To determine the expressive level of checkpoint kinase 1 (CHK1) and polo-like kinase 1 (PLK1) and to detect their clinicopathological significance in benign and malignant lesions of the stomach. Envision Tm immunohistochemistry was used to detect the expression level of CHK1 and PLK1 in conventional paraffin-embedded sections from specimens of primary foci (n=59)and metastatic foci of lymph node (n=42) of gastric cancer, peritumoral tissues (n=20), and benign lesions of the stomach (n=95). The positive rates of CHK1 were significantly higher in gastric cancer than that in different types of benign lesions(P0.05). The positive rates of CHK1 and PLK1 were significantly lower in the non-metastatic lymph node than that in the metastatic lymph node (P<0.05). The positive rate of CHK1 was significantly lower in histologic grade II than that in the histologic grade III+IV (P<0.05). Positive correlation was found between the expression of CHK1 and PLK1 in gastric cancer tissues (P<0.01). The expression level of CHK1 and /or PLK1 might be important biological markers of kinases to reflect the carcinogenesis, progression, biological behaviors, and guide clinical auxiliary treatment of gastric cancer.
To examine the association among malondialdehyde (MDA), superoxide dismutase (SOD) and incidence of apoptosis of granulosa cells in follicular fluid with the outcome of in vitro fertilization-embryo transfer (IVF-ET). We recruited 51 women undergoing an IVF-ET programme for tubal factor infertility. The women with endometriosis or endocrine diseases and those with male factor infertility were excluded. All the 51 patients underwent a long gonadotropin-releasing hormone (GnRH) agonist protocol for pituitary downregulation followed by controlled ovarian hyperstimulation with rFSH. Granulosa cells were isolated from all aspirated follicular fluid using gradient centrifugation at oocyte retrieval. The level of MDA and the activity of the SOD were measured by the thiobarbituric acid(TBA)and the chemiluminescence method, respectively. The apoptosis was studied by flow cytometry using propidium iodide. Twenty-six out of the 51 patients (51.0%) were pregnant after IVF-ET. Non-pregnant patients showed significantly higher MDA level [(1.7±0.72) nmol/(g × prot) vs. (1.1±0.56) nmol/(g × prot), P<0.05)], higher incidence of apoptosis (24.8%±6.57% vs.19.0%±5.59%, P<0.05) and lower SOD level [(3.5±1.08)×10(3)NU/(g × prot) vs. (4.4±0.99)×10(3)NU/(g × prot), P<0.05)] in the granulose cells and lower good-embryo rate (54.9±20.22% vs. 65.9±16.16%, P<0.05) compared with the pregnant patients. No correlation was detected among SOD and the number of retrieved oocytes, oocyte maturity, embryo quality, fertilization, or cleavage. A significant negative correlation was detected between MDA and fertilization rate (r=-0.425, P=0.002). No significant correlation was detected between MDA and age, the number of retrieved oocytes, oocyte maturity, cleavage, or good-embryo rate. A significant negative correlation was detected between the incidence of apoptosis and the number of retrieved oocytes (r=-0.286, P=0.042), mature oocytes (r=-0.330, P=0.020) and good-embryo rate (r=-0.311, P=0.026). There was significant negative correlation between MDA and SOD levels (r=-0.471, P<0.001); and significant positive correlation between MDA level and incidence of apoptosis (r=0.475, P<0.001). Oxidative stress may induce apoptosis in granulose cells and subsequently lower oocyte quality and lead to poor outcome of IVF-ET.
