Establishment of a screening cell model for cannabinoid type 1 receptor agonists / 药学学报

Cannabinoid receptors are one of the most expressed G protein-coupled receptors in the central nervous system, which are potential drug targets for inflammation, pain and drug abuse. Cannabinoid receptors are composed of type 1 receptor (CB1R), type 2 receptor (CB2R) and other receptors, of which CB1R plays a vital role in regulating central memory, cognition, and motor function. Therefore, screening CB1R agonists has potential value in treating nervous system diseases. In this study, the intracellular loop 3 (ICL3) domain of CB1R was replaced with a circular-permutated enhanced green fluorescent protein (cpEGFP). After infecting HEK 293T cells with lentivirus particles, we obtained a stable cell line that was overexpressed human CB1R-cpEGFP after puromycin selection. The interaction between receptor agonists and CB1R led to the change of receptor conformation, resulting in de-protonation of the EGFP, and enhancing the fluorescence intensity. Therefore, active CB1R compounds could be verified by measuring the fluorescence intensity. Using CB1R agonist arachidonyl-2′-chloroethylamide (ACEA) as a positive control to evaluate the reliability of this model, studies have shown that ACEA could induce receptor activation and increase fluorescence intensity, while antagonist rimonabant inhibited receptor activation with unchanged fluorescence intensity. In conclusion, this study successfully constructed a fluorescent probe screening model for CB1R agonists.

Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein / 北京大学学报(医学版)

OBJECTIVE@#To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs.@*METHODS@#The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot.@*RESULTS@#The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05).@*CONCLUSION@#A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.

Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer / 中山大学学报(医学科学版)

ObjectiveTo construct a human ovarian cancer cell line SKOV3 （SK-Luc-EGFP） stably co-expressing luciferase （Luc） and enhanced green fluorescent protein （EGFP） and to explore its application in ovarian cancer research both in vitro and in vivo. MethodsThe recombinant plasmid pCDH-Luc-T2A-EGFP-Puro was constructed by introducing a Luc-T2A-EGFP fusion gene fragment amplified by Overlap PCR into plasmid vector. The three-plasmid lentivirus packaging system was transfected into HEK 293T cells and the viral supernatant was harvested to infect SKOV3 cells. SK-Luc-EGFP cell line with the highest fluorescence intensity of EGFP was obtained by puromycin selection and flow cytometry assessment， and the Luc expression of the cell line was subsequently validated by in vitro bioluminescent assay. SK-Luc-EGFP cells were further explored for the following applications： distinguishing SK-Luc-EGFP cells from non-tumor cells in ascites by flow cytometry and confocal microscopy； visualizing adhesion of SK-Luc-EGFP cells to mesothelial cells or omentum by fluorescence microscopy； monitoring process of SK-Luc-EGFP tumorigenesis by in vivo bioluminescence imaging. ResultsA recombinant lentiviral expression plasmid pCDH-Luc-T2A-EGFP-Puro was constructed and packaged into lentiviral particles that were then transfected into SKOV3 cells to generate SK-Luc-EGFP cell line. The purity of SK-Luc-EGFP cells based on EGFP expression was 100% as validated by fluorescence microscopy and flow cytometry； SK-Luc-EGFP cells could be visually distinguished from non-tumor cells in ascitic fluid by flow cytometry and confocal imaging. Moreover， Luc expression in SK-Luc-EGFP cells was verified by in vitro bioluminescence assay， and a linear relationship with a correlation coefficient of 0.997 9 was found between cell number and the bioluminescent signal. Adhesion of SK-Luc-EGFP cells to mesothelial cells was directly observed by fluorescence imaging in in vitro adhesion assay； peritoneal adhesion of SK-Luc-EGFP cells to omentum was also observed after intraperitoneal （i.p.） injection of SK-Luc-EGFP cells in nude mice； in the peritoneal metastasis mouse model established by i.p. injection of SK-Luc-EGFP cells， monitoring of tumorigenesis process was achieved by in vivo bioluminescence imaging. ConclusionSK-Luc-EGFP cell line is a useful tool for investigating ovarian cancer in vitro and in vivo.

Preparation of transgenic Musca domestica by microinjection method / 生物工程学报

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.

