糖尿病血管病变的分子免疫机理研究
摘要
目的 从分子水平研究刺激性免疫分子和与细胞凋亡有关因子在糖尿病血管病变中的变化，探讨这些免疫分子之间以及这些免疫分子与糖尿病血管病变发生发展之间的相关性，为早期防治糖尿病血管并发症提供一定的理论依据。
方法 应用酶联免疫吸附实验法(ELISA)测定30例正常人和64例2型糖尿病患者血浆血小板衍化生长因子(PDGF-BB)、血管内皮生长因子(VEGF)、可溶性Fas(sFas)和可溶型补体末端复合物(sC5b-9)的含量。
结果 ①2型糖尿病组与正常对照组比较，血浆sC5b-9含量明显升高(P＜0.01)。②2型糖尿病组与正常对照组比较，血浆PDGF-BB含量明显升高(P＜0.01)。2型糖尿病有血管病变组与糖尿病无血管病变组相比，血浆PDGF-BB明显增高(P＜0.05)。③2型糖尿病组与正常对照组比较，血浆VEGF含量明显升高(P＜0.01)。2型糖尿病有血管病变组与糖尿病无血管病变组相比，血浆VEGF明显升高，差异显著(P＜0.05)。④2型糖尿病组与正常对照组比较，血浆sFas含量明显升高(P＜0.01)。2型糖尿病有血管病变组与糖尿病无血管病变组相比，血浆sFas明显升高(P＜0.05)。
结论 PDGF-BB、VEGF、sFas和补体末端复合物sC5b-9参与了糖尿病血管病变的发生和发展，其可能机制是：长期高血糖使补体调节蛋白CD59被糖化，其抑制补体末端复合物C5b-9形成的功能受损，sC5b-9生成增多。sC5b-9与血管内皮细胞和平滑肌细胞结合致使内皮细胞和平滑肌细胞损伤活化，内皮细胞和平滑肌细胞释放刺激性免疫分子，主要为PDGF-BB和VEGF，另外，PDGF-BB对VEGF的表达具有正调节作用。PDGF-BB和VEGF通过和内皮细胞、平滑肌细胞上相应受体结合，促使平滑肌细胞、单核巨噬细胞、内皮细胞增生和内膜下迁移，同时产生和分泌多种生长因子，再通过自分泌和旁分泌作用，进一步促进这些细胞增殖；内皮细胞和平滑肌细胞损伤活化高表达sFas，sFas通过与细胞膜Fas竞争与Fas配体结合拮抗Fas产生凋亡，使凋亡信号
中文摘要
减弱。刺激信号的增强、凋亡信号的减弱最终导致糖尿病血管病变的
发生和发展。
Objective To study the change of irritative immune molecules and factor about cell apoptosis and the relationship between these immune molecules and development of diabetic angiopathy and to provide academic basis for the prevention of diabetic vascular complications.
Methods Plasma sC5b-9, PDGF-BB, VEGF and sFas levels were determined by an enzyme-linked immunosorbent assay in 64 cases of non-insulin-dependent diabetes mellitus (NIDDM) and 30 cases of healthy individuals.
Results The levels of sC5b-9, PDGF-BB,VEGF and sFas in patients with NIDDM were significantly higher than those in healthy individuals(P<0.01). Levels of PDGF-BB, VEGF and sFas in DM patients with vascular disease were significantly higher than those in DM patients without vascular disease (P<0.05).
Conclusions sC5b-9, PDGF-BB, VEGF and sFas may be involved in development and progress of diabetic angiopathy. Long increased glucose level in diabetes causes complement regulatory membrane protein CD59 glycation, which impairs its function to restrict C5b-9 formation. sC5b-9 increases. Insertion of the C5b-9 into endothelial cell and smooth muscle cell membranes causes these cells activation and the release of irritative immune molecules from endothelial cells and smooth muscle cells. These immune molecules mainly contain PDGF-BB and VEGF. In addition, PDGF-BB has certain up-regulation on expression of VEGF. By binding to relevant receptors on endothelial cells and smooth muscle cells, PDGF-BB and VEGF promote proliferation of smooth muscle cells, monocyte-macrophages and endothelial cells and the release of several growth factors which then further promote these cells proliferation by the way of autocrine and paracrine pathway. Activated endothelial cells and smooth muscle cells express sFas which competit
ively bind Fas ligand with Fas to reverse apoptosis that Fas reduced. Both increased stimulation
signal and decreased apoptosis signal lead to development and progress of diabetic angiopathy.
