碱性成纤维细胞生长因子缓释微球治疗兔膝骨关节炎的实验研究

目的 研究壳聚糖包被碱性成纤维细胞生长因子(bFGF)的缓释微球对实验兔膝关节骨性关节炎的治疗作用.方法 2008年11月至2009年7月,选取健康成年新西兰大白兔54只,随机分成6组:对照组、模型组、PBS微球组(PBS-M)、bFGF溶液组(bFGF-S)、10μgbFGF微球组(10-bFGF-M)及100μg bFGF微球组(100-bFGF-M).膝关节腔内注射木瓜蛋白酶建立兔膝骨关节炎模型(对照组除外).除模型组外其余各组分别于建模后第3周和第6周于关节腔内注射1ml干预液,分别含PBS微球、bFGF溶液、10μg bFGF微球及100μg bFGF微球.于第9周处死动物后取材,通过大体及组织病理学检查评价实验结果.结果 根据Ink评分及Mankin评分结果,模型组关节软骨损伤程度重于对照组(t=8.22,P=0.00;t=17.20,P=0.00),PBS-M组、bFGF-S组的关节软骨损伤程度与模型组近似(Ink评分:t=0.26,P=0.79;t=0.80,P=0.45;Mankin评分:t=1.51,P=0.17;t=0.56,P=0.60),而10-bFGF-M组和100-bFGF-M组关节软骨损伤程度较模型组明显减轻(Ink评分:t=3.58,P=0.01;t=6.82,P=0.00;Mankin评分:t=3.41,P=0.01;t=5.00,P=0.00).100-bFGF-M组与10-bFGF-M组相比关节软骨损伤程度明显减轻(t=50.29,P=0.00;t=2.80,P=0.02).结论 bFGF缓释微球能维持bFGF关节腔内的有效浓度,可能通过加速蛋白多糖的合成并抑制其分解等作用机制逆转了关节软骨损伤的病理过程.

Abstract：

Objective To study the therapeutic effect of chitosan-coated basic fibroblast growth factor (bFGF) slow-releasing microspheres on the knee osteoarthritis in the rabbit. Methods From November 2008 to July 2009, 54 New Zealand rabbits were divided into 6 groups at random, which were the control group, the model group, the PBS-M group, the bFGF-S group, the 10-bFGF-M group and the 100-bFGF-M group, respectively. The model of knee osteoarthritis was induced by the injection of papain in the rabbit. Except the control and model groups, all the experimental groups were implanted 1 ml intervention solution at the third and sixth weeks, including the PBS microspheres, bFGF solution, 10 pμg bFGF microspheres and 100 μg bFGF microspheres, respectively. The rabbits were sacrificed at the ninth week after operation, and then articular cartilage was conducted the morphological and histopathological evaluation. Results The damage of articular cartilage in the model group was more serious than that in the control group, with statistical differences according to the Ink score (t = 8. 22, P = 0. 00) and Mankin score (t = 17. 20, P =0. 00). The damage of articular cartilage in the PBS-M and bFGF-S groups were similar with that in the model group, according to the Ink score (t =0. 26, P =0. 79; t =0. 80, P =0.45) and Mankin score (t =1.51, P=0. 17; t =0.56, P=0.60). The Ink and Mankin scores in the 10-bFGF-M and 100-bFGF-M groups were better than that in the model group (Ink score: t =3.58, P =0. 01; t =6. 82,P=0.00; Mankin score: t =3.41, P=0.01; t =5.00, P=0.00), with the 100-bFGF-M group much better (t =5.29, P =0. 00; t =2. 80, P =0. 02). Conclusions The bFGF slow-releasing microsphere can keep its effective intra-articular concentration, which may accelerate the synthesis of protcoglycan and inhibit its decomposition to reverse the damage of articular cartilage.     

壳聚糖微球介导人IL-1Ra与TGF-β1基因转染兔软骨细胞的研究

目的 探讨壳聚糖微球介导人IL-1Ra与TGF-β1基因转染兔软骨细胞.方法 制备壳聚糖微球介导IL-1Ra质粒与TGF-β1质粒转染系统,检测其载药、体外释药、降解,转染体外培养的兔膝软骨细胞,荧光显微镜、荧光定量PCR、MTT检测.结果 壳聚糖-IL-1Ra DNA和壳聚糖-TGF-β1 DNA微球平均径粒(2.8±0.2)μm和(2.6±0.1)μm,包封率(88.3±4.1)%和(87.2±2.6)%;缓释分3个阶段.荧光显微镜观察、荧光定量PCR证实软骨细胞转染基因得到30 d表达.MTT提示转染促进软骨细胞增殖.结论 壳聚糖微球介导IL-1Ra与TGF-β1基因转染软骨细胞可获得较长期目的基因表达,可促进软骨细胞增殖,为用于基因治疗软骨退变和促进软骨修复提供基础.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

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.     

Expression of MCP-1 and TGF-β1 and its significance in early stage of obstructive jaundice in rats

Objective To investigate the expression of MCP-1 and TGF-β1 in early stage of ob-structive jaundice and explore its relation to liver correlated injury indexes in rats. Methods Fifty male Wistar rats were randomized into the control group, sham-operated group and biliary obstruction group. On day 10, serum ALT and BIL-T levels were determined in inferior caval blood and hepatic MDA was estimated in liver homogenates. Meanwhile, serum TGF-β1 level was measured by ELISA and the MCP-1 infiltration determined with immunohistochemistry. Results Serum ALT and BIL-T values were elevated. Meanwhile, the liver MDA content was increased. The positive expression of MCP-1 was augmented and serum TGF-β1 was over-expressed in the early stage after obstructive jaun-dice in rats. The hepatic MCP-1 expression had a positive correlation with the elevated ALT, BIL-T and MDA levels. However, the serum TGF-β1 content only had a positive correlation with ALT and BIL-T. Conclution Hepatic MCP-1 and serum TGF-β1 expression in the early stage after biliary tract obstruction is related to liver injury and extrahepatic cholestasis. Meanwhile, the hepatic MCP-1 ex-pression is correlated with hepatic oxidation stress. They have an important significance in liver injury in rats during the early period of obstructive jaundice.     

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.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.     

Pro-inflammatory effect of transforming growth factor β1 in rat peritoneal mesothelial cells

Objective To investigate the pro-inflammatory effect of transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF-β1 (P<0.05), peak MCP-1 induction occurred at 6 h (P<0.01), and the stimulatory effect of TGF-β1 persisted through 24 h (P<0.05). MCP-1 protein levels were significantly increased after 6 h treatment with TGF-β1(P<0.05), peak MCP-1 induction occurred at 48 h(P<0.01). Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1 (P<0.05). TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.