BACKGROUND & AIMS: BACKGROUND:NFkappaB, an important transcriptional factor, has been reported to play a significant role in the coordinated transcription of cytokine and adhesion molecule genes. Therefore, blocking the NFkappaB may attenuate ischemia reperfusion injury in the myocardium. For blocking transcriptional factors, gene therapy, such as cis element "decoy," appears to be an innovative and useful therapy. This study aimed to prove the efficacy of cis element decoy against NFkappaB binding site for myocardial protection.

METHODS AND RESULTS:Rat hearts were transfected with fluorescence isothiocyanate-labeled cis element decoy against NFkappaB (NF)-binding site (NF group, n=6) and scrambled decoy (SD) group (n=6) by coronary infusion of hemagglutinating virus of Japan (HVJ)-liposome during cardioplegic arrest. Both the NF and SD groups showed marked FITC-staining in the nuclei of myocytes, demonstrating the efficacy of gene transfer into the nuclei of cardiac myocytes as compared with the control group transfected with empty liposomes. After 3 days of transfection, the NF group showed significantly higher percentages of recovery of left ventricular developed pressure (NF versus SD, 87+/-11 versus 54+/-12%) and coronary flow (97+/-16 versus 61+/-15%) than did the control hearts when exposed to ischemia (30 minutes, 37 degrees C) and reperfusion (30 minutes, 37 degrees C). The NF group showed a significantly lower percentage of neutrophil adherence to endothelial cells (38+/-6 versus 81+/-3%) and a lower tissue level of interleukin-8 (109+/-48 versus 210+/-55 ng/mg) than did the SD group.

CONCLUSION:The hearts transfected with cis element decoy against NFkappaB binding site showed significant improvement in tolerance against ischemia-reperfusion injury in association with the inhibition of neutrophil adherence and tissue IL-8 production. This suggests that NFkappaB plays a significant role in ischemia-reperfusion injury. This method, using in vivo gene transfection of cis element decoy against NFkappaB binding site, appears to be a novel and future strategy for myocardial protection.

背景与目标： 背景 : 据报道，NFkappaB是一种重要的转录因子，在细胞因子和粘附分子基因的协调转录中起着重要作用。因此，阻断NFkappaB可以减轻心肌缺血再灌注损伤。对于阻断转录因子，基因疗法 (例如顺式元件 “诱饵”) 似乎是一种创新且有用的疗法。本研究旨在证明顺式元素诱饵对NFkappaB结合位点的心肌保护作用。
方法和结果 : 用荧光异硫氰酸酯标记的顺式元素诱饵转染大鼠心脏，以对抗NFkappaB (NF) 结合位点 (NF组，n = 6) 和加扰诱饵 (SD) 组 (n = 6) 在心脏停搏期间通过冠状动脉输注日本血凝病毒 (HVJ) 脂质体。与用空脂质体转染的对照组相比，NF和SD组在心肌细胞核中均显示出明显的FITC染色，表明基因转移到心肌细胞核中的功效。转染3天后，NF组显示出明显更高的左心室发展压力恢复百分比 (NF与SD，87 +/-11对54 +/-12%) 和冠状动脉血流 (97 +/-16对61 +/-15%) 比暴露于缺血 (30分钟，37 ℃) 和再灌注 (30分钟，37 ℃) 时的对照心脏。NF组与SD组相比，中性粒细胞粘附内皮细胞的百分比显着降低 (38/-6对81/-3%)，组织interleukin-8水平降低 (109/-48对210/-55 ng/mg)。
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结论 : 用cis元件decoy转染的针对NFkappaB结合位点的心脏显示出对缺血再灌注损伤的耐受性显着改善，同时抑制了中性粒细胞粘附和组织IL-8产生。这表明NFkappaB在缺血再灌注损伤中起重要作用。该方法使用针对NFkappaB结合位点的cis元件decoy的体内基因转染，似乎是一种新颖且未来的心肌保护策略。