方法 应用酶联免疫吸附实验法(ELISA)测定30例正常人和64例2型糖尿病患者血浆血小板衍化生长因子(PDGF-BB)、血管内皮生长因子(VEGF)、可溶性Fas(sFas)和可溶型补体末端复合物(sC5b-9)的含量。
结果 ①2型糖尿病组与正常对照组比较，血浆sC5b-9含量明显升高(P＜0.01)。②2型糖尿病组与正常对照组比较，血浆PDGF-BB含量明显升高(P＜0.01)。2型糖尿病有血管病变组与糖尿病无血管病变组相比，血浆PDGF-BB明显增高(P＜0.05)。③2型糖尿病组与正常对照组比较，血浆VEGF含量明显升高(P＜0.01)。2型糖尿病有血管病变组与糖尿病无血管病变组相比，血浆VEGF明显升高，差异显著(P＜0.05)。④2型糖尿病组与正常对照组比较，血浆sFas含量明显升高(P＜0.01)。2型糖尿病有血管病变组与糖尿病无血管病变组相比，血浆sFas明显升高(P＜0.05)。
结论 PDGF-BB、VEGF、sFas和补体末端复合物sC5b-9参与了糖尿病血管病变的发生和发展，其可能机制是：长期高血糖使补体调节蛋白CD59被糖化，其抑制补体末端复合物C5b-9形成的功能受损，sC5b-9生成增多。sC5b-9与血管内皮细胞和平滑肌细胞结合致使内皮细胞和平滑肌细胞损伤活化，内皮细胞和平滑肌细胞释放刺激性免疫分子，主要为PDGF-BB和VEGF，另外，PDGF-BB对VEGF的表达具有正调节作用。PDGF-BB和VEGF通过和内皮细胞、平滑肌细胞上相应受体结合，促使平滑肌细胞、单核巨噬细胞、内皮细胞增生和内膜下迁移，同时产生和分泌多种生长因子，再通过自分泌和旁分泌作用，进一步促进这些细胞增殖；内皮细胞和平滑肌细胞损伤活化高表达sFas，sFas通过与细胞膜Fas竞争与Fas配体结合拮抗Fas产生凋亡，使凋亡信号
中文摘要
减弱。刺激信号的增强、凋亡信号的减弱最终导致糖尿病血管病变的
发生和发展。
Objective To study the change of irritative immune molecules and factor about cell apoptosis and the relationship between these immune molecules and development of diabetic angiopathy and to provide academic basis for the prevention of diabetic vascular complications.
Methods Plasma sC5b-9, PDGF-BB, VEGF and sFas levels were determined by an enzyme-linked immunosorbent assay in 64 cases of non-insulin-dependent diabetes mellitus (NIDDM) and 30 cases of healthy individuals.
Results The levels of sC5b-9, PDGF-BB,VEGF and sFas in patients with NIDDM were significantly higher than those in healthy individuals(P<0.01). Levels of PDGF-BB, VEGF and sFas in DM patients with vascular disease were significantly higher than those in DM patients without vascular disease (P<0.05).
Conclusions sC5b-9, PDGF-BB, VEGF and sFas may be involved in development and progress of diabetic angiopathy. Long increased glucose level in diabetes causes complement regulatory membrane protein CD59 glycation, which impairs its function to restrict C5b-9 formation. sC5b-9 increases. Insertion of the C5b-9 into endothelial cell and smooth muscle cell membranes causes these cells activation and the release of irritative immune molecules from endothelial cells and smooth muscle cells. These immune molecules mainly contain PDGF-BB and VEGF. In addition, PDGF-BB has certain up-regulation on expression of VEGF. By binding to relevant receptors on endothelial cells and smooth muscle cells, PDGF-BB and VEGF promote proliferation of smooth muscle cells, monocyte-macrophages and endothelial cells and the release of several growth factors which then further promote these cells proliferation by the way of autocrine and paracrine pathway. Activated endothelial cells and smooth muscle cells express sFas which competit
ively bind Fas ligand with Fas to reverse apoptosis that Fas reduced. Both increased stimulation
signal and decreased apoptosis signal lead to development and progress of diabetic angiopathy.
