Μελέτη παραγόντων βελτιστοποίησης των τεχνικών μεταφοράς και διατήρησης του αυτόλογου λίπους (fat transer)

2016 ◽  
Author(s):  
Μαργαρίτα Μουστάκη
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Adipose Derived Stem Cells ◽  
Green Fluorescent

Εισαγωγή:Ο αυτόλογος λιπώδης ιστός έχει αποδείχθηκε ένα εξαιρετικό υλικό αύξησης μαλακών μορίων. Η απομόνωση των βλαστικών κυττάρων από τον λιπώδη ιστό (Adipose Derived Stem Cells, ADSCs) μοιραία οδήγησε την έρευνα να στραφεί στη μελέτη της συνδυασμένης μεταμόσχευσης αυτόλογου λίπους και ADSCs (cell assisted lipotransfer-CAL).Σκοπός:Η συγκεκριμένη μελέτη είναι μια in vivo μελέτη σε ζωικό πρότυπο της υποβοηθούμενης με ADSCs λιπομεταφοράς. Σκοπός της μελέτης είναι να αποδείξει με ποσοτικές μετρήσεις τη δυνατότητα των ADSCs να βελτιώνουν την ποιότητα και την μακροπρόθεσμη διατήρηση του μοσχεύματος λίπους κατά τη λιπομεταφορά, συγκρίνοντας την με την παραδοσιακή λιπομεταφορά.Μέθοδοι:Για τους σκοπούς της μελέτης, βλαστικά κύτταρα απομονώθηκαν από διαγονιδιακούς μύες C57BL / 6J-GFΡ που εκφράζουν την πράσινη φθορίζουσα πρωτεΐνη (Green Fluorescent Protein, GFP) με αποτέλεσμα τα ίδια τα βλαστικά κύτταρα να φθορίζουν, γεγονός που μας επέτρεψε την παρακόλουθηση τους απεικονιστικά. Τα GFP-θετικά βλαστικά κύτταρα αφού απομονώθηκαν από το λίπος αναμείχθηκαν με τεμαχισμένο λιπώδη ιστό που συλλέχθηκε από τις βουβωνικές χώρες των C57BL / 6JolaHsd μυών και στη συνέχεια συν-εμφυτεύτηκαν σε Hsd:Athymic Nude-Foxn1nu μύες. Σε όλους τους μύες η έγχυση του ADSC-εμπλουτισμένου μείγματος έγινε στη μία πλευρά της ράχης ενώ στην αντίθετη πλευρά της ράχης πραγματοποιήθηκε η έγχυση ελέγχου (control) με ίδια ποσότητα τεμαχισμένου λιπώδους ιστού (μη εμπλουτισμένου) από τις βουβωνικές χώρες των C57BL / 6JolaHsd μυών .Η επιβίωση των εμφυτευμένων GFP-θετικών βλαστικών κυττάρων παρακολουθήθηκε με in νίνο μοριακή απεικόνιση φθορισμού 56 ημερών. Για τη συγκριτική μελέτη του ποσοστού επιβίωσης το μοσχεύματος λίπους τα μοσχεύματα αφαιρέθηκαν χειρουργικά στις 7 και στις 56 ημέρες μετά τη μεταμόσχευση, ζυγίστηκαν και υποβλήθηκαν σε ιστολογική εξέταση και ανοσοϊστοχημικό έλεγχο για τους δείκτες CD34 και Ki67.Αποτελέσματα:Τα αποτελέσματα έδειξαν καλύτερη επιβίωση του ADSC-εμπλουτισμένου λίπους σε σύγκριση με το μη εμπλουτισμένο (63% του αρχικής μάζας έναντι 33% μεγαλύτερη σε σχέση με την παραδοσιακή λιπομεταφορά,p<0.05). Η παρουσία των φθοριζόντων ADSCs μέχρι και την 56η ημέρα παρακολούθησης επιβεβαιώθηκε με την in vivo απεικόνιση φθορισμού. Η νεο-αγγειακή πυκνότητα ήταν αυξημένη στα εμπλουτισμένα με ADSCs λιπομοσχεύματα με διαφορά στατιστικά σημαντική (p<0.05)Συμπεράσματα: Ο εμπλουτισμός των λιπο-μοσχευμάτων με ADSCs είναι μια αξιόπιστη και αποτελεσματική μέθοδος βελτιστοποίησης της τεχνικής μεταφοράς αυτόλογου λίπους. Τα ex-vivo καλλιεργημένα ADSCs συμβάλλουν στη νεο-αγγείωση και αναγέννηση του ισχαιμικού ισχαιμικού λιπομοσχεύματος .

