GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.
Animals ; Cell Line ; Cytomegalovirus ; genetics ; metabolism ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Prealbumin ; genetics ; metabolism ; Promoter Regions, Genetic

OBJECTIVETo examine the activation and cytotoxicity of human peripheral blood T lymphocyte induced in vitro by human 4-1-BB ligand (4-1-BBL) gene transfected into tumor Tca8113 cells.

METHODSThe eukaryotic expression vector pEGFP-h4-1-BBL was transfected into human oral carcinoma cell line Tca8113 by Lipofectamine 2000. The transfected cells were then selected in medium containing G-418, cloned by limited dilution and named as Tca8113-4-1-BBL. Human 4-1-BBL mRNA and protein expression of transfected cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. The tumor cell vaccines (TCV) were obtained by treatment with mitomycin (MMC). Human peripheral blood mononuclear cells (PBMC) were prepared from lymphoprep, and then stimulated with anti-CD-3 mAb and incubated with non-transfected or transfected TCV-Tca8113 cells, respectively. The proliferation of T cells was evaluated by trypan blue exclusion; the CCK-8 was used to detect the cytotoxic effect of T lymphocytes. Meanwhile, the secretion of interferon-gamma (IFN-gamma) and interleukin (IL)-2 in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe Tca8113 cells transfected by pEGFP-h4-1-BBL could express human 4-1-BBL efficiently. As compared with wild type Tca8113 cells, the transfected Tca8113 cells could markedly promote proliferation, IL-2 and IFN-gamma production and cytotoxic activity of lymphocytes.

CONCLUSIONSThe transfection of human 4-1-BBL gene in Tca8113 cells is effective in enhancing its immunogenicity and inducing antitumor immune response in vitro.

4-1BB Ligand ; genetics ; Carcinoma, Squamous Cell ; genetics ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Interferon-gamma ; immunology ; Interleukin-2 ; immunology ; Lymphocyte Activation ; Mouth Neoplasms ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

Objective To establish a UPLC-MS/MS method for determination of 10 anti-rheumatic constituents illegally added in Chinese traditional medicine and health products preparation.Methods The column was ACQUITY UPLC BEHC18 (50 mm× 2.1 mm,1.7 μm),The mobile phase was acetonitrile-Ammonium acetate solution (containing 0.1％ Acetic acid) with gradient elution at a flow rate of 0.3 mL/min.The ion source was electrospray ionization (ESI),Multiple-reaction monitoring (MRM) was performed to identify and quantify 10 anti-rheumatic constituents.Results 10 linear calibration curves were obtained with r ≥ 0.996 1.The recoveries were determinated at three concentration and ranged from 92.5％ to 101.8％.The precision of the method was shown by RSD (n =5) ranged from 0.9％ to 3.1％.The ranges of limit of detection were from 0.001 5 to 0.018 μg,and quantitation were from 0.004 5 to 0.55 μg.The illegally added chemicals were detected with 10 batches of 27 batches of samples.Conclusion The method were simple,sensitivity,accurate,and can be used to detect Anti-rheumatic constituents illegally added in Chinese traditional medicine and health products.

OBJECTIVETo explore the influence of proximal-tip location on partial necrosis in distally based sural neuro fasciocutaneous flap.

METHODSFrom April 2001 to May 2009,157 distally based sural neuro fasciocutaneous flaps were conducted to repair the soft tissue defect in distal region of lower leg, ankle and feet in 153 patients. Date of the flaps and the patients were retrospectively analyzed. From the tip of lateral malleolus to the popliteal crease, posterior aspect of the lower leg was equally divided into 9 regions that were 1st to 9th region from inferiorly to superiorly, respectively. The flaps were divided into 2 groups: survival group (including uneventfully survived flaps, flaps with distally epidermal necrosis and with wound dehiscence) and partial necrosis group. Based on the location of the proximal tip of flaps, the flaps were stratified into 4 groups: flaps with the proximal tip locating in the 6th or lower region (group A), the 7th region (group B), the 8th region (group C) and the 9th region (group D). Harvesting the flaps started from exploring the perforator of peroneal vessel in the adipofascial pedicle, then the flaps were elevated retrogradely.

