【作者】 赵莹；

【导师】 王海昌； 张荣庆；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 硕士

【摘要】 研究背景冠状动脉粥样硬化性心脏病简称冠心病（coronary heart disease,CHD）,是影响人类健康的主要疾病之一,经皮冠状动脉内成形术（percutaneous transluminal coronary angioplasty,PTCA）目前已经成为一种常规有效的治疗CHD的方法。药物支架（drug-eluting stent,DES）的使用使支架置入术后支架内再狭窄（in-stent restenosis,ISR）的发生率降低到5～10%左右,但仍然无法彻底解决ISR的问题。大量的多中心、随机和双盲对照研究显示,糖尿病（diabetes millitus,DM）患者PTCA的疗效差,ISR发生率较高[1-3],具体机制的研究仍不够深入,因而严重影响其临床应用。同时有研究显示,置入体内的金属支架在体内会以“洗脱”的方式脱出Fe²⁺[4],不同类型支架“洗脱”到支架周围的Fe²⁺量并非完全一致,而且其对与之直接接触的血管平滑肌细胞（vascular smooth muscle cells,VSMC）的生物功能的影响尚不清楚。有研究认为Fe²⁺对VSMC增殖有一定程度抑制作用[5],此研究结论似乎与临床观察到的现象不相符合。为此,本研究采用原代培养的3～5代人脐静脉平滑肌细胞（human umbilical vascular smooth muscle cell,HUSMC）,参照上述研究[5]采用的Fe²⁺浓度梯度分组,分别用普通培养液模拟正常人体内环境、高糖培养液模拟人体内高糖环境、高胰岛素环境模拟人体高胰岛素环境培养HUVSMC,以研究Fe²⁺、胰岛素对其增殖以及金属蛋白酶-2（matrix metalloproteinase-2,MMP-2）分泌功能的影响,探讨血糖异常和/或胰岛素抵抗患者体内金属支架ISR发生的可能机制并找寻临床证据。研究目的1.观察Fe²⁺对葡萄糖预孵育的VSMC增殖及MMP-2分泌的影响。2.观察胰岛素对葡萄糖预孵育的VSMC增殖及MMP-2分泌的影响。3.观察Fe²⁺对葡萄糖和胰岛素预孵育的VSMC增殖、MMP-2分泌以及VSMC胞内Ca²⁺浓度的影响。4.探讨其可能的作用机制。第一部分方法健康产妇剖宫产分娩的无菌胎儿脐带分离出脐静脉,组织块法培养HUVSMC,取生长稳定的3～5代细胞,分为正常组（葡萄糖5 mmol/L）以及高糖组（葡萄糖25 mmol/L）的。1.各分为六个亚组:①空白对照组O;②Fe²⁺组A（0.00625 mg/mL）;③Fe²⁺组B（0.0125 mg/mL）;④Fe²⁺组C（0.025 mg/mL）;⑤Fe²⁺组D（0.05 mg/mL）;⑥Fe²⁺组E（0.1 mg/mL）。37℃、5%CO2培养箱内干预24 h。2. MTT比色法测定细胞增殖情况。3. ELISA试剂盒检测细胞培养上清中MPP-2含量。结果1.正常组:与O组比,B～E组对VSMC有促进增殖作用（P<0.05）;仅E组能明显促进VSMC分泌MMP-2（P<0.01）,其余各组与对照组间差异无统计学意义。2.高糖组:与O组比,B～E组,能促进VSMC增殖（P<0.01）及MMP-2的分泌,D组、E组促进作用最强。3.正常组与高糖组比较:B～E各相应亚组间,高糖组对VSMC增殖及MMP-2分泌的促进作用较正常组强（P<0.05）。结论正常及高葡萄糖环境,适当的浓度Fe²⁺能影响VSMC的生物学特性,促进VSMC的增殖与MMP-2的分泌,且高糖环境下作用更为显著。第二部分方法1.同实验一。2.各分为4个亚组:①空白对照组O;②胰岛素组A（10 mIU）;③胰岛素组B（100 mIU）;④胰岛素组C（1000 mIU）。37℃、5%CO2培养箱内干预48 h。3. MTT比色法测定细胞增殖情况。4. ELISA试剂盒检测细胞培养上清中MPP-2含量。结果1.正常组:与O组比,各亚组均可促进VSMC增殖（P<0.01）及MMP-2的分泌（P<0.01）,且B组（100 mIU）作用最强。2.高糖组:与O组比,A组、B组均促进VSMC的增殖（P<0.05）;但A、B、C三个亚组均能促进MMP-2的分泌（P<0.01）,且B组（100 mIU/L）作用最强。3.正常组与高糖组比较:A、B各相应亚组间,高糖组对VSMC增殖及分泌MMP-2作用较正常组强（P<0.05）,而相应C组间高糖组对VSMC增殖及分泌MMP-2作用较正常组弱（P<0.01）。结论正常及高葡萄糖环境,适当浓度的胰岛素能促进VSMC增殖及MMP-2的分泌,效应具有浓度依赖性;且高糖环境下促进作用更为显著。第三部分方法1.同实验一。2.各分为4个亚组:①空白对照组O（10%FBS的DMEM,72 h）;②Fe²⁺组A(10%FBS的DMEM,48 h+0.05 mg/mL Fe²⁺,24 h);③胰岛素组B（10%FBS的DMEM,24 h+100 mIU胰岛素,48 h）;④胰岛素+Fe²⁺组C(100 mIU胰岛素,48 h+100 mIU胰岛素及0.05 mg/mL Fe²⁺,24 h)。3. MTT比色法测定细胞增殖情况。4. ELISA试剂盒检测细胞培养上清中MPP-2含量。5.激光共聚焦法测定细胞内Ca²⁺浓度。结果1.正常组:与O比较,A、B、C各组VSMC内Ca²⁺浓度均升高,VSMC增殖及MMP-2分泌能力增强。其中A、B组较C组促进VSMC胞内Ca²⁺浓度升高的作用高（P<0.01）,促进VSMC增殖及MMP-2分泌作用强（P<0.05）。2.高糖组:与O比较,A、B、C各组VSMC内Ca²⁺浓度均升高,VSMC增殖及MMP-2分泌能力增强。其中A、B组促进VSMC胞内Ca²⁺浓度升高、促进MMP-2分泌作用较C组强（P<0.01）,三组促进VSMC增殖作用无明显差异（P=0.484）。3.正常组与高糖组比较:高糖组A、B各相应亚组间对VSMC增殖及分泌MMP-2作用较正常组强（P<0.05）,C组两相应亚组间无明显差异（P=0.167）。高糖组A～C各亚组细胞内Ca²⁺浓度均高于正常组（P<0.05）。结论正常及高葡萄糖环境下,适当浓度的胰岛素、Fe²⁺能促进VSMC增殖、MMP-2的分泌以及VSMC胞内Ca²⁺浓度升高,且高糖组促进作用更为明显。正常及高葡萄糖环境下,胰岛素、Fe²⁺单独作用促进MMP-2分泌、VSMC胞内Ca²⁺浓度升高作用较胰岛素、Fe²⁺共同作用时强。促进VSMC增殖作用,在正常葡萄糖环境下,胰岛素、Fe²⁺单独作用时较胰岛素、Fe²⁺共同作用时强,但在高葡萄糖环境下三者作用无明显差异。更多还原