BACKGROUND & AIMS: :There are several pathways leading to apoptosis. It is not clear whether cells choose one of them or use multiple processes when they commit to apoptosis. MOLT-4 cells undergo apoptosis after X-irradiation through the p53-dependent pathway and/or ceramide signal. To evaluate the relative contribution of these pathways, we studied effects of the expression of various levels of transfected murine mutant p53 cDNA (TGC-->CGC of codon 173, corresponding to codonl76 in human p53) on the induction of apoptosis in X-irradiated or heated MOLT-4 cells. When survival was determined by the dye-exclusion test at 24 h after irradiation, the percentage of X-ray- or heat-induced dead cells was markedly decreased, depending on the expression level of mutant p53 protein in transfected clones. The appearance of apoptotic cells as determined by morphological changes was also decreased. These inhibitions were almost complete at 24 h after irradiation with X-rays in the case of the highest-expressing clone. p21 WAF1 protein was increased in MOLT-4 cells after X-irradiation, but not in the transfectant. These results suggest that murine mutant p53 protein has a dominant-negative effect against normal p53 in MOLT-4, and that the X-ray-induced apoptosis in MOLT-4 is fully p53-dependent.

背景与目标： : 有几种导致细胞凋亡的途径。尚不清楚细胞在进行凋亡时是选择其中之一还是使用多个过程。MOLT-4细胞在x射线照射后通过p53-dependent途径和/或神经酰胺信号发生凋亡。为了评估这些途径的相对贡献，我们研究了不同水平的转染鼠突变型p53 cDNA (密码子173的TGC->CGC，对应于人p53中的密码子76) 的表达对X-辐照或加热的MOLT-4细胞中凋亡诱导的影响。当在照射后24小时通过染料排斥试验确定存活率时，x射线或热诱导的死细胞的百分比显着降低，这取决于转染克隆中突变p53蛋白的表达水平。由形态变化决定的凋亡细胞的出现也减少了。在表达最高的克隆的情况下，这些抑制作用在x射线照射后24小时几乎完成。X射线照射后，MOLT-4细胞中p21 WAF1蛋白增加，但转染剂中没有增加。这些结果表明，鼠突变型p53蛋白对MOLT-4中的正常p53具有显性负作用，并且完全p53-dependent了x射线诱导的MOLT-4中的凋亡。

BACKGROUND & AIMS: :Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique, but with defined culture medium without feeder cells, during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells, starting from 150,000 cells. However, only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining, flow cytometry, qRT-PCR, in vitro differentiation assay, and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses, thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13, chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence, this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic.

背景与目标： : 通过转染合成的编码转录因子的修饰mRNA，已成功地从体细胞成纤维细胞中衍生出诱导多能干细胞 (iPSC)。该技术避免了在细胞重编程中使用重组DNA和病毒载体。本研究通过使用类似的技术从脂肪来源的间充质干细胞 (一名50岁女性患者) 中衍生出iPSC，但在重编程和繁殖过程中使用了没有饲养细胞的确定培养基。进行克隆选择，以从150,000细胞开始，从新生重编程细胞的各个菌落产生12个推定的iPSC系。但是，在连续10次连续传代之后，只有7条线保持了其未分化状态。然后对这七个行进行了严格的分析，以确认其作为iPSC的身份。这些测试包括免疫染色，流式细胞术，qRT-PCR，体外分化测定和SCID小鼠内的畸胎瘤形成测定。在所有分析中始终观察到阳性结果，从而验证了细胞为完全重新编程的iPSC。尽管所有7个iPSC系在第13代之前均显示正常的核子图，但7个系中的4个发生了染色体异常，其体外培养范围超过24个连续传代。只有三个系保留了正常的核型46，XX。其余四行显示出正常和异常核型的镶嵌。因此，这项研究成功地从中年患者丰富且易于获取的脂肪组织中提取了iPSC; 在无饲养者条件下利用基于mRNA的无整合技术。这是在临床中将iPSC转化为个性化再生医学的一步。

BACKGROUND & AIMS: :Treatment of many pathologies of the brain could be improved markedly by the development of noninvasive therapeutic approaches that elicit robust, endothelial cell-selective gene expression in specific brain regions that are targeted under MR image guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a noninvasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce an MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e., "sonoselective" transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing MBs with pulsed low-pressure (0.1 MPa) 1.1-MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e., 0.3-MPa and 0.4-MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low-pressure regimen does not up-regulate inflammatory or immune responses. Single-cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.

