Abstract:Objective:To establish the STO cell lines expressing the recombinant porcine leukemia inhibitory factor(LIF)and try to culture the rat induced pluripotency stem cells(riPSCs)with the established STO-pLIF cells as the feeder layer. Methods:We established the stable STO cell lines(STO-pLIF cells) which express LIF gene effectively by lipofectin transfection with the pig LIF eukaryotic expression vector pCAG-pLIF. The expression level of the inserted exogenous LIF gene was tested by RT-PCR,qPCR and Western blot. The cell lines which expressed pig LIF efficiently were used as the feeder cells to culture the riPSCs. Besides the RT-PCR testing the pluripotency factors,we also undertook the growth curve and the alkaline phosphates expressions detection as well as the immunocytofluorescent to identify the pluripotency of the riPSCs. Results: The expression level of the inserted exogenous LIF gene of the STO-pLIF cell lines was higher than that of the STO (P < 0.05), and the riPSCs cultured with the STO-pLIF were close to the traditional riPSCs, and had difference with the riPSCs cultured with the N2B27 without LIF (P < 0.05).Conclusion:The stable STO cell lines effectively expressing porcine recombinant porcine leukemia inhibitory factor were established successfully and these cells can maintain the pluripotency and self renew of riPSCs as the feeder cells.

Abstract:Objective:To investigate the most effective dosage and optimal time window of Plasmocin in eliminating mycoplasma infection,and detect whether the drug exert negative effects on human pluriotent stem cell(hPSC) culture. Methods:hPSCS were treated with 25.0,12.5,8.5,5.0 μg/mL of Plasmocin. The efficiency for removing mycoplasma infection was compared by PCR. Directly differentiation of hPSC to forebrain neurons after rescued. Results:All of the concentrations for Plasmocin eradicated mycoplasma infection. However,the highest dosage (25.0 μg/mL)lead to toxic effects and the lowest dosage(5.0 μg/mL)required longer treatment time. The 12.5 μg/mL group had adverse effects on cell growth but the 8.5 μg/mL group contributed to hPSCs maintenance. After mycoplasma elimination,hPSC efficiently differentiated to forebrain neurons. Furthermore,after 9 months post-Plasmocin treatment,mycoplasam was undetected on hPSC. Conclusion:One week treatment with 8.5 μg/mL Plasmocin completely eradicated mycoplasma contamination without affecting hPSC maintenance and differentiation.

Abstract:Objective:To investigate the effect of TG-6 on myocardial ischemia reperfusion injury in rats and the underlying mechanism. Methods:The drugs were administered by intravenous injection 5 min before ischemia reperfusion(I/R)injury. Myocardial ischemia reperfusion injury(MI/RI)model was treated by 45 min of left anterior descending(LAD)occlusion followed by 24 h of reperfusion. Measurement of rat electrocardiogram(ECG),hematoxylin-eosin(HE)staining of cardiac tissue,serum lactate dehydrogenase(LDH),creatine kinase(CK),superoxide dismutase(SOD)and malondialdehyde(MDA). Serum inflammatory cytokines,such as tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin -1β(IL-1β)were measured by ELISA kits. The protein expression of TLR4,IκBα and NF-κB were measured by Western blot. Results:Compared with the I/R group,TG-6 decreased S-T elevation,reduced myocardial pathological lesions,significantly decreased serum LDH,CK,MDA,TNF-α,IL-6,IL-1β content,and increased SOD activity. TG-6 reduced TLR4,IκBα and NF-κB protein expression in cardiac tissue. Conclusion:TG-6 exerts strong favorable cardioprotective function on myocardial I/R injury, which may be related to anti-inflammatory and antioxidant activity.