Experimental observation of transplantation of rabbit corneal stromal cells with LV-EGFP<i> in vitro</i> / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the survival time and distribution of rabbit corneal stromal cells(CSCs)after transplantation of rabbit corneal <i>in vitro</i>. <p>METHODS: Primary rabbit CSCs was cultured <i>in vitro</i> and identified by immunohistochemical staining. using lentivirus(LV)with marker gene enhanced green fluorescent protein(EGFP)transfection rabbit CSCs, the growth status and fluorescence intensity of the transfected cells were observed under an inverted fluorescence microscope. The <i>in vitro</i> animal experiments were randomly divided into 2 groups. experimental group lines of LV-EGFP tag of rabbit CSCs suspension stromal injection, control group amount of normal saline injection corneal stroma, Frozen sections were taken 1wk and 1mo after surgery to observe the fluorescence of transplanted CSCs, and hematoxylin-eosin(HE)was used to observe the tissue morphology of paraffin sections. <p>RESULTS: LV-EGFP transfected rabbit CSCs showed a small amount of fluorescence after 24h under an inverted fluorescence microscope, with the strongest at 96h and 110h. There was no significant difference in the morphology of the transfected CSCs and normal CSCs. Green fluorescence can be seen in the stromal layer of the cornea in the experimental group at 1wk and 1mo, while there is no green fluorescence in the control group. Paraffin section for 1wk showed obvious epithelial cell hyperplasia and slight corneal edema in the experimental group, and a small amount of inflammatory cell infiltration. 1mo after surgery, the epithelial cell hyperplasia was weakened in the experimental group, and no corneal layer edema was observed. No obvious abnormality was found in the control group for 1wk and 1mo. <p>CONCLUSION: Extracorporeal corneal stroma transplantation of LV-EGFP labeled rabbit CSCs can survive at least 1mo in the corneal and is compatible with adjacent tissues.

Lentiviral vector mediated enhanced green fluorescent protein screening for stably transfected rabbit bone marrow mesenchymal stem cells / 西安交通大学学报(医学版)

Objective: To isolate, culture and identify rabbit bone mesenchymal stem cells (BMSCs) so as to explore the optimal conditions for lentiviral vector-mediated enhanced green fluorescent protein (eGFP) infection in rabbit BMSCs and screen stable transfected BMSCs in rabbits. Methods: BMSCs were obtained by whole bone marrow adherence method. The osteogenic, chondrogenic and adipogenic differentiation of BMSCs was made by alizarin red, toluidine blue and oil red O staining, respectively. The expressions of CD44 and CD90 were detected by immunofluorescence. The concentration of puromycin was used to screen the minimum lethal concentration of BMSCs; the lentiviral vector with multiplicity of infection (MOI) of 50, 100, 150 and 200 mediated eGFP BMSCs were infected; the fluorescence expression was observed under an inverted microscope, and the stable transformation system was screened with puromycin. Results: When MOI was 150, lentiviral vector-mediated eGFP infection of rabbit BMSCs was the most efficient. The optimum concentration of puromycin for stable transfection of rabbit BMSCs was 1.0 μg/mL. Conclusion: Rabbit BMSCs were successfully cultured in this experiment. The stem cells were labeled with lentivirus-mediated GFP and stable transfected rabbit BMSCs were screened. A simple and effective stem cell labeling method was established to label BMSCs in vivo.

Fluorescence tracing of umbilical cord blood mesenchymal stem cells in transplantation treatment of scalded wound in mice / 国际检验医学杂志

Objective Using the previously established mesenchymal stem cells strain derived from human fetal umbilical cord blood (FUCB-MSCs) to culture then label enhanced green fluorescent protein (EGFP) , and to observe skin repair effects of FUCB-MSCs by GFP tracing after exogenous FUCB-MSCs transplantation on to scald wound models of SCID mice.Methods FUCB-MSCs were labeled GFP by transfection with the recombinant retrovirus containing EGFP gen;The established SCID scald mice model were randomLy divided into 3groups, low dose group, high dose group and control group, 6rats each group, 2wounds each mouse, 12wounds in total, then were tail intravenous injected into 0.2mL 1×106, 0.2mL 2×106 GFP-FUCB-MSCs cells, and same volume of medium respectively.On 9days after transplantation, the sections from scald wound area were observed the expression of GFP under the fluorescence microscope and the others were analyzed by the bright-field microscopy after HE staining, and the area of wound surface and the number of wound cells were compared simultaneously.Results After 48h, expression of EGFP in FUCB-MSCs can be seen under the fluorescence microscope, positive rate of GFP was>80％, and after 6weeks GFP expression is still stable, besides, the positive expression of human GFP can be observed after transplantation and there were no fluorescence decay in transplantation after 3weeks.Compared with the control group, there was a significant difference in wound area and wound cell number in the low and high-dose group (P<0.05) .ConclusionGFP can be used as a tracking marker to label FUCB-MSCs during transplantation treatment.It indicates that exogenous FUCB-MSCs can migrate to the scalded wounds via blood circulation system and continuously participate in the repair through SCID mouse.

