Murine tumor necrosis factor(mTNF)cDNA was inserted into the polylinker site of MNSM retroviral vector to create pMNSM-SV40-mTNF. Albumin enhancer/promoter (Alb e/p) sequence was used to replace SV40 early region promoter of pMNSM-SV40-mTNF vector to create pMNSM-Alb e/p-mTNF recombinant retroviral vector. The retroviral constructs were introduced into amphotropic retroviral packaging cells PA317. Production of the recombinant retroviruses was accomplished by the lipofectamine-mediated gene transfer procedure. The murine tumor cells were infected with the retroviruses in the presence of polybrene. Dot hybridization of total RNA from modified tumor cell with mTNF cDNA probe and mTNF bioassay demonstrated that transcription and expression of mTNF gene drived by Alb e/p were markedly high in the hepatoma cells which produced albumin, and inhibited in the non-hepatoma tumor cells. The hepatoma cells modified with mTNF gene lost its tumorgenicity and significantly inhibited the growth of the parental hepatoma in vivo. High-titer MNSM-Alb e/p-mTNF retroviruses or the high-titer retroviruses producing packaging cells, after intra-tumoral injection, specifcally inhibited the growth of the hepatoma, and significantly prolonged the survival period of the hepatoma-loaded mice. Biopsy and immunohistochemical assay of the hepatoma during in vivo gene therapy, showed the occurrence of extensive tumor necrosis, bleeding, and CD8+, CD25+, CD4+ lymphocytic infiltration and fibrosis.

Aim: To observe the effects of sarsasapogenin ( SAR) on osteoblasts and osteoclasts cultured in vitro. Methods: Colonal murine calvarial osteoblast-like cell line MC3T3-E1 cells were cultured in vitro. MTT,p-nitropheneye phosphate and tinctorial method of alizarin Bordeaux were used to investigate the effects of SAR on the proliferation, ALP expression, and mineralization tuberculation of MC3T3-E1 cells. Mature osteoclasts were i-solated from the long bone of one-day rat. Meanwhile, marrow cells of mouse bone were cultured with induction of 1,25( OH)_2VitD_3. During the culturing of osteoclasts or marrow cells, SAR of different concentrations was added into the medium. The number of osteoclasts was recognized as tartrate resistant acid phosphatase( TRAP) ( +) multinucleate cells and the resorption lacuna on bone slice were examined with toluidine blue staining. Results: Comparing with the control group, SAR (0.01, 0. 1, 1μg/mL) significanthy increased the proliferation of MC3T3-E1 cells (P <0. 05, P <0. 01). There was no significant difference in the expression of ALP in early pro-liferating MC3T3-E1 cells exposed to SAR of 0.01,0. 1, 1μg/mL, but in the differentiation phase MC3T3-E1 cells, SAR improved ALP activity very significantly if compared with the control group, of which SAR of 1 μg/mL had the most promotion effect(P <0. 01). In addition, compared to the control group, there were, to various ex-tents, increased in the number of mineral nodes in MC3T3-E1 cells after 15day incubation with SAR of different conentrations. Furthermore, no obvious effects of 0.01-1μg/mL SAR on mature osteoclast were observed. But typical osteoclasts were formed when marrow cells were cultured with the induction of 1,25(OH)_2D_3 in medium for 7 days while little or no osteoclasts were induced from marrow cells in the presence of SAR. Conclusion: The results suggest that SAR can effectively promote the proliferation, differentiation and mineralization of osteoblasts cultured in vitro. Besides, SAR can inhibit the generation of osteoclasts from marrow cells.

BACKGROUND:Recently, electrospun materials have been extensively applied in the drug delivery system. OBJECTIVE:To overview the application prospect of electrospun materials in drug delivery systems. METHODS:A computer-based search of PubMed and NCBI databases was performed for literatures about the research progress of electrospinning in tissue engineering and chemotherapy published within the past 10 years using the keywords of“electrospinning, drug delivery system, nanofibers, electrospun materials”.RESULTS AND CONCLUSION:Compared with traditional materials, electrospun stents hold good versatility and control able parameters, thus granting its unique advantage under various physiological conditions. Current drug-loaded materials composed of natural products, synthetic polymers and blended materials;as to drugs, there are antibiotics, chemotherapy medication, DNA and protein. Electrospun materials have been used in tissue engineering, cancer chemotherapy and wound healing. We focus on not only the application progress of electrospun materials in traditional treatments, but also its usage, condition-control ed drug release and living-cel carrying. Electrospun materials combined with various drug-loaded present a broad prospect.

