放射诱导调控序列的合成及其辐射诱导特性的研究

目的　合成放射诱导调控序列并鉴定其辐射诱导特性。方法　利用人工寡核苷酸片段合成含有 6个重复CArG元件的放射诱导调控序列 ,以绿色荧光蛋白 (GFP)作为报告基因转染Tca8113细胞检测其辐射诱导特性。结果　低剂量放射线照射可诱导这种人工调控序列增强GFP在Tca8113细胞中的表达 ,且 3Gy剂量最为明显 ,提高到放射前的 16 1%。结论　人工合成放射诱导调控序列在低剂量放射线照射下能明显增强其下游外源性基因的表达 ,为进一步研究放射 -基因治疗奠定了基础。     

Distinctive population of Gfap‐expressing neural progenitors arising around the dentate notch migrate and form the granule cell layer in the developing hippocampus

In the adult hippocampus, granule cells continue to be generated from astrocyte‐like progenitors expressing glial fibrillary acidic protein (GFAP) that differ from embryonic neocortical progenitors. However, during the embryonic period, dentate granule neurons and neocortical pyramidal neurons are derived from the ventricular zone (VZ) of the pallium. Our question is when do GFAP+ progenitors of granule neurons appear in the developing hippocampus during the embryonic period, and how do they form the granule cell layer. The present analysis using Gfap‐GFP transgenic mice shows that the GFP+ distinct cell population first appears in the VZ of the medial pallium at the dorsal edge of the fimbria on embryonic day 13.5. During the perinatal period, they form a migratory stream from the VZ to the developing dentate gyrus, and establish the germinal zones in the migratory stream, and the marginal and hilar regions in the developing dentate gyrus. GFP+ cells in these regions were positive for Sox2 and Ki67, but negative for BLBP. GFP+ cells with Neurogenin2 expression were largely distributed in the VZ, whereas GFP+ cells with Tbr2 and NeuroD expressions were seen in the migratory stream and developing dentate gyrus. Prox1‐expressing GFP+ cells were restricted to the developing dentate gyrus. These results suggest that distinctive Gfap‐expressing progenitors arising around the dentate notch form germinal regions in the migratory stream and the developing dentate gyrus where they differentiate into granule neurons, indicating that distinct astrocyte‐like neural progenitors continue to generate granule neurons, from the beginning of dentate development and throughout life. J. Comp. Neurol. 522:261–283, 2014. © 2013 Wiley Periodicals, Inc.     

Characterization and gene transfer in mesenchymal stem cells derived from human umbilical-cord blood

It has been shown that the stromal-cell population found in bone marrow can be expanded and differentiated into cells with the phenotypes of bone, cartilage, muscle, neural, and fat cells. However, whether mesenchymal stem cells (MSCs) are present in human umbilical-cord blood (UCB) has been the subject of ongoing debate. In this study, we report on a population of fibroblastlike cells derived from the mononuclear fraction of human UCB with osteogenic and adipogenic potential, as well as the presence of a subset of cells that have been maintained in continuous culture for more than 6 months. These cells were found to express CD29, CD44, CD90, CD95, CD105, CD166, and MHC class, but not CD14, CD34, CD40, CD45, CD80, CD86, CD117, CD152, or MHC class II. We also compared gene expression after gene transfer using lenti- and adenoviral vectors carrying the green fluorescence protein to the MSCs derived from UCB because a reliable gene-delivery system is required to transfer target genes into MSCs, which have attracted attention as potential platforms for the systemic delivery of therapeutic genes. The lentiviral vectors can transduce these cells more efficiently than can adenoviral vectors, and we maintained transgene expression for at least 5 weeks. This is the first report showing that UCB-derived MSCs can express exogenous genes by way of a lentivirus vector. These results demonstrate that human UCB is a source of mesenchymal progenitors and may be used in cell transplantation and a wide range of gene-therapy treatments.     