Site-specific biotinylation of EGFP with double biotin molecules based on Avi-tag technology / 中国免疫学杂志

Objective:To prepare the site-specific biotinylation of enhanced green fluorescence protein with double biotin molecules using Avi-tag technology.Methods:The EGFP gene was prepared by PCR and cloned into pdi-Avitag resulting the vector pEGFP-( Avitag) 2.The fusion protein EGFP-( Avitag ) 2 was expressed in E.coli DH5αand purified by employing IMAC.The site-specific biotinylation was implemented by BirA enzyme in vitro, and then was identified by competitive ELISA and Western blot.Results:The recombinant prokaryotic expression vector pEGFP-(Avitag)2 was correctly constructed,and EGFP-(Avitag)2 fusion was successfully expressed in E.coli DH5α.The results of competitive ELISA and Western blot showed that the EGFP-( Avitag) 2 could be site-specific biotinylation with double biotin molecules based on Avi-tag technology.Conclusion: The site-specific biotinylation of EGFP with double biotin molecules is successfully prepared,and we anticipate that can be used for BAS to improve the sensitivity and specificity of immunosensors.

Weakening resistance marker for establishing a process of screening high-producing CHO cell lines / 中国药科大学学报

To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.

Recombinant adeno-associated virus serotype 9 transfection of atherosclerosis mice:determination of the optimal expression time in vivo / 中国组织工程研究

BACKGROUND:Recombinant adeno-associated virus serotype 9 has a high affinity in myocardial tissue, and the expression of recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein (rAAV9-eGFP) in the aorta of atherosclerosis mice is not clear. OBJECTIVE:To explore the optimal time point of rAAV9-eGFP expression in the aorta of atherosclerosis mice. METHODS:Atherosclerosis model was established with high-fat diet in 30 ApoE-/-mice for 16 weeks. Among them, 25 mice were injected with 5.0×1011 vg (virus genomes) rAAV9-eGFP through the tail vein, while the remaining 5 mice were injected with saline, serving as the control group. The virus-transfected mice were kil ed at 14, 21, 28, 35 and 60 days after transfection, and aortic tissue was harvested. The expression of enhanced green fluorescent protein was detected with laser scanning confocal microscope. Western blot assays were used to detect the expression of enhanced green fluorescent protein in aorta. The expression of enhanced green fluorescent protein in vivo was observed and the optimal expression time point was determined. RESULTS AND CONCLUSION:rAAV9-eGFP effectively transfected the aorta of atherosclerosis mice, enhanced green fluorescent protein was expressed in aortic tissue, and the expression intensity increased gradual y with the increasing transfection time. The highest expression level was found at 35 days after transfection and then maintained stable at 60 days. There were significant differences at different time points after transfection (P<0.001). These data indicate that rAAV9-eGFP can be effectively expressed in the aorta of atherosclerosis ApoE-/-mice and rAAV9-eGFP can be regarded as the optimal vector in the treatment of atherosclerosis.

Effects of Human Umbilical Cord Mesenchymal Stem Cells on Renal Ischaemia-reperfusion Injury in Rats

Objective This study aims to observe the function of umbilical cord-mesenchymal stem cells (UC-MSCs) labelled with enhanced green fluorescent protein (eGFP) in the repair of renal ischaemia-reperfusion (I/R) injury, to determine the effects on inflammatory cascade in an established rat model and to explore possible pathogenesis. Materials and Methods Sixty rats were randomly divided into three groups: the sham-operated, I/R and UC-MSC treatment groups. All rats underwent right nephrectomy. Ischaemia was induced in the left kidney by occlusion of the renal artery and vein for 1hour, followed by reperfusion for 24 hours or 48 hours. Kidney samples were collected to observe morphological changes. Immunohistochemistry was performed to assess the expression of intercellular adhesion molecule 1 (ICAM-1) in the renal tissue sample, as well as the number of infiltrating polymorphonuclear neutrophils (PMNLs) and UC-MSCs with positive eGFP. Results Renal histopathological damages and the expression of ICAM-1 and PMNL increased significantly in the I/R group compared with those in the sham-operated group, whereas the damages were less conspicuous in the UC-MSC treatment group. Conclusions Renal ICAM-1, which mediated PMNL infiltration and contributed to renal damage, was significantly up-regulated in the I/R group. UC-MSCs were identified to inhibit these pathological processes and protect the kidney from I/R injury. .

