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Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats.

Zhu Q, Liu Z, Wang C, Nie L, He Y, Zhang Y, Liu X, Su G - Mol Med Rep (2015)

Bottom Line: The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05).The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups.In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.

ABSTRACT
The aim of the present study was to examine the effect of growth-associated protein-43 (GAP-43) on bone marrow mesenchymal stem cell (BMSC) differentiation in a rat model of traumatic optic neuropathy (TON). GAP‑43 and short hairpin (sh)RNA‑GAP‑43 were inserted into pGLV5 and pGLV3 lentiviral vectors, respectively. The stable control, GAP‑43‑overexpression and GAP‑43‑knockdown cell lines (GFP/BMSCs, GAP‑43/BMSCs and shGAP‑43/BMSCs, respectively) were established. The expression of GAP‑43, neuron‑specific enolase (NSE), nestin, neurofilament (NF), neuron‑specific nuclear‑binding protein (NeuN) and βIII‑tubulin were detected in the GAP‑43/BMSCs and shGAP‑43/BMSCs with retinal cell‑conditioned differentiation medium using semi‑quantitative polymerase chain reaction (PCR), western blotting and cell immunofluorescence. In addition, the BMSCs were observed under fluorescence microscopy. The Sprague‑Dawley rat models of TON were established and identified by retrograde labeling of retinal ganglion cells (RGCs) with fluoroGold (FG). The lentiviral‑mediated GAP‑43‑modified BMSCs were then transplanted into the rat model of TON. The expression of GAP‑43 was detected in the retinal tissues using qPCR and western blotting. The histopathology of the retinal tissues was observed using hematoxylin and eosin (H&E) staining. The GAP‑43/BMSCs exhibited positive expression of NSE, NF, nestin and βIII‑tubulin, and exhibited a neuronal phenotype. The shGAP‑43/BMSCs markedly inhibited expression of NeuN, NSE, NF, nestin and βIII‑tubulin induced by retinal cell‑conditioned differentiation medium. The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05). The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups. In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

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Morphology and identification of BMSCs using flow cytometry. (A) Third passage BMSCs uniformly assumed a vortex-like or radiating appearance. (B) Flow cytometric analysis demonstrated that third passage BMSCs were positive for CD90 and CD44, while negative for CD11b and CD34. Scale bar=50 µm. BMSCs, bone marrow mesenchymal stem cells.
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f1-mmr-12-04-5691: Morphology and identification of BMSCs using flow cytometry. (A) Third passage BMSCs uniformly assumed a vortex-like or radiating appearance. (B) Flow cytometric analysis demonstrated that third passage BMSCs were positive for CD90 and CD44, while negative for CD11b and CD34. Scale bar=50 µm. BMSCs, bone marrow mesenchymal stem cells.

Mentions: The third passage BMSCs uniformly assumed a vortex-like or radiating appearance (Fig. 1A). The cells were positive for CD90 (94.7%) and CD44 (74.4%), and negative for CD11b (0.12%) and CD34 (2.4%; Fig. 1B). Therefore, the third passage BMSCs were used in the subsequent experiments.


Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats.

Zhu Q, Liu Z, Wang C, Nie L, He Y, Zhang Y, Liu X, Su G - Mol Med Rep (2015)

Morphology and identification of BMSCs using flow cytometry. (A) Third passage BMSCs uniformly assumed a vortex-like or radiating appearance. (B) Flow cytometric analysis demonstrated that third passage BMSCs were positive for CD90 and CD44, while negative for CD11b and CD34. Scale bar=50 µm. BMSCs, bone marrow mesenchymal stem cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581804&req=5

f1-mmr-12-04-5691: Morphology and identification of BMSCs using flow cytometry. (A) Third passage BMSCs uniformly assumed a vortex-like or radiating appearance. (B) Flow cytometric analysis demonstrated that third passage BMSCs were positive for CD90 and CD44, while negative for CD11b and CD34. Scale bar=50 µm. BMSCs, bone marrow mesenchymal stem cells.
Mentions: The third passage BMSCs uniformly assumed a vortex-like or radiating appearance (Fig. 1A). The cells were positive for CD90 (94.7%) and CD44 (74.4%), and negative for CD11b (0.12%) and CD34 (2.4%; Fig. 1B). Therefore, the third passage BMSCs were used in the subsequent experiments.

Bottom Line: The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05).The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups.In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.

ABSTRACT
The aim of the present study was to examine the effect of growth-associated protein-43 (GAP-43) on bone marrow mesenchymal stem cell (BMSC) differentiation in a rat model of traumatic optic neuropathy (TON). GAP‑43 and short hairpin (sh)RNA‑GAP‑43 were inserted into pGLV5 and pGLV3 lentiviral vectors, respectively. The stable control, GAP‑43‑overexpression and GAP‑43‑knockdown cell lines (GFP/BMSCs, GAP‑43/BMSCs and shGAP‑43/BMSCs, respectively) were established. The expression of GAP‑43, neuron‑specific enolase (NSE), nestin, neurofilament (NF), neuron‑specific nuclear‑binding protein (NeuN) and βIII‑tubulin were detected in the GAP‑43/BMSCs and shGAP‑43/BMSCs with retinal cell‑conditioned differentiation medium using semi‑quantitative polymerase chain reaction (PCR), western blotting and cell immunofluorescence. In addition, the BMSCs were observed under fluorescence microscopy. The Sprague‑Dawley rat models of TON were established and identified by retrograde labeling of retinal ganglion cells (RGCs) with fluoroGold (FG). The lentiviral‑mediated GAP‑43‑modified BMSCs were then transplanted into the rat model of TON. The expression of GAP‑43 was detected in the retinal tissues using qPCR and western blotting. The histopathology of the retinal tissues was observed using hematoxylin and eosin (H&E) staining. The GAP‑43/BMSCs exhibited positive expression of NSE, NF, nestin and βIII‑tubulin, and exhibited a neuronal phenotype. The shGAP‑43/BMSCs markedly inhibited expression of NeuN, NSE, NF, nestin and βIII‑tubulin induced by retinal cell‑conditioned differentiation medium. The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05). The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups. In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

Show MeSH
Related in: MedlinePlus