慢病毒载体介导不同内部启动子驱动绿色荧光蛋白表达效率

目的在人类免疫缺陷病毒-1(HIV-1)慢病毒载体中引入三种不同的内部启动子来驱动绿色荧光蛋白基因(GFP)的表达,初步比较内部启动子的效率。方法慢病毒载体三质粒系统中,转移质粒的构建采用限制性内切酶酶切、T4DNA连接酶连接方法和内切酶酶切鉴定。磷酸钙沉淀法将三质粒共转染293T包装细胞。病毒滴度的测定采用6孔板培养感染细胞,荧光显微镜计数GFP阳性细胞。通过荧光显微镜观察绿色荧光的表达情况判断启动子的效率。结果构建了含PPT元件和含不同内部启动子和GFP的质粒。转染293T细胞后均观察到较强的绿色荧光。表达荧光的293T细胞数以巨细胞病毒(CMV)启动子最多,肝细胞特异性启动子(LSP)较少,泛醌启动子(PUB)介于两者之间。CMV为内部启动子的慢病毒载体滴度5×106I U/ml,其他二种启动子的滴度(1~2)×105I U/ml。结论在所选择的三种内部启动子中,CMV启动子的效率最高,LSP较强,PUB介于两者之间。     

Lifespan extension by n-butanol extract from seed of Platycladus orientalis in Caenorhabditis elegans

Aim of the study

As a traditional Chinese medicine, seed of Platycladus orientalis(Linnaeus) Franco has been extensively used as a tonic and sedative remedy. The present study was conducted to investigate whether lifespan was extended and the mechanisms of n-butanol extract from seed of Platycladus orientalis (BSPO) in Caenorhabditis elegans. The findings could provide the pharmacological basis for a treatment in traditional medicine.

Materials and methods

Lifespan extension by BSPO was evaluated under normal culture conditions and in a stress test. A possible mechanism of the anti-aging effect of BSPO, a change in the stress-resistance of related proteins, was also investigated in C. elegans.

Results

It has been shown that BSPO could significantly extend lifespan of C. elegans in a concentration dependent manner under normal culture conditions and stress. Further studies demonstrated that BSPO treatment significantly decreased reactive oxygen species (ROS) accumulation, up-regulated resistance to stress of related proteins, including glutathione S-transferase-4 (GST-4) and heat shock protein-16.2 (HSP-16.2), and reduced the amount of lipofuscin in transgenic C. elegans.

Conclusion

These results indicated that BSPO extended the lifespan, which could be attributed to its direct ROS scavenging activity, reducing the amount of lipofuscin and increasing the expression of gens associated with resistance to stress. These obtained data provided valuable support for traditional clinical practice to extend lifespan and to provide tonic remedy.     

GFP小鼠对三维体外着床模型的定位及验证

目的 本研究将基于已经建立的三维小鼠体外胚胎着床模型,运用绿色荧光（GFP）小鼠胚胎替代ICR小鼠胚胎,验证三维着床模型的可行性。方法 将孕第4天GFP小鼠及ICR小鼠囊胚与ICR小鼠的内膜组织进行体外共培养。观察两组胚胎黏着率的差异。对GFP小鼠囊胚组通过荧光显微镜进行观察,共培养后进行组织学研究。结果 GFP小鼠囊胚及ICR小鼠囊胚与ICR小鼠子宫内膜组织共培养28h后的胚胎黏着率分别为46.6%及54.8%;两组黏着率之间差异无统计学意义（P=0.364）。GFP小鼠囊胚与ICR小鼠子宫内膜组织共培养28h及40h后均可在荧光显微镜下见到胚胎,组织学研究证实GFP小鼠囊胚可以黏着于ICR小鼠子宫内膜。结论 三维着床模型确实能够实现不同来源小鼠胚胎的黏附着床。运用GFP小鼠胚胎还发现了胚胎与子宫内膜组织之间存在着信息及分子联系。     

Effect of low-molecular-weight heparin on the commitment of bone marrow cells to liver sinusoidal endothelial cells in CCl(4)-induced liver injury.

