pEGFP/Ang-1转染大鼠BMSCs视网膜下移植对糖尿病大鼠视网膜血管渗漏的影响
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摘要
目的
利用毕氏酵母血管生成素1重组质粒(pEGFP/Ang-1)转染大鼠BMSCs,将转染后的BMSCs行视网膜下移植,从而确认血管紧张素1(Ang-1)转染BMSCs对糖尿病大鼠视网膜的保护作用。
方法
选取雄性SD大鼠4~6只,无菌条件下取出股骨和胫骨,剪去骨骺端,用培养基反复冲洗骨髓腔分离BMSCs,并结合反复贴壁法使其进一步纯化鉴定。利用毕氏酵母血管生成素1重组质粒(pEGFP/Ang-1)转染大鼠BMSCs,将转染后的Ang-1/BMSC视网膜下移植。另选SD大鼠96只,随机分为正常大鼠组(CON)、空白对照糖尿病组(DM1)、糖尿病大鼠+视网膜下移植BMSCs组(DM2)、糖尿病大鼠+视网膜下移植Ang-1/BMSCs组(ANG)。尾静脉注射链脲佐菌素(STZ)建立糖尿病模型,成模后DM2组视网膜下移植BMSCs,ANG组视网膜下移植Ang-1/BMSCs,分别4w、8w、12w处死动物。用伊凡思蓝方法检测视网膜的渗透量;用免疫组化观察移植细胞在视网膜中的分布融合情况,Western blot方法检测各组大鼠视网膜Ang-1的表达情况。
结果
DM1、DM2组糖尿病大鼠渗透量较大,并随时间延长渗透性逐渐增加; ANG组视网膜下移植Ang-1/BMSCs能够明显抑制这种变化。免疫组化分析证实移植Ang-1/BMSCs主要整合在糖尿病大鼠视网膜节细胞层和内颗粒层。Western blot分析证明随着糖尿病视网膜病变的发生发展,DM1、DM2组糖尿病大鼠视网膜中Ang-1的表达量显著减少,而ANG组中Ang-1表达量较DM1、DM2显著增加,与CON、DM1、DM2组比较差异有统计学意义(P < 0.05), DM1与DM2组比较差异无统计学意义(P>0.05)。
结论
1. Ang-1/BMSCs视网膜下移植后见移植细胞整合在节细胞层和内颗粒层。
2. Ang-1/BMSCs视网膜下移植可以改善糖尿病大鼠视网膜微循环。
Objective
Use of Pichia pastoris recombinant angiopoietin 1 (pEGFP/Ang-1) transfected with rat BMSCs, BMSCs transfected lines after subretinal transplantation, thus confirming the angiotensin 1 (Ang-1) transfected BMSCs on diabetes Retina in rats. Use of Pichia pastoris recombinant angiopoietin 1 (pEGFP/Ang-1) transfected with rat BMSCs, after transfection Ang-1/BMSC the subretinal transplantation.
Methods
Male SD rats were selected from 4 to 6, under sterile conditions remove the femur and tibia, cut epiphyseal end of the bone marrow cavity with repeated washing medium isolated BMSCs, combined with repeated adherence to further purification and identification. Replacement of the 96 SD rats were randomly divided into normal rats (CON), diabetic control group (DM1), subretinal transplantation in diabetic rats + BMSCs group (DM2), subretinal transplantation in diabetic rats + Ang-1 / BMSCs group (ANG). Intravenous injection of streptozotocin (STZ) model of established diabetes, DM2 group after modeling subretinal transplantation BMSCs, ANG Group subretinal transplantation Ang-1/BMSCs, respectively, 4w, 8w, 12w animals were sacrificed..Detected by Evans blue infiltration capacity of the retina. Immunohistochemistry using transplanted cells in the retina of the distribution of fusion. Western blot method to detect the retinal expression of Ang-1.
