Objective To study the effects of cripto on migration, invasion, and liver metastasis of colorectal cancer cell. Methods After human colorectal cancer cell line SW480 was transfected by cripto small interfering RNA (siRNA), the mRNA and protein level were determined by Real-time RT-PCR and Western blot, respectively. The migration and invasion ability were evaluated by wound-healing assay and boyden chamber model, respectively. Thirty nude mice model of liver metastasis from colorectal cancer was established by splenectomy. Results The siRNA could down-regulate the level of mRNA and protein of cripto in a dose- and time-dependent manner. Suppression of cripto expression could inhibit migration and invasion ability of human colorectal cancer cell in vitro. The metastastic rate and tumor nodules were lower in transfection with cripto siRNA than in two control groups in vivo. Conclusions Cripto gene might play an important role in regulation of liver metastasis from colorectal carcinoma cell, and suppression of cripto gene by siRNA can inhibit liver metastasis of colorectal cancer.

Objective To investigate the correlations of extracellular matrix and hepatic ultramicrostructural changes with clinical manifestations in patients with mild chronic hepatitis B (CHB).Methods Patients with chronic HBV infections were enrolled and were divided into mild CHB group (n=66) and HBV carrier group (n=10).Serum samples were collected from patients, and serum HBV markers, HBV DNA load and liver fibrosis indexes were measured.All subjects received liver biopsy, and the tissue samples were observed by light microscope and electron microscope.T test and χ2 test were performed for measurement data and enumeration data, respectively.Spearman test was used for ranked data.Results The differences on ALT and AST levels between mild CHB group and HBV carrier group were significant (t=12.42, 7.06, P＜0.05), but there was no significant difference on HBV DNA load between two groups (t=0.24, P ＞ 0.05).Serum liver fibrosis indexes (hyaluronic acid, type Ⅲ collagen,type Ⅳ collagen and laminin protein) in mild CHB group were not significantly higher than those in HBV carrier group (t=0.45, 0.95, 0.76 and 1.21, P ＞0.05).In mild CHB group, there were 33 patients with ≥G2 and ≥S2, but in HBV carrier group were only 2 patients (χ2=4.17, P ＜ 0.05).Seventeen patients in mild CHB group were with S3-4, while that was not observed in HBV carrier group (χ2=4.75, P ＜0.05).In mild CHB group, hepatic ultramicrostrutural changes on fat storing cell, collagen protein and portal area were correlated with fibrosis grades, and the correlation coefficients were 0.351, 0.675 and 0.301, respectively (P=0.004, 0.000 and 0.014).Conclusion Electron microscope is of higher sensitivity than light microscope in observing hepatic ultramicrostructural changes, which is effective in evaluating the severity of mild CHB.

Objective To evaluate the effect of silencing ACAT1 gene on colon cancer cells proliferation,migration,invasion and colon cancer development by using the small interference RNA (siRNA) in colon cancer cell line HT-29.Methods Acyl coenzyme A cholesterol acyltransferase 1 (ACAT1) gene was silenced in HT-29 cell lines using Hiperfect transfection reagent.The expression level of ACAT1 was detected by real time PCR.CFSE and transwell assays were used to evaluate the effect of ACAT1 gene interfering on cells proliferation,mi gration and invasion.Result ACAT1 mRNA expression decreased obviously after siRNA interference.Compared with pre-transfection,proliferation,migration and invasion of colon cancer cells have been significantly inhibited (P ＜ 0.05).Conclusion ACAT1 gene interference reduced proliferation,migration and of invasion of HT29 cells,which provide a new potential target for colon cancer treatment.

Objective To prepare serum calibrators for CRP measurement.Methods Fresh serum without infectious diseases , hemolysis, lipemia and choloplania were collected and divided into 3 groups, low, medium, and high, according to the CRP concentration.Each serum pool was mixed , filtered, sterilized and aliquoted.The materials were tested for homogeneity and stability.The values of the CRP was assigned by particle enhanced immunonephelpmetry , and calibrated with international reference materials.The expanded uncertainty was evaluated.Results The materials were tested to be homogeneous (Ubb﹤Ur, P>0.05) with Ubb values being 0, 0.125, 0, Ur values being 0.046, 0.213, 0.785, and F values being 0.803, 1.686, 0.966 in CL, CM, CH groups respectively.Stability study, where F values are 0.609, 0.259, and 1.557 at 22-25℃, 1.217, 4.583, and 0.893 at 2-8℃(P>0.05), showed that the materials were stable for at least 3 days at 22-25 ℃or 30 days at 2-8 ℃, respectively.The certified values of the 3 levels materials for CRP were ( 2.64 ±0.14 ) , ( 31.17 ±0.63 ) , ( 73.85 ±1.74 ) mg/L, respectively.Conclusion The calibrators prepared for serum CRP measurement were homogeneous , stable and accurately assigned and can be used to calibrate the CRP measure system.

