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.

Preservação da proteína verde fluorescente no tecido ósseo descalcificado / Preservation of the green fluorescent protein on decalcified bone tissue

A proteína verde fluorescente (GFP) foi originalmente descoberta no cnidário Aequorea victoria. Células-tronco GFP positivas podem ser rastreadas in vivo quando usadas na terapia de doenças. No entanto, no osso, a fluorescência gerada pela GFP pode ser perdida durante o processo de descalcificação, dificultando o rastreamento das células-tronco usadas no tratamento de doenças ou defeitos ósseos. O objetivo deste estudo foi comparar diferentes técnicas de preservação da GFP no tecido ósseo descalcificado. Foram utilizados fêmures de ratas GFP Lewis distribuídos em quatro grupos: 1) descalcificado em ácido fórmico e incluído em parafina; 2) descalcificado em ácido fórmico e submetido à criomicrotomia; 3) descalcificado em EDTA e incluído em parafina; e 4) descalcificado em EDTA com criomicrotomia. Secções de tecido ósseo de todos os grupos foram analisadas para identificação da fluorescência natural e posteriormente submetidas à imunofluorescência, sendo utilizados anti-GFP e Alexa Flúor 555. As imagens foram obtidas por microscopia confocal. Osteócitos, osteoblastos e células da medula óssea de ratos GFP somente tiveram sua fluorescência natural preservada no tecido ósseo descalcificado em EDTA e submetido à microtomia por congelação. Nos demais grupos, houve perda da fluorescência natural, e as células GFP somente puderam ser identificadas com o uso da reação de imunofluorescência com anti-GFP. Conclui-se que a descalcificação em EDTA e a criomicrotomia são as melhores técnicas para preservar a fluorescência natural das células GFP no tecido ósseo e que a visualização de células GFP em tecido ósseo descalcificado em ácido fórmico e incluído em parafina somente pode ser realizada com o uso da técnica de imunofluorescência.

Green fluorescent protein (GFP) was originally derived from the cnidarians Aequorea victoria. GFP-positive stem cells can be tracked in vivo when used in the therapy of diseases. However, in the bone, the fluorescence generated by GFP can be lost during the decalcification process, hindering the tracking of stem cells used in the treatment of diseases or bone defects. The aim of this study was to compare different techniques of preservation of GFP in the decalcified bone tissue. Femurs of female Lewis GFP rats were distributed in four groups: 1) decalcified in formic acid and paraffin-embedded; 2) decalcified in formic acid submitted to cryomicrotomy; 3) decalcified in EDTA and paraffin-embedded and 4) decalcified in EDTA with cryomicrotomy. Sections of bone tissue of all the groups were analyzed for identification of the natural fluorescence and subsequently submitted to the immunofluorescence using anti-GFP and Alexa Flúor 555. The images were obtained by confocal microscopy. Osteocytes, osteoblasts and bone marrow cells of GFP rats only had natural fluorescence preserved in the bone tissue decalcified in EDTA and submitted to cryomicrotomy. In others groups there were loss of the natural fluorescence and the GFP cells could be only identified with the use of the immunofluorescence with anti-GFP. In conclusion, the decalcification in EDTA and the cryomicrotomy are the best techniques to preserve the natural fluorescence of the GFP cells in the bone tissue and the GFP cells in bone tissue decalcified in formic acid and paraffin-embedded can be visualized only with the use of the immunofluorescence with anti-GFP.

Visualization of serotonin 1A receptor trafficking in neuron-like PC12 cells / 基础医学与临床

