Objective To research the relation of early growth response gene-1(EGR-1) and NF-κB in human T-cell leukemia virus type 1(HTLV-1) Tax protein positive cells. Methods RT-PCR was used to amplify the aimed segments EGR-1 cDNA which was then inserted into an eukaryotic expression plasmid pcDNA3.0 to construct pcDNA3.0-EGR-1. The constructed plasmid was transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, the expression levels of EGR-1, p65 and Tax mRNA in transfected cells were assay by RT-PCR after 48 h post-transfection, the proteins of EGR-1 and p65 were detected by Western blot after 48 h post-transfection too. The constructed plasmid and pNF-κB-luc reporter gene plasmid was co-transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, and the activity of luciferase was assay after 48 h post-transfection. Results The results showed that the eukaryotic expression plasmid pcDNA3.0-EGR-1 was successfully constructed. The mRNA and protein expression of EGR-1 could be promoted significantly by Tax. EGR-1 can promote the mRNA and protein expressions of p65 in TaxP cells, the activity of NF-κB was up-regulated by EGR-1 too. Conclusion EGR-1 maybe involve in adult T-cell leukemia(ATL) by increasing the activation of NF-κB.

Objective To explore the effect of HTLV-1 (human T-cell leukemia virus type 1 ) Tax protein on the DcR3 gene expression in T cells.Methods The construction of DcR3 (-1010 bp to +114 bp) luciferase reporter gene; MT2,TaxP,and Jurkat E6-1 cells were transfected with DcR3 luciferase reporter gene (pGL3-DcR3-1uc) using liposomes according to the manufacturer's instructions.For the control group,pGL3-basic replaced it respectively.At 48 h after incubation,luciferase activity was measured with a luciferase assay system; Jurkat cells were transfected with pCMV-Tax-Bam using liposomes,and total RNA was extracted from the cells at 48 h after incubation.Reverse transcription was performed using standard protocols.Then real time PCR with primers DcR3 and 3-actin was conducted;The expression of DcR3 protein was detected by flow cytometry in MT2,TaxP,and Jurkat cells.Results The construction of DcR3 luciferase reporter gene is identified correctly; The detection of luciferase activity showed that the luciferase activity of experimental group in MT2 cells was increased by (32.07±12.43)-fold,of which in TaxP cells was increased by ( 13.27±4.04)-fold,and the luciferase activity of experimental group in Jurkat cells was increased by ( 1.26±0.49 ) -fold.And there is statistic significance about the relative luciferase activity of experimental group in MT2 cells and TaxP cells compared to the relative lueiferase activity of experimental group in Jurkat cells respectively ( P＜0.01 ) ; The result of real-time PCR showed that the level of DcR3 mRNA in the experimental group was higher compared with the control group(P＜0.05) ; The result of FCM shows the expression of DcR3 protein in MT2,TaxP cells is higher than that in Jurkat cells( P＜0.05 ).Conclusion Tax protein can promote the expression of DcR3 gene in T cells.