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirns spike protein induced hormonal and cellular immune response in Balb/c mice.

Objective:To investigate the effects and mechanisms of Wnt pathway inhibitor IWR-1-endo on the biological behaviors of human hepatocarcinoma cell Huh7.Methods:Human hepatocellular carcinoma cell Huh7 was cultured in vitro, and Huh7 cells were treated with IWR-1-endo at different concentrations (0, 20, 40, 80, 160, 320 μmol/L). Scratch test was used to detect changes in cell migration ability at diffe-rent drug concentrations, plate cloning was used to detect changes in cell proliferation, Western blotting was used to detect changes in the expression of Wnt pathway related protein β-catenin, and immunofluorescence staining was used to detect the expression of β-catenin in cytoplasm and nucleus. Results:The results of the scratch test showed that the 24 h scratch healing rates of Huh7 cells treated with 0, 20, 40, 80, 160, 320 μmol/L IWR-1-endo were (20.55±0.05)%, (12.10±0.08)%, (9.36±0.10)%, (3.62±0.09)%, (0.62±0.04)% and (0.23±0.02)%, respectively, and there was a statistically significant difference ( F=230.87, P<0.001). Further pair comparison showed that there were statistically significant differences in 24 h scratch healing rates among different concentrations (all P<0.001). The 48 h scratch healing rates were (34.77±0.08)%, (17.69±0.05)%, (11.60±0.04)%, (5.68±0.07)%, (2.66±0.04)% and (1.75±0.02)%, respectively, and there was a statistically significant difference ( F=589.68, P<0.001). Further pair comparison showed that there were statistically significant differences in 48 h scratch healing rates among different concentrations (all P<0.001). After treatment with IWR-1-endo at the concentration of 0, 20, 40, 80, 160, 320 μmol/L, the clone formation rates of Huh7 cells were (61.67±0.21)%, (57.33±0.11)%, (50.00±0.25)%, (36.67±0.28)%, (23.33±0.12)% and (15.00±0.08)%, respectively, and there was a statistically significant difference ( F=403.56, P<0.001). Further pair comparison showed that there were statistically significant differences in clone formation rates among different concentrations (all P<0.001). After treatment with 0, 20, 40, 80, and 160 μmol/L IWR-1-endo for 24 h, the relative expression levels of β-catenin in Huh7 cells were 0.30±0.08, 0.25±0.07, 0.22±0.05, 0.15±0.01 and 0.06±0.02, respectively, and there was a statistically significant difference ( F=247.00, P<0.001). Compared with 0 μmol/L, the relative expression levels of β-catenin treated with 80 and 160 μmol/L had statistical significance ( P=0.014; P=0.008). Compared with 0 mol/L, immunofluorescence showed that the expressions of β-catenin in cytoplasm and nucleus were reduced after 80 μmol/L IWR-1-endo treatment. Conclusion:Wnt pathway inhibitor IWR-1-endo can inhibit the migration and proliferation of hepatocarcinoma cells Huh7 by inhibiting the activity of Wnt pathway. The above inhibitory effects are dose-dependent.

Objective:To investigate the effects of Jianpi Huatan Prescription on a rat model of polycystic ovary syndrome (PCOS) with insulin resistance (IR) induced by letrozole combined with high fat diet based on the liver kinase B1 (LKB1)/AMP activated protein kinase (AMPK) signaling pathway; To discuss its mechanism.Methods:A total of 40 SD female rats were divided into model group (30 rats) and blank group (10 rats). The model group rats were fed with high-fat feed from the first week of modeling for 8 consecutive weeks, and from the fourth week onwards, were given a daily gavage of letrozole solution for 5 consecutive weeks; the blank group was fed normally, and from the 4th week onwards, an equal amount of sodium carboxymethyl cellulose solution was administered. After the completion of modeling, the modeling group was divided into Jianpi Huatan Prescription group, Metformin group, and model group according to random number table method. The Jianpi Huatan Prescription group and the metformin group were given corresponding drug interventions, while the other two groups were simultaneously administered an equal amount of physiological saline for a total of 4 weeks. Ovarian morphological changes were observed using HE staining; intraperitoneal glucose tolerance test (IPGTT) was used to detect glucose tolerance in rats; Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E 2), total testosterone (T), fasting blood glucose (FBG), and fasting insulin (FINS) in rats; Real time fluorescence quantitative PCR and Western blot techniques were used to detect changes in LKB1, AMPK mRNA and protein expression in the LKB1/AMPK signaling pathway of rat ovarian tissue. Results:Compared with the model group, the polycystic changes in the ovaries of rats in the Jianpi Huatan Prescription group were improved, and the growth rate of body weight decreased. The levels of FBG, FINS, HOMA-IR, and IPGTT decreased ( P<0.05 or P<0.01), while serum FSH and HDL-C levels were significantly up-regulated ( P<0.05 or P<0.01). The levels of T, LH, LH/FSH, TC, TG, and LDL-C decreased ( P<0.05 or P<0.01); The mRNA and protein expression levels of LKB1 and AMPK in ovarian tissue increased ( P<0.05). Conclusion:Jianpi Huatan Prescription can improve PCOS-IR in rats caused by the combination of letrozole and high-fat diet, regulate various levels of sex hormones, and improve glucose and lipid metabolism disorders by regulating the LKB1/AMPK signaling pathway, which has a repairing effect on polycystic ovary disease in rats.