To detect whether and where brain functional connectivity exists in the resting state of patients with early-onset schizophrenia by using functional magnetic resonance imaging (fMRI). Nineteen early-onset schizophrenic patients were diagnosed with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) of American Psychiatric Association. The 19 early-onset schizophrenic patients and another 19 healthy volunteers underwent fMRI in resting state. Cingulate gyrus was selected as region of interest and the difference was analyzed in the cingulate gyrus functional connectivity pattern between the 19 patients with early-onset schizophrenia (EOS) and 19 matched controls using resting-state fMRI. A two-sample t test was performed on the individual in a voxel by voxel manner. Statistical map was set a combined threshold of P20. Functional connectivity in the resting state was abnormal in the patients,including decreased functional connectivity and increased functional connectivity. The abnormal area was distributed all over the brain. The brain area with decreased functional connectivity included bilateral posterior cerebellar lobes, superior frontal gyrus, middle frontal gyrus, gyrus rectus,hippocampus, cuneus gyrus,fusiform gyrus,middle occipital gyrus,inferior occipital gyrus, right inferior temporal gyrus,right middle temporal gyrus, and right angular gyrus. The brain area with increased functional connectivity included left middle temporal and left inferior temporal gyrus. Abnormal cingulate gyrus functional connectivity of schizophrenia might exist in the resting state. Resting state fMRI is important for the research of schizophrenia.
To determine the role of fosinopril and valsartan intervention in Klotho, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor (PAI-1) gene expression in hypertensive renal interstitial fibrosis (RIF) in the kidney tissue of spontaneously hypertensive rats (SHR). We randomly divided 20 male 22-week-old SHR into 4 groups (5 in each group): a hypertension group (SHR group), a fosinopril group [Fos group, 10 mg/( kg · d) gavage], a valsartan group [Val group, 10 mg/( kg · d) gavage], and a fosinopril plus valsartan group [Fos + Val group, fosinopril 10 mg/( kg · d) + valsartan 50 mg/( kg · d) gavage]. Another five 22-week-old male Wistar Kyoto rats (WKY) were used as controls. Through monitoring the weight of the rats, tail artery pressure, 24-hour urine protein by fosinopril and/or valsartan intervention after the 8-week trial. RT-PCR and Western blot were used to detect the mRNA and protein expression of Klotho, MMP-9, TIMP-1, and PAI-1 in the kidneys. RT-PCR showed that in the SHR group, Klotho mRNA and protein expression were significantly decreased(P<0.01), while mRNA and protein expression of MMP-9, TIMP-1, and PAI-1 were significantly higher compared with the WKY group(P<0.01). With fosinopril and / or valsartan intervention, Klotho mRNA expression in the Fos group (P<0.01), Fos + Val group (P<0.01), Val group (P<0.05), Klotho protein expression in the Fos group(P<0.05), Fos + Val group (P<0.05), Val group (P<0.01), were significantly increased compared with those in the SHR group. The mRNA and protein expression of MMP-9, TIMP-1, and PAI-1 in the Fos group, Val group, and Fos + Val group were significantly lower than those in the SHR group (P<0.01). The expression of Klotho mRNA had negative correlation with the expression of MMP-9 mRNA (r= -0.864, P<0.01), TIMP-1 mRNA (r=-0.725, P<0.01) and PAI-1 mRNA (r=-0.785, P<0.01). The Klotho protein expression had negative correlation with the expression of MMP-9 protein (r=-0.614, P<0.05), TIMP-1 protein (r=-0.579, P<0.05), and PAI-1 protein (r=-0.552, P<0.05). Anti-aging gene Klotho and the genes related with extracellular matrix degradation gene MMP-9, TIMP-1, PAI-1 are involved in hypertensive renal injury. The expression of Klotho and MMP-9, TIMP-1, and PAI-1 is closely correlated. Fosinopril and valsartan which increase the Klotho mRNA and protein expression can alter the expression of Klotho-MMPs/TIMPs, which may be the main mechanism to prevent interstitial fibrosis.
To explore the regional homogeneity of resting state brain activity in early onset schizophrenia using functional magnetic resonance imaging. Schizophrenia or schizophreniform disorder was diagnosed according to DSM-IV-TR (diagnostic and statistical manual of mental disorders, fourth edition, text revision). A total of 18 adolescents with early-onset schizophrenia (EOS; onset of psychotic symptoms by age 18) and 18 age- and gender-matched healthy volunteers were tested in a resting-state fMRI scan. Regional homogeneity approach was used to analyze the functional imaging data,and statistical parametric mapping 5 (SPM5)was used to perform t-test in ReHo maps between the patients and controls. In comparison with the controls, the early-onset patients showed significantly decreased regional homogeneity in bilateral medial prefrontal cortex(P20), but no brain regions showed significantly increased regional homogeneity in the patients. Regional homogeneity of resting state brain activities in EOS was decreased in bilateral medial prefrontal cortex. These abnormal changes may be involved in the psychopathology of schizophrenia.