This study is to investigate protective effect of safflor yellow B (SYB) against vascular endothelial cells (VECs) injury induced by angiotensin-II (Ang-II). VECs were cultured and divided into six groups: control group, Ang-II group, Ang-II + SYB (1 micromolL(-1)) group, Ang-II + SYB (10 micromolL(-1)) group, Ang-II + SYB (100 micromolL(-1)) group and Ang- II + verapamil (10 micromolL(-1)) group. Except control group, all of VECs in other groups were treated with Ang- II at the final concentration of 0.1 micromolL(-1). Mitochondria membrane potential (MMP) and free calcium concentration ([Ca2+]i) were measured by laser scanning confocal microscopy, and mitochondria complex IV activity was detected by BCA method. The levels of reactive oxygen species (ROS) in VECs were analyzed by fluorescence detector and apoptosis of VECs was observed by flow cytometer. Caspase 3 was determined by Western blotting method. Comparing with control group, Ang-II was able to increase [Ca2+]i and ROS level, decrease MMP level, inhibit complex IV activity and enhance caspase 3 activity in VECs, as a result, enhance apoptosis of VECs. But SYB could significantly reduce the result induced by Ang- II relying on different dosages (P < 0.05 or P < 0.01). SYB was able to eliminate the effect of Ang-II on VECs via regulating [Ca2+]i, mitochondrial structure and function and inhibiting apoptosis.

Objective To study the characteristics of skinfold thickness of Han adults in Jiangsu province . Methods The skinfold thicknesses of facial , subscapular , suprailiac , biceps , triceps and calf on 311 urban adults ( 157 males and 154 females) and 421 rural adults ( 213 males and 208 females ) of Han were investigated in Huaian city of Jiangsu province .Results The thickness of skinfold of urban females were thicker than that of urban males .Rural adults were the same .Han adults of Jiangsu showed the most significant differences between urban areas and rural areas .The values of six skinfold thicknesses of Jiangsu urban adults have positive correlation with age .Conclusion Han adults of Jiangsu show the most significant differences between genders .

To construct retroviral vector carrying human interleukin-3 c0mplementary DNA(HuIL-3 cDNA) under control of human a-fetoprotein gene enhancer core sequence and human SV4O pro-moter. Methods and Results: HuIL-3 cDNA was inserted into polylinker site of retroviral vector pMNSMto construct retr0viral vector pMNS-IL-3, in which the transcription of HuIL-3 cDNA was drived by SV40early region promoter. Human Q-fetoprotein gene enhancer core sequence was released from plasmidpGEM. 7Zf-AFPe and inserted into the polylinker site of pMNSM. Then human interleukin-3 cDNA wasinserted int0 p0lylinker site to construct retroviral vector pMNSA-IL-3, in which HuIL-3 cDNA transcrip-tion was drived by SV40 early region promoter and enhanced by human a-fetoprotein enhancer core se-quence- Conclusion: The vectors are of significance for hepatoma-specific gene therapy.

Objective To explore the diagnostic model and clinical application value of serum proteomic fingerprint in inflammatory bowel disease (IBD) .Methods Serum proteome profiles of 72 IBD patients (54 Crohn′s disease (CD) and 18 ulcerative colitis (UC) and 44 healthy controls were analyzed by the weak cation exchange (WCX) beads combined matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF‐MS ) technique . Among three groups , every two groups were compared .Wilcoxon rank sum test was used to screen out the peaks of difference expressed protein (P<0 .05) .Genetic algorithm combining with support vector machine (SVM ) was utilized to select the best diagnostic model .The predictive effects of this model was evaluated by leave one out method (LOO ) . Results The 10 most discriminating protein peaks were screened out between CD group and healthy control group , between UC group and healthy control group , between CD group and UC group . A diagnostic model established with four protein peaks ,the mass‐to‐charge ratio (M /Z ) of them was 3 275 .29 ,4 963 .91 ,4 980 .53 and 5 336 .90 ,could better distinguish CD and healthy controls .The specificity was 97 .7% ,and the sensitivity was 92 .6% in CD diagnosis .A diagnostic model established with four protein peaks ,the M /Z of them was 2 272 .41 ,2 660 .42 ,3 029 .77 and 5 002 .78 ,could better distinguish UC and healthy controls .The specificity was 100 .0% ,and the sensitivity was 94 .4% .A specificity was 50 .0% and sensitivity was 88 .9% in CD diagnosis with the diagnostic model of six protein peaks and the M /Z of them was 2 082 .63 ,2 210 .64 ,4 039 .02 ,4 298 .30 ,4 978 .03 ,5 002 .22 .Conclusion The diagnostic model of serum difference expressed protein in CD and UC is established by MALDI‐TOF‐MS technique and genetic algorithm combining with SVM ,which has high diagnostic value in IBD .