RESULTSOf the 157 flaps, 125 survived uneventfully,8 showed distal epidermal necrosis,wound dehiscence occurred in 6 flaps, 18 flaps (11.5%) showed distal partial necrosis. Partial necrosis occurred in zero of 19 flaps in group A (0), 1 of 44 flaps in group B (2.3% ), 7 of 62 flaps in group C (11.3% ) and 10 of 32 flaps in group D (31.3% ). The differences in partial necrosis rate between group A and group B , group B and group C, were not statistically significant (P > 0.05). Partial necrosis rate was higher in group D than in group C (P = 0.012), it was lower in group A + group B (1.6%) than in group C + group D (18. 1% ) (P = 0. 001).

CONCLUSIONSDistally based sural neuro fasciocutaneous flap can survive reliably when the proximal tip of flap is not beyond the junction between lower 7/9 and upper 2/9 of the lower leg, whereas probability of partial necrosis occurring in the flap increase significantly when the proximal tip of flap locates in upper 1/9 of the lower leg.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Leg Injuries ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Soft Tissue Injuries ; surgery ; Sural Nerve ; Surgical Flaps ; Treatment Outcome ; Young Adult

BACKGROUNDAccumulating evidence demonstrates that the microenvironment of the host has an important effect on the chemoresistance of tumors. We also found that the formation of intrinsic multidrug resistance is related to environmental factors that are common with tumor growth of hepatocellular carcinoma. The aim of this study was to explore the molecular mechanisms by which multidrug resistance of hepatocellular carcinoma is induced by the microenvironment. In particular, the regulation of nuclear transcription factor (hypoxia-inducible factor-1α, HIF-1α) activation in the process of multidrug resistance formation was investigated.

METHODSHepG2 cells were exposed to different microenvironmental conditions respectively, such as hypoxia, stimulation of glucose deprivation and transfection of plasmid PcDNA3/HBx. In the HepG2 cells, the expression of the related MDR proteins, HIF-1α protein expression and localization, activity of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) were detected. Specific inhibitor U0126 was used to block ERK/MAPK signal pathway, the alteration of HIF-1α and the related MDR proteins were investigated. Multivariate analysis of variance (MANOVA) repeated measures and one-way analysis of variance (ANOVA) followed by Tukey test or t-test were used to determine differences over time and effects of the treatments.

RESULTSThe above three microenvironment factors increase the expression of the related MDR proteins (including P-gp, LRP, and MRP1) and induce MDR of HepG2 cells. HIF-1α was induced at the protein and mRNA levels and the nuclear translocation was also increased. The activity of ERK/MAPK was also increased in HepG2 cells. But when ERK/MAPK pathway was inhibited, the mRNA and protein expression of MDR1, MRP1, and LRP was to some extent decreased. Inhibition of ERK/MAPK significantly reduced activated HIF-1α protein and the nuclear translocation of HIF-1α, whereas HIF-1α mRNA levels were not affected.

CONCLUSIONSThe microenvironmental factors could induce MDR of HepG2 cells by the activity of HIF-1α. The activity of HIF-1α is regulated by the ERK/MAPK pathway at the phosphorylation level. As an important nuclear transcription factor, HIF-1α controls the transcription of MDR-related genes and the synthesis of their corresponding proteins by ERK/MAPK signal pathway in HepG2 cells.

Drug Resistance, Neoplasm ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; physiology ; MAP Kinase Signaling System ; Tumor Microenvironment

