Objective To construct a recombinant lentiviral expression vector containing NIS and EGFP gene,and to explore the feasibility of NIS gene for monitoring the bone marrow derived mesenchymal stem cells (BMSCs) migration to the intracranial glioma.Methods The NIS and EGFP gene fragments were subcloned into lentiviral vector pLVX-puro,then packaged and amplified in HEK293T cells to obtain recombinant lentivirus pLVX-CMV-NIS-EGFP.pLVX-CMV-0-EGFP was constructed as control.BMSCs were isolated,cultured,and transfected by lentivirus.The antibiotic-resistant transfected BMSCs (BMSCs-NIS-EGFP and BMSCs-EGFP) were selected.The expression of NIS gene was examined by Western blot.Functional NIS activity was confirmed by the uptake of 125I and the inhibition effect of NaClO4.The nude mice intracranial glioma models were established.MicroSPECT was performed at 24 h post BMSCs-NIS-EGFP injection via the tail vein.Results pLVX-CMV-NIS-EGFP and pLVX-CMV-0-EGFP were successfully constructed and packaged.BMSCs were successfully isolated and cultured.Stable cell lines BMSCs-NIS-EGFP and BMSCs-EGFP were constructed after lentivirus transfection and puromycin selection.The expression of NIS gene was detected by Western blot in BMSCs-NIS-EGFP,but not in BMSCs-EGFP.BMSCs-NIS-EGFP showed significantly more uptake of 125I (nearly 10 times than the uptake in BMSCs-EGFP) and the uptake could be significantly inhibited by NaClO4.The nude mice intracranial glioma models were successfully established and the BMSCs-NIS-EGFP in glioma foci could be visualized by microSPECT imaging at 24 h post injection.Conclusions A recombinant lentivirus containing NIS gene could be successfully constructed for monitoring BMSCs migration towards intracranial glioma.It might provide evidence on the research of BMSCs and NIS gene mediated therapy for glioma.

Objective To observe the effects of microRNA-21 (miR-21) inhibitor on apoptosis of type Ⅱalveolar epithelial cells (AEC Ⅱ) in rats with hyperoxia-induced acute lung injury (HALI).Methods Eighty Sprague-Dawley (SD) rats were divided into air-control group,hyperoxia injury group,empty-virus control group (200 μL solution with lentivirus was dropped into the nasal) and miR-21 inhibitor pretreatment group (200 μL solution with lentivirus contained miR-21 inhibitor was dropped through the nasal) by random number table.After treatment,the rats in all groups were fed in the hyperoxia incubator with oxygen concentration exceeding 90％ for production of HALI model,and the rats in air-control group were fed normally without any treatment.Ten rats were selected at 0,24,48 and 72 hours after exposure in hyperoxia environment respectively,and the general changes of lung tissues were observed in light microscope.The right lung tissues were harvested to observe the pathological changes under light microscopy.The left lung tissues of other 10 rats in each group were harvested at 48 hours after execution,the miR-21 expression was determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR),the protein expression of cysteinyl aspartate-specific proteinase-3 (caspase-3) was determined by Western Bolt,and apoptosis of AEC Ⅱ was detected by TdT-mediated dUTP nick end labeling (TUNEL).Results ① No abnormal appearance in lung tissues was observed at all time points in the air-control group.In hyperoxia injury group,the lung injury would be more severe if the exposure time was longer,and lung tissues turned dark red after exposure for 72 hours,with patchy hemorrhage in several places;the structure of lung tissues was disordered,the alveolar wall was broken,the alveolar septum was significantly edematous and broadened,and there was plenty of inflammatory cell infiltration and edema fluid appeared inside the alveolar space.In miR-21 inhibitor pretreatment group,the degree of lung tissue injury was more severe than that of the hyperoxia injury group,and there was no significant change in empty-virus control group.(②) Compared with air-control group,miR-21 expression of the hyperoxia injury group was significantly decreased (2-△△Ct:0.021 ± 0.005 vs.0.037 ± 0.006),and the protein expression of caspase-3 was significantly increased (A value:0.423±0.081 vs.0.123±0.023,both P ＜ 0.05).After pretreatment with miR-21 inhibitor,the expression of miR-21 was further decreased (2-△△Ct:0.014±0.003 vs.0.021 ±0.005),while the protein expression of caspase-3was further increased (A value:0.691 ±0.085 vs.0.423 ±0.081,both P ＜ 0.05).There were no statistically significant differences in the expression of miR-21 (2-△ △ct:0.025 ± 0.007 vs.0.021 ± 0.005) and caspase-3 (A value:0.475 ± 0.062vs.0.423 ±0.081) between empty-virus control group and hyperoxia injury group (both P ＞ 0.05).(③) Compared with air-control group,the apoptosis cells in hyperoxia injury group were increased,which was further increased after pretreatment of miR-21 inhibitor,but no changes were found in empty-virus control group.Conclusion Inhibition of miR-21 expression in vivo could aggravate the injury of lung tissue in HALI rats,and increase the apoptosis of AEC Ⅱ.