Abstract:Objective:To study the effects of glibenclamide on long-term neurological deficits of cerebral ischemia/reperfusion injury in mice. Methods:C57BL/6 mice were performed to establish the cerebral ischemia/reperfusion injury models by occluding the right middle cerebral artery (MCAo). Then,we continuously treated them with glibenclamide (20 mg/kg)by intragastric administration for 5 weeks. We monitored the fasting blood sugar (FBS)and weight of mice per week. We observed the neurobehavioral changes by behavior tests including corner test,cylinder test,rotarod test and foot fault test. Moreover,we observed the changes of astrocytes by immunohistochemistry staining. Results:Continuous treatment with glibenclamide for 5 weeks had no effect on the FBS. Compared to the control group,it significantly increased the body weight at the 2nd week of treatment(P < 0.01),and at the 3rd week of treatment,it reduced the foot fault rate (P < 0.05)and prolonged the time of animals remained on the rotating rod (P < 0.05). In addition,it reduced the area of glial scar (P < 0.05). Conclusion:Continuous treatment with glibenclamide can advance long-term functional recovery of cerebral ischemia/reperfusion injury in mice.

Abstract:Objective:To investigate the role of PEA3 in albumin induced mouse proximal tubular cells(mPTCs)apoptosis and its underlying mechanism. Methods:The cells were cultured with albumin in vitro,and divided into the control group,the vehicle control group,the PEA3 overexpression group,the albumin model group and the PEA3 overexpression in albumin model group. Real-time fluorescence quantitative polymerase chain reaction was used to investigate the level of PEA3 gene transfection efficiency. Flow cytometry and Hoechst staining technique were performed to analyze the apoptosis of mPTCs. The mRNA and protein levels of E-cadherin,α-SMA and Vimentin were detected by RT-PCR and Western bolt. Results:Transfection of PEA3 over-expresed the mRNA level of PEA3 for 2.5 fold. The apoptosis of mPTCs treated with albumin was increased compared with the control group,and overexpression of PEA3 ameliorated albumin-induced apoptosis. Albumin treatment reduced the expression of E-cadherin and increased the expression of Vimentin and α-SMA. Overexpression of PEA3 reversed the reduction of E-cadherin and ameliorated the increase of Vimentin and α-SMA. Conclusion:Overexpression of PEA3 can protect mPTCs from apoptosis and epithelial-mesenchymal transition induced by albumin.

Abstract:Objective:The present study was designed to investigate the protective effect of Iptakalim against hypoxia-induced apoptosis in human pulmonary arterial endothelial cells (HPAEC)and the potential mechanisms involved. Methods:After medicine and hypoxia treatment,the MTT assay was performed to measure cell viability. Hoechst 33342 was then used to detect apoptosis. Finally,the expression of phospho-JNK,JNK and caspase-3 was assayed by Western blot. Results:Our study showed that hypoxia significantly decreased the cell viability by inducing cellular apoptosis,while Iptakalim reversed the hypoxia-induced apoptosis, which can be blocked by 5-HD. Iptakalim and SP600125 consistently inhibited the phosphorylation of c-JNK and the expression of caspase-3 induced by hypoxia,and this effect was completely blocked by the 5-HD. Conclusion:Iptakalim protects the HPAEC from hypoxia-induced apoptosis by opening the KATP channels. The possible cellular mechanisms for this protective effect may involve the inhibition of phospho-JNK and the downstream cell death pathways.

Abstract:Objective:To construct a luciferase reporter plasmid containing the splicing isoform of CD2-associated protein(CD2AP) human gene promoter and its deletants and to evaluate promoter activity in human embryonic kidney (HEK)-293 cells and HeLa cells. Methods:Extract total RNA from stable HeLa cells and make it to be cDNA by RT-PCR. The 2 200 bp fragment of CD2AP002(one of the spliceosome of CD2AP)was amplified by PCR with cDNA as a template and was directionally cloned into pGL3-Basic multiple cloning sites to construct a new luciferase reporter plasmid. We repeat the above steps to build a series of 5′ deletion promoter plasmids. Transfection of HEK 293 cells and HeLa cells with these new plasmids was performed to induce the relative luciferase activity. Results:DNA sequencing and restriction endonuelease analysis verified the successful construction of the plasmid contaning CD2AP002 human gene promoter and its deletants. Conclusion:The plasmid pGL3-CD2AP002 promoter and its deletants are successfully constructed and have some promoter activities in HEK293 cells and HeLa cells.

