Comparison of methods for stable co-expression of different subtype drug-matabolizing enzymes in HepG2 cells by piggyBac transposon / 中国药理学与毒理学杂志

OBJECTIVE To study the methodology of achieving stable co-expression of drug-metab?olizing enzymes in the HepG2 cells by the piggyBac (PB) transposon system. METHODS N-terminal attachment of enhanced green fluorscent protein plasmid (pEGFP- N2) and 2A peptide linked recombinant PB transposon plasmid containing dual-genes encoding drug metabolizing enzymes cyto?chrome P450 3A4 (CYP3A4) and CYP2C19 (pPB-CYP3A4-2A-2C19) were transfected into HepG2 cells respectively by Lipofectamine?LTX reagent, GenJetTM (Ver.Ⅱ) reagent and Neon?Transfection System reagent, which were widely used for large-sized DNA fragments transfection. 48 h later, the transfection efficiency and cell toxicity were detected and compared between the three methods so as to find a method with relatively high efficiency and low toxicity for later transfection.Then,three groups of recombinant PB transposons-single-gene transposon (PB-CYP3A4), 2A peptide linked dual-gene transposon (PB-CYP3A4-2A-2C19) and multiple single-gene transposon mixture〔PB-CYP3A4, PB-CYP2C8, PB-CYP2A6, organic anion transporting polypeptide 1B1 PB transposon (PB-OATP1B1)〕-were transfected into HepG2 cells respectively with the above established method.The puromycin (Puro)-resistant and GFP positive cell clones were picked up and further cultured. The mRNA, protein and metabolic levels of drug-metabolizing enzymes in monoclonal cell lines were detected by quantitative real-time PCR,Western blotting and high performance liquid chromatography-tandem mass spectrometry respectively after screening by Puro and green fluorescence. Comparisons of different groups were made using statistical analysis. RESULTS The comparison of three different transfection methods indi?cated that the transfection efficiency of GenJetTMwas up to(94.2±2.5)% and (89.3±3.3)%,significantly higher than those of the other two methods (P<0.01), along with lower cytotoxicity. Then GenJetTMwas chosen for later transfection. In the Puro-resistant monoclonal cell lines of single transposon PB-CYP3A4,PB-CYP3A4-2A-2C19 groups,the mRNA,protein and enzyme activity levels of drug-metabo?lizing enzymes were significantly increased respectively.The recombinant transposon (PB-CYP3A4-2A-2C19) containing 2A peptide could achieve stable and efficient co-expression of two metabolizing enzymes CYP3A4 and CYP2C19,while the expression of drug-metabolizing enzymes remained unbal?anced and random in those of multiple single-gene transposon mixture group (PB-CYP3A4, PB-CYP2C8,PB-CYP2A6,PB-OATP 1B1)(CYP3A4 was expressed in some cell clones only).CONCLUSION GenJetTM could be an effective method for the PB recombinant transposon transfection into HepG2 cells, by which the PB transposon could mediate stable expression of drug-metabolizing enzymes. In terms of multi-gene expression,a low and unbalanced expression is found by multiple transposons co-transfection method,which is different from that by virus mediated method.In contrast,mono-PB trans?poson linked by 2A peptide can achieve stable expression of multi-genes.

Nr2e1 Downregulation Is Involved in Excess Retinoic Acid-induced Developmental Abnormality in the Mouse Brain / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>This study aimed to investigate the expression pattern and function of Nuclear receptor subfamily 2 group E member 1 (Nr2e1) in retinoic acid (RA)-induced brain abnormality.</p><p><b>METHODS</b>The mouse model of brain abnormality was established by administering 28 mg/kg RA, and neural stem cells (NSCs) were isolated from the mouse embryo and cultured in vitro. Nr2e1 expression was detected by whole mount in situ hybridization, RT-PCR, and Western blotting. Nr2e1 function was determined by transducing Nr2e1 shRNA into NSCs, and the effect on the sonic hedgehog (Shh) signaling pathway was assessed in the cells. In addition, the regulation of Nr2e1 expression by RA was also determined in vitro.</p><p><b>RESULTS</b>Nr2e1 expression was significantly downregulated in the brain and NSCs of RA-treated mouse embryos, and knockdown of Nr2e1 affected the proliferation of NSCs in vitro. In addition, a similar expression pattern of Nr2e1 and RA receptor (RAR) α was observed after treatment of NSCs with different concentrations of RA.</p><p><b>CONCLUSION</b>Our study demonstrated that Nr2e1 could be regulated by RA, which would aid a better understanding of the mechanism underlying RA-induced brain abnormality.</p>

