This study was designed to measure the performance of county health systems in central and western China utilizing routine healthcare data. Drawing on a literature review and expert consultation, the study established a theoretical framework and an indicator system for performance review. Questionnaires were designed and disseminated to collect empirical data on health system performance in four counties of two central and western provinces. Quantitative data were subjected to descriptive statistical analysis through SPSS12.0. Three dimensions were introduced in the performance review framework—health outcomes, financial risk protection and consumer and provider satisfaction. Health outcomes were assessed from four secondary indicators: infant mortality rate; maternal mortality rate; under-5 child mortality rate; and the incidence of Class A and Class B notifiable diseases. Financial risk was assessed using two secondary indicators: the proportion of the cost of inpatient care that was reimbursed under the New Cooperative Medical System (NCMS) insurance scheme, and the rate of NCMS funds utilization. The assessment of satisfaction was made using two secondary indicators: the overall satisfaction of local residents with healthcare services, and the satisfaction of health practitioners at the township and village level. The study indicated better health system performance in the two counties in Chongqing than those in Shanxi. It was concluded that outcome framework scores can fairly reveal performance differences among county health systems in central and western China, and can provide practical evidence for optimizing the operation and inputs of county health systems. Caution needs to be exercised in generalizing such performance outcomes as many factors such as spending and organization that contribute to county health system performance were not included in the study.
In order to ascertain prevalence rate of premarital sexual intercourse, unintended pregnancy and abortion, and evaluate associated factors of unintended pregnancy among undergraduates from all over China, the representative sample of unmarried undergraduates was obtained by using a multi-stage, stratified, probability cluster design, and data were collected by using a survey questionnaire. 62 326 available responders were gained. 11.6% of them acknowledged having experiences of premarital sexual intercourse (standardized prevalence rate of sexual intercourse was 13.8%). 31.5% of students active in premarital sex acknowledged undergoing unintended pregnancy. 76.2% of pregnant students selected abortion to end it. Of students active in premarital sex, 46.2% used contraception at the first sexual intercourse, 28.2% replied “always” using contraception in sexual intercourse. The rate of using condoms, oral contraceptives (OCs), and withdrawal among students who had used contraception was 52.0%, 31.0%, and 27.2% respectively. “No preparation for sex” (40.3%), “pleasure decrement” (32.1%), “won’t-be-pregnancy in occasional sexual intercourse” (30.2%) were their common excuses for using no contraception. The identified risk factors for unintended pregnancy among students active in premarital sex by multivariate analysis were as follows: having no steady lover [having no steady lover vs having a steady lover: odds ratio (OR), 1.875; 95% confidence interval (CI), 1.629–2.158], unaware of the course of conception (unaware vs aware: OR, 2.023; 95% CI, 1.811–2.260), considering abortion not endanger women’s physical and mental health (no endangerment vs endangerment: OR, 2.659; 95% CI, 2.265–3.121), nonuse of contraception (never use vs always use: OR, 1.682; 95% CI, 1.295–2.185). Medical students were not less likely to experience an unintended pregnancy than nonmedical students (OR, 1.111; 95% CI, 0.906–1.287). The substantial proportion of unintended pregnancy among undergraduates indicates a need for convenient and targeted contraceptive education and services.
Allergic rhinitis (AR), with an increasing uptrend of the prevalence in many developed and developing countries, is a global health problem that affects people of all ages and ethnic groups. However, data on the prevalence of self-reported AR in western China are rare. This study investigated the epidemiological features of self-reported AR in western China. In the cross-sectional, population-based study, a validated questionnaire survey on self-reported AR was carried out in 4 major cities in western China by multistage, stratified and cluster sampling, from January to December 2008. The total prevalence rate was 34.3%, with 32.3% (Chongqing), 34.3% (Chengdu), 37.9% (Urumqi), 30.3% (Nanning), respectively. The prevalence presented to increase with age before 30 years old while decrease with age after 30 years old, and the highest prevalence was in 19–30 years group in Chongqing, Chengdu and Nanning which significantly showed “persistent and moderate-severe” type (P<0.0001); In Urumqi, there wasn’t a significant increasing or decreasing trend of prevalence rate with age but with an “intermittent and mild”predominance (P<0.0001). There were no distinct sexual differences in prevalence rates in the 4 cities. The morbidity was positively related to monthly average temperature and sunshine (r=0.76645, P=0.0036; r=0.67303, P=0.0165), but negatively associated with relative humidity (r=−0.64391, P=0.0238) in Urumqi. Interestingly, the monthly morbidity was negatively associate with average temperature, sunshine and precipitation in Nanning (r=−0.81997, P=0.0011; r=−0.60787, P=0.0360; r=−0.59443, P=0.0415). Self-reported AR is becoming common in western China with a rapid development in recent years, affecting about three persons out of ten. The climatic factors may have an indirect impact on the prevalence rate through the effects on the local allergens.