Results
DM1, DM2 and diabetic rats with a large amount of infiltration and permeability increased gradually with time; SIM group subretinal transplantation Ang-1/BMSCs can inhibit this change. Immunohistochemical analysis confirmed that the main integration Ang-1/BMSCs diabetic rats transplanted retinal ganglion cell layer and internal granular layer. Western blot analysis showed that with the development of diabetic retinopathy, STZ-induced DM1, DM2 and diabetic rat retina the expression of Ang-1 significantly reduced, while the ANG group, Ang-1 expression was significantly increased, and DM1, DM2 There was significant difference between groups (P <0.05), DM1 and DM2 group were significantly (P> 0.05).
Conclusions
1、Ang-1/BMSCs retinal integration of transplanted cells after transplantation see in the ganglion cell layer, inner granular layer.
2、Ang-1/BMSCs subretinal transplantation of retinal microcirculation of diabetic rats.
利用毕氏酵母血管生成素1重组质粒(pEGFP/Ang-1)转染大鼠BMSCs,将转染后的BMSCs行视网膜下移植,从而确认血管紧张素1(Ang-1)转染BMSCs对糖尿病大鼠视网膜的保护作用。
方法
选取雄性SD大鼠4~6只,无菌条件下取出股骨和胫骨,剪去骨骺端,用培养基反复冲洗骨髓腔分离BMSCs,并结合反复贴壁法使其进一步纯化鉴定。利用毕氏酵母血管生成素1重组质粒(pEGFP/Ang-1)转染大鼠BMSCs,将转染后的Ang-1/BMSC视网膜下移植。另选SD大鼠96只,随机分为正常大鼠组(CON)、空白对照糖尿病组(DM1)、糖尿病大鼠+视网膜下移植BMSCs组(DM2)、糖尿病大鼠+视网膜下移植Ang-1/BMSCs组(ANG)。尾静脉注射链脲佐菌素(STZ)建立糖尿病模型,成模后DM2组视网膜下移植BMSCs,ANG组视网膜下移植Ang-1/BMSCs,分别4w、8w、12w处死动物。用伊凡思蓝方法检测视网膜的渗透量;用免疫组化观察移植细胞在视网膜中的分布融合情况,Western blot方法检测各组大鼠视网膜Ang-1的表达情况。
结果
DM1、DM2组糖尿病大鼠渗透量较大,并随时间延长渗透性逐渐增加; ANG组视网膜下移植Ang-1/BMSCs能够明显抑制这种变化。免疫组化分析证实移植Ang-1/BMSCs主要整合在糖尿病大鼠视网膜节细胞层和内颗粒层。Western blot分析证明随着糖尿病视网膜病变的发生发展,DM1、DM2组糖尿病大鼠视网膜中Ang-1的表达量显著减少,而ANG组中Ang-1表达量较DM1、DM2显著增加,与CON、DM1、DM2组比较差异有统计学意义(P < 0.05), DM1与DM2组比较差异无统计学意义(P>0.05)。
结论
1. Ang-1/BMSCs视网膜下移植后见移植细胞整合在节细胞层和内颗粒层。
2. Ang-1/BMSCs视网膜下移植可以改善糖尿病大鼠视网膜微循环。
Objective
Use of Pichia pastoris recombinant angiopoietin 1 (pEGFP/Ang-1) transfected with rat BMSCs, BMSCs transfected lines after subretinal transplantation, thus confirming the angiotensin 1 (Ang-1) transfected BMSCs on diabetes Retina in rats. Use of Pichia pastoris recombinant angiopoietin 1 (pEGFP/Ang-1) transfected with rat BMSCs, after transfection Ang-1/BMSC the subretinal transplantation.