BACKGROUND/AIMS: Recently liver regeneration by bone marrow transplantation has been proposed as an alternative source of functional liver cells. We investigate commitment of bone marrow cells (BMCs) to liver regeneration and the effect of dalteparin sodium (DS) on regeneration of the damaged liver caused by carbon tetrachloride (CCl(4)) administration in the mice. METHODS: Liver injury was produced in 8-week-old mice by treating with CCl(4) for 4 weeks. Thereafter, mice received a lethal dose of irradiation (10Gy) to whole body, followed by injection of 1x10(7) green fluorescent protein (GFP)-positive BMCs via the tail vein. DS (50IU/kg, intraperitoneally) was administered daily for 28 consecutive days starting at 1 day post-BMC transplantation. Lineage marker analysis of GFP-positive liver cells was performed immunostaining with a CD31 antibody. RESULT: Four weeks after BMC transplantation, GFP-positive cells in the CCl(4)-damaged liver could be detected in the lobule displaying a meshwork architecture extending from the periportal to pericentral regions, a pattern simulating sinusoidal lining. This localization of GFP-positive cells suggested that these cells were closely associated with sinusoidal endothelial cells. By staining the GFP-positive cells for CD31, it was confirmed that the majority of the GFP-positive cells are also positive for CD31. The GFP(+)CD31(+) cells were barely detected in the control group (1.0+/-1.2 per field). In marked contrast, a numerous number of GFP(+)CD31(+) cells were detected in the liver section obtained from the CCl(4)-induced liver damage group (3.8+/-1.3 per field, P<0.05 versus control). The number of GFP(+)CD31(+) cells in CCl(4) plus DS-treated group was further increased to 8.3+/-1.3 per field (P<0.05 versus CCl(4)-induced liver damage group). CONCLUSION: The majority of GFP-positive BMCs was committed to sinusoidal endothelial cells. DS promoted BMC differentiation into sinusoidal endothelial cells in the CCl(4)-damaged liver.     

Promotion of Transplanted Bone Marrow-derived Cell Migration into the Periodontal Tissues due to Orthodontic Mechanical Stress

Background: Bone marrow-derived cells (BMCs) have abilities of cell migration and differentiation into tissues/organs in the body and related with the differentiation of teeth or periodontal tissue including fibroblasts. Then, we examined the effect of orthodontic mechanical stress to the transplanted BMC migration into periodontal tissues using BMC transplantation model.Material and Method: BMC from green fluorescence protein (GFP) transgenic mice were transplanted into 8-week-old female C57BL/6 immunocompromised recipient mice, which had undergone 10 Gy of lethal whole-body-irradiation. Five mice as experimental group were received orthodontic mechanical stress using separator between first molar (M1) and second molar (M2) 1 time per week for 5 weeks and 5 mice as control group were not received mechanical stress. The maxilla with M1 and M2 was removed and was immunohistochemically analyzed using a Dako Envision + Kit-K4006 and a primary anti-GFP-polyclonal rabbit antibody. Immunohistochemically stained was defined as positive area and the pixel number of positive area in the periodontal tissue was compared with the previously calculated total pixel number of the periodontal tissue.Results: The immunohistochemistry revealed that GFP positive cells were detected in the periodontal tissues, both in the experimental and control specimens. The ratio of pixel number in the examination group showed 5.77 ± 3.24 % (mean ± SD); and that in the control group, 0.71±0.45 % (mean ± SD). The examination group was significantly greater than that of control group (Mann-Whitney U test: p<0.001).Conclusion: These results suggest that orthodontic mechanical stress accelerates transplanted BMC migration into periodontal tissues.     

Identification of two novel type 1 peroxisomal targeting signals in Arabidopsis thaliana

Peroxisomes lack their own genetic material and must therefore import proteins encoded by genes in the nucleus. Amino acids within these proteins serve as targeting signals: they direct the delivery of the proteins to the organelle. The majority of soluble proteins destined for the peroxisomal matrix utilize a type 1 peroxisomal targeting signal (PTS1): a C-terminal tripeptide that follows the pattern small/basic/hydrophobic. We have discovered two new C-terminal tripeptides that target proteins to peroxisomes in Arabidopsis thaliana. The tripeptides PSL and KRR do not fit the major PTS1 consensus but cause green fluorescent protein to accumulate in peroxisomes of stably transformed Arabidopsis. We have identified forty-one proteins in the Arabidopsis genome that also bear these tripeptides at their C-termini and may therefore be peroxisomal.     