Objective:To explore whether microRNA-21-5p (miR-21-5p) has the effect of anti-apoptosis of human alveolar typeⅡ epithelial cells (ATⅡ).Methods:ATⅡ cells derived from the human were cultured in vitro and used for experiments when the cells were grown until the presence of lamellar bodies and microvilli were observed by light microscope. The cells were divided into blank control group (direct culture), hydrogen peroxide (H 2O 2) injury group (cultured with 0.5 mmol/L H 2O 2), and miR-21-5p overexpression group (using miR-21-5p with a multiplicity of infection (MOI) of 100 lentiviral overexpression vector with 0.5 mmol/L H 2O 2) and miR-21-5p empty virus control group (miR-21-5p lentiviral blank vector was co-cultured with 0.5 mmol/L H 2O 2). In each group, cell proliferation was detected by cell counting kit-8 (CCK-8) at 0, 12, 24, 36, and 48 hours of cell culture; cell apoptosis was detected by flow cytometry at 24 hours of culture. Results:① Cell proliferation activity test results: with the extension of cell culture time, the cell proliferation activity of the blank control group gradually increased, while the cell proliferation activity gradually decreased after the addition of 0.5 mmol/L H 2O 2. However, the cells proliferation activity in the miR-21-5p overexpression group decreased more slowly than that in the H 2O 2 injury group and the miR-21-5p empty virus control group, and the cell proliferation activity at 48 hours was significantly higher than the H 2O 2 injury group and the miR-21-5pempty virus control group ( A value: 0.295±0.005 vs. 0.184±0.005, 0.169±0.002, both P < 0.05). It showed that both H 2O 2 and lentivirus accelerated cell damage, while miR-21-5p could reduce cell apoptosis. ② Apoptosis rate test results: compared with the blank control group, the apoptosis rate increased significantly after adding 0.5 mmol/L H 2O 2; while the apoptosis rate of the miR-21-5p overexpression group was lower than that of the H 2O 2 injury group and miR-21-5p empty virus control group [early apoptosis rate: (14.31±0.12)% vs. (24.50±0.12)%, (23.41±0.13)%; late apoptosis rate: (8.12±0.13)% vs. (9.71±0.11)%, (10.41±0.15)%; overall apoptosis rate: (22.33±0.12)% vs. (34.21±0.10)%, (33.82±0.14)%; all P < 0.05], which further proved that miR-21-5p had anti-apoptotic effects. Conclusion:miR-21-5p has an anti-apoptotic effect on human ATⅡ.