糖尿病并发症相关基因醛糖还原酶酵母细胞模型的建立

目的 构建由绿色荧光蛋白（GFP）标记的糖尿病慢性并发症相关基因醛糖还原酶（AR）表达载体，并在酵母细胞中表达，建立AR抑制剂筛选模型。方法 构建AR与GFP的嵌合基因，将此AR：：GFP嵌合基因克隆到酵母表达载体pYEX-BX中，再将重组载体pYEX-BX-AR：：GFP转化至酵母宿主菌INVSC1，在SC-UD选择性培养基中表达AR：：GFP。结果 PCR扩增出约l106bp大小片段，为AR基因片段；RT-PCR在酵母细胞中检测到ARmRNA的表达；经CuS04诱导后可见AR：：GFP融合蛋白的绿色荧光；诱导剂CuSO4终浓度在100～200μmol／L范围内相对荧光强度较高，细胞密度适中；培养基SC-UD的pH值为6左右最适合细胞生长和荧光表达。结论 AR：：GFP在酿酒酵母中成功地表达，酵母细胞是研究目的基因功能与表达的良好模式菌。     

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10⁵ cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.     

Participation of bone marrow-derived cells in hippocampal vascularization after status epilepticus

PurposeDiseases such as temporal lobe epilepsy, brain trauma and stroke can induce endothelial cell proliferation and angiogenesis in specific brain areas. During status epilepticus (SE), bone marrow-derived cells are able to infiltrate and proliferate, dramatically increasing at the site of injury. However, it is still unclear whether these cells directly participate in vascular changes induced by SE.MethodTo investigate the possible role of bone marrow-derived cells in angiogenesis after seizures, we induced SE by pilocarpine injection in previously prepared chimeric mice. Mice were euthanized at 8 h, 7 d or 15 d after SE onset.ResultsOur results indicated that SE modified hippocampal vascularization and induced angiogenesis. Further, bone marrow-derived GFP⁺ cells penetrated through the parenchyma and participated in the formation of new vessels after SE. We detected bone marrow-derived cells closely associated with vessels in the hippocampus, increasing the density of blood vessels that had decreased immediately after pilocarpine-induced SE.ConclusionWe conclude that epileptic seizures directly affect vascularization in the hippocampus mediated by bone marrow-derived cells in a time-dependent manner.     

绿色荧光蛋白在成人胰腺来源的巢蛋白阳性干细胞中的表达

目的 研究绿色荧光蛋白(GFP)在成人胰腺来源的巢蛋白阳性干细胞(NPSCs)中的表达。方法 应用脂质体(lipofectin)法，将含GFP基因的质粒pEGFP—N1转染至培养的NPSCs中，用荧光显微镜观察GFP的表达。结果 大约40％的NPSCs呈现强度较均匀的绿色荧光。结论 GFP基因可以被转染至NPSCs中，并在NPSCs中表达。     

Ad-vcam-1-gfp重组腺病毒转染人脐血源基质细胞的实验研究

本研究探讨血管细胞黏附分子（vcam-1）修饰的人脐血源基质细胞（human umbilical cord blood derived strom cells，CBDSC）在造血调控中的作用。采用DNA重组技术，将目的基因vcam-1克隆至含有报告基因gfp的穿梭质粒；在BJ5183细胞中与pAdeasy—1质粒进行同源重组，产生重组腺病毒载体转染人脐血源基质细胞并进行鉴定。结果表明：vcam-1基因与pAdTrack—CMV载体成功连接，经NotⅠ／XhoⅠ双酶切后电泳可见2个大小约为9kb和2kb左右条带，经PCR法扩增后电泳可见1个600bp左右条带，证明pAdTrack—CMV—vcam-1重组质粒构建成功。pAdTrack—CMV—vcam-1质粒与pAdeasy—1质粒同源重组后，产物经PACⅠ酶切后电泳可观察到2个大小约为31kb、4kb左右条带，与预期结果相符。腺病毒载体转染人脐血源基质细胞后免疫化学、RT—PCR、荧光显微镜等方法均检测到目的基因vcam-1的表达。结论：构建ad—vcam-1-gfP重组腺病毒载体可成功转染人脐血源基质细胞并使其vcam-1表达增高。