引文
1 田凤华．中国糖尿病现状及初步分析．中华流行病学杂志 1998，19：361-362
2 潘长玉，陆菊明，田慧，等．2型糖尿病患者初诊时血管并发症患病率的调查分析．中华内分泌代谢杂志 1997，13201-205
3 Weller M, Malipiero U, Rensing-Ehl A, et al. Fas/Apol gene transfer for human malignant glioma [J]. ConneerRes, 1995,55(13):2936-2944
4 Nishigaki K, Minatoguchi S, Seishims M, et al. Plasma Fas ligand, an inducer of apoptosis and plasma soluble Fas, an inhibitor of apoptosis, in patients with chlonic congestive heart failure [J]. J A M Coll Cardiol, 1997, 29(6):1214-1220
5 UK Prospective Diabetes Study Group. Effect of intensive blood-glucise control with metformin on complication in overweight with type 2 diabetes (UKPDS 34).Lancet, 1998, 352:854
6 Acosta J, Hettinga J, Fluckiger R, et al. Molecular basis for a link between complement and the vascular complications of diabetes [J]. Proc Natl,2000,97(10): 5450-5
7 Ninomiyah, Stewart B H, Rollins S A, et al. Contribution of the linked carbohydrate of erythrocyte antigen CD59 to its complement inhibitory activity. J BioChem, 1992,267:840-4
8 Lorenzi M. Glucose toxicity in the vascular complications of diabetes: the cellular perspetive. Diabetes Metab Rev,1992,8:85-103
9 Rus HG et al. Atherosclerosis,1986;61:35-42
10 Niculescu F, Huge F, Rus Hg, et al. Quantitative evaluation of the terminal C5b-9 complement complex by ELISA in human atherosclerotic arteries. Clin Exp Immunol, 1987;69:477-483
11 Caraher EM, Conroy SJ, Newsholme P, et al. Evidence for enhanced rates of complement activation in serum from patients with newly diagnosed insulin-dependent diabetes mellitus exposed to rat islet cells and complement-dependent induction of islet cell apoptosis, J Endocrinol.1999 Jul;162 (1):143-53
12 Tedeso F, Pausa M, Nardon E, et al. J exp Med, 1997,185:1619-1627
13 周镜然，朱锡华，姜曼．补体膜攻击复合物刺激血管内皮细胞胞浆游离钙离子浓度的变化．细胞与分子免疫学杂志 2002，18(4)：342
14 Benzaquen L R, Nicholson weller A, Halperin J. Terminal complement proteins
C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial·cells[J]. J Exp Med, 1994,179(3):985-992
15 Kilgore KS et al. Am J Pathol,1996;149:953-961
16 Bui MN, Sack MN, Moutsatsos G, et al. Autoantibody titers to oxidized lowdensity lipoprotein in patients with coronary atherosclerosis. Am Heart J,1996;131:663-667
17 Bhakdi S. Arterioscler Thromb, 1992;12:536
18 Ronald AS, Charles E.A, Daniel F.BP, et al. Platelet derived growth factor in renal development and disease. Kidney Int, 1998,54:731-746
19 Dobrian A, S.S. Wade, R.L. Prewitt. PDGF A expression correlates with blood pressure and remodeling in 1KIC hypertensive rat arteries. Am Jphysiol, 1999,276 (6pt2):H2159-2167
20 Futamura A, Izumino K, Nakagawa Y, et al. Effect of the platelet derived growth factor antagonist trapidil on mesangial cell proliferation in rats. Nephron, 1999, 81:428-433
21 Beitz JG, Kim IS, Calabresi P, et al. Human microvascular endothelial cells express receptors for platelet-derived growth factor. Proc Natl Acad Sci USA,1991, 88:2021-2025
22 Thommen R, Humar R, Misevic G, et al. PDGF-BB increases endothelial migration on cord movements during angiogenesis in vitro, J Cell Biochem, 1997,64:403-413
