Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury. Although melatonin is known as an effective antioxidant, the mechanism of the protection cannot be explained merely by antioxidation. This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron survival. To find it out, an ischemia/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

By using a yeast two-hybrid system, a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins, and its effects on the growth of yeast cells and the activation of reporter genes were investigated. Total mRNA extracted from Hela cells was reversely transcribed into cDNA. Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector. The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109, respectively. After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium, their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay. The bait plasmid HPV18 E6 was successfully obtained. After being cultured in YPDA liquid medium for 16h, the A_{600 nm} values of two yeast fluids were 0.98±0.03 and 0.99±0.02, respectively. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-α-gal plates, while no colony could survive on SD/-His/-Trp/X-α-gal, SD/-Ade/-Trp/X-α-gal plates, indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly, and could not activate the transcription of reporter gene alone. The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.

KCNK17 is a member of the acid-sensitive subfamily of tandem pore K⁺ channels, which are open at all membrane potentials an red contribute to cellular resting membrane potential. Recent genome-wide study (GWA) has shown that variants within KCNK17 confer genetic susceptibility for increasing ischemic stroke. In an effort to discover additional polymorphism(s), we scrutinized the genetic polymorphisms in the KCNK17. By direct DNA sequencing in 32 individuals, we identified nine sequence variants within the 16 kb of whole KCNK17 gene: one in exon1, one in intron and seven in the promoter region. Haplotypes, their frequencies and linkage disequilibrium coefficients (D′), among polymorphisms were estimated. All the polymorphisms in the 5′-flanking region (SNP2-SNP7) being in complete (or nearly complete) association with each other in the promoter region maybe produce synergistic effect to regulate the expression of KCNK17 gene and then have an influence on the pathogenesis of cerebrovascular diseases. The common haplotypes were observed comprising 88.9% of the total haplotypes in the same block. Bioinformatic analysis predicted several potential transcriptional factors binding sites by SNP −95, −134, −596 and −846. However, these binding sites need to be experimentally verified. The information concerning genetic polymorphisms of KCNK17 gene might provide valuable information for future genetic studies of diseases.

Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133⁺ enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs. PBMCs from healthy subjects were used either directly for cell culture or for CD133⁺ sorting. The two groups of cells were cultured in complete medium 199 (M199) for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo. The results showed that PBMCs produced more colony-forming units (CFU) than CD133⁺ enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144⁺ cells in CD133⁺ group were less than in PBMCs group (P<0.01). PBMCs group secreted more VEGF than CD133⁺ group on the day 7 (P<0.01). As compared with CD133⁺ group, PBMCs group had more potent potential of proliferation and vascularization in vitro. It was concluded that CD133⁺ sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.

In order to investigate the anti-tumor angiogenesis activity with a recombinant Ag43/FGFR1 chimeric protein (AF) vaccine in a mouse H22 hepatoma model, tumor volume and survival rate of the mice were studied at a 3-day interval. Microvessel density (MVD) was detected by immunohistochemistry. The endothelial deposition of autoantibodies within tumor tissues was examined by immunofluorescent staining, and anti-FGFR1 antibody-producing B cells (APBCs) were tested by enzyme-linked immunospot (ELISPOT) assay. Compared with the three control groups, the tumor volume was significantly decreased and the survival time was significantly prolonged in AF-immunized group (P<0.05). The number of APBCs in AF-immunized mice (129.6±10.9) was more than in controls [6.2±1.1 (FGFR1), 6.0±1.2 (Ag43) and 5.2±1.4 (NS), P<0.01]. Moreover, the endothelial deposition of autoantibodies was found in tumor tissues from AF-immunized mice, but not in control groups. MVD in AF-immunized group was significantly lower than in FGFR1-immunized group, Ag43-immunized group and NS group (10.3±3.1 vs 39.4±8.6 vs 42.3±9.8 and 43.6±10.6, P<0.01). These findings demonstrated that the AF protein vaccine effectively inhibited tumor angiogenesis and growth via production of autoantibodies against self-FGFR1.