Effects of Transplantation with Marrow-Derived Mesenchymal Stem Cells Modified with Survivin on Renal Ischemia-Reperfusion Injury in Mice

PURPOSE: To determine whether renal injury induced by ischemia-reperfusion (I/R) could be further improved by mesenchymal stem cells (MSCs) modified with survivin. MATERIALS AND METHODS: Lentiviral vectors were used to introduce the survivin gene into MSCs and the MSCs modified with survivin were transplanted into established mice models of renal I/R injury. Seven days later, serum creatinine (Scr) and blood urea nitrogen (BUN) were measured and the survival of MSCs was determined. Hematoxylin and eosin staining was used to assess renal pathological change. The expressions of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) in kidney tissue were detected by western blot. RESULTS: Mice transplanted with survivin-modified MSCs demonstrated good renal function recovery with Scr and BUN decline close to normal levels and improvement of renal I/R injury repair. Additionally, the survival of transplanted MSCs modified with survivin was enhanced and the expression of HGF and bFGF in kidney tissue was increased. CONCLUSION: Our results demonstrated that MSCs engineered to over-express survivin could enhance their therapeutic effect on renal I/R injury in mice, probably via the improved survival ability of MSCs and increased production of protective cytokines in ischemic tissue.

Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling / 国际口腔科学杂志·英文版

Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as "bait": Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans (S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the "bait" oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The "prey" bacterial mixtures (including known species or natural saliva samples) were added, unbound cells were washed off after the incubation period and the remaining cells were eluted using 0.2 mol x L(-1) glycine. Genomic DNA was extracted, subjected to 16S rRNA PCR amplification and separation of the resulting PCR products by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F. nucleatum adhered to a variety of bacterial species including uncultivable and uncharacterized ones. This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

Transurethral prostatectomy with the bipolar plasma kinetic technique for benign prostate hyperplasia: a report of 297 cases / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the effect and safety of transurethral prostatectomy with the bipolar plasma kinetic technique (PKRP) in the treatment of benign prostate hyperplasia(BPH).</p><p><b>METHODS</b>Two hundred and ninty-seven BPH patients underwent transurethral prostatectomy with the bipolar plasma kinetic technique. The preoperative estimated weight of the prostate ranged from 35 g to 102 g, averaging 52 g.</p><p><b>RESULTS</b>The operation lasted 40 approximately 65 min, averaging 51 min. The resected tissues weighed 40 approximately 80 g, averaging 46 g. During the operation no transurethral resection (TUR) syndrome occurred. The catheter was removed 4 approximately 5 days after the operation, all with fluent urination. The patients were followed up for 2 approximately 33 months. IPSS decreased from average 31.5 preoperatively to average 6.8 postoperatively (P < 0.001). Average maximum flow-rate (Q(max)) decreased from 6.3 ml/s preoperatively to 18.6 ml/ s postoperatively (P < 0.001). Preoperative average residual urine was 97 ml and reduced to average 9 ml after the operation. Temporary incontinence occurred in 4 cases, perioperative hemorrhage in 2, and urethral stricture in 1.</p><p><b>CONCLUSION</b>Transurethral prostatectomy with the bipolar plasma kinetic technique is a safe and effective means for the treatment of BPH.</p>

Cell Transplantation to Improve Heart Function: Cell or Matrix

Current attempts to regenerate the damaged myocardium after myocardial infarction have primarily focused on therapies directed at increasing regional perfusion and reducing cell loss. Accumulating evidence suggests that implantation of healthy muscle cells into the damaged myocardium replaces the fibrotic tissue. In addition to muscle cells, stem cells in circulation, from bone marrow or in the myocardium, have recently been documented to have great potential to differentiate into myogenic cells. These neo-myogenic cells in the myocardial scar tissue prevented ventricular dilatation and delayed cardiac dysfunction. Early clinical trials show encouraging data for cellular cardiomyoplasty. Although the beneficial effects of cell therapy for myocardial regeneration after an infarction have lead to phase I clinical trials, the mechanism of the novel therapy is often questioned. Replacing the scar tissue with muscle cells and stimulating neo-vessel formation in the implanted area have been proposed. However, a number of studies recently demonstrated that the survival rate of implanted cells was too low and that number of implanted cells decreased with time after transplantation. The number of surviving cells may not be enough to form adequate new muscle tissue to repair the damaged myocardium. We recently found that extracellular matrix in the myocardium plays an important role in maintaining the ventricular chamber size, and disruption of the matrix network may contribute to the apoptosis of cardiomyocytes leading to dilated cardiomyopathy. We implanted smooth muscle cells into the heart with dilated cardiomyopathy prior to ventricular dilatation. We found that implanted cells survived in the implanted area and altered myocardial matrix metabolism both within and remote from the region of implantation. Matrix metalloproteinase activity decreased in the transplanted group as compared with control group. The matrix structure was maintained and ventricular dilatation was prevented. These data suggest that implanted cells prevented ventricular dilatation through the alteration of matrix metabolism, which is a possible mechanism for implanted cells to improve heart function.