Abstract:Objective:To investigate short-term effect of ketogenic diet on metabolism of mice. Methods:BALB/c mice were divided into two groups and placed on normal diet or ketogenic diet for 4 days. Body weight and blood glucose were monitored everyday. On the fourth day,the blood samples and livers were gathered and liver weight/body weight were calculated. Serum triglycerides(TG),total cholesterol(TC),free fatty acids(FFA),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and beta hydroxybutyric acid(β-HB)were tested. The content of TG,cholesterol and glycogen in liver were detected. The expression levels of genes related to metabolism in livers were detected by real-time PCR. Liver pathology was also examined by HE staining. Results:Short-term ketogenic diet significantly reduced blood glucose and body weight of mice. Liver weight/body weight of short-term ketogenic mice was higher than that of control mice. A four-day feeding of ketogenic diet resulted in increased TC and β-HB in blood. Meanwhile,more TG and less glycogen were detected in the liver. Real-time PCR demonstrated that hepatic genes involved in fatty acid oxidation,oxidative phosphorylation and ketogenesis were increased. Accumulation of lipid was observed in the liver of ketogenic mice under a microscope. Conclusion:Short-term ketogenic diet induced fatty acid oxidation and ketogenesis in mice. Different from long-term ketogenic mice,elevated blood cholesterol and FAS mRNA levels were observed in short-term ketogenic mice without obvious liver damage.

Abstract:Objective:To explore the expression of microRNA-224 (miR-224)in non-small cell lung cancer (NSCLC)and its effect proliferation and apoptosis of NSCLC cell. Methods:Real-time quantitative PCR(qPCR)was used to detect and compare the expression of miR-224 in 20 pairs of NSCLC,adjacent normal tissue,NSCLC cell line A549 and normal epithelium cell line 16HBE. The expression of miR-224 in A549 cell was down-regulated by anti-miR-224 transient transfection,and the effect was identified by qPCR. The influence of miR-224 on A549 cell proliferation was detected by MTT assay and colony formation assay. Flow cytometry was used to test cell cycle and apoptosis. The expression of Bim was tested by anti-miR-224 transient transfection,and the effect was identified by qPCR and western blot. Results:Compared with adjacent normal tissues,miR-224 were significantly up-regulated in NSCLC tissue and cell.(P < 0.05). Down-regulating miR-224 can inhabits proliferation and accelerates apoptpsis as well as increasing the expression of Bim. Conclusion:The expression of miR-224 is up-regulated in NSCLC. MiR-224 can promotes proliferation and inhabits apoptosis by down-regulating Bim.

Abstract:Objective:To detect the expression level of HOXA transcript at the distal tip (HOTTIP)in esophageal squamous cell cancer(ESCC)tissues and cell lines and investigate the potential underlying molecular mechanism. Methods:qRT-PCR was used to determine the relative HOTTIP expression levels in ESCC tissues and cell lines. Small interfering RNA(si-HOTTIP)was transfected into ESCC cells to knock down HOTTIP expression. CCK-8 assay and Transwell assay were performed to evaluate the proliferation and invasion ability of ESCC cells. Results:Compared with controls,HOTTIP expression levels were significantly up-regulated in ESCC tissues and cell lines. Higher expression level of HOTTIP was found associated with larger tumor size and more lymph node metastasis. CCK-8 assay and Transwell assay showed that silence of HOTTIP inhibited the proliferation and invasion of Eca-109 and TE-13 cells in vitro. Conclusion:HOTTIP plays crucial role in proliferation and invasion of ESCC.

Abstract:Objective:To select the optimal internal reference genes for gene expression analysis in colorectal tumor tissues. Methods:The total RNA of colorectal cancer tissues from 10 patients weve studied. The mRNA transcription profiles of five frequently used housekeeping genes,including GAPDH,ACTINB,UBC,HRPT1,and 18S rRNA,were tested by real-time quantitative RT-PCR. The stability of these control genes was analyzed by geNorm and NormFinder softwares. Results:The M value of GAPDH and ACTINB was the least and identified as the suitable and stable internal reference gene combinations using geNorm software. The M volue of 18S rRNA was the highest and its stability was the lowest. Furthermore,GAPDH was the most stable reference gene,which was followed by UBC using NormFinder software. Conclusion:The combination of GAPDH and ACTINB,but not 18S rRNA and HRPT1,is the most reliable reference gene group for normalization of real-time RT-PCR in colorectal tumor tissues.