背景与目标： : 通过开发非侵入性治疗方法，可以显着改善许多脑部疾病的治疗方法，这些方法在MR图像指导下靶向的特定大脑区域中引起强大的内皮细胞选择性基因表达。虽然聚焦超声 (FUS) 与充满气体的微气泡 (MBs) 结合已成为MR图像指导的基因传递到大脑的一种非侵入性方式，但它仅用于瞬时破坏血脑屏障 (BBB)，可能会引起无菌炎症反应。在这里，我们介绍了一种MR图像引导的FUS方法，该方法引起脑血管系统的内皮选择性转染 (即 “超声选择性” 转染)，而无需打开BBB。我们首先确定，用脉冲低压 (0.1 MPa) 1.1-MHz FUS激活循环的，带有阳离子质粒的MBs促进了超声选择性基因递送到内皮，而没有MRI可检测到的BBB破坏。内皮选择性程度与FUS压力成反比变化，较高的压力 (即0.3-MPa和0.4-MPa FUS) 一致地诱导BBB开放和血管外转染。大量RNA测序分析表明，超声选择性低压方案不会上调炎症或免疫反应。单细胞RNA测序表明，超声选择性转染的脑内皮细胞的转录组不受治疗的影响。此处开发的方法允许靶向基因递送到血管，可用于促进血管生成，释放内皮细胞分泌的因子以刺激神经再生，或招募神经干细胞。

BACKGROUND & AIMS: :Five star polymers based on the positively ionizable hydrophilic 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the hydrophobic but hydrolyzable tetrahydropyranyl methacrylate (THPMA) were prepared by group-transfer polymerization (GTP) using ethylene glycol dimethacrylate (EGDMA) as the coupling agent. In particular, four isomeric star copolymers (one heteroarm, two star block, and the statistical star), all with a 3:1 DMAEMA:THPMA molar ratio, plus one star homopolymer of DMAEMA, with degrees of polymerization of the arms equal to 15, were synthesized. After star polymer preparation and preliminary characterization, the THPMA units were hydrolyzed to negatively ionizable hydrophilic methacrylic acid (MAA) untis, thus yielding star polyampholytes. All the star polyampholytes as well as the commercially available transfection reagent SuperFect were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 bearing the enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was affected by star architecture. The DMAEMA15-star-MAA5 polyampholyte presented the highest transfection efficiency of all the star polymers tested but lower than that of SuperFect at its optimum conditions. All four star copolymers showed decreased toxicity compared to the DMAEMA star homopolymer for the same amounts of star polymer tested and also compared to the SuperFect at its optimum conditions.

背景与目标： : 以乙二醇二甲基丙烯酸酯 (EGDMA) 为偶联剂，通过基团转移聚合 (GTP) 制备了基于正离子化亲水性甲基丙烯酸2-(二甲基氨基) 乙酯 (DMAEMA) 和疏水但可水解的甲基丙烯酸四氢吡喃基酯 (THPMA) 的五星聚合物。特别是，合成了四种同质星形共聚物 (一个杂臂，两个星形嵌段和统计星形)，均具有3:1的DMAEMA:THPMA摩尔比，加上一种DMAEMA的星形均聚物，臂的聚合度等于15。经过星形聚合物的制备和初步表征，将THPMA单元水解为可负离子的亲水性甲基丙烯酸 (MAA)，从而产生星形多两性电解质。评估了所有星形多两性电解质以及市售转染试剂SuperFect用带有增强绿色荧光蛋白 (EGFP) 作为报告基因的修饰质粒pRLSV40转染人子宫颈HeLa癌细胞的能力。转染效率受到星形结构的影响。DMAEMA15-star-MAA5在所有测试的star聚合物中，聚两性电解质具有最高的转染效率，但在最佳条件下低于SuperFect。对于相同数量的star聚合物，与DMAEMA star均聚物相比，所有四种star共聚物均显示出降低的毒性，并且在最佳条件下与SuperFect相比。