Objective To explore the mechanisms of trafficking and signaling of serotonin 1A receptor(5-HT_(1A))and its spatiotemporal distribution in living cells.Methods The mouse 5-HT_(1A) gene amplified by RT-PCR was recombined into pEGFP-N1 vector and the EGFP coding sequence was located in-frame at the C-terminal end of the 5-HT_(1A) receptor.The 5-HT_(1A)-EGFP was transfected into neuron-like PC12 cells as well as HEK293.The transfected cells were visualized using confocal microscopy,the mobility of 5-HT_(1A)-EGFP was monitored by live measurements and fluorescence recovery after photobleaching.Results The 5-HT_(1A) gene was identitical with the published gene sequence NM_008308.4 and a 5-HT_(1A)-EGFP fusion construct was created.After stable transfection of the plasimd into a PC12 cell line and analysis with a confocal laser scanning microscopy,the EGFP-tagged 5-HT_(1A) was predominantly associated with the plasma membrane,but some intracellular vesicles in the perinuclear region also contained the fusion protein.The predominant localization of 5-HT_(1A)-EGFP at the plasma membrane was confirmed in transiently transfected HEK293 cells.Bleached fluorescence was partialy recovered in 100 seconds,indicating that the 5-HT_(1A)-EGFP was mobiled on the membrane.Conclusion Spatiotemporal distribution and mobility of 5-HT_(1A) tagged with EGFP can be monitored in the 5-HT_(1A)-EGFP stable PC12 cell line,which could be an excellent neuron-like experimental cell model for research of 5-HT_(1A) trafficking and signaling.

Expression Patterns of Injected Adenovirus in the Nasal Cavity and Paranasal Sinus Mucosa of the Rat / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: A potential use of gene therapy to deliver therapeutic peptides to the nasal mucosa has not been investigated. The purpose of this study was to assess the feasibility of using a recombinant adenovirus vector to target the nasal cavity and paranasal sinus mucosa of the rat. MATERIALS AND METHOD: Ten male Sprague Dawley rats were used in this experiments, and adenovirus vectors containing green fluorescent protein (GFP) were injected into rat via tail vein. On fourteen days after gene transfer, 10 rats were sacrificed. The turbinate, nasal septum and paranasal sinus mucosa were examined by fluorescent microscope, and immunohistochemical analysis using anti-GFP antibody was performed. RESULTS: The turbinate and septal mucosa after injection of adenovirus vector expressing GFP showed diffused distribution of fluorescent produced by GFP. Immunohistochemical analysis showed that GFP expression in the turbinate mucosa was localized to the largely ciliated colmnar epithelium and to some lamnina propria, and that in the septal mucosa, it was localized to the ciliated columnar epithelium. Expression in the turbinate mucosa was more obvious than that in the nasal septal mucosa. There was no expression in the paranasal sinus mucosa. CONCLUSION: This study suggests that recombinant adenovirus vectors can be used to transfer genes to turbinate and nasal septal mucosa. Gene therapy targeting mucosal epithelium can be a helpful method to treat patients with chronic inflammatory disease of the nasal mucosa such as allergic rhinitis or neoplasm of the nasal cavity.

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene / 中国病毒学

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain,seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI,which contained the full-length cDNA of the NDV ZJI strain.The pNDV/ZJI,with three helper plasmids,pCIneoNP,pCIneoP and pCIneoL,were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase.After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock,an infectious NDV ZJI strain was successfully rescued.Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP.After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells,the recombinant NDV,NDV/ZJIGFP,was rescued.Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection,indicating that the GFP gene was expressed at a relatively high level.NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route.Four days post-infection,strong green fluorescence could be detected in the kidneys and tracheae,indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

CULTIVATION AND OSTEOBLASTIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS ON GFP-EXPRESSING MOUSE IN VITRO / 解剖学报

Objective To isolate the bone marrow mesenchymal stem cells(MSCs) from the GFP-expressing mouse, and to study the osteoblastic differentation of the cells. Methods MSCs were isolated by density gradient centrifugation, then the clutrued cells were induced to osteoblastic differentiation using the conditional medium. We detectd the expression of GFP and MSCs differentiation into osteoblasts by histochemistry and immunochemistry. Results The MSCs maintained the expression of GFP during expanded and induced process. After induced for 10 days, lots of alkaline phosphatase and osteoclcin staining positive cells were observed.Conclusion The MSCs of GFP-expressing mouse were successfully isolated and differentiated into ostoblasts. It may be valuable for tracing the seeding cells in tissue engineering bone.