【Abstract】 BackgroundCoronary heart disease is one of the major diseases that threatens human health. Percutaneous transluminal coronary angioplasty（PTCA）is becoming a popular and effective treatment for coronary heart disease. The use of drug-eluting stent help to reduce the incidence of in-stent restenosis（ISR） to 5～10%, but still can’t completely solve the ISR promble. Lots of multi-center, double-blind and randomized controlled study showed that the patients with diabetes have not as good effect and higher ISR rate than patients without diabetes[1-3]. For the mechanisms are still not fully understood, its clinical application is seriously affected. At the same time, some studies showed that ion leakage ftom metallic implant stens[4], and the dose of ion are various from type to type. The effect of the ion on adjacent vascular smooth muscle cells（VSMC） is still unknown. There was a study which found out that Fe²⁺ may extently inhibites the proliferation of VSMC[5], but it seems that this conculsion doesn’t apparently comply with clinical observation. So we utilized primarily cultured human umbilical vein smooth muscle cells （HUVSMC）（Passage3～5）, and the Fe²⁺concentration gradient group which adopted from that study to carry out a study. We use used low glucose DMEM（5.5 mmol/L）to simulate the general human body microenvironment, DMEM with high concentration glucose（25 mmol/L） and high insulin to simulate the human body microenvironment under diabetes conditions, in order to elucidate the effects of Fe²⁺, insulin on proliferation and secretion of MMP-2, and to investigate the possible mechanisms of the ISR in patients with abnormal blood glucose and/or abnormal glucose tolerance, and look for clinical evidence.AimsThe aims of our present study were:1. to elucidate effects of Fe²⁺ on proliferation and secretion of MMP-2 in VSMC inculated with glucose.2. to elucidate effects of insulin on proliferation and secretion of MMP-2 in VSMC inculated with glucose.3. to elucidate effects of Fe²⁺ on proliferation and secretion of MMP-2 in VSMC incubated with insulin and glucose.4. to investigate the possible mechanism.Part OneMethodsThe umbilical vein was obtained by a sterile procedure. HUVSMC were derived following standard culture methods, and then divided to normal glucose（5 mmol/L, groupⅠ）and high glucose（25 mmol/L, groupⅡ）incubation. 1. VSMCs were divided into 6 sub-groups:①control group O;②Fe²⁺group A（0.00625 mg/mL）;③Fe²⁺group B（0.0125 mg/mL）; ④Fe²⁺group C（0.025 mg/mL）;⑤Fe²⁺group D（0.05 mg/mL）;⑥Fe²⁺group E（0.1 mg/mL）. Cells were intervened for 24 h in 37℃and 5%CO2 incubator.2. VSMC proliferation was assessed with MTT method.3. MMP-2 secretion was measured with ELISAResults1. Normal glucose groupⅠ: compared to the control group O, proliferation in group B～Eincreased, P<0.05; the secretion of MMP-2 was promoted only in group E, P<0.01.2. High glucose groupⅡ: compared to the control group O, the proliferation