Methods
Male SD rats were selected from 4 to 6, under sterile conditions remove the femur and tibia, cut epiphyseal end of the bone marrow cavity with repeated washing medium isolated BMSCs, combined with repeated adherence to further purification and identification. Replacement of the 96 SD rats were randomly divided into normal rats (CON), diabetic control group (DM1), subretinal transplantation in diabetic rats + BMSCs group (DM2), subretinal transplantation in diabetic rats + Ang-1 / BMSCs group (ANG). Intravenous injection of streptozotocin (STZ) model of established diabetes, DM2 group after modeling subretinal transplantation BMSCs, ANG Group subretinal transplantation Ang-1/BMSCs, respectively, 4w, 8w, 12w animals were sacrificed..Detected by Evans blue infiltration capacity of the retina. Immunohistochemistry using transplanted cells in the retina of the distribution of fusion. Western blot method to detect the retinal expression of Ang-1.
Results
DM1, DM2 and diabetic rats with a large amount of infiltration and permeability increased gradually with time; SIM group subretinal transplantation Ang-1/BMSCs can inhibit this change. Immunohistochemical analysis confirmed that the main integration Ang-1/BMSCs diabetic rats transplanted retinal ganglion cell layer and internal granular layer. Western blot analysis showed that with the development of diabetic retinopathy, STZ-induced DM1, DM2 and diabetic rat retina the expression of Ang-1 significantly reduced, while the ANG group, Ang-1 expression was significantly increased, and DM1, DM2 There was significant difference between groups (P <0.05), DM1 and DM2 group were significantly (P> 0.05).
Conclusions
1、Ang-1/BMSCs retinal integration of transplanted cells after transplantation see in the ganglion cell layer, inner granular layer.
2、Ang-1/BMSCs subretinal transplantation of retinal microcirculation of diabetic rats.
引文
1 Fong DS, Aiello L, Gardner TW, et al. Diabetic retinopathy. Diabetes Care,2003; 26(25) : 226-229.
2 Tomita M. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells,2002, 20(4):279-283.
3牟大鹏,苏冠方,徐春玲,等.骨髓间充质干细胞向视网膜神经节样细胞的诱导分化.眼科新进展Yanke Xin jin zhan, 2009, 29(36) : 94-97.
4张杰,单清,马萍,等.骨髓间充质干细胞在激光损伤大鼠视网膜下分化的观察.中华医学杂志, 2003, 83 (22) : 1993-1998.
5王陆飞,石艳,苏冠方,等.骨髓间充质干细胞在大鼠眼内分化情况的研究.中国实验诊断学, 2009,13(2):1007-1009.
6 Tomita M,Adachi Y,Yamada H,et al.Bone marrow-derived stem cells Can diferentiate into retinal cells in injured rat retina.Stem Cells,2002,20(4):279-283.
7 Minoru T, YasushiAd, Haruhiko Y, et al. BoneMarrow-derived stem cells can differentiate into retinal cells in injured rat retina. StemCells, 2002, 20(6): 200-204.
8 Lou J, Xu F, Merkel K, et al. Gene therapy: adenovirus mediated human bone morphogenetic protein-gene transfer induces mesenchymal progenitor cell proliferation and in vitro and bone formation in vivo.J Orthop Res, 1999, 17(9): 43-47.
9 Yoo JU, Mandell I,Angele P, et al. Chondrogenitor cells and gene therapy.Clin Orthop, 2000, 379(30): 164-168.
10管怀进,龚启荣.现代基础眼科学.北京人民军医出版社,1998;285-290.
11 Xu Q ,Qaum T ,Adamis AP. Sensitive blood-retinal barrier breakdownquantitation using Evans blue. Invest Ophthalmol Vis Sci, 2001; 42(3) :789～794.
12 Ishida S,Usui T,Yamashiro K,et al. VEGF164 is proinflammatory in the diabetic retina. Invest Ophthalmol Vis Sci, 2003, 44(5) :2155～2162.
13 Maisonpierre PC,Suri C,Jones PF,et al. Angropoietin-2, anature antagonist for Tie2 that disrupt in vjvo angiogenesis.Science,1997,27(7):55-60.
