Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

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.

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.

Construction of ATMT System for Clonostachys rosea and Its Conditional Optimization of Colonization into Glycyrrhiza uralensis Seeds / 中国实验方剂学杂志

ObjectiveAgrobacterium tumefaciens-mediated transformation （ATMT） of Clonostachys rosea， an endophytic fungus of Glycyrrhiza uralensis seeds， was established and optimized， and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds， so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects， including the concentration of acetosyringone， co-culture time and the concentration of conidia of recipient fungi. Then， high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature， co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1， acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h， the transformation efficiency of C. rosea was the highest， which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein （GFP） and β-glucuronidase （GUS） staining. When co-culture temperature was 25 ℃， co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1， the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest， which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea， but also of colonization of the transformants into G. uralensis seeds， which can lay a foundation for the development of biofertilizer of G. uralensis.

A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing / 药物分析学报

Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1-6)for 2 h,under 20％ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.

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.

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

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.

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.

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.

Construction ,Expression and Characteristic Analysis of Recombinant Human TIM-4-EGFP Fusion Protein / 华中科技大学学报(医学版)

Objective The present study aimed to explore the expression and purification of a fusion protein of human TIM-4 and EGFP in Escherichiacoli(E.coli)and evaluate its bioactivity.Methods The cDNA fragments of human TIM-4 and EG-FP genes were respectively amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a. The constructed re-combinant plasmid pET-28a-TIM-4-EGFP was transformed to E. coli BL21 (DE3)for the expression under the induction of IPTG.The expressed protein was purified by Ni-NTA resin.Recombinant protein was analyzed by SDS-PAGE and Western blotting ,and its binding activity to the apoptotic cells was detected under the fluorescence microscope.Results The TIM-4-EG-FP vector was constructed and expressed in E. coli. The TIM-4-EGFP fusion protein was identified and verified by SDS-PAGE and Western blotting.Our results demonstrated that all the TIM-4-EGFP fusion proteins recognize and bind directly to apoptot-ic cells ,but not to viable cells.We further verified that the interactions of TIM-4-EGFP with apoptotic cells were blocked by TIM-4-Ig fusion proteins.Conclusion We successfully constructed a fusion protein encoding human TIM-4 and EGFP ,and ex-pressed it in E.coli. The fusion protein shows a readily obtainable source of biologically active TIM-4 ,which has considerable potential for further studies on human TIM-4 and its receptor.

Preparation and in vitro evaluation of redox-sensitive polypeptide vector for co-delivery of gene and chemotherapeutic drugs / 第二军医大学学报

Objective To prepare a lipoic acid (LA) modified intrinsically disordered protein-cytosol-localizing internalization peptide 6 (CL) nanocomplex (LA-CL) entering cells by non-endocytosis mechanism for co-delivery of gene and chemotherapeutic drugs, and to investigate its transfection efficiency and cellular uptake on human embryonic kidney cell line HEK293 cells and its release behavior in vitro. Methods We synthesized four disulfide cross-linked lipoic acid modified LA-CLss(1-4) at different cross-linked degrees using different mass fractions (2.5%, 5%, 10% and 20%) of cysteine as cross-linking agent. The construction of LA-CLss was characterized by1H nuclear magnetic resonance (1HNMR) and gel permeation chromatography. The LA-CLss/plasmid enhanced green fluorescent protein (pEGFP) nanocomplexes were self-assembled with LA-CLss and pEGFP at different nitrogen/phosphorus (N/P) ratios (2.5, 5, 10, 20, 40 and 80). The size and zeta potential of LA-CLss/pEGFP nanocomplexes were determined by particle size analyzer, and the pEGFP enrichment capacity of LA-CLss was determined by agarose gel electrophoresis. The docetaxel (DTX)-loaded micelles were prepared by ultrasonic emulsification, and the critical micelle concentration of LA-CLss3 was determined by pyrene fluorescence probe spectroscopy. The LA-CLss/pEGFP nanocomplexes were co-cultured with HEK293 cells, and the transfection efficiencies of LA-CLss/pEGFP nanocomplexes at different cross-linked degrees were investigated. Results1 HNMR results showed the LA-CLss was successfully synthesized. When N/P ratio was 40, the transfection efficiency of LA-CLss3/pEGFP nanocomplex by HEK293 cells was significantly higher than that of LA-CL/pEGFP, LA-CLss1/pEGFP, LA-CLss2/pEGFP and LA-CLss4 nanocomplexes. The encapsulation efficiency and drug loading of docetaxel-loaded micelles prepared by ultrasonic emulsification were (85.25±0.04)% and (8.81±0.02)%, respectively. Cellular uptake test showed that the gene could be effectively delivered into the HEK293 cells by the LA-CLss micelles. In vitro release experiments showed that the LA-CLss micelles had redox-responsive drug release behavior. Conclusion The prepared LA-CLss/DTX/pEGFP nanocomplex is expected to become an efficient vector for co-delivery of gene and chemotherapeutic drugs.

Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro

BACKGROUND Gene reporter-fluorescent cells have emerged as alternative method for drug screening. OBJECTIVE Achievement of constitutive expression of fluorescent protein GFP by Leishmania braziliensis as alternative method for drug screening. METHODS L. braziliensis-GFP was generated using Leishmania tarentolae pLEXSY-egfp for constitutive expression of GFP. Fluorescent cells were selected and subjected to standardisation tests of anti-promastigote and anti-intracellular amastigote assays. FINDINGS Our results showed that L. braziliensis-GFP method is faster and more sensitive than Allamar Blue-resazurin. MAIN CONCLUSION Transfected parasites maintained stable fluorescence after successive in vitro passages and pLEXSY system can be used to achieve non-L. tarentolae fluorescent cells.

Expression of GFP as a tracer marker of ucMSCs in cerebral ischemia-reperfusion injury rats / 国际检验医学杂志

Objective To investigate the feasibility of green fluorescent protein (GFP) as a marker to trace the transplanted um-bilical cord mesenchymal stem cells (ucMSCs) in rats with cerebral ischemia-reperfusion .Methods ucMSCs were transfected by GFP-adenovirus .The rats were subjected to left middle cerebral artery occlusion using suture method .1 × 106 GFP-ucMSCs were transplanted with cerebral stereotaxic technique .Frozen sections of brain tissue were made at 7 d after cerebral ischemia .The ex-pression of GFP was observed by fluorescence microscope .Results In vitro ,the morphology of GFP in ucMSCs was fibrous ,and when fused ,the clusters were arranged in a radial or whirlpool shape ,The morphological feature of transfected ucMSCs was similar to that un-transfected ucMSCs .Under the fluorescence microscope ,the positive rate of GFP was more than 80% .In addition ,GFP could spread to the whole cells and show the completed form .No rejection was observed in the rats after transplantation ,and the GFP was found near the site of transplantation after 7 d ,and the fluorescence was not attenuated .Conclusion GFP is an effective tracer maker for ucMSCs transplantation in the treatment of ischemia-reperfusion ,which could provide experimental method for fur-ther study .

Expression of GFP as a tracer marker of ucMSCs in cerebral ischemia-reperfusion injury rats / 国际检验医学杂志

Objective To investigate the feasibility of green fluorescent protein (GFP) as a marker to trace the transplanted um-bilical cord mesenchymal stem cells (ucMSCs) in rats with cerebral ischemia-reperfusion .Methods ucMSCs were transfected by GFP-adenovirus .The rats were subjected to left middle cerebral artery occlusion using suture method .1 × 106 GFP-ucMSCs were transplanted with cerebral stereotaxic technique .Frozen sections of brain tissue were made at 7 d after cerebral ischemia .The ex-pression of GFP was observed by fluorescence microscope .Results In vitro ,the morphology of GFP in ucMSCs was fibrous ,and when fused ,the clusters were arranged in a radial or whirlpool shape ,The morphological feature of transfected ucMSCs was similar to that un-transfected ucMSCs .Under the fluorescence microscope ,the positive rate of GFP was more than 80% .In addition ,GFP could spread to the whole cells and show the completed form .No rejection was observed in the rats after transplantation ,and the GFP was found near the site of transplantation after 7 d ,and the fluorescence was not attenuated .Conclusion GFP is an effective tracer maker for ucMSCs transplantation in the treatment of ischemia-reperfusion ,which could provide experimental method for fur-ther study .