Objective To construct a recombinant lentivirus vector containing the human NIS gene and HIF-1α with the myosin light chain-2v(MLC-2v) as a promoter and to investigate the specific expression and feasibility of NIS as a reporter gene in cardiomyocytes.Methods The target gene HIF-1α and NIS were subcloned into the lentivirus (Lv)-elongation factor (EF)1-HIF-1α-internal ribosome entry site (IRES)-NIS and Lv-MLC-HIF-1α-IRES-NIS lentivirus vectors.The recombinated vectors were transfected into Hela cells by lipofectamine 2000.The expression of HIF-1α and NIS in the transfected Hela cells was detected by indirect immunofluorescence and Western blot.The H9C2 cells were exposed to different multiplicities of infection (MOI; 5,10,20,40) with packaged virus particles.The infection efficiency was detected by Western blot.MOI 20 was used for H9C2,NIH-3T3 and L6 cell lines and the specificity of the MLC-2v promoter was detected by the count of NIS protein in the 3 different cell lines with Western blot.The function and features of NIS protein were evaluated by dynamic iodine uptake and NaClO4 iodine uptake inhibition tests in vitro.Two-sample t test was used to analyze the data.Results The two recombinant lentivirus vectors were constructed successfully.The HIF-1α protein was expressed in the cytoplasm and the NIS protein was expressed on the cell membrane in Hela cells.The grey levels of NIS and HIF-1α proteins in the positive control were 69.8 and 71.9,respectively,which were 109.4 and 92.7 after being prompted by EF1,and 141.9 and 132.4 by MLC-2v.The expression of these proteins was much higher by EF1 promoter than that by MLC-2v promoter.The optimal MOI for the Lv-MLC-HIF-1α-IRES-NIS virus to infect H9C2 cells was 20.With the MOI of 20,the grey levels of NIS protein promoted by EF1 were 23.4,29.8 and 28.6 for H9C2,NIH-3T3 and L6 cells infected with Lv-EF1-HIF-1α-IRES-NIS virus,respectively.The expression of NIS protein promoted by MLC-2v was much higher in H9C2 cells than the other two cell lines.The grey level of NIS protein was 157.9 in H9C2 cells,178.8 in L6 cells and 217.3 in NIH-3T3 cells.The NIS protein expressed in infected H9C2 cells showed high radioiodine uptake.The peak of iodine uptake was 4 287.2 counts · min-1 at 40 min which was 16.85 times of the control group (254.4 counts · min-1) (t=5.34,P＜ 0.01).The inhibition rate of iodine uptake was up to 85.5％ (3 666.4/4 287.2,t=21.3,P＜0.01) by NaClO4.Conclusions MLC-2v promoter allows specific expression of the external gene HIF-1α and NIS in myocardium.The cardiomyocytes transfected with NIS gene acquires the function of iodine uptake.Therefore,NIS may have a potential to be the reporter gene to monitor the external gene therapy in ischemic cardiomyopathy.

Adenocarcinoma ; genetics ; pathology ; surgery ; Adult ; Aged ; Aged, 80 and over ; Biopsy ; methods ; Bronchoscopy ; Carcinoma, Large Cell ; genetics ; pathology ; surgery ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; surgery ; Carcinoma, Squamous Cell ; genetics ; pathology ; surgery ; Female ; Gene Amplification ; Humans ; Lung Neoplasms ; genetics ; pathology ; surgery ; Male ; Middle Aged ; Polyploidy ; Receptor, Epidermal Growth Factor ; genetics ; Young Adult