To explore the protective effect of sodium tanshinone IIA sulfonate (STS) on microcirculatory disturbance of small intestine in rats with sepsis, and the possible mechanism, a rat model of sepsis was induced by cecal ligation and puncture (CLP). Rats were randomly divided into 3 groups: sham operated group (S), sepsis group (CLP) and STS treatment group (STS). STS (1 mg/kg) was slowly injected through the right external jugular vein after CLP. The histopathologic changes in the intestinal tissue and changes of mesenteric microcirculation were observed. The levels of tumor necrosis factor-α (TNF-α) in the intestinal tissue were determined by using enzyme-linked immunoabsorbent assay (ELISA). The expression of intercellular adhesion molecule-1 (ICAM-1) in the intestinal tissue was detected by using immunohistochemisty and Western blot, that of nuclear factor κB (NF-κB) and tissue factor (TF) by using Western blot, and the levels of NF-κB mRNA expression by using RT-PCR respectively. The microcirculatory disturbance of the intestine was aggravated after CLP. The injury of the intestinal tissues was obviously aggravated in CLP group as compared with S group. The expression levels of NF-κB p65, ICAM-1, TF and TNF-α were upregulaed after CLP (P<0.01). STS post-treatment could ameliorate the microcirculatory disturbance, attenuate the injury of the intestinal tissues induced by CLP, and decrease the levels of NF-κB, ICAM-1, TF and TNF-α (P<0.01). It is suggested that STS can ameliorate the microcirculatory disturbance of the small intestine in rats with sepsis, and the mechanism may be associated with the inhibition of inflammatory responses and amelioration of coagulation abnormality.
The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro, as well as the roles of nuclear factor-kappa B (NF-κB) and IκBα was investigated. The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay. Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay. The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy. The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells. Triptolide at nM range induced proliferation inhibition in a dose- and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells. Besides, we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol. These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.
Associations between “lipid-related” candidate genes, blood lipid concentrations and coronary artery disease (CHD) risk are not clear. We aimed to investigate the effect of three newly identified lipids loci from genome-wide association studies on CHD and blood lipid levels in Chinese Han population. The genotypes of SNPs at three newly identified lipid loci and blood lipids concentrations were examined in 1360 CHD patients and 1360 age- and sex-frequency matched controls from an unrelated Chinese Han population. Allele T of rs16996148 occurred less frequently in CHD patients with the odds ratio (OR) being 0.64 (95% CI 0.50 to 0.81), after adjusting for conventional risk factors and was associated with a 33% decreased CHD risk (P<0.01) comparing with the major allele G. Individuals with GT genotype had the lowest CHD risk. No associations were found between the polymorphisms of other two loci with CHD risk and all three SNPs had no effect on lipid profile in this population. SNP rs16996148 on chromosome 19p13 is significantly associated with lower risk for CHD in Chinese Han population. However, it remains unresolved why these lipid-related loci had significantly less effects than the correspondingly expected effects on blood lipids levels in this population.
In order to evaluate the effectiveness of everolimus vs. rapamycin in the treatment of diabetic nephropathy, 8-week old diabetic (db/db) mice received everolimus (2 mg/kg every day) or rapamycin (2 mg/kg every day) for 4 weeks or 12 weeks respectively. Blood and 24-h urine samples were collected for biochemical tests. One kidney from each mouse was homogenized for protein analysis and the other was removed for histological analysis. The expression levels of transforming growth factor-β1 (TGF-β1)and phospho-p70s6k were detected by using ELISA and Western blot, respectively in the renal tissue as well as in mesengial cell culture samples. Everolimus was significantly more effective than rapamycin in improving indexes of renal function and glomerular hypertrophy, and in decreasing accumulation and expansion of the extracellular matrix. However, everolimus inhibited TGF-β1 secretion and p70s6k phosphorylation induced by high glucose in vitro less efficiently than rapamycin at the same dose. Everolimus was more effective than rapamycin in preventing diabetic nephropathy in vivo, which may be contributed to the fact that everolimus has better bioavailability and a higher oral absorption rate.
