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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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Overexpression of GAP-43 promotes the differentiation of BMSCs into neuron-like cells. (A) Western blot analysis demonstrated that the protein expression of the GAP-43 was significantly increased in the LV5-GAP-43 group, compared with the NC and Blank groups. (B) BMSCs were observed using microscopy and it was revealed that BMSCs exhibited a neuronal phenotype following transduction with LV5-GAP-43. (C) BMSCs expressed positive specific neural markers for NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group, determined using semi-quantitative polymerase chain reaction detection. Scale bar=50 µm. GAP-43, growth-associated protein-43; BMSCs, bone marrow mesenchymal stem cells; NSE, neuron-specific enolase; NF, neurofilament; NC, transduced with negative control scramble lentiviral vector; LV5-GAP-43, transduced with LV5-GAP-43; M, marker.
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f2-mmr-12-04-5691: Overexpression of GAP-43 promotes the differentiation of BMSCs into neuron-like cells. (A) Western blot analysis demonstrated that the protein expression of the GAP-43 was significantly increased in the LV5-GAP-43 group, compared with the NC and Blank groups. (B) BMSCs were observed using microscopy and it was revealed that BMSCs exhibited a neuronal phenotype following transduction with LV5-GAP-43. (C) BMSCs expressed positive specific neural markers for NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group, determined using semi-quantitative polymerase chain reaction detection. Scale bar=50 µm. GAP-43, growth-associated protein-43; BMSCs, bone marrow mesenchymal stem cells; NSE, neuron-specific enolase; NF, neurofilament; NC, transduced with negative control scramble lentiviral vector; LV5-GAP-43, transduced with LV5-GAP-43; M, marker.

Mentions: Western blot analysis revealed that the protein expression of GAP-43 was significantly increased in the LV5-GAP-43 group, compared with in the NC and blank groups (P<0.05; Fig. 2A), suggesting that the GAP-43/BMSC line was successfully established. BMSCs were observed under fluorescence microscopy 3, 5 and 7 days after transduction with LV5-GAP-43. The bodies of the BMSCs were contracted with protruding processes at day 3, and the typical neuron-like cells appeared at day 5. On day 7, the majority of the BMSCs exhibited a neuronal phenotype and the prominences formed a network structure (Fig. 2B). According to semi-quantitative PCR, the BMSCs exhibited positive expression of NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group (Fig. 2C).


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)

Overexpression of GAP-43 promotes the differentiation of BMSCs into neuron-like cells. (A) Western blot analysis demonstrated that the protein expression of the GAP-43 was significantly increased in the LV5-GAP-43 group, compared with the NC and Blank groups. (B) BMSCs were observed using microscopy and it was revealed that BMSCs exhibited a neuronal phenotype following transduction with LV5-GAP-43. (C) BMSCs expressed positive specific neural markers for NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group, determined using semi-quantitative polymerase chain reaction detection. Scale bar=50 µm. GAP-43, growth-associated protein-43; BMSCs, bone marrow mesenchymal stem cells; NSE, neuron-specific enolase; NF, neurofilament; NC, transduced with negative control scramble lentiviral vector; LV5-GAP-43, transduced with LV5-GAP-43; M, marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-12-04-5691: Overexpression of GAP-43 promotes the differentiation of BMSCs into neuron-like cells. (A) Western blot analysis demonstrated that the protein expression of the GAP-43 was significantly increased in the LV5-GAP-43 group, compared with the NC and Blank groups. (B) BMSCs were observed using microscopy and it was revealed that BMSCs exhibited a neuronal phenotype following transduction with LV5-GAP-43. (C) BMSCs expressed positive specific neural markers for NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group, determined using semi-quantitative polymerase chain reaction detection. Scale bar=50 µm. GAP-43, growth-associated protein-43; BMSCs, bone marrow mesenchymal stem cells; NSE, neuron-specific enolase; NF, neurofilament; NC, transduced with negative control scramble lentiviral vector; LV5-GAP-43, transduced with LV5-GAP-43; M, marker.
Mentions: Western blot analysis revealed that the protein expression of GAP-43 was significantly increased in the LV5-GAP-43 group, compared with in the NC and blank groups (P<0.05; Fig. 2A), suggesting that the GAP-43/BMSC line was successfully established. BMSCs were observed under fluorescence microscopy 3, 5 and 7 days after transduction with LV5-GAP-43. The bodies of the BMSCs were contracted with protruding processes at day 3, and the typical neuron-like cells appeared at day 5. On day 7, the majority of the BMSCs exhibited a neuronal phenotype and the prominences formed a network structure (Fig. 2B). According to semi-quantitative PCR, the BMSCs exhibited positive expression of NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group (Fig. 2C).

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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Related in: MedlinePlus