23 Eriksson A, Siegbahn A, estermark B, et al. PDGF alpha- and beta- receptors activae unique and common signal transduction pathways. EMBO J,1992,11:543-550
24 Hermann S et al. Science,1995;267(10):1445
25 Benzaquen L R et al. J Exp Med,1994;179:985
26 季军，方卫华，司履生．兔髂动脉内膜损伤后PDGF-B mRNA在血管壁原位表达增强．中国病理生理杂志 2000，16(7)：603-605
27 任雨笙，贾国良．血小板衍化生长因子对血管组织细胞生物学作用的研究．心功能杂志 1999；11(2)：83-84
28 Kelly JD et al. J Biol Chem,1994;266(14):8987-8992
29 Failer DV et al. J Biol Chem,1994;269(17):5022-5026
30 Cantley LC et al. Cell, 1991;64:281-302
31 Hunter T et al. Cell,1991;64:249-260
32 Aleria V, Pajusola K, Kumar V, et al. Circulation, 1996,93:1493.
33 Soker S, J BioChem,1993,268:7685-7691
34 Liu Y, Circulation, 1995,77:638-643
35 Ryuto M, Ono M, Izumi H, et al. J Biol Chem, 1996,271:28220-28228
36 Gille J, Swerlick RA, Caughman SW, et al. EMBO J,1997,16:750-759
37 Hammes HP, Jihong LIN, Bretxel RG, et al. Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental baokground diabetic retiopathy of the rat [J] Diabetes, 1998,47(3):401-406
38 Spiriabetic human retinasn KS, Saghizadeh M, Lewin SL, et al. Basement membrane and growth factor gene expression in normal and di. Curr Eye Res, 1999,18 (6):490-499
39 张曼，许华林，张敏，等．高糖刺激血管平滑肌细胞血管内皮生长因子的表达．中华内分泌代谢杂志 2002；18(3)：232
40 Vriese AS, Tilton RG, Elger Ⅱ, et al. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes.[J] J Am Soc Nephrol,2001,12(5):993-1000
41 Cha DR, Kim NH, Yoon JW, et al. Role of vascular endothelial growth factor in diabetic nephropathy. [J] Kedney Int,2000,58(Supp17):S104-S112
42 Blann AD, Belgore FM, McCollum CN, et al. Vascular endothelial growth factor and its receptor, Fit-1, in the plasma of patients with coronary or peripheral atherosclerosis, or Type Ⅱ diabetes.[J] Clin Sci(Lond).2002 Feb;102(2):187-94
43 Tanaka M, Suda T, Takahashi T, et al. EMBO J,1995;14:1129
44 Moiler P, Koretz K, Leithhauser F, et al. Expression of APO1(CD95)、a member of the NGF/TNF receptor super family, in normal and enoplastic colon epithelium[J]. Int J Cancer, 1994,57(3):371
45 仇生龙，彭志海，王裕发．大肠癌患者血清可溶性Fas(sFas)水平表达．肿瘤 2000，20(3)：195-197
46 Cheng J, Zhou T, Liu C, et al. Protection from Fas mediated apoptosis by a soluble form of the Fas molecule. Science, 1994,263:1759-1762
47 Guillot R, Bringuier AF, Porokhov B, et al. Increased levels of soluble Fas in serum from diabetic patients with neuropathy. Diabetes Metab.2001;27(3):315-
321
48 Amasaki Y, Kobayashi S, Takeda T, et al. UP-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erthe matosus (SLE): a possible mechanism for lymphapenia. Clin Exp Immuno,1995;99:245
49 Matsui T. Two platelet-derived growth factor receptors in vascular smooth muscle cells. Jpn Circ J, 1991,55:1027
50 Poptic EJ, Dicorleto PE. Cultured bovine aortic endothelial cells (BAEC) secrete a homodimeric BB form of PDGF. FASEB J,1990,4:A488
51 Engerman RL, Plaffenbach D, Dares MD. Cell turnover of capillaries [J]. Lab Invest, 1967,17:739-743
2 潘长玉，陆菊明，田慧，等．2型糖尿病患者初诊时血管并发症患病率的调查分析．中华内分泌代谢杂志 1997，13201-205