14李国灏,杨为民.血管生成素的作用、表达及其肿瘤血管生长的关系.临床外科杂志,2004,5(12) : 311-312.
15 Suri C, Jones PF, Patan S, et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor.during embryonic angiogenesis Cell, 1996, 87(12) : 1171-1180.
16 Thurston G, Rudge JS, Loffe E, et al.Angiopoietin-1 protects the adult vasculature against plasma leakage Nat Med, 2000, 6(4) : 460-463.
17左中夫,刘学政. Ang/Tie-2信号传导系统与视网膜微血管形成.国际眼科学杂志, 2006, 6 ( 4) : 857-859.
18 Janssen AM, Poulaki V, Tsujikawa A, et al. Suppression of diabetic retinopathy with angiopoietin-1. Am J Pathol, 2002, 160(5) : 1683-1693.
19 Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.Stem Cells, 2002, 20(4) : 279-283.
20 Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.Stem Cells, 2002, 20(4) : 279-283.
21 Anthony K, Shen WY, Wilson AS,et al. Differentiation of marow stromal cells into photoreceptors in the rat eye.J Neurosci, 2003, 23(21) : 7742-7749.
22 Kicic A, Shen WY, Wilson AS, et al. Differentiation of marrow stromal cells into photoreceptors in the rat eye.J Neurosci, 2003, 23(21) : 7760-7768.
23 Arnhold S, Absenger Y, Klein H, et al. Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse.Graefes Arch Clin Exp Ophthalmol, 2007, 24(5) : 414-422.
24 Boettcher C, Ulbricht E, Helmlinger D, et al. Long-term engraftment of systemically transplanted, gene-modified bone marrow-derived cells in the adult mouse retina.Br J Ophthalmol, 2003,12(21) : 740-745.
25计菁,罗敏,冯霞.大鼠胚胎视网膜中神经干细胞的体外培养和鉴定.眼科新进展, 2008; 28(6) : 429-431.
26张琨,卢光琇,高铃,等.大鼠胚胎视网膜干细胞的分离培养.眼科新进展, 2006, 26(9) : 646-649.
27 Lew D, Parker SE, Latimer T, et al. Cancer gene therapy using plasmid DNA:Pharma- cokimetic study of DNA following injection in mice. Hum GeneTher,1995, 6 (12) : 553-564.
28袁伟,路磊.绿色荧光蛋白转染骨髓间充质干细胞及体外向神经样细胞的分化.中国组织工程研究与临床康复, 2008, 47(12) : 9253-9256.
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18张杰,单清,马萍,等.骨髓间充质干细胞在激光损伤大鼠视网膜下分化的观察.中华医学杂志, 2003, 83(22): 1993-1998.
19王陆飞,石艳,苏冠方,等.骨髓间充质干细胞在大鼠眼内分化情况的研究.中国实验诊断学, 2009, 13(24) : 1007-1009.
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21 Tomita M,Adachi Y,Yamada H, et a1. Bone marrow-derived stem cells Can diferentiate into retinal cells in injured rat retina.Stem Cells,2002,20(4) : 279-283.
22 Minoru T, YasushiAd, Haruhiko Y, et al. BoneMarrow-derived stem cells can differentiate into retinal cells in injured rat retina. StemCells, 2002, 20(6): 200-204.
23 Anthony K,Shen W Y,Wilson AS, et al.Differentiation of marow stromal cells into photoreceptors in the rat eye.J Neurosci,2003,23(21) : 7742-7749.
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26 A rnhold S, He iduschka P, Klein H, Absenger Y, B asnaoglu S,Kreppel F, et al. Adenovirally transduced bone marrow cells differentiate in to pigment epithelial cells and induce rescue effectsion RCS rats. Invest Ophthalm of VisSci, 2006, 47( 9 ) : 4121-4129.