BACKGROUND & AIMS: :Survivin, an inhibitor of apoptosis (IAP) protein detected in many tumors but not in most normal differentiated tissues, has been widely recognized as an attractive target for cancer therapy. We previously showed that survivin expression is associated with cell proliferation. Although liposome-mediated transfection of survivin-specific siRNA decreases survivin expression and cell proliferation, these effects are limited in part by the low efficiency of the transfection. In the present study we therefore investigated the possibility of better suppressing survivin expression and cell growth by using treatments combining survivin-specific siRNA and the topoisomerase I inhibitor topotecan. Survivin-specific siRNA and topotecan given simultaneously inhibited survivin expression and cell proliferation synergistically, but topotecan alone or topotecan and siRNA given metachronously did not alter survivin expression. We hypothesized that topotecan increases the efficiency of siRNA transfection by increasing cellular uptake, and we confirmed this hypothesis by using fluorescein-labeled siRNA. Combination therapy using survivin-specific siRNA and topotecan should thus show a synergistic effect due to increased cellular uptake of siRNA and offer an attractive approach for treatment of advanced renal cancer.

背景与目标： Survivin是一种在许多肿瘤中检测到但在大多数正常分化组织中未检测到的凋亡抑制剂 (IAP) 蛋白，已被广泛认为是癌症治疗的有吸引力的靶标。我们先前显示survivin表达与细胞增殖有关。尽管脂质体介导的survivin特异性siRNA的转染会降低survivin的表达和细胞增殖，但这些作用在一定程度上受到转染效率低下的限制。因此，在本研究中，我们研究了通过使用结合survivin特异性siRNA和拓扑异构酶I抑制剂拓扑替康的治疗方法更好地抑制survivin表达和细胞生长的可能性。Survivin特异性siRNA和拓扑替康同时协同抑制survivin表达和细胞增殖，但单独给予拓扑替康或异时给予拓扑替康和siRNA不会改变survivin表达。我们假设拓扑替康通过增加细胞摄取来提高siRNA转染的效率，并且我们通过使用荧光素标记的siRNA证实了这一假设。因此，使用survivin特异性siRNA和拓扑替康的联合治疗应显示出协同作用，这是由于siRNA的细胞摄取增加，并为晚期肾癌的治疗提供了一种有吸引力的方法。

BACKGROUND & AIMS: :The Drosophila Schneider S2 (S2) Expression System enables expression of recombinant proteins constitutively, as well as inductively. This system can establish both transient and stable transformants with various selection markers. The generation of stable cell lines for increased expression or large scale expression of the desired protein is currently accomplished by cotransfection of both the expression and selection vectors. The selection vectors, pCoHYGRO and pCoBLAST, are commercially available using hygromycin-B and blasticidin S, respectively. Recently, we generated a plasmid, pCoPURO, for selection of transfected S2 cells using puromycin, which allows significant acceleration of the selection time. Although co-transfection of the selection marker with the plasmid for heterologous protein expression is functional in stable expression at short culture periods, the expression levels of stable transformants are continuously decreased during long culture times. To overcome this limitation, we generated pMT-PURO, a new plasmid that contains both the expression cassette and puromycin selection marker in a single plasmid. This system allows rapid selection and maintenance of the transformed S2 lines for extended culture periods.

背景与目标： : 果蝇Schneider S2 (S2) 表达系统可组成性和诱导性表达重组蛋白。该系统可以通过各种选择标记建立瞬态和稳定的转化子。目前，通过共转染表达和选择载体来实现用于所需蛋白质的增加表达或大规模表达的稳定细胞系的产生。选择载体pCoHYGRO和pCoBLAST分别使用潮霉素B和blasticidin S可商购。最近，我们生成了一个质粒pCoPURO，用于使用嘌呤霉素选择转染的S2细胞，这可以显着加快选择时间。尽管将选择标记物与用于异源蛋白表达的质粒共转染在短培养期间具有稳定表达的功能，但在长培养时间内，稳定转化体的表达水平会持续降低。为了克服这一限制，我们产生了pMT-PURO，这是一种新的质粒，在单个质粒中同时包含表达盒和嘌呤霉素选择标记。该系统可以快速选择和维护转换后的S2品系，以延长培养时间。

BACKGROUND & AIMS: :Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.