Objective With Pregnant women weight correction for serum marker Multiple of Median (MoM) of First trimester and second-trimester,integrated screen-ing for Down's syndrome (DS),can reduce the false positive rate.Methods The same pregnant woman were taken venous blood vessels with sterile vacuum during the first trimester (11-13 W(+ 6) d) and the second trimester (15-20 W(+ 6) d),Alpha-fetoprotein (AFP),serum free beta-human chorionic gonadotrophin (Free beta hCG) and pregnancyassociated protein-A (PAPP-A) of three kinds of serum marker screening indicators were assayed by Using Time-resolved fluoroimmunoassay (TRFIA).Screening for risk assessment software was used to calculate serum marker Multiple of Median,To assess the risks of 7 997 cases of local pregnant women DS,To construct the weight equation of local population using nonlinear weighted regression method,With maternal weight correction for serum marker Multiple of Median (MoM) of local pregnant women,Comparing the changes of screening index MoM before and after correction,chi square test to compare the detection rate and false positive rate.Results MoM values of three kinds of serum markers (al-pha-fetoprotein,free beta subunit of human chorionic gonadotropin,pregnan-cy-associated plasma protein A) decreased with the weight increasing,Screening index MoM after correction weight equation,the screening of false positives for crowd from 4.12％ down to 3.86％ (x2 =0.021,P ＞ 0.05).Setting threshold (cut-off) at 1/270,and no change detection rates were 71.4％ the local population before and after correction weight equation.Conclusion Maternal weight may affect the results of Down's syn-drome sereening.When screening proposal to set up,it is worth making weight cor-rections for serum maker multiple of median in order to get accurate risk calculation results.

Objective To construct a recombinant baculovirus dual expression vector containing NIS gene under the control of human telomerase reverse transcriptase (hTERT) promoter and plasminogen kringle 5 (K5) gene driven by early growth response 1 (Egr1) promoter,and to explore the feasibility of targeting both tumor and tumor vessel with combination of radioiodide and antiangiogenic therapy.Methods The hTERT-NIS gene and Egr1-K5 gene fragments were subcloned into baculovirus vector,then packaged and amplified in the sf9 cells to obtain recombinant baculovirus Bac-hTERT-NIS-Egr1-K5.Bac-CMV-NISEgr1-K5,Bac-hTERT-0-Egr1-K5 and Bac-hTERT-NIS-Egr1-0 were constructed as controls.The expression of NIS and K5 genes in human cervix cancers cells (HeLa) was examined by Western blot and quantitative real-time PCR.Functional NIS activity was confirmed by the uptake of 125I,the inhibition of NaClO4 and the cytotoxicity of 131I.The apoptotic effect of 131I-inducedK5 on human umbilical veins endothelial cells (HUVEC)was analyzed by an apoptosis assay using flow cytometry.Statistical analysis was performed using the analysis of variance.Results The recombinant baculovirus Bac-hTERT-NIS-Egr1-K5 was successfully constructed.The NIS gene under the control of hTERT promoter was specifically expressed in HeLa cells.The baculovirusinfected HeLa cells showed a significant increase of 125I uptake,which was significantly inhibited by NaClO4(F199.296,P＜0.05).Furthermore,a notable decreased cell survival rate (38.3％) was found after 131I treatment.The expression of K5 gene induced by 131I was elevated in a dose or time dependent manner and resulted in obvious inhibition with cell survival rate of 30.8％ in baculovirus-infected HUVEC cells,which was significantly higher than that in the control groups (11.2％ and 10.9％ respectively,F=19.926,45.409;both P＜0.05).Conclusions A recombinant baculovirus dual expression vector containing the NIS and K5 genes has been successfully constructed.This study suggests the feasibility of a synergistic strategy of NISbased raidoiodide therapy and K5-based antiangiogenic therapy in vitro,and make it possible to perform in vivo study in the near future.

