Fe~（2+）和/胰岛素对葡萄糖预处理的人脐静脉平滑肌细胞增殖及MMP-2分泌的作用
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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Fe~(2+)[4],不同类型支架“洗脱”到支架周围的Fe~(2+)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Fe~(2+)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Fe~(2+)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Fe~(2+)、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Fe~(2+)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Fe~(2+)对葡萄糖和胰岛素预孵育的VSMC增殖、MMP-2分泌以及VSMC胞内Ca~(2+)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;②Fe~(2+)组A(0.00625 mg/mL);③Fe~(2+)组B(0.0125 mg/mL);④Fe~(2+)组C(0.025 mg/mL);⑤Fe~(2+)组D(0.05 mg/mL);⑥Fe~(2+)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Fe~(2+)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);②Fe~(2+)组A(10%FBS的DMEM,48 h+0.05 mg/mL Fe~(2+),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+Fe~(2+)组C(100 mIU胰岛素,48 h+100 mIU胰岛素及0.05 mg/mL Fe~(2+),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Ca~(2+)浓度。
结果
1.正常组:与O比较,A、B、C各组VSMC内Ca~(2+)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Ca~(2+)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Ca~(2+)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Ca~(2+)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Ca~(2+)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、Fe~(2+)能促进VSMC增殖、MMP-2的分泌以及VSMC胞内Ca~(2+)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、Fe~(2+)单独作用促进MMP-2分泌、VSMC胞内Ca~(2+)浓度升高作用较胰岛素、Fe~(2+)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、Fe~(2+)单独作用时较胰岛素、Fe~(2+)共同作用时强,但在高葡萄糖环境下三者作用无明显差异。
Background
Coronary 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 Fe~(2+) 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 Fe~(2+)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 Fe~(2+), 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.
Aims
The aims of our present study were:
1. to elucidate effects of Fe~(2+) 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 Fe~(2+) on proliferation and secretion of MMP-2 in VSMC incubated with insulin and glucose.
4. to investigate the possible mechanism.
Part One
Methods
The 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;②Fe~(2+)group A(0.00625 mg/mL);③Fe~(2+)group B(0.0125 mg/mL); ④Fe~(2+)group C(0.025 mg/mL);⑤Fe~(2+)group D(0.05 mg/mL);⑥Fe~(2+)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 ELISA
Results
1. 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. Conclusions
These results demonstrate that Fe~(2+)could stimulated the proliferation of VSMC, and increase the secretion of MMP-2. Part Two
Methods
1. 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 ELISA
Results
1. 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. Conclusions
These results demonstrate that insulin could stimulated the proliferation of VSMC, and increase the secretion of MMP-2 dose-dependent. Part Three
Methods
1. The same as part one.
2. Divided into 4 sub-group:①control group O(10%FBS DMEM, 72 h);②Fe~(2+) group A(10%FBS DMEM,48 h+0.05 mg/mL Fe~(2+), 24 h);③Fe~(2+) group B(10%FBS DMEM, 24h+100 mIU Insulin, 48 h);④Fe~(2+) group C(100 mIU Insulin, 48 h+100 mIU Insulin and 0.05 mg/mL Fe~(2+), 24 h).
3. VSMC proliferation was assessed with MTT method.
4. MMP-2 secretion was measured with ELISA.
5. The intracellular Ca~(2+) concentration was assessed by laser confocal microscopy.
Results
1. Normal glucose groupⅠ: compared to the control group O, the intracellular Ca~(2+) 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 Ca~(2+) 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 Ca~(2+) concentration of VSMC in high glucose groupⅡwas higher than normal glucose groupⅠ, P<0.05.
Conclusions
These results demonstrate that Fe~(2+) could stimulated the proliferation of VSMC, and increase the intracellular Ca~(2+) as well as the secretion of MMP-2.
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Fe~(2+)[4],不同类型支架“洗脱”到支架周围的Fe~(2+)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Fe~(2+)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Fe~(2+)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Fe~(2+)、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Fe~(2+)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Fe~(2+)对葡萄糖和胰岛素预孵育的VSMC增殖、MMP-2分泌以及VSMC胞内Ca~(2+)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;②Fe~(2+)组A(0.00625 mg/mL);③Fe~(2+)组B(0.0125 mg/mL);④Fe~(2+)组C(0.025 mg/mL);⑤Fe~(2+)组D(0.05 mg/mL);⑥Fe~(2+)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Fe~(2+)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);②Fe~(2+)组A(10%FBS的DMEM,48 h+0.05 mg/mL Fe~(2+),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+Fe~(2+)组C(100 mIU胰岛素,48 h+100 mIU胰岛素及0.05 mg/mL Fe~(2+),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Ca~(2+)浓度。
结果
1.正常组:与O比较,A、B、C各组VSMC内Ca~(2+)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Ca~(2+)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Ca~(2+)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Ca~(2+)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Ca~(2+)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、Fe~(2+)能促进VSMC增殖、MMP-2的分泌以及VSMC胞内Ca~(2+)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、Fe~(2+)单独作用促进MMP-2分泌、VSMC胞内Ca~(2+)浓度升高作用较胰岛素、Fe~(2+)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、Fe~(2+)单独作用时较胰岛素、Fe~(2+)共同作用时强,但在高葡萄糖环境下三者作用无明显差异。
Background
Coronary 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 Fe~(2+) 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 Fe~(2+)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 Fe~(2+), 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.
Aims
The aims of our present study were:
1. to elucidate effects of Fe~(2+) 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 Fe~(2+) on proliferation and secretion of MMP-2 in VSMC incubated with insulin and glucose.
4. to investigate the possible mechanism.
Part One
Methods
The 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;②Fe~(2+)group A(0.00625 mg/mL);③Fe~(2+)group B(0.0125 mg/mL); ④Fe~(2+)group C(0.025 mg/mL);⑤Fe~(2+)group D(0.05 mg/mL);⑥Fe~(2+)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 ELISA
Results
1. 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. Conclusions
These results demonstrate that Fe~(2+)could stimulated the proliferation of VSMC, and increase the secretion of MMP-2. Part Two
Methods
1. 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 ELISA
Results
1. 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. Conclusions
These results demonstrate that insulin could stimulated the proliferation of VSMC, and increase the secretion of MMP-2 dose-dependent. Part Three
Methods
1. The same as part one.
2. Divided into 4 sub-group:①control group O(10%FBS DMEM, 72 h);②Fe~(2+) group A(10%FBS DMEM,48 h+0.05 mg/mL Fe~(2+), 24 h);③Fe~(2+) group B(10%FBS DMEM, 24h+100 mIU Insulin, 48 h);④Fe~(2+) group C(100 mIU Insulin, 48 h+100 mIU Insulin and 0.05 mg/mL Fe~(2+), 24 h).
3. VSMC proliferation was assessed with MTT method.
4. MMP-2 secretion was measured with ELISA.
5. The intracellular Ca~(2+) concentration was assessed by laser confocal microscopy.
Results
1. Normal glucose groupⅠ: compared to the control group O, the intracellular Ca~(2+) 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 Ca~(2+) 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 Ca~(2+) concentration of VSMC in high glucose groupⅡwas higher than normal glucose groupⅠ, P<0.05.
Conclusions
These results demonstrate that Fe~(2+) could stimulated the proliferation of VSMC, and increase the intracellular Ca~(2+) as well as the secretion of MMP-2.
引文
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