The changes in the tau protein phosphorylation and expression of bcl-2, and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion (I/R) were explored, and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer’s disease. The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of tau protein phosphorylation at Ser396, Ser404, Tyr231, Ser199/202 sites and the expression of bcl-2, bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot. The relationship between the tau protein phosphorylation and the expression of bax, or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method. The results showed that the level of tau hyperphosphorylation at Ser199 / 202, Ser396, Ser404, Tyr231 sites and the expression levels of bcl-2, and bax were significantly higher in I/R group than in the sham group, but the ratio of bcl-2/bax was decreased. Neuronal apoptosis, bax expression and the tau protein hyperphosphorylation were co-localized. It is suggested that Alzheimer’s disease-like pathological changes occur after cerebral I/R. The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis. The cerebral infarction may contribute to Alzheimer’s disease occurrence and development.

Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from peripheral blood and bone marrow (BM) in AA patients. In the current study, BM CD4⁺ T cells were isolated from AA patients and healthy controls with immunomagnetic beads sorting, and proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells were compared between them. By ³H-TdR method, CD4⁺ T cells in AA group presented more enhanced proliferative activity. The stimulation index in control group and AA group was 1.47±0.24, and 2.51±0.34 respectively (P<0.01). After BM CD4⁺ T cells were induced by high concentration of CD3 monoclonal antibody for 18 h, evident apoptosis cells could be seen under the electron microscope in both control group and AA group. Flow cytometry revealed that apoptosis rates in the early and late stages of AA group were significantly higher than in control group (P<0.01). Early-stage apoptosis rate in control and AA groups was (6.85±1.48)% and (16.98±4.40)%, and late-stage apoptosis rate in control group and AA group was (2.65±1.57)% and (7.74±0.83)%, respectively (P<0.01). The CFU-GM count in AA group and control group was (74.50±9.50)/10⁴ cells and (124.25±19.80)/10⁴ cells respectively under an inverted microscope (P<0.01), and the expression levels of CyclinD3 mRNA and protein in cord blood CD34⁺ cells were both down-regulated induced by BM CD4⁺ T cell culture supernatant in AA patients. These results indicate that BM CD4⁺ T cells of AA patients are likely in an abnormally proliferative, and activated state which can correlate intimately with AA hematopoiesis damage. BM CD4⁺ T cells in AA patients can secret some soluble cytokines that can inhibit proliferation of hematopoietic stem cells by suppressing the expression of Cyclin D3, resulting in hematopoiesis failure.

The binding function of EGF1 domain peptide with tissue factor (TF) and its ability of triggering coagulation were explored. The TF expression model in vitro was established by lipopolysaccharide induction. The affinity of EGFP-EGF1 and TF expressing cells was analyzed by fluorescence microscopy and flow cytometry (FCM). The affinity of EGFP-EGF1 and rat soluble TF was quantitated by surface plasmon resonance (SPR). The ability of EGFP-EGF1 in triggering coagulation was tested by prothrombin time assay. The FCM results showed recombinant factor VII (rFVII) could definitely depress the integration of EGFP-EGF1 with recombinant TF (rTF) (68.65%±3.86% vs 57.98%±4.71%, P<0.01). The SPR results indicated the association constant ka of EGFP-EGF1 proteins was higher than rFVII (8.29±1.39 vs 3.75±0.32, P<0.01). However, the EGFP-EGF1 protein lost the activity of triggering coagulation as compared with blood plasma of normal SD rats (56.8±3.2 s vs 17.8±3.4 s, P<0.01). It was concluded that the rat EGF1 peptide could specifically bind to TF without the ability of triggering coagulation. EGF1 peptide may be a good target head for delivering drugs to TF in anticoagulation therapy.