BACKGROUND: Until now, there are no reliable methods for the treatment of urinary incontinence after radical prostatectomy. Some limitations exist in drug therapy, mid-urethral suspension, and filling agent treatment. Therefore, the use of autologous adipose-derived stem cells (ADSCs) is expected to become a first-line treatment strategy for urinary incontinence after radical prostatectomy. OBJECTIVE: To report our initial experience with transurethral injection of autologous ADSCs for the treatment of urinary incontinence after radical prostatectomy. METHODS: Patients and their families were informed of possible risks and benefits prior to the participation in the trial. After providing written informed consent, six patients with persistent urinary incontinence after radical prostatectomy were enrolled in the study. Under general anesthesia, about 50 mL of adipose tissue was obtained from each patient by liposuction. ADSCs were obtained by separation with centrifugation using the Celution cell-processing device. A mixture of ADSCs and adipose tissue was transurethrally injected into the submucosal space of the membranous urethra. Functional and anatomical improvement was assessed through a 24-hour pad test, validated patient questionnaire, urethral pressure profile, and magnetic resonance imaging (MRI) through 12-week follow-up. RESULTS AND CONCLUSION: Urine leakage volume was improved with time in all patients in the 24-hour pad test, with the exemption of temporal deterioration in two patients at the first 2 weeks post-injection. Subjective symptoms and quality of life assessed on the basis of questionnaire results showed similar improvement. The mean maximum urethral closing pressure increased from 4.312 kPa to 6.223 kPa at 12 weeks after cell injection. MRI results showed an increase in functional profile length (from 6.1 to 8.3 mm) between the lower rim of the pubic bone and the bladder neck. Adverse events, such as pelvic pain, inflammation, or de novo urgency, were undetected in any case during the follow-up. To conclude, the transurethral injection of autologous ADSCs can be a safe and effective treatment for urinary incontinence after radical prostatectomy.

The microRNA (miRNA) regulation mechanisms associated with atherosclerosis are largely undocumented. Specific selection and efficient validation of miRNA regulation pathways involved in atherosclerosis development may be better assessed by contemporary microarray platforms applying cross-verification methodology. A screening platform was established using both miRNA and genomic microarrays. Microarray analysis was then simultaneously performed on pooled atherosclerotic aortic tissues from 10 Apolipoprotein E (apoE) knockout mice (apoE-/-) and 10 healthy C57BL/6 (B6) mice. Differentiated miRNAs were screened and cross-verified against an mRNA screen database to explore integrative mRNA-miRNA regulation. Gene set enrichment analysis was conducted to describe the potential pathways regulated by these mRNA-miRNA interactions. High-throughput data analysis of miRNA and genomic microarrays of knockout and healthy control mice revealed 75 differentially expressed miRNAs in apoE-/- mice at a threshold value of 2. The six miRNAs with the greatest differentiation expression were confirmed by real-time quantitative reverse-transcription PCR (qRT-PCR) in atherosclerotic tissues. Significantly enriched pathways, such as the type 2 diabetes mellitus pathway, were observed by a gene-set enrichment analysis. The enriched molecular pathways were confirmed through qRT-PCR evaluation by observing the presence of suppressor of cytokine signaling 3 (SOCS3) and SOCS3-related miRNAs, miR-30a, miR-30e and miR-19b. Cross-verified high-throughput microarrays are optimally accurate and effective screening methods for miRNA regulation profiles associated with atherosclerosis. The identified SOCS3 pathway is a potentially valuable target for future development of targeted miRNA therapies to control atherosclerosis development and progression.
Animals ; Aorta/metabolism/pathology ; Apolipoproteins E/*deficiency/metabolism ; Atherosclerosis/genetics/pathology ; Down-Regulation/genetics ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Regulatory Networks/genetics ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/*genetics/metabolism ; Models, Biological ; *Oligonucleotide Array Sequence Analysis ; RNA, Messenger/genetics/metabolism ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction/*genetics ; Suppressor of Cytokine Signaling Proteins/genetics/metabolism ; Up-Regulation/genetics

OBJECTIVETo investigate the feasibility of transfection of recombinant human endothelial nitric oxide synthase (eNOS) into human hypertrophic scar fibroblasts (HSFbs), and to observe NO secretion and the synthesis of collagen I and III.

METHODSRecombinant human eNOS with karyocyte expressive vector was constructed in vitro, then was transfected into HSFbs which was isolated from hypertrophic scar tissues and cultured in vitro (T group). The HSFbs untransfected (normal culture) or transfected with empty-vector was used as control group and empty-vector group respectively. The mRNA expression of eNOS, collagen I and III was determined by Realtime PCR. The content of NO was determined by NO assay kit.