3 Weller M, Malipiero U, Rensing-Ehl A, et al. Fas/Apol gene transfer for human malignant glioma [J]. ConneerRes, 1995,55(13):2936-2944
4 Nishigaki K, Minatoguchi S, Seishims M, et al. Plasma Fas ligand, an inducer of apoptosis and plasma soluble Fas, an inhibitor of apoptosis, in patients with chlonic congestive heart failure [J]. J A M Coll Cardiol, 1997, 29(6):1214-1220
5 UK Prospective Diabetes Study Group. Effect of intensive blood-glucise control with metformin on complication in overweight with type 2 diabetes (UKPDS 34).Lancet, 1998, 352:854
6 Acosta J, Hettinga J, Fluckiger R, et al. Molecular basis for a link between complement and the vascular complications of diabetes [J]. Proc Natl,2000,97(10): 5450-5
7 Ninomiyah, Stewart B H, Rollins S A, et al. Contribution of the linked carbohydrate of erythrocyte antigen CD59 to its complement inhibitory activity. J BioChem, 1992,267:840-4
8 Lorenzi M. Glucose toxicity in the vascular complications of diabetes: the cellular perspetive. Diabetes Metab Rev,1992,8:85-103
9 Rus HG et al. Atherosclerosis,1986;61:35-42
10 Niculescu F, Huge F, Rus Hg, et al. Quantitative evaluation of the terminal C5b-9 complement complex by ELISA in human atherosclerotic arteries. Clin Exp Immunol, 1987;69:477-483
11 Caraher EM, Conroy SJ, Newsholme P, et al. Evidence for enhanced rates of complement activation in serum from patients with newly diagnosed insulin-dependent diabetes mellitus exposed to rat islet cells and complement-dependent induction of islet cell apoptosis, J Endocrinol.1999 Jul;162 (1):143-53
12 Tedeso F, Pausa M, Nardon E, et al. J exp Med, 1997,185:1619-1627
13 周镜然，朱锡华，姜曼．补体膜攻击复合物刺激血管内皮细胞胞浆游离钙离子浓度的变化．细胞与分子免疫学杂志 2002，18(4)：342
14 Benzaquen L R, Nicholson weller A, Halperin J. Terminal complement proteins
C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial·cells[J]. J Exp Med, 1994,179(3):985-992
15 Kilgore KS et al. Am J Pathol,1996;149:953-961
16 Bui MN, Sack MN, Moutsatsos G, et al. Autoantibody titers to oxidized lowdensity lipoprotein in patients with coronary atherosclerosis. Am Heart J,1996;131:663-667
17 Bhakdi S. Arterioscler Thromb, 1992;12:536
18 Ronald AS, Charles E.A, Daniel F.BP, et al. Platelet derived growth factor in renal development and disease. Kidney Int, 1998,54:731-746
19 Dobrian A, S.S. Wade, R.L. Prewitt. PDGF A expression correlates with blood pressure and remodeling in 1KIC hypertensive rat arteries. Am Jphysiol, 1999,276 (6pt2):H2159-2167
20 Futamura A, Izumino K, Nakagawa Y, et al. Effect of the platelet derived growth factor antagonist trapidil on mesangial cell proliferation in rats. Nephron, 1999, 81:428-433
21 Beitz JG, Kim IS, Calabresi P, et al. Human microvascular endothelial cells express receptors for platelet-derived growth factor. Proc Natl Acad Sci USA,1991, 88:2021-2025
22 Thommen R, Humar R, Misevic G, et al. PDGF-BB increases endothelial migration on cord movements during angiogenesis in vitro, J Cell Biochem, 1997,64:403-413
23 Eriksson A, Siegbahn A, estermark B, et al. PDGF alpha- and beta- receptors activae unique and common signal transduction pathways. EMBO J,1992,11:543-550
