels of cytochrome c and activated caspase-3; and (4) significantly up-regulated Bax protein with down-regulated Bcl-2. These effects of aldosterone were significantly inhibited after co-administration with eplerenone but not with hydralazine. It was concluded that aldosterone induced vascular cell apoptosis by its direct effect on the aorta via mineralocorticoid receptors and independently of blood pressure, which may contribute to aldosterone-mediated vascular injury.

The effects of combined RNA interference (RNAi) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) genes on telomerase activity in a bladder cancer cell line (BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer (BTCC). Three TR-specific double-stranded small interfering RNAs (siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA. The phTR-siRNA, phTERT-siRNA, and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells. The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction, and a telomeric repeat amplification protocol was applied to detect telomerase activity. Growth inhibition of BIU-87 cells was measured by MTT assay. Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro. The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-III, pRNAT-hTR-III, and pRNAT-hTR-III+hTERT-III in BIU-87 cells. The inhibition efficiency of pRNAT-hTERT-III, pRNAT-hTR-III, pRNAT-hTERT-III+pRNAT-hTR-III was 67% for TERT mRNA, 41% for TR mRNA, 57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively. The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased, especially in the cells treated with combined RNAi-hTR and -hTERT. Gene chip analysis revealed that 21 genes were down-regulated (ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PNN, SERPINE1, THBS1, TNFRSF1A, and UCC1). The results indicated that hTR-siRNA and hTERT-siRNA, especially their combination, siRNA hTR+hTERT, specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity. Molecular biological mechanism by which combined siRNA-TR and -TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.

Our study investigated the neurotoxicity of quinolinic acid (QA) to spiral ganglion cells (SGCs), observed the protective effects of N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and magnesium ions on the QA-induced injury to SGCs, and analyzed the role of QA in otitis media with effusion (OME)-induced sensorineural hearing loss (SNHL). After culture in vitro for 72 h, SGCs were exposed to different media and divided into 4 groups: the blank control group, the QA injury group, the MK-801 treatment group, and the MgCl₂ protection group. The apoptosis rate of SGCs was analyzed by Annexin V and PI double staining under the fluorescence microscopy 24 h later. SGCs were cultured in vitro for 72 h and divided into four groups: the low concentration QA group, the high concentration QA group, the MK-801 group, the MgCl₂ group. The transient changes of intracellular calcium concentration were observed by the laser scanning confocal microscopy. Apoptosis rate in QA injury group was higher than that in blank control group and MgCl₂ protection group (both P<0.05), but there was no significant difference between MK-801 treatment group and blank control group (P>0.05). In high concentration QA group, there was an obvious increase of the intracellular calcium concentration in SGCs, which didn’t present in low concentration QA group. In MgCl₂ group, the peak values of the intracellular calcium concentration in SGCs were reduced and the duration was shortened, but the intracellular calcium concentration in SGCs had no significant change in MK-801 group. It was concluded that QA could injure SGCs by excessively activating NMDA receptors on the cell membrane, which might be the mechanism by which OME induced SNHL, while Mg²⁺ could protect the SCGs from the neurotoxicity of QA.

Radiofrequency thermocoagulation (RFT) of the gasserian ganglion is a routine and effective technique for the treatment of classical trigeminal neuralgia (CTN). In this study we compared its efficacy in patients with CTN and atypically symptomatic or mixed trigeminal neuralgia (MTN). Fifty-seven patients were treated with RFT for trigeminal neuralgia from June 2006 to February 2009. Thirty patients had CTN, and 27 had MTN. Outcomes were measured by using the visual analog pain scale (VAS) and patients’ reports of quality of life (QOL), medication usage, and complications over a follow-up period of up to 3 years. Our results showed that the patients with MTN were younger, tended to have bilaterial involvement of the first division, and were unresponsive to treatment. All surgeries were completed smoothly. About 86.7% CTN patients and 48.1% MTN patients responded immediately to RFT. The VAS scores were significantly higher in the CTN group than in MTN group (P<0.05). Kaplan-Meier curves showed that 1-year, 2-year, and 3-year pain relief rates were 76.7%, 73.3%, and 73.3% in the CTN group and 46.6%, 41.4%, and 41.4% in the MTN group, respectively. The rates of pain relief for both groups leveled off at 2 years. Complications included numbness, dysesthesia, and anesthesia dolorosa. RFT did not cause any deaths and complications were low. The treatment was very effective for CTN and, to some degrees, effective for MTN. If numbness, dysesthesia, and anesthesia dolorosa are limited to the trigger area, QOL will be greatly improved.

In this case-control study, the relationship between M196R (676 T→G) variant in exon 6 of tumor necrosis factor receptor type 2 ( TNFR2 ) gene and genetic susceptibility of acne vulgaris in Han Chinese was investigated. A total of 93 acne vulgaris patients and 90 healthy subjects from Han Chinese ethnic group were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was adopted to analyze the single nucleotide polymorphisms (SNPs) of TNFR2 M196R gene, and to examine the association between acne vulgaris and the polymorphisms in TNFR2 M196R gene. The relationship between different genotypes and the susceptibility of acne vulgaris was analyzed. The results showed that there was significant difference in the frequency of the genotype M/R+R/R in the TNFR2 M196R genetic polymorphisms between acne vulgaris patients and healthy controls (χ²=4.343; P=0.037; OR=1.899; 95% CI: 1.036–3.445); and there was significant difference in the allele (R) frequency between acne vulgaris patients and healthy controls (χ²=5.588; P=0.018; OR=1.838; 95% CI: 1.105–3.057). It was concluded that the high frequency of 196R allele in the functional M196R polymorphism of TNFR2 is a risk factor for acne vulgaris in Han Chinese.

Solitary fibrous tumor (SFT) in bladder is extremely rare. In this study, we reported one case of bladder SFT and reviewed the only ten cases of the disease that had been reported so far. The patient suffered from residual urine sensation and urethral pain. Cystoscopy revealed a 7-cm protruding mass at the dome of the bladder, and bladder mucosa biopsy showed normal differentiation of the bladder mucosa with a small amount of inflammatory cells. Radical resection of the tumor was performed in this patient. Pathological examination found uniform, haphazardly arranged spindle cells, the majority of which were CD34-positive and Vimentin-positive and proved that the mass was a solitary fibrous tumor. Within a period of 9 months of follow-up, no reoccurrence was found.