The expression of CD8+CD25+FoxP3+ regulatory T cells (CD8+Tregs) in the peripheral blood of patients with stable chronic obstructive pulmonary disease (COPD), and the effect of muscarinic cholinergic receptor antagonist tiotropium bromide on the expression of CD8+Tregs were investigated. Twenty-three patients with moderate to severe stable COPD were enrolled in this study. All patients inhaled tiotropium bromide (18 μg daily) for 3 months. Before and after inhalation of tiotropium bromide, peripheral blood samples were collected from the patients, and T cells were labeled by three-color labeled monoclonal antibodies. Flow cytometry was used to detect the quantity and percentage of CD8+T cells, CD8+CD25+T cells, CD8+Tregs, CD4+T cells, CD4+CD25+T cells and CD4+CD25+FoxP3+ regulatory T cells (CD4+Tregs) respectively. The percentage of CD4+T cells was increased from (27.82±2.18)% to (35.53±1.3)% (t=3.20, P=0.004) in the peripheral blood of patients with stable COPD after inhalation of tiotropium bromide for 3 months, that of CD4+CD25+T cells was decreased from (10.03 ±1.42)% to (4.21 ±0.65)% (t=3.78, P=0.001), and that of CD8+Tregs was increased from (8.41 ±1.68)% to (21.34 ±4.20)% (t=2.72, P=0.013). At baseline, CD8+T cells, CD8+CD25+T cells and CD4+Tregs were detectable in the peripheral blood, but no significant changes were observed after treatment. Linear correlation analysis revealed that the difference before and after treatment in CD4+T cells and CD4+CD25+T cells was negatively correlated with the ratio of change in CD8+Tregs before and after treatment (r=−0.61, P=0.013; r=-0.72, P=0.001 respectively). In the peripheral blood of patients with stable COPD, there was the expression of CD8+Tregs and CD4+Tregs. Muscarinic receptor antagonist, tiotropium bromide, can promote the amplification of CD4+T cells, inhibit the expression of CD25+T cells, and enhance the expression of CD8+Tregs. CD8+Tregs and CD4+Tregs can be used as new indicators to understand the immune status of patients. They are helpful in judging the treatment efficacy and disease immunophenotype.
Recently, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is suggested as a new agent in the fighting against fibrogenesis. In tumor, DJ-1 is identified as a negative regulator of PTEN. But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear. Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model. Human proximal tubular epithelial cells (HKC) were treated with transforming growth factor-beta 1 (TGF-β1), or transfected with DJ-1 or PTEN. Confocal microscope was used to investigate the localization of DJ-1 and PTEN. The selective phosphoinositide-3 kinase (PI3K) inhibitor, LY294002, was administered to inhibit PI3K pathway. The DJ-1 and PTEN expression, markers of epithelial-mesenchymal transition (EMT) and Akt phosphorylation were measured by RT-PCR, Western blotting or immunocytochemistry. In vitro, after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h, the expression of DJ-1 was increased, and that of PTEN was decreased. In vivo, the same results were identified in 5/6-nephrectomized rats. In normal HKC cells, most of DJ-1 protein localized in cytoplasm, and little in nucleus. TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei. In contrary, TGF-β1 emptied cytoplasmic PTEN protein into nucleus. Overexpression of DJ-1 decreased the expression of PTEN, promoted the activation of Akt and the expression of vimentin, and also led to the loss of cytoplasmic PTEN. Contrarily, overexpression of PTEN protected HKC cells from TGF-β1-induced EMT. In conclusion, DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.
Systemic lupus erythematosus (SLE) is a multiple organ autoimmune disorder, including the liver, but the possible reason in impairment in the liver is still unclear. Our present study assessed alterations of transcription factor Foxp3+ regulatory T cells (Tregs) and several other immune molecules [programmed cell death 1 and its ligand (PD1 and PD-L1), and interleukin 10 (IL-10) and transform growth factor β (TGF-β)] in the liver and other major organs of lupus-prone BXSB mice by flow cytometry, real-time quantitative reverse transcription PCR, and enzyme-linked immunosorbent assay. Results showed that both frequency and number of Foxp3+ Tregs were dramatically reduced in the thymus, spleen and kidney of the BXSB mice (P<0.05), but those in the liver were kept in nearly normal range, when compared to negative control C57BL/6 mice. In comparison to control mice, the mRNA levels of Foxp3, PD1 and PD-L1 were significantly decreased in the kidneys of BXSB mice (P<0.05), but there was no significant difference in the livers of the BXSB mice (P>0.05). Protein levels of IL-10 and TGF-β in serum showed no significant difference between BXSB and C57BL/6 mice, but were significantly increased in the kidneys and livers of BXSB mice as compared with those in C57BL/6 mice (P<0.05). These results suggest that reduced Foxp3+ Tregs are involved in the pathogenesis of SLE in BXSB mice, and relatively higher number of these cells in the livers than in the other target organs could constitute a protective mechanism against hepatic lesions in lupus-prone mice, which may provide insights into development of new therapeutic approaches in SLE patients.