RESULTSThe expression of eNOS mRNA in T group was 5.92 +/- 0.21, which was obviously higher than that in empty-vector group (0.98 +/- 0.13, P < 0.05). The expression of collagen I mRNA (0.76 +/- 0.15), and collagen III (0.79 +/- 0.08) in T group was significantly lower than those in empty-vector group (0.98 +/- 0.15, 1.02 +/- 0.12, P < 0.05, respectively). The content of NO in T group (36.1 +/- 0.8 micromol/L) was obviously higher than that in empty-vector group (28.4 +/- 1.0 micromol/L, P < 0.01) and control group (27.7 +/- 1.3 micromol/L, P < 0.01).

CONCLUSIONHSFbs can be the target cells for eNOS gene transfection. The transfected cells can express eNOS and produce NO, which inhibit the synthesis of collagen.

Cicatrix, Hypertrophic ; metabolism ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; metabolism ; Humans ; In Vitro Techniques ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; RNA, Messenger ; metabolism ; Transfection

OBJECTIVETo elucidate intracellular signal pathway in formation of multidrug resistance (MDR) of hepatocellular carcinoma (HCC) induced by its microenvironment, and to explore the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.

METHODSActivity of ERK/MAPK was examined by Western blot technique through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein in HepG2 cells exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX. After being treated by the specific ERK/MAPK pathway inhibitor U0126, Western blot technique was used to analyze the alterations of the expression of P-gp, MRP1, LRP and HIF-1alpha at protein level. RT-PCR was used to analyze the alterations of the expression of HIF-1alpha mRNA. Cellular location of HIF-1alpha protein was determined by immunocytochemistry after being treated by U0126.

RESULTSThe activations of ERK/MAPK determined by the ratio of phosphorylated ERK/MAPK to the total ERK/MAPK were increased in varying degrees in HepG2 cells respectively exposed to different microenvironment. After being treated by U0126 for 12 h, the expressions of mdr1, MRP1, LRP genes and protein in those cells were decreased to some extent. However, the gene expression of HIF-1alpha was not influenced and only its protein was decreased. HIF-1alpha protein was reversely translocated into cytoplasm from nucleus after being treated by U0126.

CONCLUSIONSERK/MAPK pathway is involved in the course of the formation of MDR of HCC induced by microenvironment.

Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Immunohistochemistry ; Liver Neoplasms ; genetics ; metabolism ; pathology ; MAP Kinase Signaling System ; physiology ; Mitogen-Activated Protein Kinases ; metabolism ; Multidrug Resistance-Associated Proteins ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the effect of Shenfu injection (SFI) in treating non small cell lung cancer (NSCLC) patients on quality of life with gemcitabine (GEM) plus cisplatin (GP) regimen.

METHODSThirty-four patients were ready to receive GP regimen chemotherapy for treating NSCLC disease, according to lot-drawing, they were divided into SFI pre-treatment group (18 cases) and SFI post-treatment group (16 cases). SFI pre-treatment group: During the first treatment course, chemotherapy was begun with SFI 60 ml, intravenous dripping on the 3rd day, once daily, consecutively for 10 days; on the 1st day, GP regimen (GEM 1250 mg/m(2), intravenous dripping, on the 1st and 8th day; cisplatin 70 mg/m(2) on the 2nd day; 21 days as one cycle) was carried out; in the second treatment course GP regimen was merely given to serve as the self-control. SFI post-treatment group: the medicament sequence order was reversed from that of pre-treatment group. Using dual international quality of life (QOL) scores, the effect of SFI on the patients' QOL was observed through randomized self pre- and post-crossover control.

RESULTSThe QOL in the 34 patients after being treated by SFI in combination with GP chemotherapy regimen in one group, and GP chemotherapy regimen alone in the other, was improved in different degrees, with significant difference (P < 0.01); comparison of SFI combined with GP chemotherapy regimen with GP chemotherapy alone showed that QOL in patients was significantly different (P < 0.01).

CONCLUSIONSFI could improve QOL in patients with NSCLC who were treated with GP regimen.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Cisplatin ; administration & dosage ; adverse effects ; Cross-Over Studies ; Deoxycytidine ; administration & dosage ; adverse effects ; analogs & derivatives ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Lung Neoplasms ; drug therapy ; Male ; Middle Aged ; Quality of Life