Identification of the expression of recombinated plasmid pLNCX-EGFP-C1-hri in PA317 packaging cell line / 中国医学装备

Objective:To identify the expression of plasmid pLNCX-EGFP-C1-hri targeting the gene of Human ribonuclease inhibitor (hri) in PA317 cells which is capable of expression in mammalian cells.Methods: The vector of pLNCX-EGFP-C1-hri was transfected into PA317 cells by Lipofectamine 2000 and then the expression of recombinated plasmid was verified in living cells by observing the transcription level of egfp-hri fusion gene mRNA with RT-PCR method and the expression level of egfp-fusion hRI protein with western blotting method respectively.Results: Both RT-PCR and western blotting showed the egfp-hri fusion gene was obviously expression in PA317 cells.Conclusion: The plasmid of pLNCX-EGFP-C1-hri targeting hRI is successfully constructed and the protein of hRI can be expressed in PA317 cells correctly.

Study on NIH3T3 cells infected by retrovirus encoding EGFP fusion of human ribonuclease inhibitor protein / 中国医学装备

Objective:To identify the expression of egfp-hri fusion on NIH3T3 cells which is carried by recombinant retrovirus. Methods:The egfp-hri fusion gene was integrated intoNIH3T3 cells by the infection of recombinant viral supernatant from the G418 resistent PA317 cells transfected stably with the plasmid of pLNCX-EGFP-C1-hri. The expression effect of egfp-hri fusion was visualized under the fluorescent microscope, while the expression levels of egfp-hri fusion protein by Western blot. Results:The observation from fluorescent microscope showed the green fluorescent was obviously observed in the cytoplasm , western-blotting showed the expression level of egfp-hri fusion gene increased by 83%and 81%, as compared with those uninfected cells and in the cells infected with empty vector, respectively. Conclusion:The infected NIH3T3 cells can express egfp-hri fusion protein successfully.

Gene transfer characteristics and cell tropism of recombinant adenovirus in the hippocampus of adult mouse / 中华神经医学杂志

Objective To explore the gene transfer characteristics and different cell tropism of the recombinant adenovirus serotype 5 (rAd5) driven by murine cytomegalovirus (mCMV) immediate-early promoter in the hippocampus of adult mouse,and provide a basis for adenovirus-mediated transgene research in the hippocampus of mouse in the future.Methods Forty-two male healthy adult C57BL/6 mice were injected with the titre and volume matched rAd5 vectors which contained enhanced green fluorescent protein (EGFP) sequences as a reporter gene.Then animals (n=6 for each point) were killed 1,3,7,14,30,45,60 days after injection.The coronal cryosections of brains were processed,and EGFP expression was observed with fluorescence microscopy.Double-label immunofluorescence method was used to observe the EGFP expression in different types of cells to identify the cell tropism in the hippocampus.Results The expression of EGFP carried by rAd5 vectors was detected on the first day after the transfection,with peak expression between days 3 and 45,while EGFP activity decreased sharply 60 days after transfection.EGFP highly expressed in the dentate subgranular zone (SGZ) of the hippocampus but rarely in the granule cell layer (GCL) of the dentate gyrus.Moreover,no EGFP was detected in the pyramidal cell layers of CA1 to CA3 area.The counterstaining showed that rAd5 mainly transduced neuronal precursor cells in the SGZ of the hippocampus and rarely transduced neurons in the GCL.Conclusion The rAd5-mediated gene transfer is rapid and efficient and can keep for a long time in suitable titre; moreover,rAd5 has a highly specific tropism with the neuronal precursor cells in the SGZ of the hippocampus in mice.