Tubulointerstitial fibrosis (TIF) is a common pathological feature of end-stage kidney disease. Previous studies showed that upregulation of TGFβ1 notably contributed to the chronic renal injury and irbesartan halted the development of TIF in rats with 5/6 renal mass reduction. This study was to investigate the effects of irbesartan on chronic TIF and the mechanism involved TGFβ1 in the rodent model of chronic renal failure involving 5/6 nephrectomy. The results showed that irbesartan significantly attenuated the rise in blood pressure and tubulointerstitial injury observed in this model. Masson staining of the renal tissue revealed that there appeared severe renal tubule atrophy and fibrosis in operation group, but the lesion was attenuated mostly in irbesartan-treated group. Immunohistochemistry showed that irbesartan treatment apparently decreased the protein expression of TGFβ1 which was up-regulated in operation groups. Western blot showed that irbesartan treatment down-regulated the expression of TGFβ1, phosphorylated smad2 (p-smad2), AT1R and phosphorylated p38 (p-p38) MAPK, but significantly up-regulated the protein expression of smad6 as compared with operation group. These findings suggest that irbesartan attenuates hypertension and reduces the development of TIF in rats with 5/6 renal mass reduction via changes in the expression of these proteins at least including smad6, TGF-β1, p-smad2, AT1 and p-p38 MAPK.

The present study attempted to examine the effects of bile acid pool size on liver regeneration after hepatectomy. The rats were fed on 0.2% cholic acid (CA) or 2% cholestyramine for 7 days to induce a change in the bile acid size, and then a partial hepatectomy (PH) was performed. Rats fed on the normal diet served as the controls. Measurements were made on the rate of liver regeneration, the labeling indices of PCNA, the plasma total bile acids (TBA), and the mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), farnesoid X receptor (FXR), and transcription factor c-Jun or c-fos. As compared with the normal and CA groups, the rate of liver regeneration was decreased on the day 3, and 7 after PH; the peak of the labeling indices of PCNA was delayed and the labeling indices were significantly reduced on the day 1; the TBA were also decreased on the day 1; the expression of FXR decreased but that of CYP7A1 increased at any given time; at the 1st, and 3rd h, the expression of c-Jun was declined in the cholestyramine group. The reduction in the bile acid pool size was found to delay the liver regeneration, which may be caused by the down-regulation of FXR and c-Jun expression.

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine™ 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by LipofectamineTM 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.

We studied the regulatory effects of the estragen receptorβ (ERβ) gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved. A human ERβ gene recombinant expression plasmid, pEGFP-C1-ERβ, was constructed and transfected into the Caco-2 colon cancer cell line, a line with low ERβ gene expression. The expression of ERβ mRNA and protein was detected 72 h after transfection. RT-PCR was used to examine the expression levels of the progesterone recepror (PR) gene containing the classic estrogen response element (ERE), the C-fos oncogene containing the AP-1 site (a non-classical ER binding site), the epigenetic modifying genes, such as Dnmt1, Dnmt3a, Dnmt3b, and histone methyltransferase (HMT), and the human mismatch repair gene hMLH1. Methylation-specific PCR was used to detect the changes in the methylated sites of the CpG islands in the promoters of the ERβ, PR, and C-fos genes. The results indicated that the human ERβ gene recombinant expression plasmid pEGFP-C1-ERβ was successfully constructed and transfected into Caco-2 cells. As compared with the control group, the mRNA and protein expression of ERβ gene was increased significantly 72 h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells. As compared with the control group, the mRNA expression of the PR, C-fos, Dnmt3a and Dnmt3b genes was increased significantly 72 h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells, but the mRNA expression of the Dnmt1, HMT, and hMLH1 genes decreased significantly (P<0.05). As compared with the control group, different degrees of demethylation occurred in the promoters of the ERβ, progesterone receptor (PR), and C-fos oncogene 72 h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells. The methylation index of the estrogen signal transfection pathway in Caco-2 cells was decreased significantly following the expression restoration of ERβ gene (P<0.05). It is concluded that the restoration or up-regulation of the ERβ gene in Caco-2 cells may significantly activate the expression of the related target genes in the downstream estrogen signal transfection pathway and may result in the demethylation changes of the pathway. During the process, the expression level and activity of the epigenetic modifying genes and the human mismatch repair gene have changed simultaneously. The regulatory effect of the ERβ gene on the estrogen signal transfection pathway to a certain extent partly involves demethylation.