背景与目标： : 尽管有各种基因递送技术，但尚未对其在斑马鱼细胞培养中的应用进行广泛研究。在这里，我们报告了与其他非病毒基因递送方法相比，斑马鱼原代胚胎成纤维细胞的核转染导致更高的转染效率。使用不同大小的绿色荧光蛋白 (GFP) 基因构建体进行转染。用4.9-kb质粒获得最大的DNA摄取，导致43% GFP阳性细胞。用7.4-kb pH2B-GFP质粒进行核转染，然后进行遗传蛋白 (G418) 选择，成功地用于建立表达核组蛋白2B-GFP融合蛋白的细胞系。通过核转染有效转染斑马鱼成纤维细胞提供了质粒递送的非病毒技术，可用于在这些细胞中过表达感兴趣的基因。

BACKGROUND & AIMS: :Sirtuin 3 (SIRT3) is a deacetylase important for antioxidant protection, cell longevity, and aging. We hypothesized that SIRT3 improve oxidative resistance of aged cells and improve cell therapy in aged patients. In vitro, the proliferation and oxidative resistance of human mesenchymal stem cells (hMSCs) significantly declined with age. The expression and activity of antioxidant enzymes, including catalase (CAT) and manganese superoxide dismutase (MnSOD), increased after transfection of SIRT3 in hMSCs from older donors (O-hMSCs). The protein level of Forkhead box O3a (FOXO3a) in nucleus increased after SIRT3 overexpression. The antioxidant capacity of O-hMSCs increased after SIRT3 overexpression. 3-Amino-1,2,4-triazole (3-AT, CAT inhibitor) or diethyldithiocarbamate (DETC, SOD inhibitor) that was used to inhibit CAT or SOD activity significantly blocked the antioxidant function of SIRT3. When two inhibitors were used together, the antioxidant function of SIRT3 almost disappeared. Following myocardial infarction and intramyocardial injections of O-hMSCs in rats in vivo, the survival rate of O-hMSCs increased by SIRT3 transfection. The cardiac function of rats was improved after SIRT3-overexpressed O-hMSC transplantation. The infarct size, collagen content, and expression levels of matrix metalloproteinase 2 (MMP2) and MMP9 decreased. Besides, the protein level of vascular endothelial growth factor A and vascular density increased after cell transplantation with SIRT3-modified O-hMSCs. These results indicate that damage resistance of hMSCs decline with age and SIRT3 might protect O-hMSCs against oxidative damage by activating CAT and MnSOD through transferring FOXO3a into nucleus. Meanwhile, the therapeutic effect of aged hMSC transplantation can be improved by SIRT3 overexpression.

背景与目标： : Sirtuin 3 (SIRT3) 是一种对抗氧化保护，细胞寿命和衰老很重要的脱乙酰基酶。我们假设SIRT3改善老年细胞的氧化抗性并改善老年患者的细胞治疗。在体外，随着年龄的增长，人间充质干细胞 (hMSCs) 的增殖和氧化抗性显着下降。在年龄较大的供体 (O-hmsc) 的hmsc中转染SIRT3后，抗氧化酶 (包括过氧化氢酶 (CAT) 和锰超氧化物歧化酶 (MnSOD)) 的表达和活性增加。SIRT3过表达后，细胞核中叉头盒O3a (FOXO3a) 的蛋白水平增加。SIRT3过表达后，O-hMSCs的抗氧化能力增加。用于抑制CAT或SOD活性的3-氨基-1,2，4-三唑 (3-AT，CAT抑制剂) 或二乙基二硫代氨基甲酸酯 (DETC，SOD抑制剂) 显着阻断了sirt3的抗氧化功能。当两种抑制剂一起使用时，SIRT3的抗氧化功能几乎消失。在大鼠体内进行心肌梗塞和心肌内注射O-hMSCs后，SIRT3转染可提高O-hMSCs的存活率。SIRT3-overexpressed o-hmsc移植后大鼠的心功能得到改善。梗死面积，胶原含量以及基质金属蛋白酶2 (MMP2) 和MMP9的表达水平降低。此外，SIRT3-modified O-hMSCs移植后，血管内皮生长因子A的蛋白水平和血管密度增加。这些结果表明，hMSCs的抗坏性随着年龄的增长而下降，SIRT3可能通过将FOXO3a转移到细胞核中激活CAT和MnSOD来保护O-hMSCs免受氧化损伤。同时，SIRT3过表达可以改善老年hMSC移植的治疗效果。

