SV40 vectors carrying minimal sequence of viral origin with exchangeable capsids

Polyomaviral vectors are generated by transfecting 293T cells with three sets of DNAs: DNA for the expression of simian virus 40 (SV40) T antigen; DNA for the expression of SV40 capsid proteins, and vector DNA harboring a reporter gene expression cassette carrying a SV40 origin. The vector DNA harbors a minimal sequence originating from SV40, and thus can carry a longer transgene. Moreover, the viable recombinants are not detectable in the vector preparation, and the vectors can transduce the DNA with efficiency similar to that of virions. Vector particles bearing capsid proteins of BK virus, JC virus, and B-lymphotropic papovavirus instead of SV40 were prepared, and they exhibited differential efficiency of gene transduction to the target cells. This method can be used to develop a surrogate system to study the functions of capsid proteins of polyomaviruses and to generate a set of polyomaviral vectors targeted at specific cell types.     

The proportionator: unbiased stereological estimation using biased automatic image analysis and non-uniform probability proportional to size sampling

The proportionator is a novel and radically different approach to sampling with microscopes based on the well-known statistical theory (probability proportional to size-PPS sampling). It uses automatic image analysis, with a large range of options, to assign to every field of view in the section a weight proportional to some characteristic of the structure under study. A typical and very simple example, examined here, is the amount of color characteristic for the structure, marked with a stain with known properties. The color may be specific or not. In the recorded list of weights in all fields, the desired number of fields is sampled automatically with probability proportional to the weight and presented to the expert observer. Using any known stereological probe and estimator, the correct count in these fields leads to a simple, unbiased estimate of the total amount of structure in the sections examined, which in turn leads to any of the known stereological estimates including size distributions and spatial distributions. The unbiasedness is not a function of the assumed relation between the weight and the structure, which is in practice always a biased relation from a stereological (integral geometric) point of view. The efficiency of the proportionator depends, however, directly on this relation to be positive. The sampling and estimation procedure is simulated in sections with characteristics and various kinds of noises in possibly realistic ranges. In all cases examined, the proportionator is 2-15-fold more efficient than the common systematic, uniformly random sampling. The simulations also indicate that the lack of a simple predictor of the coefficient of error (CE) due to field-to-field variation is a more severe problem for uniform sampling strategies than anticipated. Because of its entirely different sampling strategy, based on known but non-uniform sampling probabilities, the proportionator for the first time allows the real CE at the section level to be automatically estimated (not just predicted), unbiased-for all estimators and at no extra cost to the user.     

p53基因导致心肌细胞凋亡及其机制探讨

目的 探讨野生型ｐ５３基因与心肌细胞凋亡的关系及其可能的发生机制。方法 把野生型ｐ５３基因重组腺病毒转入体外培养的心肌细胞中,采用流式细胞计数仪（ＦＣＭ）,透射电镜等实验方法检测细胞凋亡并分析其作用机制。结果 重组腺病毒能有效地将ｐ５３基因转入心肌细胞中;转入的ｐ５３基因可以导致心肌细胞体积变小、胞浆浓缩、核固缩、染色质边集,引起细胞Ｇ１期阻滞。结论 野生型ｐ５３基因可以导致心肌细胞凋亡。并能影响细胞周期,以上结果为进一步研究心肌疾病的发病机制及其治疗提供了重要实验依据。     

pcDNA 3.1-AKR1B10真核表达载体的构建

从胰腺癌PaTu8988S细胞中提取总RNA,并克隆AKR1B10基因,构建pcDNA 3.1-AKR1B10真核表达载体。转化感受态大肠杆菌DH5α细胞,提质粒,酶切及测序鉴定,pcDNA 3.1-AKR1B10真核表达载体构建成功。     

组蛋白变异体macroH2A1真核表达载体构建及其细胞内定位的研究

目的构建带有血凝素（HA）标签的小鼠组蛋白变异体macroH2A1（mH2A1）的真核表达载体,并观察其在人胚肾293T细胞中的表达定位情况。方法提取内毒素休克的BALB/c小鼠肝脏组织的总RNA,通过逆转录-聚合酶链反应获得内毒素休克小鼠肝脏组织的cDNA,以cDNA为模板使用PCR方法扩增得到mH2A1编码序列,并将其酶切后连接至带有HA标记的载体pcDNA3-HA上;对阳性克隆进行酶切、PCR和测序鉴定。随后将重组质粒瞬时转染293T细胞,利用荧光显微镜观察。结果 PCR、双酶切和测序鉴定表明pcDNA3-mH2A1-HA真核表达质粒构建正确;经转染实验发现,该质粒能够在293T细胞中表达,表达产物主要定位在细胞核中。结论 mH2A1真核表达载体pcDNA3-mH2A1-HA的成功构建及明确其在哺乳动物细胞中的具体核定位,为进一步研究mH2A1作用细胞的信号通路提供了一个重要的工具。     

Dimensionality reduced cortical features and their use in the classification of Alzheimer's disease and mild cognitive impairment

Features defined on the cortical surface derived from magnetic resonance imaging provide important information to distinguish normal controls from Alzheimer's disease (AD) and mild cognitive impairment (MCI). We adopted cortical thickness and sulcal depth, parameterized by three dimensional meshes, as our feature. The cortical feature is high dimensional and direct use of it is problematic in a modern classifier due to small sample size problem. We applied manifold learning to reduce the dimensionality of the feature and then tested the usage of the dimensionality reduced feature with a support vector machine classifier. A leave-one-out cross-validation was adopted for quantifying classifier performance. We chose principal component analysis (PCA) as the manifold learning method. We applied PCA to a region of interest within the cortical surface. Our classification performance was at least on par for the AD/normal and MCI/normal groups and significantly better for the AD/MCI groups compared to recent studies. Our approach was tested using 25 AD, 25 MCI, and 50 normal control patients from the OASIS database.     