Objective To investigate the relationship between changes of ankle brachial index (ABI) and adverse cardiovascular events. Methods Baseline survey was conducted in 4 160 forty-year-old or older citizens living in Yunyan District of Guiyang City from May to August of 2011, which was in the way of cluster sampling to obtain their ABI and to collect information related to physical and blood biochemical examination and disease history. These citizens were conducted a follow-up survey for (39.29±1.47) months from July to December of 2014. Based on the change of ABI (ΔABI) from initial survey to follow-up survey, participants were subsequently divided into three groups: ΔABI>0.15 group,-0.15≤ΔABI≤0.15 group and ΔABI<-0.15 group. The adverse cardiovascular events during follow-up survey were compared between three groups. The risk factors affecting the adverse cardiovascular events were analyzed. Results Follow-up surveys were completed in 3 220 citizens in 3 years. The follow-up rate was 77.4%. Eighty-two new cases (2.5%) of adverse cardiovascular events were found in 3 220 cases in follow-up. The incidence rates of adverse cardiovascular events were higher inΔABI<-0.15 group compared with those of-0.15≤ΔABI≤0.15 group (8.3%vs. 2.4%, P<0.016 7). Logistic regression analysis indicated that age, hypertension history, and ΔABI<-0.15 were risk factors for adverse cardiovascular events. Exercise was the protective factor for adverse cardiovascular events. Conclusion Subjects withΔABI<-0.15 are at high risk for adverse cardiovascular events. The ΔABI can be used as a means of monitoring of adverse cardiovascular event, which provides certain forecast value for determining the possibility of adverse cardiovascular event.

Objective To investigate the relationship between brachial ankle pulse wave velocity and the incidence of cardio-cerebral events in people aged over 40. Methods Cluster sampling method was used to prospectively study 4 380 residents aged over 40 in Guiyang City District from May to August in 2011. Data of ba-PWV were collected. The follow-up examination was conducted from July to November of 2014. According to ba-PWV values, participants were divided into three groups:<14 m/s (control, n=1 039) group, 14-17.9 m/s group (n=1 393) and≥18 m/s group (n=809). Multi-factor Logistic regression model was used to analyze the relationship between ba-PWV values and risk factors of cardio-cerebral events. Results After three-year follow-up, a total of 3 241 participants were included in the final analysis. The cardio-cerebral events were identified in 63 (2.0%) cases, which were 0.6%, 2.2%and 3.2%in control group, 14-17.9 m/s group and≥18 m/s group. The value of ba-PWV increased significantly in those two groups compared with that of normal group. Logistic regression analysis showed that the incidence rates of cardiovascular and cerebralvascular events in 14-17.9 m/s group and≥18 m/s group were 2.777 (1.123-6.864) and 2.786 (1.032-7.526) times of control group after adjusting age, gender, systolic blood pressure, risk factors of diabetes, hypertension and blood lipids. Conclusion There is higher incidence rate of cardio-cerebral events in people aged over 40 in higher ba-PWV group. The value of Ba-PWV can be used to predict the occurrence of cardio-cerebral events.

Objective To explore the relationship between metabolic syndrome(MS) and glomerular filtration rate(GFR).Methods A total of 10 140 adults aged 40 years and older inhabitants in Zhaiji community of Guiyang urban areas were investigated from May 2010 to August 2010 by adopting stratified cluster sampling method.The venous blood sample was drawn for the measurements of serum creatinine(Cr), fasting plasma glucose(FPG), OGTT 2hPG, fasting insulin, triglyceride(TG), total cholesterol(TC), high-density lipoprotein-cholesterol(HDL-C), low-density lipoprotein-cholesterol(LDL-C), and fasting plasma insulin.The definition of MS in our study was modeled after the Adult Treatment Panel Ⅲ(ATP-Ⅲ).Decreased GFR was defined as an estimated GFR<60 ml·min-1·(1.73 m2)-1.Results The prevalence of GFR less than 60 ml·min-1·(1.73 m2)-1 were 3.0% and 1.2% in participants with and without MS, respectively.The multivariate-adjusted odds ratios[95% confidence interval(CI)] of MS, which were independently associated with decreased GFR, were with elevated blood pressure, higher TG, lower HDL-C, and elevated FPG, their statistically odds ratios were 1.78, 2.96, 1.06, and 1.22, respectively.The prevalence of GFR decreased with the increase of MS components by 0.56%, 1.10%, 1.50%, 2.87%, 3.23%, and the odds ratios were 1.00, 1.57, 1.93, 3.07, and 2.89, respectively.Conclusion With the increase of MS components the risk of GFR decline increased.The occurrence of chronic renal dysfunction(CKD) might integrate multiple different risk factors of MS.