BACKGROUND:Under the in vitro conditions of cell harvesting, culture, and transplantation, whether bone marrow stromal cells (BMSCs) can be effectively applied in local gene therapy remains unclear.OBJECTIVE: To construct a recombinant eukaryotic expression plasmid carrying human bone morphogenetic protein-7 (hBMP-7) gene, and to expect to enhance osteoinductive properties of rabbit BMSCs transfected.DESIGN, TIME AND SETTING: A cell-genomics in vitro observation was performed at the Laboratory of Scientific Research, Second Affiliated Hospital of Harbin Medical University between July 2006 and July 2007.MATERIALS: Human healthy fresh placental tissue was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from the women. One healthy male New Zealand rabbit was provided by the Laboratory Animal Center, Harbin Medical University.METHODS: hBMP-7 gene was cloned from human placental tissue to construct a recombinant eukaryotic expression plasmid carrying hBMP-7 gene by conjugating with eukaryotic expression vector pcDNA3.1. BMSCs were isolated from rabbit bone marrow and cultured in vitro. Then they were divided into 3 groups: pcDNA3.1-hBMP-7-transfected, pcDNA3.1 -transfected, and untransfected. 5×106 BMSCs were inoculated into a 60 mm3 flask containing antibiotic-free medium 1 day prior to transfection.MAIN OUTCOME MEASURES: RT-PCR and immunohistochemistry were employed to detect hBMP-7 expression in BMSCs, alkaline phosphatase activity, hydroxypreline content, and osteocalcin production in each group. RESULTS: After 72-hour transfection, a 1.3 kb fragment was seen in the pcDNA3.1-hBMP-7-transfected group, showing brown granules in the endochylema, but not seen in the pcDNA3.14ransfected and untransfected groups. ALP activity in the pcDNA3.1-hBMP-7-transfected group significantly increased at 2 days after transfection, peeked at 8 days, and still increased at 10 days. At each time point, alkaline phosphatase activity, hydroxyproline content, and osteocalcin production were significantly higher in the pcDNA3.1-hBMP-7-transfected group than in the pcDNA3.1 -transfected and untransfected groups (P<0.05 or P<0.01).CONCLUSION: Recombinant eukaryotic expression vector pcDNA3.1- BMP-7 was constructed successfully. Results indicated that hBMP-7 was expressed in BMSCs sufficiently and was involved in inducing differentiation of BMSCs into osteoblasts. The method would provide substantial basement for hBMP-7 gene therapy.

Objective:To preliminarily study the characteristics of ventricular synchronicity in patients of idiopathic pulmonary arterial hypertension (IPAH) with different heart function.
Methods:Our research included in 2 groups:IPAH group, n=40 patients intermittently collected in our hospital from 2010-10 to 2014-12 and Control group, n=23 normal subjects from the same period of time. Tissue Doppler echocardiography was conducted to compare interventricular systolic and diastolic time to peak (Ts and Te), their differences (Te-Ts) and (Te-Ts) of left ventricle (LV), right ventricle (RV), interventricular septum (IVS) between 2 groups.
Results:Ts and Te of RV were longer than Ts and Te of LV and IVS in both groups, P<0.05. Compared with Control group, by heart function reducing, IPAH group showed different Ts and Te of LV and IVS, P<0.05 and Ts was gradually prolonged, Te was gradually shorter;while Te of RV became longer firs tand then became shorter thereafter. In IPAH group, the patients with heart function grade IV had the longer Ts of LV and IVS than those with heart functionII, III;the patients with heart function IV had the shorter Te of LV, RV and IVS than those with heart function II, III, all P<0.05. In IPAH group with different heart function, Te between RV-IVS and RV-LV were different from Control group, all P<0.05;Te between LV-IVS in heart function IV patients had the largest difference from Control group, P<0.05. Compared with Control group, IPAH group had gradually decreased Te-Ts of LV and IVS by heart function reducing asgrade II>III>IV, all differences had statistic meaning.
Conclusion:IPAH patients with different right heart function may have interventricular systolic and diastolic de-synchronicity;by reduced heart function, interventricular Te-Ts could be specifically shortened.