Host genetic, environmental and viral factors are classified as three categories that determine clinical outcomes of hepatitis B virus (HBV) infection. The objective of this study was to detect the associations between polymorphisms rs346473 and rs346482 in Rho GTPase-activating protein 24 (ARHGAP24) gene and disease progression of HBV infection in Han Chinese population. These two SNPs were found by our DNA pooling using Affymetrix Genome-Wide Human Mapping SNP6.0 Array in HBV carriers, and verified by using TaqMan 7900HT Sequence Detection System with 758 progressed HBV carriers versus 300 asymptomatic HBV carriers (AsC) in a discovery phase and 971 progressed HBV carriers versus 328 AsC in a replication phase. Multivariable logistic regression revealed that individuals with genotype TT at variant rs346473 displayed remarkable correlations with disease progression of HBV infection both in the discovery phase (OR, 2.693; 95% CI, 1.928–3.760; P=6.2×10−9; additive model) and the replication phase (OR, 1.490; 95% CI, 1.104–2.012; P=9.0×10−3; additive model). These two SNPs were in strong linkage disequilibrium with D′=0.99 and r2=0.951, and haplotype TT disclosed an increased susceptibility to HBV progression (OR, 1.980; 95% CI, 1.538–2.545; P=8.1×10−8). These findings suggest that polymorphism rs346473 in the ARHGAP24 gene might be a part of the genetic variants underlying the susceptibility of HBV carriers to disease progression.
Multidrug resistance (MDR) plays a major obstacle to successful gastric cancer chemotherapy. The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine (Nef) in adriamycin (ADM) resistant human SGC7901/ADM gastric cancer cells. The MDR cells were heated at 42°C and 45°C for 30 min alone or combined with 10 μg/mL Nef. The cytotoxic effect of ADM was evaluated by MTT assay. Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique. Intracellular accumulation of ADM was monitored with high performance liquid chromatography. Mdr-1 mRNA, P-glycoprotein (P-gp), γH2AX expression and γH2AX foci formation were determined by real-time PCR, Western blot and immunocytochemical staining respectively. It was found that different heating methods induced different cytotoxic effects. Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells. The water submerged hyperthermia increased the cell membrane fluidity. Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM. The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp. The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells. The higher temperature was, the worse effect was. Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.
SDF-1α, a ligand for the chemokine receptor CXCR4, is well known for mediating the migration of breast cancer cells. In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4 (NT21MP) derived from the viral macrophage inflammatory protein II could antagonize tumor growth in vivo by inhibiting cellular proliferation and inducing apoptosis in breast cancer cells. However, the role of SDF-1α in the signaling pathways underlying the proliferation of human breast cancer cells and associated signaling pathways and inhibiting signal pathways of NT21MP remained unclear. The present study investigated the mechanism of NT21MP on anti-tumor in breast cancer in vitro. The effect of NT21MP on the viability of cells was determined by the MTT assay. Annexin V-FITC and PI staining was performed to detect early stage apoptosis in SKBR3 cells treated with SDF-1α and AMD3100 or NT21MP. Western blotting techniques were used to assay the composition of phosphoproteomics and total proteins present in the SKBR3 breast cancer cells. RT-PCR and Western blotting technique were used to detect the effect of NT21MP and AMD3100 on Bcl-2 and Bax expression. The results indicated that SDF-1α prevented apoptosis and promoted the proliferation of SKBR3 human breast cancer cells. As compared with untreated SKBR3 cells, Treatment with SDF-1α significantly increased cell viability, and NT21MP abolished the protective effects of SDF-1α dose-dependently (P<0.05). There was a significant decrease in the percentage of apoptotic cells after SDF-1α treatment as compared with control group (2.7%±0.2% vs. 5.7%±0.4%, P<0.05). But pretreatment of SKBR3 cells with NT21MP significantly attenuated the antiapoptotic effects of SDF-1α as compared with SKBR3 cells without NT21MP pretreatment. The proliferative and anti-apoptotic effects of SDF-1α in SKBR3 cells were associated with an increase in AKT and ERK1/2 phosphorylation as well as a decrease in Bax expression and an increase in Bcl-2 expression. These changes in intracellular processes were blocked by NT21MP in a dose-dependent manner(P<0.05). In conclusion, NT21MP efficiently inhibits SDF-1α-induced proliferation and antiapoptosis in SKBR3 cells by reducing the levels of phosphorylated AKT and ERK1/2, as well as decreasing the ratio of expression of Bcl-2 relative to Bax.