Abstract:Objective:To detect, compare and analyze the activity and significance of CREB1 protein in the hippocampus of Alzheimer’s disease (AD) and wild type control mice. Methods:Continuous intraperitoneal injection of D-galactose 90 mg/(kg·d) and AlCl3 40 mg/(kg·d) for 90 days was performed to mice to make AD mouse model. Preliminary appraise of AD model was performed by sucrose preference test and Y-maze test. CREB1, p-CREB1 protein expression levels in the hippocampal CA1, CA3 and DG areas of AD and wild type control mice were compared by immunohistochemical detection, respectively. Results:Compared with the control group, the sucrose preference percentage of AD mice decreased significantly (P < 0.01). The spatial discrimination ability of the AD group was significantly lower than that of the wild-type control group (P < 0.01). Compared with the wild type control group, the positive expressions of CREB1 and p-CREB1 proteins in the hippocampal CA1, CA3 and DG areas were significantly decreased in AD mice (P < 0.01). Conclusion:By inhibiting the activity of key nuclear transcription factor CREB1 in hippocampal tissues, D-galactose combined with AlCl3 affect neuronal survival, post injury repair and regeneration of nerve cells, resulting in ability of learning and memory impaired, thereby causing the occurrence and development of AD.

Abstract:Objective:To investigate the role of silent mating type information regulation 2 homolong1(SIRT1)activator SRT1720 in deoxycorticosterone acetate(DOCA)salt-sensitive hypertensive rats(DHR). Methods:A total of 28 male Sprague-Dawley rats underwent left nephrectomy were randomly divided into three groups:control group,model group,and SRT1720 treatment group. Control group(n=8):drinking water; model group(n=10)and SRT1720 treatment group (n=10):DOCA and silicone rubber medium at the concentration of 200 mg/kg was placed subcutaneously in rats on both sides of the scapula and given 1% NaCl drinking water. SRT1720 treatment group was given intragastric administration of SRT1720 for 4 weeks at the concentration of 100 mg/(kg·d). Systolic blood pressure(SBP)was measured once a week. After 4 weeks,central arterial pressure and pulse wave velocity (PWV)were monitored,and then rats were sacrificed. Thoracia aorta was taken for HE staining to observe the thickness and vascular caliber change. Results:In model group,SBP,central aortic pressure and PWV were significantly increased. HE staining showed marked vascular smooth muscle cell(VSMC)hypertrophy,and marked thickening of elastic fiber layer and aortic media,but a significantly decreasing in vessel diameter. Meanwhile,SRT1720 treatment group reversed these changes. Conclusion:SRT1720 could inhibit VSMC hypertrophy,reduce aortic media thickness,thereby expend blood vessel diameter,and reduce central arterial pressure and peripheral blood pressure in DOCA salt-sensitive hypertensive rats.

Abstract:Objective:To evaluate the efficacy and safety of double-dose clopidogrel compared with routine-dose dual anti-platelet treatment (DAPT)in patients with clopidogrel low response (CLR)after percutaneous coronary intervention (PCI). Methods:Ninety-two CLR patients were screened by light transmission aggregometry (LTA)after PCL and randomly divided into the clopidogrel routine-dose group and the clopidogrel double-dose group after taking routine-dose aspirin and clopidogrel for more than five days. Platelet aggregation was determined by LTA one-month post-randomization. The patients were followed up and all clinical events were recorded for one year. Results:There was no significant difference of adenosine diphosphate induced platelet aggregation (PLADP)between the two groups at baseline (P > 0.05). However,the PLADP level of the double-dose group was significantly lower than that of the routine-dose group at one-month follow-up (P < 0.001). The major adverse event rates of the two groups were both 4.3%. The double-dose group presented less secondary adverse events compared with the double-dose group,mainly attributed to cardiac rehospitalization (P < 0.01). The two groups showed comparable major bleed events (0% vs. 0%),as well as minor and minimal bleeding events (0% vs. 0% and 10.9% vs. 10.9%,respectively). Conclusion:Double-dose clopidogrel can significantly improve the ADP-induced platelet aggregation during the first month and may lower the cardiac rehospitalization event without excessive risk of bleeding in CLR patients undergoing PCI during one-year follow-up.