Endostatin neoadjuvant gene therapy extends survival in an orthotopic metastatic mouse model of renal cell carcinoma

Despite recent advances in targeted therapy, renal cell carcinoma (RCC) remains one of the most lethal urologic malignancies. Approximately 30% of patients with localised RCC will develop metastases after curative surgery. Presurgical therapy has been explored for treatment of localised RCC. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential use of an antiangiogenic agent as a neoadjuvant therapy in an orthotopic metastatic mouse model of RCC. BALB/c mice bearing Renca cells were treated before nephrectomy with NIH/3T3-LendSN cells. At the end of the experiment, ES serum levels were measured. Primary and metastatic tumour area and microvascular area were determined. In the survival studies, mice were monitored daily until they died. ES serum levels in treated mice were higher in the control group (P < 0.05). The median primary tumour area and the mean microvascular area were significantly lower in the ES-treated group compared to control group (P < 0.05). The proliferation of Renca cells in the ES-treated group was significantly reduced compared with the control group (P < 0.01). ES therapy led to a significant reduction in the number of pulmonary metastatic nodules compared with the control group (P < 0.01). Kaplan–Meier survival curves showed that the probability of survival was significantly higher in mice receiving ES therapy (P = 0.0243, Log-Rank test). Our results indicated that neoadjuvant ES gene therapy has the potential to decrease tumour burden, extend survival, and may have clinical benefit in the management of RCC.     

多功能靶向纳米载体的制备及其对神经母细胞瘤的靶向性和MRI示踪性观察

目的 制备集靶向siRNA传递及MRI显像功能于一体的多功能靶向纳米载体,并验证其对神经母细胞瘤的靶向性及MRI示踪性.方法 制备PEG-PEI-SPION,scAbGD2 -PEG-PEI-SPION并检测其基本特性.利用凝胶阻滞检测纳米载体与siRNA的复合能力;以CLSM及MRI验证纳米载体的靶向性.结果 PEG-PEI-SPION,scAbGD2-PEG-PEI-SPION平均粒径分别为(60.0±2.3)nm及(90.0±7.8)nm.氮磷比(N/P)≥2.2时siRNA与PEG-PEI-SPION,scAbGD2 -PEG-PEI-SPION完全复合.CLSM实验及MRI结果证实scAbGD2-PEG-PEI-SPION具有靶向性.结论 scAbGD2-PEG-PEI-SPION对神经母细胞瘤具有靶向传递siRNA及MRI显影的功能.     

速度向量成像评价左室型单心室短轴收缩功能

目的 观察左室型单心室短轴运动情况,评价速度向量成像(velocity vector imaging,VVI)技术估测左室型单心室患儿心室短轴收缩功能的价值.方法 左室型单心室患儿14例(单心室组)及正常儿童14例(对照组)为研究对象,应用VVI技术对主心腔短轴乳头肌水平室壁周向和径向应变及应变率进行分析.结果 单心室组前间隔、前壁、侧壁、下壁、后间隔节段的周向应变较对照组相应节段明显减低(均P ＜0.05).单心室组前间隔、前壁、侧壁、后壁、后间隔节段的周向应变率较对照组相应节段明显减低(均P ＜0.05).对照组的室间隔应变及应变率显著高于其余节段,差异有统计学意义(P＜0.05).单心室组各节段的径向应变及应变率较对照组相应节段明显减低,两组间差异具有统计学意义(P ＜0.05).对照组乳头肌水平各节段径向收缩期峰值应变及应变率差异无统计学意义(P＞0.05).结论 左室型单心室患儿存在周向和径向收缩功能不全,VVI技术能定量评价左室型单心室短轴节段室壁的收缩功能.     

3种血小板生成的关键因子共转染人骨髓基质细胞的研究

目的构建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)3个真核表达载体,并观察其在共转染的人骨髓基质细胞中的表达情况。方法应用基因重组技术,构建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)3个真核表达载体,在脂质体介导下将其导入体外培养的人骨髓基质细胞内,24 h后观察TPO、IL-6、IL-11瞬时表达情况、蛋白含量及表达,并与未转染组、空载体转染组做对比。结果 1)酶切及测序结果均可证实TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)3个真核表达载体的正确性。2)细胞转染24 h后,荧光显微镜下可观察到pEGFP-N1的表达,25%细胞发出绿色荧光;免疫细胞化学染色检测出IL-6及IL-11转染后能在细胞内表达。3)转染48 h后,Western blot检测出骨髓基质细胞内TPO、IL-6及IL-11的蛋白表达量。结论 TPO、IL-6、IL-11这3个基因可共转染人骨髓基质细胞,并在细胞内有效表达。