Therapeutic evaluation of systemic photodynamic therapy in a rat model for acute myeloid leukemia / 国际生物医学工程杂志

Objective To evaluate the efficacy of the systemic photodynamic therapy (SPDT)for treating leukemia using a Brown Norway myeloid leukemia (BNML) rat model.Methods The BNML rat model was established by injecting green fluorescent protein (GFP)-LT12 cells into the tail vein.After GFP-LT12 injection,the early-SPDT group,mid-SPDT group and late-SPDT group were treated with SPDT at 5,10 and 15 days,the negative control group was fed as usually,and the Ara-c positive control group was treated with Ara-c at 7 days.The GFP-LT12 cells were traced by a fluorescence imaging system.The GFP-LT12 cells in the tissues and organs were detected by flow cytometry.The levels of IFN-γ,IL-1α,IL-1β,IL-2,IL-4,IL-6,IL-10 and TNF-α in serum were detected by milliplex rat cytokine 9 kits.Results Compared with the negative control group,the survival times of the rats in the earlySPDT group,mid-SPDT group and the late-SPDT group were prolonged (all P＜0.05).The ratios of GFP-LT12 cells in pulp and liver were decreased in the late-SPDT group.The levels of IL-1β,IL-10,TNF-α and IFN-γin serum of the late-SPDT group were decreased (all P＜0.05).Conclusion The SPDT is an effective method for the treatment of leukemia,and the anti-tumor immune effect may play a key role in this process.

Experimental study of glioma stem cell-mediated immune tolerance in tumor microenvironment / 中华肿瘤杂志

Objective@#To investigate the tumor microenvironment of immune tolerance induced by glioma stem cells (GSC).@*Methods@#Human GSC SU3 cells transfected with red fluorescent protein (SU3-RFP) gene were implanted into the brain, subcutis (armpit and foot), liver and abdominal cavity of transgenic green fluorescence protein (GFP) nude mice to establish RFP+ /GFP+ dual fluorescence solid tumor model. The re-cultured cells derived from implanted tumor tissues, SU3-RFP cells co-cultured with peritoneal fluid of transgenic GFP nude mice and malignant ascites of tumor-bearing mice were observed by fluorescence microscopy and real-time video image tracing to analyze the microenvironment of immune tolerance mediated by RFP+ /GFP+ implanted tumor.@*Results@#Dual fluorescence labeled frozen section showed that all of cells in the tumor microenvironment were GFP+ , while the pressed tissue-patch showed that the tumor blood vessels exhibited a RFP+ /GFP+ double-positioning yellow. In the GFP single fluorescence labeled tumor tissue, all of cells in the microenvironment were green, including tumor edge, necrotic foci and blood vessel. Among them, CD68+ , F4/80+ , CD11c+ , CD11b+ and CD80+ cells were observed. In the dual fluorescence labeled co-cultured cells, the phagocytosis and fusion between green host cells and red tumor cells were also observed, and these fusion cells might transfer to the malignant dendritic cells and macrophages.@*Conclusions@#The tumor microenvironment of immune tolerance induced by GSC is not affected by the tissue types of tumor-inoculated sites, and the immune tolerance mediated by inflammatory cells is associated with the inducible malignant transformation, which may be driven by cell fusion.