BACKGROUND & AIMS: AIM:To evaluate the acting mechanism of bone morphogenetic protein-7 in anti-ischemic protective effect, we investigate the effects of BMP-7 transfection on cell apoptosis, NF-kappaB activity, and downstream genes in neonatal rat cardiomyocytes during simulated ischemia-reperfusion. METHOD:In vitro cultured neonatal rat cardiomyocytes were divided into four groups: normal control group (Group C), simulated ischemia-reperfusion group (Group IR: cultured cardiomyocytes were subjected to 2 hours hypoxia followed by 4 hours reoxygenation), transfected group (Group BT: after transfection with pcDNA3.1-BMP-7 plasmid, cardiomyocytes were subjected to 2 hours hypoxia/4 hours reoxygenation), and empty vector control group (Group ET: same as group BT except that cells were transfected with empty pcDNA3.1 vector). Malondialdehyde content, superoxide dismutase activity, and Ca(2+) concentration were measured. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining and fluorescence activated cell sorting assay were applied to determine the apoptotic rate of cardiomyocytes and immunocytochemistry and Western blot were used to detect nuclear expression of NF-kappaB in cardiomyocytes. Reverse transcription-polymerase chain reaction was used to detect mRNA expression of monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1. RESULTS:Compared with Group IR, malondialdehyde content of Group BT significantly decreased, whereas superoxide dismutase activity significantly elevated. In addition, intracellular Ca(2+) concentration of Group BT significantly reduced and apoptotic cells were significantly less. Decreased optical density ratio of NF-kappaB in the nucleus and reduced monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1 mRNA expression were also detected. CONCLUSION:These results suggest that rat recombinant BMP-7 transfection provides sustained support for the repair of myocardium from ischemia injury through reducing cell apoptosis, inhibiting lipid peroxidation, antagonizing intracellular Ca(2+) overload, and preventing the activation of downstream signaling factors in signal transduction pathway mediated by NF-kappaB.

背景与目标：

BACKGROUND & AIMS: BACKGROUND:Hydrophobic bile acids lead to the generation of oxygen free radicals in mitochondria. Accordingly, this study is to investigate if gene delivery of superoxide dismutase (SOD) will reduce hepatocyte injury caused by experimental cholestasis. METHODS:The recombinant of pLNCX-SOD gene packaged with lipofection of AMINE was transfected into hepatocytes in vitro, which stably expressed the SOD gene. RESULTS:After transfection, hepatocytes enhanced the protective effect against injury to bile and the toxicity of serum in obstructive jaundice. The inhibition of bile at the concentration of 2% (v:v, bile: DMEM 1:50) decreased obviously from (78.80+/-12.35)% to (43.35+/-9.69)% in 12 hours, from (82.55+/-11.27)% to (-26.64+/-7.66)% in 24 hours, and from (83.83+/-18.69)% to (-19.27+/-14.38)% in 48 hours, compared with that of the untransfected cells (P<0.01). The inhibition of serum at the obstructive jaudice concentration of 2.5% was obviously decreased from (89.72+/-1.52)% to (14.68+/-14.33)% in 12 hours, from (92.2+/-11.27)% to (41.39+/-7.66)% in 24 hours, and from (94.25+/-8.96)% to (22.71+/-4.38)% in 48 hours (P<0.01). CONCLUSION:Hepatocytes transfected with the pLNCX-SOD gene could obviously be resistant to the toxicity of bile and serum from rat with obstructive jaundice.