This study examined the effects of ω-3 polyunsaturated fatty acid (ω-3PUFA) on the expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and some related inflammatory factors in peripheral blood mononuclear cells (PBMCs) of patients with early-stage severe multiple trauma. Thirty-two patients who were admitted to the Department of Traumatic Surgery, Tongji Hospital (Wuhan, China) between May 2010 and November 2010, and diagnosed as having severe multiple trauma with a injury severity score (ISS) no less than 16, were enrolled in the study and divided into two groups at random (n=16 in each): ω-3PUFA group and control group in which routine parenteral nutrition supplemented with ω-3PUFA or not was administered to the patients in two groups for consecutive 7 days. Peripheral blood from these patients was collected within 2 h of admission (day 0), and 1, 3, 5 and 7 days after the nutritional support. PBMCs were isolated and used for detection of the mRNA and protein expression of TLR2 and TLR4 by using real-time PCR and flow cytometry respectively, the levels of NF-κB by quantum dots-based immunofluorescence assay, the levels of TNF-α, IL-2, IL-6 and COX-2 by ELISA, respectively. The results showed that the mRNA and protein expression of TLR2 and TLR4 in PBMCs was significantly lower in ω-3PUFA group than in control group 5 and 7 days after nutrition support (both P<0.05). The levels of TNF-α, IL-2, IL-6 and COX-2 were found to be substantially decreased in PBMCs in ω-3PUFA group as compared with control group at 5th and 7th day (P<0.05 for all). It was concluded that ω-3PUFA can remarkably decrease the expression of TLR2, TLR4 and some related inflammatory factors in NF-κB signaling pathway in PBMCs of patients with severe multiple trauma, which suggests that ω-3PUFA may suppress the excessive inflammatory response meditated by the TLRs/NF-κB signaling pathway.
Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including cancer. We found miR-146b-5p significantly dysregulated in human pancreatic cancer cells by qRT-PCR. To demonstrate its function and regulation mechanism, we overexpressed miR-146-5p by transfecting the mimics. Our data showed that miR-146b-5p overexpression significantly reduced the abilities of migration and invasion of MIA PaCa-2 pancreatic cancer cells. Furthermore, we found that matrix metalloproteinase 16 (MMP16) was a downstream target of miR-146b-5p by dual-luciferase reporter assay. Altogether, our findings suggest that miR-146b-5p may be involved in pancreatic cancer cell migration and invasion by targeting MMP16, and miR-146b-5p may be a potential therapeutic target for the pancreatic cancer.
There has been an ongoing search for clinically acceptable methods for the accurate, efficient and simple diagnosis and prognosis of hepatocellular carcinoma (HCC). Optical spectroscopy is a technique with potential clinical applications to diagnose cancer diseases. The purpose of this study was to obtain the optical properties of HCC tissues and non-tumorous hepatic tissues and identify the difference between them. A total of 55 tissue samples (HCC tissue, n=38; non-tumorous hepatic tissue, n=17) were surgically resected from patients with HCC. The optical parameters were measured in 10-nm steps using single-integrating-sphere system in the wavelength range of 400 to 1800 nm. It was found that the optical properties and their differences varied with the wavelength for the HCC tissue and the non-tumorous hepatic tissue in the entire wavelength range of research. The absorption coefficient of the HCC tissue (1.48±0.99, 1.46±0.88, 0.86±0.61, 2.15±0.53, 0.54±0.10, 0.79±0.15 mm−1) was significantly lower than that of the non-tumorous hepatic tissue (2.79±1.73, 3.13±1.47, 3.06±2.79, 2.57±0.55, 0.62±0.10, 0.93±0.16 mm−1) at wavelengths of 400, 410, 450, 1450, 1660 and 1800 nm, respectively (P<0.05). The reduced scattering coefficient of HCC tissue (5.28±1.70, 4.91±1.54, 1.26±0.35 mm−1) and non-tumorous hepatic tissue (8.14±3.70, 9.27±3.08, 2.55±0.57 mm−1) was significantly different at 460, 500 and 1800 nm respectively (P<0.05). These results show different pathologic liver tissues have different optical properties. It provides a better understanding of the relationship between optical parameters and physiological characteristics in human liver tissues. And it would be very useful for developing a non-invasive, real-time, simple and efficient way for medical management of HCC in the future.
The Leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2) gene expression in pituitary adenoma and its correlation with tumor invasiveness were studied. The expression of LRIG2 mRNA and protein in human pituitary adenoma obtained surgically was detected by RT-PCR (39 cases) and immunohistochemical staining (30 cases). It was found that LRIG2 was mostly localized at the nucleus of the pituitary adenoma cells. Its expression was significantly higher in the invasive cases than in the non-invasive cases. LRIG2 protein was positive in 14 cases out of 21 cases of invasive adenoma, but only 2 cases were positive in 9 cases of non-invasive adenoma. The positive expression rate of LRIG2 mRNA was 91.3% in invasive cases (total 23 cases) and 62.5% in non-invasive cases (total 16 cases), respectively. LRIG2 gene is overexpressed in invasive pituitary adenoma. It may play an important role in pituitary adenoma invasiveness and further studies are necessary to elucidate the mechanism under this phenomenon.