To observe the effect of autophagy on paclitaxel-induced CaSki cell death through the regulation of the expression of autophagy gene Beclin1, and to explore the interaction and relationship between autophagy and apoptosis. Eukaryotic expression vector pcDNA3.1-Beclin1 and RNA interference vector pSUPER-Beclin1 were transfected into human cervical cancer CaSki cells in vitro and screened for stable expression cell lines. The formation of autophagic vacuoles was observed with an electronic microscope. The expression of Beclin1 and LC3 was measured by Western blot. After being treated with paclitaxel, the change of cell proliferation was assessed by MTT assay, the percentage of apoptotic cells and autophagic cells were analyzed by flow cytometry. A lot of autophagic vacuoles were observed in pcDNA3.1-Beclin1 cells by electronic microscopy. Beclin1 and LC3 protein expression was up-regulated in CaSki cells transfected with pcDNA3.1-Beclin1, and was inhibited in cells transfected with pSUPER-Beclin1. MTT assay revealed the survival rate of CaSki cells was significantly decreased after being transfected with pcDNA3.1-Beclin1. After being treated with paclitaxel, the percentages of apoptotic cells and autophagic cells were both increased in pcDNA3.1-Beclin1 group compared with that of the blank control group especially the increase of apoptosis was particularly evident. Autophagy and apoptosis have different roles in the process of paclitaxel-induced cervical cancer CaSki cell line death. Overexpression of Beclin1 in CaSki cells may enhance the apoptosis induced by paclitaxel.
To determine the effect of ghrelin on protecting the human umbilical vein endothelial cells (HUVEC) from injury by angiotensin II (Ang II) in vitro. (1) HUVEC was incubated for 24 h with AngII whose final concentration in the medium varied from 10⁻⁹ to 10⁻⁶ mol/L or pretreated with 10⁻⁹ to 10⁻⁶ mol/L ghrelin for 2 h before incubation for 24 h with Ang II whose final concentration in the medium was 10⁻⁶ mol/L. HUVECs were harvested to measure the cell vitality and cell apoptosis. The cell vitality was determined by MTT and cell apoptosis rates were measured by Annexin V-FITC apoptosis detection kit. (2) HUVECs were incubated for 3, 6, 12, or 24 h with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L Ang II, respectively. Before HUVECs were incubated with 10⁻⁶ mol/L Ang II for 24 h, ghrelin (10⁻⁹, 10⁻⁸, 10⁻⁷, and 10⁻⁶ mol/L) was used to pretreat the cells for 2 h. Growth hormone secregogue receptor 1a blocker [D-Lys³]GHRP-6 was added to the cells which were incubated for 24 h with 10⁻⁶ mol/L Ang II and pretreated with 10⁻⁶ mol/L ghrelin for 2 h. Cell reactive oxygen species were measured by dichlorofluorescin (DCF) fluorescence probe method. (3) HUVECs were incubated for 24 h with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L Ang II and ghrelin, respectively,and then were incubated with 10⁻⁶ mol/L of Ang II for 3, 6, 12, or 24 h. Furthermore, HUVECs were pretreated with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L ghrelin for 30 min,1 h, or 2 h, and then were incubated with the inhibitor of mitogen-activated protein kinase /extracellular regulated kinase (MAPK/ERK1/2),PD98059, the inhibitor of phosphoinositide-3-kinase/serine threonine kinase( PI3K/Akt)wortmannin, and [D-Lys³]GHRP-6 for 24 h. NO production was compared among groups. HUVECs were pretreated with ghrelin, PD98059, wortmannin, and [D-Lys³]GHRP-6 for 2 h and co-cultured with 10⁻⁶ mol/L Ang II for 24 h, or pretreated with ghrelin plus PD98059, wortmannin, and [D-Lys³]GHRP-6 before incubation with Ang II for 24 h. NO was measured in the endothelial cell supernatants by Griess method. (4) HUVECs were cultivated with blank or Ang II with or without pretreatment with ghrelin or both ghrelin and wortmannin. The protein expression of eNOS and phospho-protein expression of Akt were measured by Western blot analysis. Ang II injuried the endothelial cell vitality,increased the cell apoptosis rates in HUVECs, and decreased NO production in HUVEC supernatants,whereas ghrelin protected HUVECs from Ang II injury. Ghrelin decreased the reactive oxygen species in HUVECs induced by Ang II. The effect could be attenuated by [D-Lys³]GHRP-6 pretreatment; PD98059 alleviated Ang II inhibition of NO production in HUVEC supernatants. Wortmannin and [D-Lys³]GHRP-6 could abolish protection of ghrelin from reducing NO production in HUVEC supernatants. Ang II reduced the expression of eNOS,but ghrelin increased eNOS expression. Wortmannin could cancel this effect of ghrelin. Ghrelin increased p-Akt expression and reached the peak in 10 and 20 min. Ghrelin may protect HUVECs from Ang II induced injury, which is related to decreasing oxidative stress, increasing the protein expression of eNOS, and activating PI3K/Akt signal pathway through GHSR1a receptor.