Construction of recombinant baculovirus carriers of Ac-EGFP srnd Ac-HGF regulated by doxycycline / 第二军医大学学报

Objective To construct novel recombinant baculoviruses with Tet-On system and enhanced green fluorescent protein (EGFP) or hepatic growth factor (HGF) which could be regulated by different concentrations of doxycycline (DOX). Methods The recombinant plasmids pFast-Tet, pTRE-EGFP and pTRE-HGF were digested. The target fragments were collected and connected to pFast-Tet, the resultants wereused to transform DH10Bac competent cells containing AcMNPV Bacmid and helper plasmid, and the Bacmid DNA were identified (named Ac-EGFP and Ac-HGF) after selection and extraction. Ac-EGFP and Ac HGF were then transfected into bone mesenchymal stem cells (BMSCs), and the expression of EGFP and HGF were regulated by different concentrations of DOX (EGFP:0, 200, 500, and 1 000 ng/mL; HGF; 0, 10, 100, 500, 1 000, and 1 200 ng/mL); EGFP expression was observed under fluorescence microscope and the level of HGF expression was detected by ELISA. Results It was verified that Tet-On system was successfully constructed in a baculovirus vector with EGFP or HGF, and they were highly transfected into BMSCs. EGFP and HGF were highly expressed when exposed to high concentrations of DOX. And the expression of EGFP and HGF were gradually decreased at low concentration or absence of DOX. Conclusion Tet-On system can be used to construct a new recombinant baculovirus vector containing EGFP or HGF and it can stably and highly transfect BMSCs; different concentrations of DOX can lead to different expression of EGFP and HGF, and they are in low background expression without DOX.

Genotype and expression of the enhanced gree fluorescent protein in LEW-Tg (EGFP) F455.5/Rrrc rats / Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.

A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.

Bone marrow-derived stem cells promote glomerular repair and regeneration in progressive glomerulosclerosis rat model / 中华肾脏病杂志

Objective To investigate the protective role of bone marrow-derived stem cells (BMDSCs) in progressive glomerulosclerosis rats,and to observe whether BMDSCs promote glomenlar repair and regeneration.Methods Progressive glomerulosclerosis was induced in enhanced green fluorescent protein (EGFP) bone marrow chimeric rats by injecting with anti-Thy1.1 antibody,followed by unilateral nephrectomy.Subsequently,these rats were treated with either BMDSCs infusion (treatment group,10 rats) or phosphate-buffered saline (untreated group,1O rats).Renal function and histological alterations were examined at week 12 after Thy1.1 antibody injection.Repair and regeneration of glomerular endothelial cells was detected by immunofluorescence.Results Only 3 rats survived in untreated group,other 7 rats died at week 2,7,9,11 after antibody injection.In treatment group,9 rats survived at week 12,only 1rat died at week 10.The BUN and Scr were significantly lower in treatment group as compared to untreated group [BUN(43.55±29.06) vs (76.58±66.19) mmol/L,Scr (138.79±75.14) vs (233.38±164.43) μmol/L].Proteinuria was not significantly different between two groups at day 3,7,14 and 28,while it was significantly decreased at day 42,56 and 84 in treatment group compared with untreated group.Light microscopy showed that there was severe diffuse mesangial cell proliferation,mesangial matrix expansion and glomerulosclerosis in untreated group,and such changes were ameliorated in treatment group.The mesangial expansion index and glomerular sclerosis index in untreated group was significantly higher than those in treatment group.More BMDCs were recruited into the glomeruli and differentiated into glomerular endothelial cells in treatment group as compared with untreated group.Double immunofluorescence stain also demonstrated that BMDSCs infusion promoted glomerular capillary repair and regeneration.Conclusion BMDSCs infusion can improve renal function and histological changes,and promote the repair and regeneration of glomerular capillary.

Reconstruction of corneal surface layer using human amniotic epithelial cells modified by lentiviral vector-mediated EGFP gene / 中华实验眼科杂志