Auditory circuit in the Drosophila brain

Most animals exhibit innate auditory behaviors driven by genetically hardwired neural circuits. In Drosophila, acoustic information is relayed by Johnston organ neurons from the antenna to the antennal mechanosensory and motor center (AMMC) in the brain. Here, by using structural connectivity analysis, we identified five distinct types of auditory projection neurons (PNs) interconnecting the AMMC, inferior ventrolateral protocerebrum (IVLP), and ventrolateral protocerebrum (VLP) regions of the central brain. These auditory PNs are also functionally distinct; AMMC-B1a, AMMC-B1b, and AMMC-A2 neurons differ in their responses to sound (i.e., they are narrowly tuned or broadly tuned); one type of audioresponsive IVLP commissural PN connecting the two hemispheres is GABAergic; and one type of IVLP-VLP PN acts as a generalist responding to all tested audio frequencies. Our findings delineate an auditory processing pathway involving AMMC→IVLP→VLP in the Drosophila brain.     

Site‐specific cytomorphology of disseminated PC‐3 prostate cancer cells visualized in vivo with fluorescent proteins

Circulating tumor cells (CTC) may reach multiple organ sites. However, CTC seeding and growth in distant organs is not random. Each metastatic site may contain a specific subpopulation of the original metastatic tumor capable of growing at that site. The fluorescent orthotopic prostate cancer model (PC‐3‐GFP) model was used for immunomagnetic capture of CTC. The captured CTC were efficiently cultivated in vitro. PC‐3‐GFP cells were also isolated from various metastatic sites, grown in vitro and examined under fluorescence microscopy. The differential morphology was compared of primary tumor cells, CTC and disseminated (DTC) from multiple metastatic sites, from nude mice with orthotopic PC‐3‐GFP. The cultured captured CTC and DTC from various organs have distinctive morphologies. Distinct cancer cell morphologies were observed at different metastatic sites as well as among CTC. The distinct morphologies were maintained during in vitro culture. The results demonstrate extensive tumor heterogeneity that could account for the widely different behavior of cancer cells in a single tumor. Further hetereogeneity testing would be a big promise for personalizing the cancer treatment in the future. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

A histological survey of green fluorescent protein expression in 'green' mice: implications for stem cell research

AIMS: The transgenic enhanced green fluorescent protein (EGFP) expressing 'green' mouse (C57BL/6-TgN(ACTbEGFP)1Osb) is a widely used tool in stem cell research, where the ubiquitous nature of EGFP expression is critical to track the fate of single or small groups of transplanted haematopoietic stem cells (HSC). Our aim was to investigate this assumed ubiquitous expression by performing a detailed histological survey of EGFP expression in these mice. METHODS: Fluorescent microscopy of frozen tissue sections was used to perform a detailed histological survey of the pattern of EGFP expression in these mice. Flow cytometry was also used to determine the expression pattern in blood and bone marrow. RESULTS: Three patterns of EGFP expression were noted. In most tissues there was an apparently stochastic variegation of the transgene, with individual cell types demonstrating highly variable rates of EGFP expression. Certain specific cell types such as pancreatic ductal epithelium, cerebral cortical neurones and glial cells and glomerular mesangial cells consistently lacked EGFP expression, while others, including pancreatic islet cells, expressed EGFP only at extremely low levels, barely distinguishable from background. Lastly, in the colon and stomach the pattern of EGFP expression was suggestive of clonal inactivation. Only cardiac and skeletal muscle showed near ubiquitous expression. CONCLUSIONS: These findings raise questions regarding the 'ubiquitous' expression of EGFP in these transgenic mice and suggest caution in relying overly on EGFP alone as an infallible marker of donor cell origin.