Objective To investigate the clinical significance of serum anti-Saccharomyces cerevisias antibody (ASCA),anti-outer membrane porin C (anti-OmpC),antibody to Pseudomonas fluorescens-associated sequence I2 (anti-I2 )and antibody to bacterial flagellin (anti-CBirl )in the diagnosis and treatment of inflammatory bowel disease (IBD).Methods From 2011 to 2013,87 patients with IBD were enrolled and divided into Crohn′s disease (CD)group (66 cases)and ulcerative colitis (UC)group (21 cases).A total of 62 age and gender matched healthy individuals were enrolled as the control group. Fasting blood samples (2 mL)of the subjects were collected.The expression of ASCA,anti-OmpC,anti-I2 and anti-Cbirl antibodies was detected with enzyme-linked immunosorbent assay (ELISA)kits.The diagnosis value of each antibody in IBD and the differential diagnostic value of in UC and CD were compared by receiver operating characteristic (ROC)curve.Results The area under the curve (AUC)of ASCA between IBD and the healthy control group,between CD group and UC group was 0.580 and 0.512, respectively;the accuracy in diagnosis was low.The AUC of anti-CBirl between IBD and the healthy control group was 0.617.There was no differential diagnosis significance of the other antibodies.The positive rate of ASCA in IBD group was 62.1 % (54/87),which was significantly higher than that in the control group (38.7%,24/62).The positive rates of anti-OmpC and anti-I2 in IBD group was significantly lower than those in the control group and the differences were statistically significant (both P <0.05).No difference was observed in positive rates of serum antibodies among the others groups (all P >0.05).The specificity,sensitivity,positive predictive value (PPV)and negative predictive value (NPV)of ASCA in differential diagnosis of CD and UC was 52.4%,66.7%,81 .48% and 33.33%,respectively.The specificity and sensitivity of anti-OmpC,anti-I2 and anti-CBirl in differential diagnosis of CD and UC was 81 .0% to 100.0% and 9.1 % to 37.9%,respectively.The specificity,sensitivity,PPV and NPV of double-positive ASCA and anti-I2 in the diagnosis of CD was 57.1 %,86.4%,82.6% and 50.0%, respectively.The positive rate of ASCA and anti-I2 in CD group was significantly higher than that in UC group (84.8%(56/66)vs 57.1 % (12/21 );χ2 =5 .633,P =0.018 ).Conclusions Positive ASCA has some significance in the diagnosis of patients with IBD in our country.The detection of anti-I2 can help to diagnose ASCA negative CD.Because of low sensitivity and positive rate,anti-OmpC and anti-CBirl have limited value in the diagnosis of IBD and the differential diagnosis of UC and CD in our country.

