Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Erbin在肾间质纤维化中的表达和作用

Objective To investigate the expression of Erbin in renal interstitial fibrosis (RIF) and the effect of over-expression of Erbin on transforming growth factor β1 (TGF-(β1)-induced epithelial-mesenchymal transition (EMT) in NRK52E cells. Methods In vivo, the model of renal fibrosis was induced by 5/6 subtotal nephrectomy in rat. Scr and BUN was detected and Masson staining was used to evaluate the level of renal tissue fibrosis. The location and expression of Erbin in renal tissue were detected by immunohistochemistry and Western blotting. In vitro, after NRK52E cells were treated by TGF-β1 (10 μg/L) for 72 h, immunofluorescence and Western blotting were used to obverse the expression and distribution of E-cadherin and α-SMA. The expression of Erbin mRNA and protein were detected by RT-PCR and Western blotting respectively. NRK52E cells were transiently transfected with Prk5-myc-Erbin plasmid via lipofectamine 2000, then the expressions of Erbin, E-cadherin and α-SMA were detected by Western blotting. Results (l)Compared to sham group with Scr (33.96±7.28) μmol/L and BUN (8.11±2.55) mmol/L, rats in 5/6 nephrectomy model with Scr (140.52±61.11) μmol/L and BUN (34.23±7.66) mmol/L revealed renal dysfunction. Masson staining indicated kidney interstitial fibrosis, and the expression of Erbin was significantly increased in renal tissue(2.9 folds), especially in tubular epithelia. (2)In vitro, the expressions of Erbin and α-SMA were markedly increased (2.3 folds and 2.1 folds, P<0.05, respectively) and the expression of E-cadherin was dramatically decreased in NRK52E cells stimulated by TGF-β1, which were consistent with immunofluorescence results. TGF-β1-induced E-cadherin suppression and a-SMA induction could be efficiently blocked by over-expression of Erbin (all P <0.05). Conclusions Erbin is up-regulated in renal interstitial fibrosis, and over-expression of Erbin can partly inhibit renal EMT induced by TGF-β1, which indicates Erbin playing an protective role in renal fibrosis.     

Erbin在肾间质纤维化中的表达和作用

Objective To investigate the expression of Erbin in renal interstitial fibrosis (RIF) and the effect of over-expression of Erbin on transforming growth factor β1 (TGF-(β1)-induced epithelial-mesenchymal transition (EMT) in NRK52E cells. Methods In vivo, the model of renal fibrosis was induced by 5/6 subtotal nephrectomy in rat. Scr and BUN was detected and Masson staining was used to evaluate the level of renal tissue fibrosis. The location and expression of Erbin in renal tissue were detected by immunohistochemistry and Western blotting. In vitro, after NRK52E cells were treated by TGF-β1 (10 μg/L) for 72 h, immunofluorescence and Western blotting were used to obverse the expression and distribution of E-cadherin and α-SMA. The expression of Erbin mRNA and protein were detected by RT-PCR and Western blotting respectively. NRK52E cells were transiently transfected with Prk5-myc-Erbin plasmid via lipofectamine 2000, then the expressions of Erbin, E-cadherin and α-SMA were detected by Western blotting. Results (l)Compared to sham group with Scr (33.96±7.28) μmol/L and BUN (8.11±2.55) mmol/L, rats in 5/6 nephrectomy model with Scr (140.52±61.11) μmol/L and BUN (34.23±7.66) mmol/L revealed renal dysfunction. Masson staining indicated kidney interstitial fibrosis, and the expression of Erbin was significantly increased in renal tissue(2.9 folds), especially in tubular epithelia. (2)In vitro, the expressions of Erbin and α-SMA were markedly increased (2.3 folds and 2.1 folds, P<0.05, respectively) and the expression of E-cadherin was dramatically decreased in NRK52E cells stimulated by TGF-β1, which were consistent with immunofluorescence results. TGF-β1-induced E-cadherin suppression and a-SMA induction could be efficiently blocked by over-expression of Erbin (all P <0.05). Conclusions Erbin is up-regulated in renal interstitial fibrosis, and over-expression of Erbin can partly inhibit renal EMT induced by TGF-β1, which indicates Erbin playing an protective role in renal fibrosis.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.