Some studies indicate that adipose derived stem cells (ADSCs) can differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells in vitro. However, whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated. In this study, the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene, and then the cells were induced for neural differentiation. The morphology of those ADSCs began to change within two days which developed into characteristics of round cell bodies with several branching extensions, concomitantly expressing EGFP fluorescence. Approximately 60% of the total cell populations were bipolar or multipolar in shape. Some of them appeared to make contact with their neighboring cells. RT-PCR, Western blot and Immunocytochemistry revealed that the expression levels of the markers of neurons and oligodendrocytes such as MAP2, NF-70, Neu N and RIP upon neural induction were increased, but the expression of the special marker of astrocytes, GFAP, was undetectable until 96 h after induction when a small signal was observed. It was concluded that the ADSCs transfected with EGFP possessed the ability to undergo morphologic and phenotypic changes consistent with neural differentiation in vitro. It suggests that these cells might provide an ideal source for further stem cell research with possible therapeutic application for spinal cord injury.

The cancer stem cells (CSCs) from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified. The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension, and mixed homogeneously into 1.2% alginate gel. Single-cell alginate gel was cultured with serum-free DMEM/F12 medium. Epirubicin (0.8 μg/mL) was added to the medium to enrich CSCs. After cultured conventionally for 7 to 10 days, most of cells suspended in alginate gel were killed by epirubicin. But few cells survived and some single-cell cloning spheres formed. Immunofluorescent staining for Oct3/4 and Nanog was implemented to find cells with properties of self-renewal and multi-potential differentiation. Cells from cloning spheres were transplanted into BALB/c mice to detect the tumorigenicity in vivo. The results showed that some cells positive for Oct3/4 (TRITC) and Nanog (TRITC) were found in single-cell cloning spheres, and most of positive cells were concentrated in the core of sphere. Cells from spheres could form osteosarcoma in the body of mice. It was concluded that cells from single-cell cloning spheres had the properties of the expression of parts of stem cell genes (Oct3/4 and Nanog), resisting anti-cancer drugs, and tumorigenicity in vivo. To sum up, it is believed that cells obtained from osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs are cancer stem cells.

The efficacy of injecting sclerosing agent next to transverse process of cervical vertebra to induce vertebral artery type of cervical syndrome (CSA) was observed. Twenty rabbits were randomly divided into two groups: the model group and the control group. The rabbits in the model group were injected with sclerosing agent next to transverse process of cervical vertebray, on the contrary, the rabbits in the control group were injected with nothing. Transcranial Doppler (TCD) was used to detect the average speed of blood (Vm), pulsatility index (Pi) and the resistant index (Ri) of the vertebral artery, hematoxylin and eosin (HE) staining was used to observe the morphological changes, and immunohistochemistry was used to detect the expression of α-smooth muscle actin (α-SMA) and matrix metalloproteinase-2(MMP-2). TCD showed increased Pi, Ri and decreased Vm in the model group (P<0.05) compared with the control group. HE staining revealed hyperplasia and hypertrophied smooth muscle cells in the model group (P<0.05). Immunohistochemistry displayed up-regulation of α-SMA and MMP-2 in the model group (P<0.05). It was concluded that injecting sclerosing agent next to transverse process of cervical vertebra induces remodeling of vertebral artery in rabbits, suggesting it is a practical method to establish CSA animal model.