Autologous Bone Marrow Cell Transplantation Combined with Off-Pump Coronary Artery Bypass Grafting in Human Ischemic Myocardium

Recently, autologous bone marrow cell transplantation (CTx) for angiogenesis and myogenesis in ischemic myocardium has been extensively investigated to improve heart function. This study was designed to evaluate the effects of CTx with off-pump coronary artery bypass grafting (OPCAB) in patients who were not feasible for complete revascularization. Seven male patients underwent CTx combined with OPCAB in 5, CTx only in 1, and mitral valve repair in 1 patient simultaneously. Bone marrow was aspirated from iliac bone. Mean 1.5 x109 mononuclear cells including mean 7.3 x106 CD34+ cells and 2.4 x106 AC133+ cells were obtained and concentrated with 10cc. These cells were transplanted into non-graftable ischemic myocardium. Heart function was evaluated in all patients using MIBI scan, echocardiogram and heart magnetic resonance imaging (MRI) preoperatively. The effect of CTx was evaluated using MIBI scan, echocardiogram, and MRI postoperatively. An average of 2 grafts were bypassed. Other territories were transplanted with isolated mononuclear cell. All patients had an uncomplicated postoperative course. After 2 to 7 months follow-up, there was improvement in symptom, ejection fraction (from 43% to 47%) on echocardiogram and myocardial perfusion on MIBI scan and MRI in all patients. These preliminary data showed improvement of heart function and myocardial perfusion and also showed the feasibility and safety of combined therapy with OPCAB and CTx in ischemic myocardium. However, the effectiveness of CTx alone cannot be readily assessed. Further randomized, controlled studies are required to evaluate the effectiveness of CTx alone.

The combined therapy of intervention, operation and biology in patients with middle-advanced renal cell carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the effect of the combined therapy of intervention, operation and biology in patients with middle-advanced renal cell carcinoma.</p><p><b>METHODS</b>Combined therapy was used in 52 cases as combined care groups and the single operational therapy was used in 50 cases as control group. Compare their resection rates, surgical risks and 3, 5 years survival rates.</p><p><b>RESULTS</b>In the combined care group, the excision rate was 100%, the average amount of blood transfusion during the operation was 280 ml, the average operation time was 100 minutes, and the 3, 5 years survival rates were 73% and 50%; While in the control group, the results were 90%, 396 ml, 130 minutes, 55% and 27% respectively. There were significant differences between 2 groups (P < 0.05).</p><p><b>CONCLUSION</b>Combined therapy could elevate resection rates and 3, 5 years survival rates and decrease surgical risks.</p>

Smooth Muscle Cells Transplantation is better than Heart Cells Transplantation for Improvement of Heart Function in Dilated Cardiomyopathy

Muscle cell transplantation may delay or prevent cardiac dilation in dilated cardiomyopathy. The present study was designed to compare the effects of the heart function of smooth muscle cell (SMCs) auto-transplantation and heart cell (CMs) allo-transplantation in dilated cardiomyopathic hamsters, and to determine which cells are better for cell transplantation. CMs and SMCs were isolated from BIO 53.58 hamsters, and cultured for transplantation. CMs, SMCs (4 X 10(6) cells each) or culture medium were transplanted into 17 weeks old BIO 53.58 hamsters to achieve CM transplantation (CMTx), SMC transplantation (SMCTx), and controls (Con) (N=10 each). Cyclosporine (5 mg/Kg) was administered subcutaneously to CMTx. Healthy hamsters (sham, N=6) were used to compare heart functions. Four weeks after transplantation, heart function was evaluated in all groups using a Langendorff perfusion apparatus. Histology demonstrated severe focal myocardial necrosis in the dilated cardiomyopathic hearts. CMTx and SMCTx formed huge muscle tissue in the dilated myocardium. Sham, SMCTx, and CMTx had a better heart function than Con (p < 0.01), and SMCTx had a better peak systolic pressure (p < 0.05) and developed pressure (p < 0.05) than CMTx at any balloon volume. However, sham and SMCTx were not statistically different. SMCTx and CMTx formed muscle tissue and produced better heart function in the cardiomyopathic hearts, and SMCTx showed better systolic and developed pressures than CMTx, even though they were similar in other functions. Significantly, SMCTx had heart functions, which were similar to those of healthy hamster's hearts.