and secretion of MMP-2 in group B～Eincreased, P<0.05, and the group D and Ewere the most evident.3. Normal glucose groupⅠvs High glucose groupⅡ: the proliferation and secretion level of MMP-2 in high glucose groupⅡ（sub-group B～E）, was higher than normal glucose groupⅠ, P<0.05. ConclusionsThese results demonstrate that Fe²⁺could stimulated the proliferation of VSMC, and increase the secretion of MMP-2. Part TwoMethods1. The same as part one.2. Divided into 4 sub-group:①control group O;②Insulin group A（10 mIU）;③Insulin group B（100 mIU）;④Insulin group C（1000 mIU）. Cells were intervened for 24 h in 37℃and 5%CO2 incubator. 3. VSMC proliferation was assessed with MTT method.4. MMP-2 secretion was measured with ELISAResults1. Normal glucose groupⅠ: compared to the control group O, proliferation and secretion of MMP-2 increased in all group, P<0.01, and was most evident in group B.2. High glucose groupⅡ: compared to the control group O, the proliferation in group A～B increased, P<0.05; and secretion of MMP-2 in all group increased, P<0.01; and was most evident in group B.3. Normal glucose groupⅠvs high glucose groupⅡ: the proliferation and secretion level of MMP-2 was higher in high glucose groupⅡ（sub-group A～B）, P<0.05, but was lower in sub-group C than normal glucose groupⅠ. ConclusionsThese results demonstrate that insulin could stimulated the proliferation of VSMC, and increase the secretion of MMP-2 dose-dependent. Part ThreeMethods1. The same as part one.2. Divided into 4 sub-group:①control group O（10%FBS DMEM, 72 h）;②Fe²⁺ group A(10%FBS DMEM,48 h+0.05 mg/mL Fe²⁺, 24 h);③Fe²⁺ group B（10%FBS DMEM, 24h+100 mIU Insulin, 48 h）;④Fe²⁺ group C(100 mIU Insulin, 48 h+100 mIU Insulin and 0.05 mg/mL Fe²⁺, 24 h).3. VSMC proliferation was assessed with MTT method.4. MMP-2 secretion was measured with ELISA.5. The intracellular Ca²⁺ concentration was assessed by laser confocal microscopy.Results1. Normal glucose groupⅠ: compared to the control group O, the intracellular Ca²⁺ concentration of VSMC was augmented in all sub-groups, and was higher in the group A and B than in group C, P<0.01. The proliferation as well as secretion of MMP-2 were also increased in all subgroups, P<0.05.2. High glucose groupⅡ: compared to the control group O, the proliferation and secretion of MMP-2, as well as the intracellular Ca²⁺ concentration of VSMC was increased in all groups, and appearing the most prominent in group C.3. Normal glucose groupⅠvs high glucose groupⅡ: in the sub-group A and B in high glucose groupⅡ, the the proliferation and secretion level of MMP-2 was higher than normal glucose groupⅠ, P < 0.05. The intracellular Ca²⁺ concentration of VSMC in high glucose groupⅡwas higher than normal glucose groupⅠ, P<0.05.ConclusionsThese results demonstrate that Fe²⁺ could stimulated the proliferation of VSMC, and increase the intracellular Ca²⁺ as well as the secretion of MMP-2.更多还原