In order to investigate the strength, structure and cell cytocompatibility of injectable thermosensitive chitosan (CS)/poly(vinyl alcohol) (PVA) composite hydrogel, chitosan hydrochloride solution was transferred to a neutral pH and mixed with different proportions of PVA, then the gelation time and strength of these different hydrogels were tested and spatial structures were observed under a scanning electron microscopy (SEM) after freeze-drying. The cytocompatibility of the hydrogels was evaluated through cytotoxicity test and three-dimensional culture with bone marrow mesenchymal stem cells. The results showed that the CS/PVA solution kept in liquid state at low temperature (0–4°C) and turned into transparent elastomer about 15–20 min at 37°C. Gelation time was prolonged, the strength increased and porous structure became dense with the PVA content increased in the mixed hydrogel. The cytotoxicity grades of these gels were from 0 to 1. Rabbit bone marrow mesenchymal stem cells could survive and proliferate in the gel within 3 weeks, and the gel had good cytocompatibility. It was concluded that thermosensitive CS/PVA composite hydrogel not only has interpenetrating network structure and better mechanical strength, but also has good cytocompatibility, and may be used as an injectable scaffold for tissue engineering.

Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV. Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient. Cells and purity were identified by using immunocytochemistry assay with anti-CK7, Vim and β-hCG antibodies. HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection (p.i.). The results showed that highly purified trophoblast cells were obtained. Specific virus replication was increased dramatically at the 24th h p.i., and then increased slowly during 48 h and 72 h. Apoptosis rate of trophoblast cells infected with HCMV was (34.68±3.14)% at 24th h p.i., while that in control group was (15.32±2.34)% (P<0.05). It was suggested that highly purified trophoblast cells can be isolated by the simplified cell purification method. HCMV can infect human trophoblast cells, and be quickly replicated, resulting in the accelerated apoptosis of human trophoblast cells during early time.

The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.

Even though mutations in LMNA have been reported in patients with typical dilated cardiomyopathy (DCM) and atrioventricular block (AVB) previously, the purpose of this study was to disclose this novel genetic abnormality in one Chinese family with the atypical phenotype of progressive AVB followed by DCM with normal QRS interval. Genome-wide linkage analysis mapped the AVB gene in this family to a marker at chromosome 1q21.2, where the LMNA gene was located. Direct DNA sequence analysis revealed a heterozygous G to A transition at nucleotide 244 in exon 1 of LMNA, which resulted in an E82K mutation. The E82K mutation co-segregated with all affected individuals in the family, and was not present in 200 normal controls. Further clinical evaluation of mutation carriers showed that 5 of 6 AVB patients exhibited mild DCM with a late onset of age in the fourth and fifth decades. Ejection fractions were documented in 5 patients with DCM, but 4 showed a normal value of ⩾50%. Echocardiography showed that atrial dilatation occurred earlier than ventricular dilatation in the patients. This study suggests that progressive AVB with normal QRS interval and accompanying DCM at later stages may represent a distinct type of DCM. The molecular mechanism by which the E82K mutation causes AVB as the prominent phenotype in DCM may be a focus of future studies.

In order to investigate the inhibitory effects of Endostar (rh-endostatin, YH-16) in combination with radiotherapy on lung adenocarcinoma A549 in mice and the interaction mechanisms of combined therapy, the transplantation tumor models of A549 lung adenocarcinoma were established. When the largest diameter of tumor reached 1.0 cm, all nude mice were randomly divided into 4 groups: Endostar group, radiotherapy group, radiotherapy plus Endostar (combined treatment) group, and control group (n=6 in each group). The largest diameter and the vertical diameter of tumor were measured at different time points. At the 16th day, mice were executed, and the tumors were applied to analysis of rate of tumor cell apoptosis, and the expression levels of basic fibroblast growth factor (bFGF) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and those of vascular endothelial growth factor (VEGF) by immunohistochemistry. The results demonstrated that the rate of tumor inhibition in combined treatment group was higher than that in other groups. And the rate of tumor cell apoptosis in combined treatment group was also higher than that in other groups. Meanwhile, the levels of bFGF mRNA and VEGF expression in combined treatment group were lower than those in other groups. It was concluded that Endostar obviously enhanced the curative effectiveness of radiotherapy on lung adenocarcinoma A549 in mice. The underlying mechanisms may involve the down-regulation of bFGF mRNA and VEGF expression to inhibit angiogenesis by Endostar and the cooperative effect of Endostar and radiotherapy to synergistically promote tumor cell apoptosis. And Endostar inhibits angiogenesis by down-regulating the expression of bFGF mRNA and VEGF.