Objective:To establish a conditional knockout mouse model of polycystic kidney disease 1 ( Pkd1) gene based on CRISPR/Cas9 and Cre-loxP gene editing technology, and to provide an animal model for in-depth research on the role of Pkd1 gene in the development of polycystic kidney disease. Methods:In-Fusion technology was used to construct a targeting vector. Corresponding gRNAs, Cas9 mRNAs, and donor vectors carrying the loxP site were prepared based on the Pkd1 gene, and injected into the fertilized eggs of C57BL/6N mice. The fertilized eggs were transferred to the fallopian tubes of female mice with pseudopregnancy. After the newborn mice were identified by PCR and sequencing analysis, Pkd1 flox/flox F0 generation positive mice were selected. The F0 generation positive mice were bred with wild-type mice, and F1 generation heterozygous mice with Pkd1 flox/+ genotype were selected for offspring. F2 generation homozygous mice with Pkd1 flox/flox genotype were obtained through internal expansion, and then hybridized with Cre positive Ggt1/ Cre mice. F3 generation mice with Pkd1 flox/+Ggt1 Cre genotype were obtained. F4 generation mice with Pkd1 flox/flox Ggt1 Cre genotype were obtained by self crossing or backcrossing with F2 generation Pkd1 flox/flox, namely kidney-specific Pkd1 gene knockout mice ( Ggt1-cKO mice). PCR method was used to identify the genotype of mice, and then the mice were divided into wild-type control (WT) group ( n=6), Pkd1 homozygous control (PKD) group ( n=6), and Ggt1-cKO knockout validation (CKO) group ( n=6) according to the gene identification results. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of Pkd1 mRNA in the kidneys and other organs of mice in each group. HE staining was used to detect the pathological changes in renal tissues of mice in each group. The automatic biochemical detector was used to detect the blood urea nitrogen and serum creatinine levels of mice, and the kidney coefficient was calculated. Results:The PCR detection results showed that the genotype of offspring mice in CKO group was consistent with Pkd1floxflox Ggt1 Cre. Pkd1 gene was only specifically expressed in the kidney, but not in other tissues. The RT-qPCR results showed that the relative expression of Pkd1 mRNA in the renal medulla of CKO group was significantly lower than that of WT and PKD groups. The kidney volume of the CKO group had increased by about twice compared to the WT group. Under the microscope, it could be observed that there were multiple vacuoles of varying sizes and shapes in the kidneys of the CKO group, and there was a significant increase in the interstitial space of the medullary tissue. The kidney coefficient, blood urea nitrogen, and serum creatinine in the CKO group were significantly higher than those in the WT and PKD groups (all P<0.05). Conclusion:Based on CRISPR/Cas9 and Cre-loxP gene editing technology, Pkd1 gene kidney conditional knockout mice can be successfully constructed, providing an animal model for further studying the action mechanism of Pkd1 gene in polycystic kidney disease.

Objective To observe the value of multimodal imaging for diagnosis of cardiac space-occupying lesions.Methods Data of 70 patients with cardiac space-occupying lesions who underwent echocardiography and cardiac CT(CCT)were retrospectively analyzed,among them 35 also underwent cardiac MRI(CMRI).The value of multimodal imaging for diagnosis of cardiac space-occupying lesions were explored according to the results of surgical pathology or clinical diagnosis.Results Among 70 cases,benign tumors were confirmed by surgical pathology in 43 cases,while malignant tumors were confirmed by surgical pathology in 3 cases and clinically diagnosed in 1 case.Meanwhile,non-tumor-occupying lesions were clinically diagnosed in 23 cases,all obviously shrunken after treatments.Among 70 cases,echocardiography correctly diagnosed 57 cases,misdiagnosed 8 cases and unclearly diagnosed 5 cases,with diagnostic accuracy rate of 81.43%(57/70).CCT correctly diagnosed 63 cases,misdiagnosed 4 cases but missed 3 cases,with diagnostic accuracy rate of 90.00%(63/70).CMRI outcomes in all 35 cases were consistent with surgical pathologic results,with diagnostic accuracy rate of 100%(35/35).Conclusion Multimodal imaging might provide objective evidences for diagnosis and treatment of cardiac space-occupying lesions.