This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts. hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system. pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines. The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM) components was detected after transfection by real-time quantitative PCR, ELISA, immunochemistry and Western blotting, respectively. The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing. Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs. Real-time quantitative PCR, immunochemistry and Western blotting showed that the cartilage-specific ECM markers, i.e., cartilage oligomeric matrix protein (COMP), Aggrecan, collagen type X and II were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells. It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.
The anti-tumor activity of curcumin against androgen-independent prostate cancer cells in vitro and the possible mechanism were investigated. After curcumin treatment, the effect of curcumin on the proliferation of prostate cancer PC-3 cells was assessed by CFSE staining. Flow cytometery (FCM) was performed to analyze the cell cycle and the induction of apoptosis of tumor cells. A luciferase reporter gene assay was used to determine the effects of curcumin on the activities of intracellular NF-κB and AP-1 signaling pathways. The results showed curcumin could effectively inhibit the proliferation of PC-3 cells in vitro (P<0.05). Cells were arrested at G2/M phase. After curcumin treatment, the percentage of apoptotic cells was significantly higher than in control group (P<0.05). The results of the luciferase assay revealed that curcumin selectively inhibited the activities of the NF-κB and AP-1 signaling pathways in PC-3 cells significantly. It was suggested that curcumin could exert anti-tumor activity against androgen-independent prostate cancer cells in vitro by inhibiting cellular proliferation and inducing apoptosis, which was probably contributed to the inhibition of transcription factors NF-κB and AP-1.
The debate exists whether or not gonadotropin-releasing hormone (GnRH) analogs used in controlled ovarian hyperstimulation (COH) impair endometrial receptivity. Homeobox A11 (Hoxa11), Meis homeobox 1 (Meis1), cadherin 1 (Cdh1), and catenin beta 1 (Ctnnb1) are well known to be involved in successful implantation. In this study, the endometrial expression of Hoxa11, Meis1, Cdh1, and Ctnnb1 during the peri-implantation period was investigated in an in vitro fertilization (IVF) mouse model by real-time RT-PCR and Western blot to evaluate the relationship between Hoxa11, Meis1, Cdh1, and Ctnnb1 expression and the impact of the COH on endometrial receptivity. The mimic COH protocols included GnRH agonist plus human menopausal gonadotropin (HMG) (GnRH agonist group), GnRH antagonist plus HMG (GnRH antagonist group), and HMG alone (HMG group). The expression levels of Hoxa11, Meis1, Cdh1, and Ctnnb1 mRNA and protein were decreased in all of the COH groups. The expression levels of Hoxa11 and Ctnnb1 were the lowest in the GnRH agonist group, and those of Meis1 and Cdh1 were lower in the GnRH analog groups than the HMG group. There were positive correlations between the expression of Hoxa11 and Ctnnb1, as well as the expression of Meis1 and Cdh1 among all the groups. In conclusion, the COH protocols, particularly with GnRH analogs, suppressed Hoxa11, Meis1, Ctnnb1 and Cdh1 expression, in mouse endometrium during the peri-implantation period. Our data reveal a novel molecular mechanism by which the COH protocols might impair endometrial receptivity.
Chemotherapy is the preferred therapeutic approach for advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This study is to investigate the promoting effects and mechanism of miR-125b involved in the development of chemoresistance in ovarian cancer. The different expression of miR-125b in cisplatin-sensitive ovarian cancer cell line (OV2008) and its resistant variant (C13*) was identified by real-time PCR. An in vitro cytotoxicity assay and apoptosis assay using CCK-8 assay and flow cytometry, were carried out to detect the effect of miR-125b and Bak1 on cisplatin resistance of cells. Real-time PCR, Western blotting and luciferase reporter assay were used to detect whether Bak1 is a target of miR-125b. As compared with OV2008 cells, the expression levels of miR-125b in C13* cells were increased. It was found that the up-regulation of microRNA-125b caused a marked inhibition of cisplatin-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to cisplatin in OV2008 and C13* cells. Moreover, Bak1 was a direct target of miR-125b, and down-regulation of Bak1 suppressed cisplatin-induced apoptosis and led to an increased resistance to cisplatin. Our study indicates that miR-125b has a significantly promoting effect on chemoresistance of C13* cells and up-regulation of miR-125b expression contributes to cisplatin resistance through suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming cisplatin resistance in ovarian cancer.