Objective To investigate the effects of Jianpi Huatan Fang on lipid metabolism and expression levels of FOXO1 and PDK4 in rats with polycystic ovary syndrome(PCOS)and insulin resistance(IR).Methods Forty female rats were divided randomly into a blank control group(n=10)and a model group(n=30).A PCOS-IR rat model was established using a high-fat diet(8 weeks)combined with letrozole(added at weeks 4～8).Thirty successfully modeled rats were then divided randomly into a model control group,a Jianpi Huatan Fang group(11.07 g/kg),and a metformin group(0.2 g/kg)(n=10 rats per group).Drug intervention was given for 4 weeks.The general status,Traditional Chinese medicine syndrome score,and weight changes were observed in each group.Histopathological changes in the ovary were detected by hematoxylin and eosin staining.Fasting blood glucose and blood lipids were measured using a blood biochemical analyzer.Levels of sex hormones including follicle-stimulating hormone(FSH),luteinizing hormone(LH),testosterone(T),estradiol(E2),and insulin were determined by enzyme linked immunosorbent assay and changes in the expression of FOXO1 and PDK4 in the ovaries were determined by Western Blot and real-time fluorescence quantitative polymerase chain reaction.Results Rats in the model control group exhibited symptoms of spleen deficiency and phlegm-dampness and showed significant weight gain,sex hormone disorders,polycystic ovarian changes,and dysregulation of glucose and lipid metabolism,compared with rats in the blank control group(P＜0.01).In addition,weight increase was slower in rats in the Jianpi Huatan Fang group and the metformin group compared with that in the model control group,and follicle development was improved.Serum LH,T,LH/FSH,fasting blood glucose,insulin,homeostatic model assessment for IR,cholesterol,triglycerides,and low-density lipoprotein cholesterol decreased,estradiol and FSH increased,and ovarian FOXO1 and PDK4 mRNA and protein expression levels were down regulated in the Jianpi Huatan Fang group compared with the findings in the model control group(P＜0.05,P＜0.01).Serum high-density lipoprotein cholesterol content was also increased,but the difference was not significant(P＞0.05).Conclusions Jianpi Huatan Fang can effectively regulate sex hormone secretion,improve ovarian reproductive function,and regulate glucose and lipid metabolism in obese PCOS-IR rats,possibly via inhibiting the FOXO1/PDK4 pathway.

Objective:To explore the relationship between triglyceride-glucose (TyG) index with plaque components, plaque burden and characteristics of vulnerable plaque using coronary plaque analysis based on coronary artery computed tomography (CCTA).Methods:A total of 498 patients(male 296, female 202), the age ranged from 33 to 87 (63±9) years who underwent CCTA from January 2020 to September in Shandong Provincial Hospital Affiliated to Shandong First Medical University were included. The enrolled patients were divided into three groups according to the tertiles of TyG index: T 1 group (the lowest one-third), T 2 group (middle one-third) and T 3 group (the highest one-third). The plaque burden, volume and ratio of calcified, lipid and fibrous components of plaques as well as the incidence of vulnerable plaques were measured based on CCTA images. Chi-square test, ANOVA and Kruskal-Wallis test were used to compare whether the differences of indexes among the three groups were statistically significant. Multiple stepwise regression was used to analyze the influencing factors of coronary atherosclerotic plaque burden and multiple logistic regression was used to analyze the risk factors of CT high-risk plaque. Finally, ROC curve was used to evaluate the value of TyG index in the diagnosis of CT high-risk plaque, and the best diagnostic threshold of TyG index was determined. Results:The plaque burden, non-calcified plaque volume and ratio had positive relationship with TyG index ( P<0.001).TyG index was significantly correlated with plaque burden according to multiple stepwise regression analysis (regression coefficient 7.267, P<0.001). The results of CT vulnerable characteristics of plaques showed that positive remodeling, low-attenuation plaque sign and the incidence of vulnerable plaque increased with TyG index ( P<0.05). Multivariate Logistic regression analysis showed that TyG index was an independent risk factor for CT vulnerable plaque(OR=2.324,95 %CI 1.533-3.523, P<0.001). The cut-off value of TyG index that can predict vulnerable plaque was 8.43(sensitivity 77.24%, specificity 45.60%, AUC 0.645, P<0.001). Conclusions:With the increase of TyG index, the burden of coronary atherosclerosis plaque and the incidence of CT vulnerable plaque increased. TyG index is expected to be a simple and effective predictor of cardiovascular disease and adverse cardiovascular events.