背景与目标：

BACKGROUND & AIMS: :Simple and efficient transfection methods for genetic manipulation of Plasmodium falciparum are desirable to identify, characterize and validate the genes with therapeutic potential and better understand parasite biology. Among the available transfection techniques for P. falciparum, electroporation-based methods, particularly electroporation of ring-infected RBCs is routinely used. Nonetheless, transfection of P. falciparum remains a resource-intensive procedure. Here, we report a simple and economic transfection method for P. falciparum, which is termed as the lyse-reseal erythrocytes for transfection (LyRET). It involved lysis of erythrocytes with a hypotonic RBC lysis buffer containing the desired plasmid DNA, followed by resealing by adding a high salt buffer. These DNA-encapsulated lyse-reseal erythrocytes were mixed with P. falciparum trophozoite/schizont stages and subjected to selection for the plasmid-encoded drug resistance. In parallel, transfections were also done by the methods utilizing electroporation of DNA into uninfected RBCs and parasite-infected RBCs. The LyRET method successfully transfected 3D7 and D10 strains with different plasmids in 63 of the 65 attempts, with success rate similar to transfection by electroporation of DNA into infected RBCs. The cost effectiveness and comparable efficiency of LyRET method makes it an alternative to the existing transfection methods for P. falciparum, particularly in resource-limited settings.

背景与目标： : 用于恶性疟原虫遗传操作的简单有效的转染方法是鉴定，表征和验证具有治疗潜力的基因并更好地了解寄生虫生物学的理想方法。在针对恶性疟原虫的可用转染技术中，常规使用基于电穿孔的方法，尤其是环状感染的红细胞的电穿孔。尽管如此，恶性疟原虫的转染仍然是一个资源密集型的过程。在这里，我们报告了一种简单且经济的恶性疟原虫转染方法，该方法被称为用于转染的裂解-再红细胞 (LyRET)。它涉及用含有所需质粒DNA的低渗RBC裂解缓冲液裂解红细胞，然后通过添加高盐缓冲液重新密封。将这些DNA包裹的裂解再红细胞与恶性疟原虫滋养体/裂殖体阶段混合，并进行质粒编码的耐药性选择。同时，还通过利用DNA电穿孔到未感染的红细胞和寄生虫感染的红细胞中的方法进行转染。LyRET方法在65次尝试中的63次中成功转染了具有不同质粒的3D7和D10菌株，其成功率与通过将DNA电穿孔到感染的红细胞中的转染相似。LyRET方法的成本效益和可比效率使其成为恶性疟原虫现有转染方法的替代方法，尤其是在资源有限的环境中。

BACKGROUND & AIMS: :Several methods for DNA-mediated cell transfection were tested to determine the optimal conditions for transfection of human epidermal keratinocytes. The following methods were compared: electroporation, lipofection, Ca3(PO4)2 co-precipitation, DEAE-dextran, and polybrene-mediated transfection. The transfected DNA included human keratinocyte-specific promoter for keratin K14 as well as SV40 and RSV viral promoters. Enzyme assays and in situ staining were used to evaluate both quantitative and qualitative aspects of transfection, and both subconfluent and post-confluent, stratifying keratinocytes were examined. Lipofection, Ca3(PO4)2 co-precipitation, and polybrene methods transfect very efficiently, but lipofection is expensive and Ca++ in the co-precipitation procedure induces keratinocytes to differentiate. We have found that polybrene-mediated transfection followed by a 27% DMSO shock is optimal for introducing DNA into human epidermal keratinocytes.