Inflammation and infection play an important role in the pathogenesis of many cancers. Toll-like receptors (TLRs) are a class of pattern recognition receptors that recognize conserved components of microbes and trigger the immune response against invading microorganisms. Toll-like receptor 9 (TLR9) recognizes non-methylated cytosine-phosphateguanosine (CpG) DNA sequences which are the surrogate for viral DNA. TLR9 may react to tumor development and progression during chronic inflammation that involves the tumor microenvironment. In order to study the role of TLR9 in cervical cancer, we analyzed the TLR9 expression in different types of HPV infection cervical cancer cells. Then we detected if CpG sequences influenced the TLR9 expression and the sensitivity to cisplatin (DDP) of these cervical cancer cells in vitro. The expression of TLR9 mRNA and protein in SiHa, Hela and C33A cells was detected by RT-PCR and Western blotting. Real-time PCR was used to examine the TLR9 expression changes induced by CpG. Chemosensitivity of the cervical cancer cells to cisplatin (DDP) was measured by MTT. It was observed that the expression of TLR9 mRNA and protein was increased gradually in SiHa (HPV16+), Hela (HPV18+) and C33A (HPV−) cells. Low doses of CpG increased the TLR9 expression only in C33A (HPV−) cells, but not in SiHa (HPV16+) and Hela (HPV18+) cells. Furthermore, low dose of CpG significantly increased the sensitivity of C33A (HPV−) cells, but not that of SiHa (HPV16+) and Hela (HPV18+) cells. These results indicated that TLR9 may serve as a protective agent in HPV negative cervical cancer cells. It was concluded that TLR9 could improve the sensitivity to DDP in HPV negative cervical cancer cells and might represent a potential therapeutic option in clinical practice.
The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells (LECs) and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract. The cultured LECs of second and third passages were collected and treated with rapamycin. The LECs were transferred into 96-well culture plates and divided into 6 groups, and each group was set to have 8 duplicate wells. In the negative control group, the LECs were given culture medium only, and in the blank control group, only culture medium was given. In the four rapamycin-treated groups, different concentrations (20, 40, 60 and 80 ng/mL) of rapamycin were given. After treatment for 24, 48 and 72 h, the absorbance (A) values in each well were determined by MTT assay. The cell cycles of all groups were detected by using flow cytometry. Real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR) and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively. MTT assay showed that rapamycin could inhibit proliferation of LECs in a time- and dose-dependent manner. Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase, resulting in the increase of cells in G1 phase and the decrease of the cells in S phase. RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA, but up-regulate the expression of Bax mRNA, suggesting it could induce apoptosis of LECs. Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein, but promote the expression of Bax protein. It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle, but also by promoting the induction of apoptosis.
The relationship between the expression of vascular endothelial growth factor (VEGF) and microvascular density (MVD) marked by CD105 (CD105-MVD), and that between CD105-MVD and the clinicopathological characteristics of primary pterygium were investigated. The streptavidin-biotin complex (SABC) immunohistochemical staining in paraffin-embedded tissues was used to detect the expression of VEGF in 23 cases of primary pterygia and 7 normal conjunctival specimens. The antibody against CD105 was used to display vascular endothelial cells, and MVD was examined by counting the CD105-positive vascular endothelial cells. The correlations of VEGF and CD105-MVD, and those of CD105-MVD and clinicopathological data were analyzed by using SPSS 12.0. The expression of VEGF was significantly increased in epithelia (P=0.000), endothelia and stroma cells (P=0.005) in primary pterygia as compared with normal conjunctivae. The CD105-MVD in pterygia (mean 19.22±6.68) was higher than that in normal conjunctivae (mean 4.00±2.15, P=0.000). MVD in pterygia was significantly associated with the Tan classification (P=0.000) and the VEGF expression level in the stroma (P=0.020), but not with sex (P=0.61), age (P=0.150) or the VEGF expression level in the epithelia (P=0.518). Our results suggest that over-expression of VEGF and high CD105-MVD in primary pterygium may contribute to the progression by increasing angiogenesis and growth of primary pterygium.
The effect of siRNA-mediated Sox4 gene silencing on Wnt/β-catenin signaling pathway of human malignant melanoma cell line A375 was investigated. Two types of dsRNA targeting Sox4 were constructed and transfected into A375 cells, and untreated cells and cells transfected with scramble RNA were used as blank control and negative control respectively. The expression levels of mRNA and protein of Sox4, Wnt3a, β-catenin and Wnt/β-catenin signaling target gene Survivin were detected after real-time PCR and Western blot respectively. MTT assay was used to measure cell proliferation after Sox4 knockdown. β-catenin/TCF transcription reporter assay was used for assessing Wnt/β-catenin signaling pathway activity. Our results showed that the two types of Sox4 siRNA were transfected into A375 cells successfully. As compared with untreated cells, Sox4 siRNAs had no significant influence on Wnt3a expression, and Sox4 siRNAs led to the decrease of β-catenin at protein level. Wnt/β-catenin signaling pathway activity was inhibited significantly. As a target of Wnt/β-catenin signaling, Survivin was decreased at both mRNA and protein levels, and cell proliferation was attenuated. Our study suggests that Sox4 may play an important role in Wnt/β-catenin signaling pathway in human malignant melanoma cells by regulating β-catenin protein level, indicating that Sox4 is involved in the progression of malignant melanoma through Wnt/β-catenin signaling pathway.