scholarly journals The EZC-Prostate Model: Noninvasive Prostate Imaging in Living Mice

2004 ◽  
Vol 18 (3) ◽  
pp. 722-732 ◽  
Author(s):  
Xiaoming Xie ◽  
Zheng Luo ◽  
Kevin M. Slawin ◽  
David M. Spencer
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Firefly Luciferase ◽  
In Vivo Models ◽  
Charge Coupled Device ◽  
Prostate Disease ◽  
Green Fluorescent

Abstract Recently, progress in the development of prostate-specific promoters and high resolution imaging techniques has made real-time monitoring of transgenic expression possible, opening a vista of potentially important in vivo models of prostate disease. Herein, we describe a novel prostate reporter model, called the EZC-prostate model that permits both ex vivo and in vivo imaging of the prostate using a sensitive charge-coupled device. Firefly luciferase and enhanced green fluorescent protein were targeted to the prostate epithelium using the composite human kallikrein 2 (hK2)-based promoter, hK2-E3/P. In EZC-prostate mice, the ventral and dorsal/lateral prostate lobes were brilliant green under fluorescence microscopy, with expression localized to the secretory epithelium. In contrast, enhanced green fluorescent protein was undetectable in the anterior lobes of prostate, seminal vesicles, testes, liver, lung, and brain. The kinetics of luciferase activity in intact and castrated living mice monitored with the IVIS charge-coupled device-based imaging system confirmed that firefly luciferase expression was largely prostate restricted, increased with age up to 24 wk, and was androgen dependent. Decreases in reporter expression after 24 wk may reflect well known, age-related decreases in androgen signaling with age in humans. Ex vivo imaging of microdissected animals further confirmed that the luminescence detected in living mice emanated predominately from the prostate, with minor signals originating from the testes and cecum. These data demonstrate that the hK2-E3/P promoter directs strong prostate-specific expression in a transgenic mouse model. Multigenic models, generated by crosses with various hyperplastic and neoplastic prostate disease models, could potentially provide powerful new tools in longitudinal monitoring of changes in prostate size, androgen signaling, metastases, or response to novel therapies without sacrificing large cohorts of animals.

Neural Development by Transplanted Human Embryonal Carcinoma Stem Cells Expressing Green Fluorescent Protein

2005 ◽  
Vol 14 (6) ◽  
pp. 339-351 ◽  
Author(s):  
R. Stewart ◽  
M. Lako ◽  
G. M. Horrocks ◽  
S. A. Przyborski
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Neural Development ◽  
Molecular Mechanisms ◽  
Embryonal Carcinoma ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Green Fluorescent

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein

2021 ◽  
Vol 33 (2) ◽  
pp. 180
Author(s):  
Q. Xu ◽  
D. J. Milner ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Green Fluorescent Protein ◽  
Cell Sorting ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Label ◽  
Adipose Derived Stem Cells ◽  
Egfp Gene ◽  
Green Fluorescent

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

scholarly journals In Vivo and In Vitro Tissue-Specific Expression of Green Fluorescent Protein Using the Cre-Lox System in Mouse Embryonic Stem Cells

Stem Cells ◽  
2005 ◽  
Vol 23 (1) ◽  
pp. 10-15 ◽  
Author(s):  
Jan Schindehütte ◽  
Hidefumi Fukumitsu ◽  
Patrick Collombat ◽  
Gundula Griesel ◽  
Christopher Brink ◽  
...  
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Green Fluorescent

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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