In order to investigate the inhibitory effects of all trans-retinoitc acid (ATRA) on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization (TACE) on rat Walker-256 transplanted hepatocarcinoma, Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations. After culture for 48 h, the inhibitory rate of cell proliferation was determined by MTT assay; the changes of Fas and Bcl-2 mRNA expression were determined by RT-PCR, and the expression levels of Caspase3 and Caspase8 proteins were detected by Western blot. Twenty-seven Wistar rat models of hepatocarcinoma were set up successfully by implanting Walker-256 cell lines. The tumor volume at the 11th day after implantation (V_preoperation) was measured by magnetic resonance imaging (MRI). The 27 rats were randomly and equally divided into three groups, and the therapy scheme was performed as follows: group A (ATRA 0.1 mg+mitomycin 0.05 mL+lipiodol 0.05 mL+gelfoam powder 0.025 mg); group B (mitomycin 0.05 mg+lipiodol 0.05 ml+gelfoam 0.025 mg; group C (0.9% NaCl 0.2 mL). After another 11 days, MRI was performed once again to measure the tumor volume (V_{postoperation}). The expression of factor and Ki VIII -67 in the tumor tissues was detected by immunohistochemistry. The results showed that ATRA could suppress proliferation of Walker-256 cell lines. After treatment of Walker-256 cell lines with ATRA, the expression of Fas mRNA was significantly up-regulated and the Bcl-2 mRNA was significantly down-regulated by ATRA at the concentration of 10 μmol/L as compared with the control group (P<0.05). After treatment with 10 μmol/L ATRA for 48 h, the Caspase3 and Caspase8 were significantly activated as compared with the control group (P<0.05). Significant difference existed in growth rate among the three groups (P<0.01) and between either two groups (P<0.05). The expression rate of factor VIII and Ki-67 was gradually increased from group A, group B to group C. The study suggests that ATRA could inhibit the proliferation of Walker-256 cells and the effectiveness of the combined therapy (ATRA+TACE) for treating transplanted hepatoma of rats is superior to that of TACE alone.

Progesterone has nongenomic effects on inducible nitric oxide synthase (iNOS), which is mediated by mitogen activated protein kinase (MAPK) pathways. This effect is supposed to have some potential association with asymptomatic gonococcal infections in women by immunological depression. In this study, polymorphonuclear leukocytes (PMNs) challenged by gonococci were used to study the nongenomic effects of progesterone. The activation of iNOS was assessed by measuring [³H] L-arginine converses to [³H] L-citrulline, and the activity of MAPK was detected by Western blot. It was found that the activity of iNOS and the yields of NO were enhanced significantly in gonococci-challenged PMNs compared with the controls (P<0.01). Progesterone could repress the activation of iNOS through P38MAPK pathway within PMNs (P<0.05), which could be blocked by SB203580 (P<0.01), but not by actinomycin D (P>0.05). It was also found subsequently that in the serum specimens collected from gonococci-infected but asymptomatic women, the progesterone level was higher than that in women with severe symptoms (P<0.01). Moreover, the yield of NO had an inverse correlation with progesterone. With these results it suggested that the rapid nongenomic effects of progesterone may inhibit iNOS activation and NO yields mediated by P38MAPK pathways, which were supposed to be concerned with asymptomatic women infected with gonococci.