24 Hermann S et al. Science,1995;267(10):1445
25 Benzaquen L R et al. J Exp Med,1994;179:985
26 季军，方卫华，司履生．兔髂动脉内膜损伤后PDGF-B mRNA在血管壁原位表达增强．中国病理生理杂志 2000，16(7)：603-605
27 任雨笙，贾国良．血小板衍化生长因子对血管组织细胞生物学作用的研究．心功能杂志 1999；11(2)：83-84
28 Kelly JD et al. J Biol Chem,1994;266(14):8987-8992
29 Failer DV et al. J Biol Chem,1994;269(17):5022-5026
30 Cantley LC et al. Cell, 1991;64:281-302
31 Hunter T et al. Cell,1991;64:249-260
32 Aleria V, Pajusola K, Kumar V, et al. Circulation, 1996,93:1493.
33 Soker S, J BioChem,1993,268:7685-7691
34 Liu Y, Circulation, 1995,77:638-643
35 Ryuto M, Ono M, Izumi H, et al. J Biol Chem, 1996,271:28220-28228
36 Gille J, Swerlick RA, Caughman SW, et al. EMBO J,1997,16:750-759
37 Hammes HP, Jihong LIN, Bretxel RG, et al. Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental baokground diabetic retiopathy of the rat [J] Diabetes, 1998,47(3):401-406
38 Spiriabetic human retinasn KS, Saghizadeh M, Lewin SL, et al. Basement membrane and growth factor gene expression in normal and di. Curr Eye Res, 1999,18 (6):490-499
39 张曼，许华林，张敏，等．高糖刺激血管平滑肌细胞血管内皮生长因子的表达．中华内分泌代谢杂志 2002；18(3)：232
40 Vriese AS, Tilton RG, Elger Ⅱ, et al. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes.[J] J Am Soc Nephrol,2001,12(5):993-1000
41 Cha DR, Kim NH, Yoon JW, et al. Role of vascular endothelial growth factor in diabetic nephropathy. [J] Kedney Int,2000,58(Supp17):S104-S112
42 Blann AD, Belgore FM, McCollum CN, et al. Vascular endothelial growth factor and its receptor, Fit-1, in the plasma of patients with coronary or peripheral atherosclerosis, or Type Ⅱ diabetes.[J] Clin Sci(Lond).2002 Feb;102(2):187-94
43 Tanaka M, Suda T, Takahashi T, et al. EMBO J,1995;14:1129
44 Moiler P, Koretz K, Leithhauser F, et al. Expression of APO1(CD95)、a member of the NGF/TNF receptor super family, in normal and enoplastic colon epithelium[J]. Int J Cancer, 1994,57(3):371
45 仇生龙，彭志海，王裕发．大肠癌患者血清可溶性Fas(sFas)水平表达．肿瘤 2000，20(3)：195-197
46 Cheng J, Zhou T, Liu C, et al. Protection from Fas mediated apoptosis by a soluble form of the Fas molecule. Science, 1994,263:1759-1762
47 Guillot R, Bringuier AF, Porokhov B, et al. Increased levels of soluble Fas in serum from diabetic patients with neuropathy. Diabetes Metab.2001;27(3):315-
321
48 Amasaki Y, Kobayashi S, Takeda T, et al. UP-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erthe matosus (SLE): a possible mechanism for lymphapenia. Clin Exp Immuno,1995;99:245
49 Matsui T. Two platelet-derived growth factor receptors in vascular smooth muscle cells. Jpn Circ J, 1991,55:1027
50 Poptic EJ, Dicorleto PE. Cultured bovine aortic endothelial cells (BAEC) secrete a homodimeric BB form of PDGF. FASEB J,1990,4:A488
51 Engerman RL, Plaffenbach D, Dares MD. Cell turnover of capillaries [J]. Lab Invest, 1967,17:739-743