水压转染法在小鼠不同肝叶的表达效率

为了解水压转染法(hydrodynamics-based transfection,HDT)在小鼠肝脏不同肝叶的转染效率,将容量为小鼠体重的10%,绿色荧光蛋白基因质粒pEGFP-C1含量为35μg/只的生理盐水溶液以0.4 mL/s的速度从小鼠尾静脉注射,于注射后不同时间取小鼠各肝叶制备冰冻切片,在荧光显微镜下观察,计数各肝叶的绿色荧光蛋白表达情况。结果显示注射后12 h,绿色荧光蛋白阳性细胞比例最高,从24 h起表达量逐渐减少,至48 h时各肝叶均基本难以检测出绿色荧光蛋白阳性细胞。在12 h观察各肝叶的转染效率如下:右叶、蒂状叶的绿色荧光蛋白阳性细胞约为22%,左叶、中叶、尾状叶约为15%取材部位不同会造成数据分析的极显著差异。     

用于哺乳动物细胞转染的高纯度质粒DNA的制备

目的：建立简便高效、成本低廉和安全无污染的高纯度质粒提取方法。方法：在乙酸铵方法的基础上加以改进,主要改进之处在于增加了用聚乙二醇纯化质粒的步骤,并对溶液Ⅱ和溶液Ⅲ的成分和具体实验参数也做了合理的改进,以最少的步骤,充分去除了残存杂质,保证了质粒的超纯状态。结果：用本方法提取的质粒与用QIAGEN plasmid midi Kit提取的质粒在理化指标上没有差别,对哺乳动物胞具有同样的转染效率。结论：本方法可完全取代QIAGEN公司的试剂盒用于提取超纯质粒。     

Turtle Lung Cells Produce a Melanization-Stimulating Activity That Promotes Melanocytic Differentiation of Avian Neural Crest Cells

We found previously that neural crest cells in turtle embryos migrated into the lung buds and melanocytes were located in the lungs. The finding suggested to us that the lungs provide a stimulatory factor(s) to the differentiation of neural crest cells into melanocytes. We have established lung cell lines to facilitate analysis of the interactions of neural crest cells with the environment in melanocyte development. One cell line, TLC-2, was found to produce a putative melanization-stimulating activity (MSA), which promoted the melanocyte differentiation in vitro of avian neural crest cells. The TLC-2-derived MSA was different from that of basic fibroblast growth factor (bFGF), α-melanocyte stimulating hormone (α-MSH), and steel factor (SLF). Its molecular weight was estimated to be within the range of 150 kD. Our findings suggest that MSA may be a novel factor exercising a positive control over melanocyte differentiation.     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.

The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Development and characterization of cell lines from heart, liver, spleen and head kidney of sea perch Lateolabrax japonicus

Five cell lines (LJHK, LJS, LJL, LJH-1 and LJH-2) were established from the head kidney, spleen, liver and heart of sea perch Lateolabrax japonicus . The cell lines LJHK, LJS, LJL, LJH-1 and LJH-2 were subcultured 46, 32, 32, 36 and 34 times in minimum essential medium (MEM) supplemented with foetal bovine serum (FBS), sea perch serum and 10 ng ml⁻¹ basic fibroblast growth factor (bFGF). Morphology of primary cultures and subcultures of the five cell lines were observed continuously by microscopy. The suitable temperature for growth was 18 to 30° C for all of these cell lines with the optimum growth at 24° C and a reduced growth rate <18° C. The optimum concentration of FBS was found to be 10% and addition of bFGF to the medium significantly increased the growth rate of the cells. The doubling time of LJS, LJH-1, LJL, LJH-2 and LJHK cells was determined to be 52·7, 54·9, 57, 58·7 and 66 h at a plating density of 1 × 10⁵ cells ml⁻¹ at 24° C, respectively. Chromosome analysis revealed that 42, 48, 38, 43 and 45% cells maintained normal diploid chromosome number (48) in the LJH-1, LJH-2, LJHK, LJL and LJS cell lines, respectively. The LJHK cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies. Furthermore, treatment of the LJHK cells with lipopolysaccharide led to increased expression of IL-1β, demonstrating that LJHK cells might be a valuable tool for studying the expression and function of immunomodulatory gene in fishes.