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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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Ability of BMSCs to differentiate into neuron-like cells is significantly weakened when the expression of GAP-43 in BMSCs is inhibited. (A) Western blot analysis demonstrated that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43. 1, transduced with LV5-GAP-43; 2, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-844; 3, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-1215; 4, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-678; 5, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-619. (B) Western blot analysis revealed that the protein expression of the GAP-43 was significantly decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups. (C) BMSCs exhibited a neuronal phenotype in the NC and Blank groups; however, the majority of BMSCs exhibited no marked morphological changes. (D) Western blot analysis demonstrated that the expression levels of NSE, NF, nestin and βIII-tubulin were decreased in the LV3-shRNA-GAP-43-619 group compared with NC and Blank groups. (E) Cell immunofluorescence analysis revealed that the expression of GAP-43, nestin and NeuN were decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC group under induction conditions. Scale bar=50 µm. *P<0.05, vs. Blank group; #P<0.05, vs. NC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; LV3-shGAP-43, transduced with LV3-shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; siRNA, small interfering RNA; NSE, neuron-specific enolase; NF, neurofilament; NeuN, neuron-specific nuclear-binding protein.
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f3-mmr-12-04-5691: Ability of BMSCs to differentiate into neuron-like cells is significantly weakened when the expression of GAP-43 in BMSCs is inhibited. (A) Western blot analysis demonstrated that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43. 1, transduced with LV5-GAP-43; 2, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-844; 3, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-1215; 4, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-678; 5, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-619. (B) Western blot analysis revealed that the protein expression of the GAP-43 was significantly decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups. (C) BMSCs exhibited a neuronal phenotype in the NC and Blank groups; however, the majority of BMSCs exhibited no marked morphological changes. (D) Western blot analysis demonstrated that the expression levels of NSE, NF, nestin and βIII-tubulin were decreased in the LV3-shRNA-GAP-43-619 group compared with NC and Blank groups. (E) Cell immunofluorescence analysis revealed that the expression of GAP-43, nestin and NeuN were decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC group under induction conditions. Scale bar=50 µm. *P<0.05, vs. Blank group; #P<0.05, vs. NC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; LV3-shGAP-43, transduced with LV3-shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; siRNA, small interfering RNA; NSE, neuron-specific enolase; NF, neurofilament; NeuN, neuron-specific nuclear-binding protein.

Mentions: Western blot analysis revealed that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43 (Fig. 3A) and was used for the subsequent investigations. Following induction by retinal cell-conditioned differentiation medium, western blot analysis demonstrated that GAP-43 was expressed in the NC and Blank groups, but not in the LV3-shRNA-GAP-43-619 group (Fig. 3B), suggesting that the shGAP-43/BMSC line was successfully established. The BMSCs exhibited a neuronal phenotype in the NC and Blank group; however, the majority of BMSCs exhibited no marked morphological changes (Fig. 3C) in the LV3-shRNA-GAP-43-619 group, suggesting that neuron-like cells were successfully induced by retinal cell-conditioned differentiation medium. The expression levels of NSE, NF, nestin and βIII-tubulin were significantly reduced in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups (P<0.05; Fig. 3D). In addition, cell immunofluorescence analysis revealed that ~80% of BMSCs expressed GAP-43, nestin and NeuN in the NC group under induction conditions, whereas this value was <30% in the LV3-shRNA-GAP-43-619 group (Fig. 3E).


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)

Ability of BMSCs to differentiate into neuron-like cells is significantly weakened when the expression of GAP-43 in BMSCs is inhibited. (A) Western blot analysis demonstrated that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43. 1, transduced with LV5-GAP-43; 2, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-844; 3, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-1215; 4, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-678; 5, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-619. (B) Western blot analysis revealed that the protein expression of the GAP-43 was significantly decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups. (C) BMSCs exhibited a neuronal phenotype in the NC and Blank groups; however, the majority of BMSCs exhibited no marked morphological changes. (D) Western blot analysis demonstrated that the expression levels of NSE, NF, nestin and βIII-tubulin were decreased in the LV3-shRNA-GAP-43-619 group compared with NC and Blank groups. (E) Cell immunofluorescence analysis revealed that the expression of GAP-43, nestin and NeuN were decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC group under induction conditions. Scale bar=50 µm. *P<0.05, vs. Blank group; #P<0.05, vs. NC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; LV3-shGAP-43, transduced with LV3-shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; siRNA, small interfering RNA; NSE, neuron-specific enolase; NF, neurofilament; NeuN, neuron-specific nuclear-binding protein.
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Related In: Results  -  Collection

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f3-mmr-12-04-5691: Ability of BMSCs to differentiate into neuron-like cells is significantly weakened when the expression of GAP-43 in BMSCs is inhibited. (A) Western blot analysis demonstrated that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43. 1, transduced with LV5-GAP-43; 2, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-844; 3, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-1215; 4, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-678; 5, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-619. (B) Western blot analysis revealed that the protein expression of the GAP-43 was significantly decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups. (C) BMSCs exhibited a neuronal phenotype in the NC and Blank groups; however, the majority of BMSCs exhibited no marked morphological changes. (D) Western blot analysis demonstrated that the expression levels of NSE, NF, nestin and βIII-tubulin were decreased in the LV3-shRNA-GAP-43-619 group compared with NC and Blank groups. (E) Cell immunofluorescence analysis revealed that the expression of GAP-43, nestin and NeuN were decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC group under induction conditions. Scale bar=50 µm. *P<0.05, vs. Blank group; #P<0.05, vs. NC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; LV3-shGAP-43, transduced with LV3-shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; siRNA, small interfering RNA; NSE, neuron-specific enolase; NF, neurofilament; NeuN, neuron-specific nuclear-binding protein.
Mentions: Western blot analysis revealed that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43 (Fig. 3A) and was used for the subsequent investigations. Following induction by retinal cell-conditioned differentiation medium, western blot analysis demonstrated that GAP-43 was expressed in the NC and Blank groups, but not in the LV3-shRNA-GAP-43-619 group (Fig. 3B), suggesting that the shGAP-43/BMSC line was successfully established. The BMSCs exhibited a neuronal phenotype in the NC and Blank group; however, the majority of BMSCs exhibited no marked morphological changes (Fig. 3C) in the LV3-shRNA-GAP-43-619 group, suggesting that neuron-like cells were successfully induced by retinal cell-conditioned differentiation medium. The expression levels of NSE, NF, nestin and βIII-tubulin were significantly reduced in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups (P<0.05; Fig. 3D). In addition, cell immunofluorescence analysis revealed that ~80% of BMSCs expressed GAP-43, nestin and NeuN in the NC group under induction conditions, whereas this value was <30% in the LV3-shRNA-GAP-43-619 group (Fig. 3E).

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