Several studies have demonstrated the role of 4-1BBL in T cell activation. Furthermore, enhanced 4-1BB/4-1BBL interaction has been shown to amplify T-cell-mediated antitumor immunity in several mouse models. However, when applied in humans, it was difficult to generate sufficient T cells ex vivo and whole cell vaccines to transfer back into patients. To overcome this difficulty, we have focused on producing the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein. In this report, PCR and overlap PCR were used to construct the human 4-1BBL extracellular domain/anti-CD20 Fab' expression vector. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC; its antigen binding activity was examined by rosetting assay. The data of DNA sequence showed that the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 microg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells and Raji cells as shown by cellular rosetting. In conclusion, the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was induced to express in E. coli 16C9. The results of some biological activity experiments indicated that the fusion protein could bind to stimulated Jurkat cells and Raji cells. Furthermore, 4-1BBL-negative tumors can be converted into 4-1BBL-positive tumors by the fusion protein without the need for 4-1BBL gene transfer to the malignant cells.
4-1BB Ligand ; biosynthesis ; genetics ; Antibodies, Bispecific ; immunology ; Antigens, CD20 ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; genetics ; Immunotherapy ; methods ; Lymphoma, Non-Hodgkin ; therapy ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Objective:To explore whether microRNA (miRNA) -181b-5p inhibits the proliferation and invasion of cutaneous melanoma cells by targeting pleckstrin (PLEK) .Methods:Bioinformatics methods were used to analyze cutaneous melanoma-associated core genes; dual-luciferase reporter assay was performed to verify the targeted interaction between miRNA-181b-5p and PLEK. Oligo RNA and small interfering RNA (siRNA) were used to regulate the expression of miRNA-181b-5p and PLEK in A375 cells respectively in this experiment, and A375 cells were divided into the following groups in detail: mimic negative control group, miRNA-181b-5p mimic group, inhibitor negative control group, miRNA-181b-5p inhibitor group, PLEK siRNA group, siRNA negative control group, miRNA-181b-5p inhibitor + control siRNA co-transfection group and miRNA-181b-5p inhibitor + PLEK siRNA3 co-transfection group. After 48-hour treatment, qPCR was performed to determine the mRNA expression of miRNA-181b-5p and PLEK in A375 cells, Western blot analysis to determine the PLEK protein expression, and Transwell assay to assess the invasive ability of A375 cells; after additional 24-96 hours of culture, cell counting kit-8 (CCK8) assay was conducted to assess the proliferative ability of A375 cells.Results:PLEK was the core gene for cutaneous melanoma. PLEK expression in the cutaneous melanoma in situ tissues was significantly higher than that in the paracancerous tissues ( P = 0.031) , but lower than that in the metastatic tissues ( P = 0.001) . Compared with human epidermal melanocytes HEMa-LP, the mRNA and protein expression of PLEK significantly increased in A375 cells (mRNA: 3.884 ± 0.156 vs. 0.997 ± 0.010, t = 18.48, P < 0.001; protein: 2.840 ± 0.301 vs. 1.029 ± 0.094, t = 5.47, P = 0.005) , but the miRNA-181b-5p expression significantly decreased in A375 cells (0.333 ± 0.042 vs. 0.967 ± 0.069, t = 7.83, P = 0.001) . Dual-luciferase reporter assay showed targeted binding of miRNA-181b-5p to PLEK. Compared with the mimic negative control group, the miRNA-181b-5p mimic group showed significantly decreased survival rate of A375 cells (48 hours: t = 7.96, P = 0.015; 72 hours: t = 7.50, P = 0.002; 96 hours: t = 7.96, P = 0.001) , and significantly decreased invasive ability of A375 cells ( t = 5.07, P = 0.007) ; on the contrary, the survival rate and invasive ability of A375 cells were significantly higher in the miRNA-181b-5p inhibitor group than in the inhibitor negative control group (survival rate: 24 hours, t =5.38, P = 0.013; 48 hours, t = 5.36, P = 0.013; 72 hours, t =7.63, P = 0.005; 96 hours, t = 5.99, P = 0.004; invasive ability: t = 7.24, P = 0.002) ; compared with the siRNA negative control group, the proliferative and invasive ability of A375 cells significantly decreased in the PLEK siRNA group (proliferative ability: 48, 72, 96 hours, P = 0.015, 0.011, 0.001, respectively; invasive ability: t = 4.93, P = 0.008) ; compared with the miRNA-181b-5p inhibitor + control siRNA co-transfection group, the miRNA-181b-5p inhibitor + PLEK siRNA co-transfection group showed significantly decreased proliferation rate and invasive ability of A375 cells (proliferation rate: 24, 48, 72, 96 hours, P = 0.042, 0.042, 0.037, 0.017, respectively; invasive ability: t = 8.52, P = 0.001) . Conclusion:miRNA-181b-5p can inhibit the proliferation and invasion of cutaneous melanoma A375 cells, likely by down-regulating the PLEK expression.