Abstract:Objective:To analyze the clinical characters of six patients of multiple endocrine neoplasia type 1 (MEN1)and their family members and investigate the features of MEN1 gene mutation in these families. Methods:Clinical data of MEN1 patients and their family members was collected. Genomic DNA was extracted from peripheral blood of six patients and their respective family members (thirteen in total),then the 9 exons in coding sequences of MEN1 gene was amplified by PCR and subjected to direct sequencing. Results:We detected a heterozygous mutation within exon 10 (c.1378C>T)in two patients and two relatives in pedigree 1,a heterozygous mutation within exon 2 (c.80C>G)in one of patients in pedigree 2 and a heterozygous mutation within exon 9(c.1225T>C)in the proband and her mother in pedigree 3,respectively. No mutation was found in other patients or relatives. The detected c.80C>G and c.1225T>C were two novel types of MEN1 mutation,while the mutation of c.1378C>T in MEN1 gene was already known. Conclusion:Analysis of MEN1 gene mutation contributes to early diagnosis of MEN1 patients and screening of family members. The two novel types of MEN1 gene mutation in this study may increase our knowledge concerning genetic feature of MEN1.

Abstract:Objective:To evaluate the significance of hTERC gene amplification and CD105 expression in cervical condyloma acuminateum (CA). Methods:Fluorescence in situ hybridization (FISH)and imunohistochemical staining were used to detect the expression of hTERC gene and CD105 in 67 cases of cervical CA,including 31 cases without CIN,19 CA-CIN grade Ⅰ,11 CA-CIN grade Ⅱ,and 6 CA-CIN grade Ⅲ; 35 cases of cervical cancer and 24 normal cervical mucosa were performed as controls. Results:The rates of hTERC gene amplification were 0%,25.4% and 80% in normal cervical mucosa,cervical CA and cervical cancer,respectively(P < 0.01). Microvessel density(MVD) marked by CD105 antibody also had significant difference (P < 0.01). In cervical CA,hTERC gene amplification and MVD showed a positive correlation (P < 0.05)and increased with increasing CIN grade. There were significant differences between CA-CIN Ⅰ and CA-CIN Ⅱ(P < 0.01),as well as between the recurrence group and the non recurrence group(P < 0.05). Conclusion:The expression of hTERC gene and CD105 may play an important role in the recurrence of cervical CA,and in the process of conversion to the high grade CIN. The detection of hTERC gene and CD105 is helpful in the clinical screening of high-risk patients with cervical CA.

Abstract:Objective:To explore the significance and expression of iASPP in colorectal cancer and its adjacent tissues. Methods:We usd immunohistochemical method to detect the expression of iASPP in 54 cases of colorectal cancer tissue and 47 cases of para colorectal cancer tissues,and analyzed the correlations between the expressions of iASPP and the pathologic typing,infiltrative depth,clinical stage and lymph node metastasis of colorectal cancer. Results:The positive expression rate of iASPP was significantly higher in colorectal cancer tissues than that in adjacent tissues (P < 0.01). The expressions of iASPP in colorectal cancer tissues were related to the degree of tumor cells,infiltrative depth of tumor and lymph node metastasis(all P < 0.01). Conclusion:The expression levels of iASPP in colorectal cancer tissues was higher than that in colorectal cancer adjacent tissues. It may be related with tumor differetiation,infiltrative depth and lymph node metastasis. This may be useful for the diagnosis of colorectal malignant tumor.