Quantification of right ventricular (RV) volume and function remains a challenge because of RV complex geometry by conventional echocardiography. The purpose of this study was to assess RV global longitudinal function in patients with tetralogy of Fallot (TOF) by 2-dimensional ultrasound speckle tracking imaging (STI). Thirty-eight patients with TOF were enrolled in this study and divided into child group (n=25) and adult group (n=13) according to age. Thirty-eight age- and sex-matched normal subjects were selected as control groups including child control group (n=25) and adult control group (n=13). RV global longitudinal peak systolic strain (GLS), strain rate (GLSR_s), early diastolic strain rate (GLSR_e) and late diastolic strain rate (GLSR_a) were measured in all subjects by STI from the apical 4-chamber view and compared between groups. Furthermore, the main factors affecting the RV global longitudinal functional parameters were assessed. Compared with those in controls, RV GLS, GLSR_s and GLSR_e were significantly reduced in patients with TOF (P<0.05 for all). RV GLSR_s was significantly decreased in adult patients with TOF as compared with that in child patients (P<0.05). There was no significant difference in RV GLS, GLSR_e and GLSR_a between child and adult TOF groups (P>0.05). The diameter of right ventricle, main pulmonary artery and ventricular septum defect had correlations with RV GLSR_s (r₁=−0.490, r₂=0.580, r₃=−0.528, respectively, P<0.05 for all). Tricuspid annular plane peak systolic velocity (Sm) was the independent predictor of RV global strain and strain rate (β₁=0.355, P₁=0.031, β₂=0.307, P₂=0.021). RV global longitudinal function is decreased in patients with TOF, especially in adult patients. STI is a sensitive and accurate technique in RV global functional assessment.

The epidemic tendency of dental caries of school students over the past 15 years in China was analyzed in order to provide bases for prevention of dental caries. Data sets of boys and girls at the age of 7, 9, 12 years (deciduous caries) and 7, 9, 12, 14, 17 years (permanent caries) were collected from the series of Chinese National Surveillance on Students’ Constitution and Health (CNSSCH) between 1991 to 2005, a survey that covers 30 provinces of and autonomous regions of China, with Tibet Autonomous Region and Taiwan Province excluded owing to unavailability of related data. According to their socioeconomic status, three urban regions and three rural regions (province capital must be included) were selected in each province, where its socioeconomic status and sports exercise can represent the general level of the locality. All subjects were Han students. Prevalence of deciduous and permanent caries was measured. In 2005, the prevalence of deciduous caries at 7, 9, 12 years was 52.87%, 52.04% and 9.80% in urban boys, 59.37%, 58.68% and 12.18% in rural boys, and that of permanent caries in boys at 7, 9, 12, 14, and 17 years was 2.35%, 5.63%, 13.94%, 19.05% and 21.29% in urban boys, 3.42%, 7.26%, 13.35%, 16.16%, 18.55% in rural boys, while in girls at the same age groups, the prevalence of deciduous caries was 53.53%, 49.48%, 7.92% in urban girls, 59.95%, 56.13%, 8.85% in rural girls; the prevalence of permanent caries was 3.61%, 8.99%, 19.32%, 24.75%, 28.62% in urban girls, and 3.47%, 10.65%, 17.66%, 22.52%, 24.27% in rural girls. The epidemic manifested a gradient distribution in age groups, which was closely related to region of the study population. A dramatic decreasing trend was witnessed among all sex-age subgroups in the rural groups between 1990 and 2005, and such a trend was stronger in deciduous caries than in permanent caries, however, this kind of decreasing trend was not steady, because there was bounce during some year. Although the morbidity of dental caries in China is lower than the standard set by the World Health Organization, and the prevalence of dental caries decreased from 1991 to 2005, the prospect of epidemic dental caries in China is in no way optimistic. The total population with dental caries is still very large and the prevalence decreased unstably. Therefore, financial support on school oral health care should be increased, and a national school oral health service network should be set up.

This study explored the accuracy of using visual evoked potentials (VEP) technology for visual acuity estimation. The enrolled 726 patients with post-traumatic unilateral decrease in visual acuity included the injured eyes served as the experimental group, and the healthy eyes as the control group. The least signal visual angle (LSVA), and amplitude and latency of P₁₀₀ were chosen as test indexes. The results under different experimental conditions were recorded by PRVEP technology. All data collected were processed and analyzed by SPSS software. The results showed that the coincidence between subjective and VEP visual acuity was 96.7% in control group, but there was very significant difference in experimental group. It was concluded that with the regression formulation for the amplitude of P₁₀₀ and vision under LSVA, visual acuity can be estimated more accurately and impartially.