To investigate the relation between brain natriuretic peptide (BNP) rs198388 polymorphism and the susceptibility of essential hypertension in Han population of Hunan. A total of 567 patients with hypertension (the hypertension group) and 555 healthy volunteers (the control group) were enrolled. Gender, age, smoking and drinking history of the 2 groups were not significantly different. Blood pressure was measured in the 2 groups. After fasting for 12 h or more, blood glucose, total cholesterol, triglycerides, high density lipoprotein cholesterol and low density lipoprotein cholesterol were measured. DNA polymorphism analysis was done by polymerase chain reaction-restriction fragment length polymorphism method, and genotype was determined by agarose gel electrophoresis. The GG, GA, and AA genotypes were detected.The frequencies of GA and AA genotypes and A allele were significantly lower in the hypertension group (GA and AA:12.3%;A:6.9%) than those in the control group (GA and AA:18.4%; A:9.7%; P=0.009, and P=0.014, respectively). BNP rs198388 polymorphism may be associated with essential hypertension in Han people in Hunan. Carrying rs198388 GA and AA genotypes and A allele may be the reason for low risk of hypertension.
To explore the effect of tanshinone IIA (TanIIA) on calcium current induced by beta-amyloid protein 25-35 (Abeta25-35) in neurons of nucleus basalis of Meynert (nbM). Cell acute dissociated technique and the whole-cell recording model of patch-clamp technique of single-cell were used. The voltage-dependent calcium current in neurons of nbM was recorded in SD rats first. Then the effect of TanIIA on the voltage-dependent calcium current in the neurons was assayed. The change of calcium current induced by Abeta25-35 as well as the effect of TanIIA on the change of calcium current induced by Abeta25-35 in neurons of nbM were analyzed. Extracellular fluid containing different concentrations of TanIIA was irrigated, respectively. The peak current did not change obviously. There was no difference in current density between the TanIIA group and the control group at 0 mV (P>0.05). Extracellular fluid containing 200 nmol/L Abeta25-35 was irrigated after the normal calcium current recorded under whole patch clamp, and the peak current changed obviously. There was distinct difference in the current density between the Abeta group and the control group at 0 mV (P0.05). In vitro, TanIIA could inhibit the calcium current amplification induced by Abeta25-35 in neurons of nbM. TanIIA may protect neurons against the toxicity of Abeta and decrease the inward flow of Ca(2+).