Detection of circulating tumor cells by recombinant virus / 中华实验和临床病毒学杂志

The detection and molecular characterization of circulating tumor cells(CTCs) is one of the most important tool for liquid biopsy, which has the potential to enable non-invasive diagnostic tests for personalized medicine. Commercial platforms represented by CellSearch, the first FDA approved assay, have been considered to be valid for CTCs detection. However, special equipment and consumptive materials are required in the techniques listed above. Besides, most of them can not differentiate between apoptotic and viable cells, which indicates the portion of active and functional CTCs. Therefore, how to develop novel method for CTCs enrichment with metastatic potential has great significance in clinical routine. Telomerase-specific replication-selective oncolytic viruses expressing green fluorescent protein(GFP), including herpes simplex virus and adenovirus, allow the detection for human CTCs in the peripheral blood. After 24 h of transfection with recombinant virus, the tumor cells stably express GFP, and it could be used for CTCs counting by fluorescent microscopy or flow cytometry. Moreover, downstream analysis would be achieved by combination with PCR or DNA sequencing. Recombinant virus enables early detection of metastatic tumor cells, because the fluorescent signal is amplified only in viable, infected CTCs, by viral replication. This GFP-expressing virus-based method is remarkably sensitive, simple, and feasible, and it offers a new opportunity to detect and characterize CTCs in clinical routine.

Protective mechanism of ginsenoside Rg1 on hippocampus of aging mice induced by D-galactose / 中草药

Objective To investigate the protective mechanism of ginsenoside Rg1 on hippocampus of aging mice induced by D-galactose. Methods Forty nestin-green fluorescent protein (GFP) transgenic mice, aged 6-8 weeks, were randomly divided into four groups: control group, ginsenoside Rg1 control group, ginsenoside Rg1 therapy group, and model group. Learning and memory abilities were measured by Morris water maze after the modeling completed. Frozen sections were made to survey the hippocampus fluorescence intensity. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to detect the aging level of hippocampus. The activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and contents of malonaldehyde (MDA) in hippocampus were tested by chromatometry. Enzyme-linked immunosorbent assay (ELISA) was used to test the levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α proinflammatory cytokins in hippocampus. The levels of p53 and p21 were detected by Western blotting. Results The learning and memory capacities of the aging model group were decreased compared with those of the drug therapy group; The fluorescence intensity in the dentat gyrus (DG) of hippocampus of the drug therapy group was increased compared with that of the model group; The SA-β-Gal positive granules in section of brain tissue of the aging model group were increased compared with those of the drug group and drug therapy group; The activitives of SOD and T-AOC of the drug therapy group were increased compared with those of the aging model group while the content of MDA was decreased. The levels of IL-1β, IL-6, and TNF-α were decreased in the drug therapy group compared with those in the aging model group. The levels of p53 and p21 were decreased in the drug therapy group compared with those in the aging model group. Conclusion Ginsenoside Rg1 can antagonistic D-galactose and delay the aging of hippocampus. In addition, improvement of anti-oxidant ability and regulation of the level of p53-p21 pathway may be the underlying anti-aging mechanism of ginsenoside Rg1.

Cardiac fibroblasts of the rat transfected by lenti virus-stromal cell-derived factor-la-green fluorescent protein vector / 解剖学报

Objective To investigate the effect of lentivirus-stromal cell-derived factor-1α-green fluorescent protein(LV-SDF-1α-GFP) on the cardiac fibroblasts, the optimum conditions of infection, the expression and secretion of the target protein. Methods The cardiac fibroblasts of neonatal rats were primarily isolated and cultured by differential adherence methods, and were observed and identifi with immunofluorescence. LV-SDF-1α-GFP with different titers and conditions was transfected into cardiac fibroblasts. The expression of fluorescence and the optimal transfection conditions were observed. LV-SDF-1α-GFP target gene virus and negative control C0N145 virus were transfected into cardiac fibroblasts. The growth curve was drawn, and the effect of transfection on the proliferation of cardiac fibroblasts was explored. The cardiac fibroblasts were transfected with the optimum transfection dose, and the expression of SDF-1α was detected by Dot-blotting. The measurement data underwent statistical analysis. Results There was no statistical difference between the cardiac fibroblasts with SDF-1α transfected lentivirus and without no-transfected SDF-1α lentivirus. The peak of the expression of SDF-1α appeared in culture day 4 and statistical analysis showed significantly difference (P<0.05). Conclusion The LV-SDF-1α-GFP vector is of higher transfection efficiency to cardiac fibroblasts with the both low cytotoxicity and ability of secreting SDF-la protein.