Background Aluminum can cause synaptic plasticity damage in the hippocampus, probably due to blocked interneuronal signal transmission. MicroRNA-29a (miR-29a) can target phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and participate in the generation of neuronal networks, and may be involved in the effect of aluminum on the electrical activity of neuronal networks. Objective To study the role and mechanism of miR-29a-targeted PTEN in aluminum-induced neuronal network injury in primary hippocampal neurons of ICR mice treated with maltol aluminum [Al(mal)₃] in vitro. Methods Primary hippocampal neurons of ICR mice born within 24 h were cultured in vitro. The purity of neurons was determined by labeling neuron-specific microtubule-associated protein 2 (MAP2) by immunofluorescence staining on day six of the culture; neurons were treated with different concentrations of Al(mal)₃, and divided into a control group, and 10, 20, and 40 μmol·L⁻¹ Al(mal)₃ groups, and neuronal cell viability was detected by CCK-8 method. Al(mal)₃ at 20 μmol·L⁻¹ was selected for subsequent experiments to establish a neuronal network injury model for intervention. The lentivirus infection method was used to transfect miR-29a into neurons, which were divided into mNG, mNG+20 μmol·L⁻¹ Al(mal)₃, miR-29a, and miR-29a+20 μmol·L⁻¹ Al(mal)₃ groups, and micro-electrode array (MEA) was used to analyze the firing of neuronal network. The expressions of miR-29a and PTEN mRNA in each group were detected by real-time PCR (RT-PCR), and the expression of PTEN protein in each group was detected by Western blotting. Results The purity of primary mouse hippocampal neurons was greater than 90%, and the viability of the neurons was above 80% in all groups. At 48 h of the designed Al(mal)₃ treatments, the changes in spike frequency, burst frequency, network burst frequency, and synchrony index of neurons cultivated on MEA plates in the control group were 207.56%±38.70%, 73.19%±46.43%, 75.42%±33.04%, and 117.13%±15.54%, respectively; the Al(mal)₃ groups’ neuronal network electrical activity showed a decreasing trend. Compared with the control group, the spike frequency, burst frequency, network burst frequency, and synchrony index of the 20 and 40 μmol·L⁻¹ Al(mal)₃ groups significantly decreased (The changes were 171.70%±28.08%, 49.20%±23.23%, 50.20%±18.18%, and 85.45%±20.30%; 150.68%±26.15%, 43.43%±15.54%, 52.05%±26.31%, and 26.80%±8.29%, respectively, P < 0.05). Compared with the control group (1.00), the miR-29a relative expression levels were significantly decreased in the 20 μmol·L⁻¹ Al(mal)₃ group (0.74±0.09) and the 40 μmol·L⁻¹ Al(mal)₃ group (0.62±0.12) (P < 0.05); the relative expression levels of PTEN mRNA were significantly increased in the 20 μmol·L⁻¹ Al(mal)₃ group (1.32±0.12) and the 40 μmol·L⁻¹ Al(mal)₃ group (1.48±0.11) (P < 0.05); the PTEN protein relative expression levels (1.29±0.12 and 1.82±0.10, respectively) were also significantly increased (P < 0.05). By overexpressing miR-29a in mouse primary hippocampal neurons, the spike frequency, burst frequency, and network burst frequency were significantly higher in the miR-29a group compared with the mNG group (The changes were 252.80%±62.03%, 171.65%±56.30%, and 197.75%±27.12%, respectively, P<0.05). The mNG+20 μmol·L⁻¹ Al(mal)₃ group showed a significant decrease in all indicators of neuronal network electrical activity (The changes were 123.28%±47.31%, 66.62%±31.53%, 70.60%±12.48%, and 52.86%±20.26%, respectively, P < 0.05). Compared with the mNG+20 μmol·L⁻¹ Al(mal)₃ group, the electrical activity indicators of neuronal network were significantly higher in the miR-29a+20 μmol·L⁻¹ Al(mal)₃ group (The changes were 161.41%±42.13%, 101.16%±30.63%, 127.02%±29.58%, and 109.73%±15.61%, respectively, P < 0.05). Compared with the mNG group (1.00), the neuronal PTEN mRNA relative expression (0.67±0.11) and the PTEN protein expression (0.75±0.08) were decreased in the miR-29a group (P < 0.05); the PTEN mRNA relative expression (1.32±0.12) and the PTEN protein relative expression (1.46±0.15) in the mNG+20 μmol·L⁻¹ Al(mal)₃ group were increased (P < 0.05). Compared with the mNG+20 μmol·L⁻¹ Al(mal)₃ group, the PTEN mRNA relative expression (0.93±0.06) and the PTEN protein relative expression (0.92±0.09) were decreased in the miR-29a+20 μmol·L⁻¹ Al(mal)₃ group (P < 0.05). Conclusion Aluminum significantly inhibits the electrical activity of hippocampal neuronal networks, and miRNA-29a may be involved in the aluminum-induced impairment of hippocampal neuronal network electrical activity by regulating PTEN expression.