To compare the fluorescence intensity and duration of qdots streptavidin conjugate (QDs-SA) with Cy3 as the molecular probe of β-amyloid precursor protein (APP), and to provide evidence for early molecular imaging and diagnosis of Alzheimer's disease (AD). With the help of laser scanning confocal microscope and flow cytometry, the flurescence probe based on the QDs-SA was used to detect APP in HEK293 cells stably transfected pcDNA3.1/APP, and to compare with conventional fluroimmunoassay Cy3. The immunofluorescence staining detection indicated APP expression was mainly located in the plasma membrane. The mean fluorescence intensity of QDs-SA (34.2336±4.2455) was greater than that of Cy3 (21.6023±3.0102)under the confocal fluorescence microscope (P<0.05). After persistent exciting for 12 min, the fluorescence intensity of APP stained by QDs-SA decreased by 27.87%. The other stained by Cy3 decreased by 79.60%. The positive rate of APP staining had no significant difference between the QDs-SA(54.4700±3.4433)% and Cy3 (54.3800±8.5229)% by flow cytometry, but the mean fluorescence intensity had statistical significance(P<0.05). The QDs-SA (1 045.4167±47.3623) was significantly higher than the mean fluorescence intensity of Cy3 (658.5467±55.0591). QDs-SA fluorescence probes can effectively recognize APP and are sensitive and exceptionally photostable, suggesting that QDs-SA fluorescence probes could be a potential method in APP detection and offer a novel way for the diagnosis of Alzheimer's disease.
To explore the value of elastography score and strain rate ratio in the diagnosis of small breast malignant focus. We retrospectively analyzed 22 patients with breast small malignant foci less than 10 mm. Ultrasound characteristics were summed up in breast small cancer. On elastogram, 2 patients scored 3, 14 scored 4 and 6 scored 5.The average strain rate ratio of all foci was 4.76, and there was correlation between it and elastography scores. Ultrasonic elastography has important value in the diagnosis of breast small cancer.
To establish the cell line from specimens of resectable human gastrointestinal stromal tumors (GIST) and to verify the characteristics of cell biology in vitro. The tissues from biopsies of human GIST were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. After growing to 90% confluence, the cells were detached for subculture and their characteristics, including morphology, growth kinetics, karyotype analysis, immunohistochemical analysis and tumorigenicity in nude mice were determined. GIST named GIST-H1 was successfully established. The cell line was passaged for more than 60 times 1 year. The characteristics demonstrated: The population doubling time calculated in the log phase of growth was 47.5 h. The cloning efficiency in the soft agar averaged 24.8%. Electronmicroscopically, there were rich ribosomes and mitochondrion in the cytoplasm. Immunohistochemical analysis showed CD117(+), SMA(+), dog-1(+), CD34(-), and S-100(-). Karyotype analysis illustrated aneuploidy with the modal chromosomal number 60-98. The GIST cells transplanted in nude mice had high tumorigenicity. The immortalized GIST cells are developed in vitro and have specific characteristics of GIST.
To explore the reversal effect of mifepristone(MIF) on adriamycin(ADM) resistance in human breast cell line MCF-7/ADM in vitro and in vivo. The transplantable models of MCF-7 cells resisting against adriamycin were established in nude mice by subcutaneous implantation to observe the reversal effect of MIF in vivo. The mice were randomly divided into 4 groups: a control group(treated with saline water 0.2 mL intraperitoneally and edible oil 0.5 mL orally), an MIF group (treated with mifepristone 30 mg/kg orally and saline water 0.2 mL intraperitoneally), an ADM group (treated with adriamycin 5 mg/kg intraperitoneally and edible oil 0.5 mL orally) and an ADM+MIF group (treated with ADM 5mg/kg intraperitoneally and mifepristone 30 mg/kg orally every 3 days). Tumor changes were investigated after different drug treatments. The reversal effect of 5 micromol/L MIF in vitro on the ADM resistance cell line MCF-7/ADM and non ADM resistance cell line MCF-7 was determined by 4,5-dimethylthiazol-2-yl (MTT) assay. (1) The inhibitory rate of 5 micromol/L of MIF for both cell lines MCF-7 and MCF-7/ADM was less than 5%, and it had no statistical difference compared with the group that was not treated with MIF(P > 0.05). (2) ADM could inhibit the growth of both MCF-7 and MCF-7/ADM,but the inhibition concentration 50 (IC(50)) of MCF-7 (0.42 mg/L) was obviously less than that of MCF-7/ADM(17.21 mg/L) (P < 0.05). (3) IC(50) of MCF-7/ADM of MIF+ADM group was 1.96 mg/L in vitro, which was significantly less than that in ADM alone group(17.21 mg/L) (P < 0.05), and 5 micromol/L of MIF reversed ADM resistance with fold-reversal of 8.78. (4) MIF had some effect on the inhibition of MCF-7/ADM cell growth in vivo, the xenograft volume in the MIF+ADM group [(232.5149 +/- 309.2377) mm(3)] was significantly smaller than that in the control group[(962.2309 +/- 261.1313) mm(3) ] after the 4 week treatment(P<0.05), and also smaller than that in the MIF group [(778.2846 +/- 42.6919) mm(3)] and in the ADM group [(508.9648 +/- 16.2609) mm(3)](P < 0.05). There was significant inhibition on xenograft weight after MIF combined with ADM treatment in vivo, and the inhibitory rate was 78.0%. MIF can effectively reverse ADM resistance in human breast cancer cell line MCF-7/ADM both in vitro and in vivo.