27 Arnhold S,Absenger Y,Klein H,et al.Transplantation of bone marrow.derired mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse.Graefes Arch Clin Exp Ophthalmol, 2007, 245(3) : 414-422.
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2 Tomita M. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells,2002, 20(4):279-283.
3牟大鹏,苏冠方,徐春玲,等.骨髓间充质干细胞向视网膜神经节样细胞的诱导分化.眼科新进展Yanke Xin jin zhan, 2009, 29(36) : 94-97.
4张杰,单清,马萍,等.骨髓间充质干细胞在激光损伤大鼠视网膜下分化的观察.中华医学杂志, 2003, 83 (22) : 1993-1998.
5王陆飞,石艳,苏冠方,等.骨髓间充质干细胞在大鼠眼内分化情况的研究.中国实验诊断学, 2009,13(2):1007-1009.
6 Tomita M,Adachi Y,Yamada H,et al.Bone marrow-derived stem cells Can diferentiate into retinal cells in injured rat retina.Stem Cells,2002,20(4):279-283.
7 Minoru T, YasushiAd, Haruhiko Y, et al. BoneMarrow-derived stem cells can differentiate into retinal cells in injured rat retina. StemCells, 2002, 20(6): 200-204.
8 Lou J, Xu F, Merkel K, et al. Gene therapy: adenovirus mediated human bone morphogenetic protein-gene transfer induces mesenchymal progenitor cell proliferation and in vitro and bone formation in vivo.J Orthop Res, 1999, 17(9): 43-47.
9 Yoo JU, Mandell I,Angele P, et al. Chondrogenitor cells and gene therapy.Clin Orthop, 2000, 379(30): 164-168.
10管怀进,龚启荣.现代基础眼科学.北京人民军医出版社,1998;285-290.
11 Xu Q ,Qaum T ,Adamis AP. Sensitive blood-retinal barrier breakdownquantitation using Evans blue. Invest Ophthalmol Vis Sci, 2001; 42(3) :789～794.
12 Ishida S,Usui T,Yamashiro K,et al. VEGF164 is proinflammatory in the diabetic retina. Invest Ophthalmol Vis Sci, 2003, 44(5) :2155～2162.
13 Maisonpierre PC,Suri C,Jones PF,et al. Angropoietin-2, anature antagonist for Tie2 that disrupt in vjvo angiogenesis.Science,1997,27(7):55-60.
14李国灏,杨为民.血管生成素的作用、表达及其肿瘤血管生长的关系.临床外科杂志,2004,5(12) : 311-312.
15 Suri C, Jones PF, Patan S, et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor.during embryonic angiogenesis Cell, 1996, 87(12) : 1171-1180.
16 Thurston G, Rudge JS, Loffe E, et al.Angiopoietin-1 protects the adult vasculature against plasma leakage Nat Med, 2000, 6(4) : 460-463.
17左中夫,刘学政. Ang/Tie-2信号传导系统与视网膜微血管形成.国际眼科学杂志, 2006, 6 ( 4) : 857-859.
18 Janssen AM, Poulaki V, Tsujikawa A, et al. Suppression of diabetic retinopathy with angiopoietin-1. Am J Pathol, 2002, 160(5) : 1683-1693.
19 Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.Stem Cells, 2002, 20(4) : 279-283.
20 Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.Stem Cells, 2002, 20(4) : 279-283.
21 Anthony K, Shen WY, Wilson AS,et al. Differentiation of marow stromal cells into photoreceptors in the rat eye.J Neurosci, 2003, 23(21) : 7742-7749.
22 Kicic A, Shen WY, Wilson AS, et al. Differentiation of marrow stromal cells into photoreceptors in the rat eye.J Neurosci, 2003, 23(21) : 7760-7768.
23 Arnhold S, Absenger Y, Klein H, et al. Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse.Graefes Arch Clin Exp Ophthalmol, 2007, 24(5) : 414-422.
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