背景与目标： : 测试了几种DNA介导的细胞转染方法，以确定转染人表皮角质形成细胞的最佳条件。比较了以下方法: 电穿孔，脂转染，Ca3(PO4)2共沉淀，DEAE-右旋糖酐和聚布林介导的转染。转染的DNA包括角蛋白K14的人角质形成细胞特异性启动子以及SV40和RSV病毒启动子。酶测定和原位染色用于评估转染的定量和定性方面，并检查了亚融合和融合后分层的角质形成细胞。脂质转染，Ca3(PO4)2共沉淀和多brene方法非常有效地转染，但是脂质转染很昂贵，并且在共沉淀过程中Ca诱导角质形成细胞分化。我们已经发现，聚布林介导的转染随后进行27% DMSO休克是将DNA引入人表皮角质形成细胞的最佳选择。

BACKGROUND & AIMS: :Background: Previous studies have suggested a close association between REarranged during Transfection (RET) c.73 + 9277T > C and c.135G > A polymorphisms and Hirschsprung disease (HSCR) susceptibility. The results are inconsistent and contradictory. Thus, we performed a meta-analysis to evaluate the association of RET c.73 + 9277T > C and c.135G > A polymorphisms with risk of HSCR.Methods: The eligible literatures were searched by PubMed, Google Scholar, EMBASE, and CNKI up to August 5 2019.Results: A total of 20 studies including 10 studies with 1136 cases and 2420 controls on c.73 + 9277T > C and 10 studies with 917 cases and 1159 controls on c.135G > A were selected. Pooled ORs revealed that c.73 + 9277T > C and c.135G > A polymorphisms were significantly associated with an increased risk of HSCR. Moreover, stratified analysis revealed that c.73 + 9277T > C and c.135G > A polymorphisms were associated with HSCR risk in Asian, Caucasian and Chinese populations.Conclusions: This meta-analysis result indicated that the RET c.73 + 9277T > C and c.135G > A polymorphisms were associated with susceptibility to HSCR.

背景与目标： 背景: 先前的研究表明，转染过程中的重排 (RET) c.73   +   9277T  > C和c.135G  > A多态性与Hirschsprung病 (HSCR) 易感性密切相关。结果是前后矛盾的。因此，我们进行了荟萃分析，以评估RET c.73   +   9277T  > C和c.135G  > A多态性与HSCR风险的关系。方法: 通过PubMed，Google Scholar，EMBASE和CNKI检索符合条件的文献，至2019年8月5日。结果: 共选择了20项研究，包括10项1136例和2420例对照的c.73   +   9277T  > C研究和10项917例和1159例对照的c.135G  > A研究。汇总ORs发现，c.73   +   9277T  > C和c.135G  > A多态性与HSCR风险增加显著相关。此外，分层分析显示，在亚洲、高加索和中国人群中，c.73   +   9277T  > C和c.135G  > A多态性与HSCR风险相关。meta分析结果表明，RET c.73   +   9277T  > C和c.135G  > A多态性与HSCR易感性相关。

BACKGROUND & AIMS: :Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-β1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.

背景与目标： : 在出生后发育阶段，鼠第一磨牙牙胚中簇蛋白 (Clu) 的表达显着增加。如前所述，该基因的表达时程与其他编码齿状细胞分泌阶段涉及的蛋白质的基因的表达时程平行。鼠磨牙牙胚中clusterin的免疫组织化学研究表明，该蛋白位于釉质外上皮，退化的釉质器官，分泌成釉细胞以及在萌发初期将牙齿连接到口腔上皮的牙齿上皮中。转化生长因子 β1 (TGF-β1) 的免疫标记显示其位于聚集蛋白附近。用anti-miR-214体内转染后，Clu和Tgfb1的表达水平显着降低。相反，通过这种处理增加了与生长和发育调节相关的几个基因的表达。我们建议簇蛋白在分泌型牙本质发生和早期爆发阶段具有功能。用anti-miR-214治疗后的生物信息学分析表明，尽管与牙齿矿化和萌发相关的细胞活动受到抑制，但与替代发育活动 (即收缩蛋白的生物合成) 相关的活动似乎受到刺激。这些变化可能是通过共同的转录因子簇介导的调节而发生的，并支持microRNAs (miRNAs) 作为牙形成过程中分化的调节因子非常重要的建议。