To study the proteins related to paclitaxel-resistant of ovarian cancer cell line. The total proteins of paclitaxel-resistant and paclitaxel-sensitive human ovarian cancer cell lines were separated by 2-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using image analysis software. The differential proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot was used to determine the differential expression levels of the 2 proteins. Forty differentially expressed proteins were found by image analysis software, and 24 differential proteins were identified by mass spectrometry. These proteins included proliferation cell nuclear antigen (PCNA), nm23, prohibitin (PHB), annexin, alpha-enolase, heat shock protein (HSP), and so on. Twenty-four proteins in human ovarian cancer cell lines of paclitaxel-resistant and paclitaxel-sensitive are found by proteomic techniques, which may be involved in the paclitaxel-resistance of human ovarian cancer cells.
To explore the distribution pattern of G protein-coupled receptor family C, group 6, subtype A (GPRC6A) mRNA in adult mice. The distribution of GPRC6A mRNA in paraffin embedded adult mouse tissues was determined by highly sensitive nonradioactive cRNA probe in situ hybridization (ISH). We compared ISH with and without addition of tyramide signal amplification (TSA). GPRC6A wild-type and littermate GPRC6A null mice tissue sections were investigated by ISH. TSA greatly increased the sensitivity of ISH to detect GPRC6A mRNA in wild type mouse tissues. There was no detection of GPRC6A mRNA in GPRC6A gene specific knockout tissue in paraffin embedded tissue section. The mRNA of GPRC6A was detectable in the digestive gland or accessory digestive gland including salivary gland and pancreas, as well as in the tissues including kidney, testis, brain, muscle, and fat. The mRNA distribution pattern of GPRC6A gene is compatible with the phenotype of GPRC6A knockout mice.
To explore the association of Ezrin and E-cadherin expression with the invasion, metastasis and prognois of nasopharyngeal carcinoma. Imunohistochemical SP staining was used to detect the expression of Ezrin and E-cadherin in 42 nasopharyngeal carcinoma and 10 chronic nasopharyngitis specimens. Ezrin protein expression in the nasopharyngeal carcinoma tissues was significantly higher than that in the chronic nasopharyngitis tissues (P<0.05). E-cadherin expression in the nasopharyngeal carcinoma tissues was significantly lower than that in the chronic nasopharyngitis tissues (P<0.05). Expressions of both Ezrin and E-cadherin of nasopharyngeal carcinoma were closely associated with T staging,the cervical lymph node metastases and clinical staging (P<0.05). A negative correlation was found between Ezrin and E-cadherin expression in the nasopharyngeal carcinoma tissues (r=-0.450, P<0.05). Survival analysis showed that the abnormal expression of Ezrin and E-cadhrin, clinical staging and the cervical lymph node metastases were associated with the survival rate of patients with nasopharyngeal carcinoma. A negative correlation is found between Ezrin and E-cadherin expression in the nasopharyngeal carcinoma tissues. Ezrin and E-cadherin are closely related to clinical staging and the cervical lymph node metastases of nasopharyngeal carcinoma,suggesting that they may be important tumor markers for nasopharyngeal carcinoma. Combined detection of the expressions of Ezrin and E-cadherin is helpful for clinical doctors to determine the prognosis of patients with nasopharyngeal carcinoma.