Mental retardation is defined by significant limitations in intellectual function and adaptive behavior that occur before 18 years of age. Many chromosomal diseases come with mental retardation. We reported two Chinese families with partial trisomy 9p and other chromosome partial monosomy, clinical features of mental retardation and mild facial and pinkie anomalies. In the family 1, we showed that the proband carried a trisomy 9p21.3→pter and monosomy 21q22.3→qter by using fluorescence in situ hybridization analysis. Molecular genetic analysis defined the precise breakpoint on chromosome 9p between markers D9S1846 and D9S171, an interval of about 2.9 Mb on 9p21.3, and the breakpoint on chromosome 21q between markers D21S1897 and D21S1446, a region of about 1.5 Mb on 21q22.3. In the family 2, a patient with trisomy 9p21.3→pter and monosomy 5p15.33→pter, and a de novo maternal balanced translocation between chromosomes 5 and 9 was identified in his mother. Cytogenetic and molecular genetic analysis defined the precise breakpoints on chromosome 9p21.3 and chromosome 5p15.33. Further clinical investigation found that any individual had no refractoriness eczema disease except the proband in this family. These results further implicate that trisomy 9p is associated with mental retardation, and that there may be key gene duplication on chromosome 9p21.3→9pter responsible for mental retardation and mild facial anomaly. This result has been applied successfully in prenatal diagnosis of the second family.
Brain iron deposition has been proposed to play an important role in the pathophysiology of Alzheimer disease (AD). The aim of this study was to investigate the correlation of brain iron accumulation with the severity of cognitive impairment in patients with AD by using quantitative MR relaxation rate R2′ measurements. Fifteen patients with AD, 15 age- and sex-matched healthy controls, and 30 healthy volunteers underwent 1.5T MR multi-echo T2 mapping and T2* mapping for the measurement of transverse relaxation rate R2′ (R2′=R2*−R2). We statistically analyzed the R2′ and iron concentrations of bilateral hippocampus (HP), parietal cortex (PC), frontal white matter (FWM), putamen (PU), caudate nucleus (CN), thalamus (TH), red nucleus (RN), substantia nigra (SN), and dentate nucleus (DN) of the cerebellum for the correlation with the severity of dementia. Two-tailed t-test, Student-Newman-Keuls test (ANOVA) and linear correlation test were used for statistical analysis. In 30 healthy volunteers, the R2′ values of bilateral SN, RN, PU, CN, globus pallidus (GP), TH, and FWM were measured. The correlation with the postmortem iron concentration in normal adults was analyzed in order to establish a formula on the relationship between regional R2′ and brain iron concentration. The iron concentration of regions of interest (ROI) in AD patients and controls was calculated by this formula and its correlation with the severity of AD was analyzed. Regional R2′ was positively correlated with regional brain iron concentration in normal adults (r=0.977, P<0.01). Iron concentrations in bilateral HP, PC, PU, CN, and DN of patients with AD were significantly higher than those of the controls (P<0.05); Moreover, the brain iron concentrations, especially in parietal cortex and hippocampus at the early stage of AD, were positively correlated with the severity of patients’ cognitive impairment (P<0.05). The higher the R2′ and iron concentrations were, the more severe the cognitive impairment was. Regional R2′ and iron concentration in parietal cortex and hippocampus were positively correlated with the severity of AD patients’ cognitive impairment, indicating that it may be used as a biomarker to evaluate the progression of AD.
Intravenous leiomyomatosis (IVL) is a rare benign neoplasm which originates from the smooth muscle cells and is usually confined to the pelvic venous system. Rarely, intracaval and intracardiac extension has been described. Death can occur as a result of intracardiac involvement. We reported 4 cases of IVL with right heart involvement (intracardiac leiomyomatosis, ICL). Three of them suffered recurrent sudden syncope, and the other one was totally asymptomatic. All of them were successfully treated through one-stage operation under extracorporeal circulation.
The etiology of deep vein thrombosis (DVT) is still not elucidated nowadays. Based on the accordance between DVT incidence and the anemophilous pollen concentration in the air, we proposed the hypothesis that allergic reaction induced by anemophilous pollen may cause “idiopathic” DVT, and proinflammatory factors may play an important role in the thrombosis process.