Background Studies demonstrated that human amniotic epithelial cells (AECs) have some characteristics of embryonic stem cells and they were used to re-establish the surface of eyes. Human AECs may serve as new seed cells in tissue engineering for corneal epithelium reconstitution in the future. Objective The present study was to investigate the application value of human amniotic epithelium cells transfected by lentiviral vectormediated enhanced green fluorescent protein (EGFP) gene as new seed cell source for engineering the corneal surfacelayer. Methods Lentiviral vector carrying the objective gene EGFP was transfected into human amniotic epithelial cells (pLenti6/V5-DEST),and the transient expression of the transgene in the human amniotic epithelial cells was observed under the fluorescence microscope. Flow cytometry was used to detect the positive expression rates of EGFP in transfected cells. The transfected human amniotic epithelial cells were seeded onto the fresh corneal stromal surface of New Zealand white rabbit and cultured in vitro. The stem cell deficiency ( SCD ) models were established by cutting off the limbus of cornea in 20 eyes of New Zealand white rabbits, and the model rabbits were then divided into 2 groups randomly. The transplanted grafts carrying the pLenti6/V5-DEST-EGFP gene-transferred human amniotic epithelium cells were regarded as the pLenti6/V5-DEST-EGFP group, and the corneal stroma graft without any epithelial cell served as the control group. The opacity of stroma and corneal conjunctivalization and vascularization were observed daily. The rabbits' eyes were extracted one month after operation. The expression of EGFP in the cornea was detected under the fluorescence microscope, and the expression of CK8, CK18 and CK12 in cornea was detected by immunohistochemical staining. Results The shape of the transferred human amniotic epithelial cells resembled normal human amniotic epithelial cells. 48 hours after the transient transfection of EGFP presented with the highest expression level throughout the observation duration, with a positive expression rate of EGFP of 61.5% ,showing significant differences in comparison with that of 12 ( 5.24% ) , 24 ( 38.27% ) or 96 ( 39. 10% ) hours ( P ＜0. 05) post-transfection; but no obvious difference was found in the positive rate of transiently transfected EGFP between 48 hours and 72 hours ( 58.36% ) ( P＞0. 05 ). Six cornea grafts were clear in 1 month and two corneas were rejected during the observation period in the pLenti6/V5-DEST-EGFP group. A few new blood vessels were seen around the graft. Ten corneas of the control group became opaque and cloudy with new blood vessels growth around the grafts. Imunohistochemistry revealed the positive expressions of CK8, CK1 8 and CK12 in the corneal epithelial layer in the pLenti6/V5-DEST-EGFP group. However,the expression of CK12 was absent in the control group. Conclusion Human amniotic epithelium cells transfected with the pLenti6/V5-DEST-EGFP gene is a new and ideal feed cell type to reconstruct the corneal surface layer. Lentivirus is a relatively safe gene transfection vector.

Neurogenic Differentiation of Murine Adipose Derived Stem Cells Transfected with EGFP in vitro / 华中科技大学学报（医学）（英德文版）

Some studies indicate that adipose derived stem cells(ADSCs)can differentiate into adipogenic,chondrogenic,myogenic,and osteogenic cells in vitro.However,whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated.In this study,the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene,and then the cells were induced for neural differentiation.The morphology of those ADSCs began to change within two days which developed into characteristics of round cell bodies with several branching extensions,concomitantly expressing EGFP fluorescence.Approximately 60% of the total cell populations were bipolar or multipolar in shape.Some of them appeared to make contact with their neighboring cells.RT-PCR,Western blot and Immanocytochemistry revealed that the expression levels of the markers of neurons and oligodendrocytes such as MAP2,NF-70,Neu N and RIP upon neural induction were increased,but the expression of the special marker of astrocytes,GFAP,was undetectable until 96 h after induction when a small signal was observed.It was concluded that the ADSCs transfected with EGFP possessed the ability to undergo morphologic and phenotypic changes consistent with neural differentiation in vitro.It suggests that these cells might provide an ideal source for further stem cell research with possible therapeutic application for spinal cord injury.

In vitro and in vivo models of osteosarcoma for research of enhanced green fluorescent protein expression / 中国癌症杂志

Background and purpose: Mouse osteosarcoma model was widely used in osteogenic malignant tumor research, and it was helpful for studying the invasion and metastasis of the tumor cells when it was well marked in vivo. The purpose of this study was to establish mouse sarcoma cell lines (S180) that were infected with enhanced green fluorescent protein(EGFP). Methods: EGFP-S180 expressing strong EGFP fluorescence was acquired by electroblot, and supplemented with G418 (800 mg/mL), c-Myc was detected by laser scanning confocal microscopy. Meanwhile, the cancer-bearing model was established subcutaneously within the abdominal cavity. Results: EGFP-S 180 cells were cloned. There was no significantly difference between c-Myc expressions in S180 cells and those in EGFP-S180 cells (P>0.05), and between the cancer-bearing time subcutaneously and the time within abdominal cavity (P>0.05). Conclusion: According to in vitro and in vivo assay, it showed that EGFP-expressing S180 cells could be used for studying further the tumor biological behavior with fluorescence technology.