Abstract:Objective:To compare nutritional status of elder and youth-middle-aged surgical patients both on admission and at discharge,and investigate the rationality of nutritional therapy. Methods:Nutritional risk screening (NRS2002)was applied to investigate 167 cases of newly admitted patients. Then we divided patients into two groups by 65-year-old,including the youth-middle-aged group and the elder group. NRS2002≥3 was set as nutrition risk. The rationality of nutritional support and prognosis were investigated as well. Results: ①NRS score and the proportion of the patients whose NRS≥3 in the elder group were higher than those in the youth-middle-aged group both on admission and at discharge. The patients both in the two groups had poorer nutritional status at discharge. ②23 cases(20%)in the youth-middle-aged group and 12 cases(22%)in the elder group received parenteral nutrition(PN) respectively.The calories total parenteral nutrition(TPN) provided both in the two groups were insufficient. The ratios of insufficient calories providing were 60% in the youth-middle-aged group and 67% in the elder group. The ratio of calories to nitrogen was low. In the patients whose NRS≥3,there were 12 cases (24%)and 11 cases (34%)who received PN support between the two groups respectively. The calories TPN provided both in the two groups were insufficient too. The ratios were 63% in the youth-middle-aged group and 73% in the elder group respectively. The ratio of calories to nitrogen was low too.③Compared with the patients whose NRS≥3 in the youth-middle-aged group,the patients whose NRS≥3 in the elder group had longer hospital stay. Conclusion:The nutritional support therapy should be strengthened while hospitalized. Nutrition support treatment should be supplied to the patients with nutritional risk according to NRS score. The calories TPN provided were insufficient. and the composition was not reasonable. There was a still asingle amino acid infusion in PN.

Abstract:Objective:To explore the correlation between Lp-PLA2 combined with modified ABCD2 score and early neurological deterioration(END) in patients with mild ischemic stroke. Methods:A total of 180 patients with acute mild ischemic stroke were recruited in this study. All patients were evaluated by improved ABCD2 score for risk stratification,and the serum Lp-PLA2 level was detected by ELISA. Results:The level of Lp-PLA2 was significantly increased compared with that in the non END group (P < 0.05) . Lp-PLA2 was significantly related with improved ABCD2 score (P < 0.05). The Cox regression logistic regression model indicated that ABCD2 score (≥4),large artery stenosis (≥75%) and Lp-PLA2 (>200 ng/mL) were the predictive factors of END composite end points in mild cerebral ischemic stroke (P < 0. 05). Conclusion:Lp-PLA2 combined with modified ABCD2 score can improve the ability to predict the risk of END events in mild ischemic stroke.

Abstract:Objective:To prove that it is feasible to diagnoze discoid lateral meniscus in radiographs by analysis of plain radiographic findings of discoid lateral menisci with matched controls in a quantitative method.Methods:Total 122 consecutive patients,who were diagnosed with discoid lateral meniscus (discoid group)by magnetic resonance imaging(MRI)and arthroscopy,and 122 age-and sex-matched controls with normal medial and lateral menisci on the basis of MRI findings were included. Each plain radiograph was evaluated from the anteroposterior view for the following variables:height of the fibular head(A),lateral jointspace distance (B),height of the lateral tibial spine (C),obliquity of the lateral tibial plateau (α),obliquity of the lateral femoral condyle (β),distance from the lateral tibial spine to the lateralfemoral condyle (D),height of the medial tibial spine(E),chordal distance of the femoral condyle (F,G),the A/B,B/C and the F/G. Statistical analyses were used to determine the differences between the two groups. Results:A significant difference was found in the A,B,C,D,F,A/B and B/C between the two groups. The difference in cut-off values of A/B and B/C was most significant. At the same time,the roc curve area of the A/B and B/C ranked highest among all the results. Conclusion:There were significant differences in the variables,especially the A/B and the B/C,between plain radiographic findings of discoid lateral meniscus patients and normal controls. The results of the A/B and the B/C showed a positive impact on the diagnosis of discoid lateral meniscus in this research. These findings enable radiographs to screen for discoid lateral meniscus.