超声诱导培养平滑肌细胞凋亡研究
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摘要
目的：研究不同剂量超声对培养大鼠肺动脉血管平滑肌细胞（VSMC）凋亡的影响，为超声治疗的临床应用提供实验依据。观察超声对培养大鼠血管平滑肌细胞内Ca2+的影响，探讨超声诱导细胞凋亡是否与钙信号介导有关。
方法：不同剂量、频率为650KHz的超声波辐照体外培养的大鼠血管平滑肌细胞。照射后24h将细胞收集、涂片并固定，用 TUNEL标记法及电镜观察凋亡细胞，用免疫组织化学方法检测B细胞淋巴瘤-2伴x蛋白(Bax蛋白)表达：用荧光指示剂Fura-2/AM于单波长荧光分光光度仪上测定细胞内钙离子浓度。
结果:
1 TUNEL检测：0.25W/cm2×2min组超声辐照血管平滑肌细胞后24h，TUNEL阳性细胞率为5.4%，对照组为4.7%，无显著差异（P>0.05）；1.0W/cm2×5min组及0.5W/cm2×5min组超声辐照细胞后24h，TUNEL阳性细胞率明显升高，分别为20.2%和15.6%，显著高于0.25W/cm2×2min组及对照组（P<0.05）。
2 电镜观察：1.0W/cm2×5min组及0.5W/cm2×5min组超声在辐照后24h，均有细胞质基质电子密度深，有较多小空泡和髓样结构及凋亡小体形成，以1.0W/cm2×5min组更为明显。
3 Bax蛋白表达：在超声辐照细胞后24h，0.25W/cm2×2min组细胞Bax蛋白表达率与对照组无显著差异（P>0.05）；1.0W/cm2×5min组及0.5W/cm2×5min组Bax蛋白表达率明显升高，显著高于0.25W/cm2×2min组及对照组（P<0.05）。
4 细胞内钙离子浓度测定：0.25W/cm2×2min组，在无钙测定液条件下，
细胞内Ca2+为138.5±18.7nmol/L，与静息态无性差异（P>0.05）；在有钙测定液条件下，细胞内Ca2+为281.4±32.2nmol/L，与静息态有显著性差异（P<0.05）。
本课题受国家自然基金主任基金（30040030）资助
0.5W/cm2×5min 组：不论测定液是否有钙，细胞内Ca2+均显著升高（P<0.05），分别升为523.7±70.9nmol/L和265.1±49nmol/ L ，且在有钙测定液条件下明显高于无钙测定液条件下测定的细胞内Ca2+（P<0.05）。
结论：声强为0.5W/cm2 及1.0W/cm2超声辐照培养的大鼠血管平滑肌细胞，5min能诱导其凋亡，1.0W/cm2×5min组及0.5W/cm2×5min组Bax蛋白表达率明显升高；0.5W/cm2超声辐照5min 能引起细胞外钙内流及细胞内钙的释放，而使细胞胞浆内游离钙离子浓度升高，提示，0.5W/cm2超声诱导细胞凋亡可能与钙信号介导有关。
Objective: To study the induction of apoptosis of rat vascular smooth muscle cell cultured in vitro irradiated with various intensity and exposure time ultrasound ,and to provide experimental bases for its clinical application in future.we observe the effects of ultrasound exposure on intracellular free calcium of rat vascular smooth muscle cell cultured in vitro,trying to seek if cell apoptosis by ultrasound induction mediates by intracellular calcium signaling.
Methods: Rat vascular smooth muscle cells were cultured in vitro with DMEM. Cells were irradiated by ultrasound with dosage series and a frequency of 650 KHz . Apoptosis was detected 24 hours after irradiation by TUNEL marking method and electronic microscope respectively, Immunohistochemical method was used to evaluate the expressions of Bax;Concentrations of intracellular calcium in cultured smooth muscle cells were measured by calcium sensitive fluorescence indicator Fura-2-/AM technique.
Results:
1 Apoptosis was detected 24 hours after irradiation by TUNEL marking method.The ratio of positive cells marked cell by TUNEL was 5.4%( 0.25W/cm2×
2min) no more than in the controlled group 4.7% (P>0.05). The ratio of positive cells marked cell by TUNEL was 20.2%(1.0W/cm2×5min)and 15.6%(0.5W/cm2×5min) respectively higher than in the controlled group statistically(P<0.05).
2 Electron-microscopical observations:In experimental gorups(1.0W/cm2×5min and 0.5W/cm2×5min),many apoptotic phenomena were observed:electronic density of cytoplasm was high, showing many vesiculated and the form of apoptotic body,especially in 1.0W/cm2×5min group.
3 Bax protein expression:Except 0.25W/cm2×2min group,the positive rates of bax protein in experimental group were all increased than in the controlled group statistically (P<0.05).
4 In 0.25W/cm2×2min group,ultrasound increased [Ca2+]i in vascular smooth muscle cell in presence of calcium in medium ,but did not in absence of calcium in medium. In 0.5W/cm2×5min group,ultrasound increased [Ca2+]i in vascular smooth muscle cells in either presence or absence of calcium in medium,but [Ca2+]i was significantly higher (523.7±70.9nmol/L) presence of calcium than that (265.1±49.3nmol/L) in absence of calcium statistically (P<0.05).
Conclusions: We found that ultrasound can induce apoptosis of rat vascular smooth muscle cells cultured in vitro in 1.0W/cm2×5min and 0.5W/cm2×5min group. In 1.0W/cm2×5min and 0.5W/cm2×5min group, the positive rates of bax protein in experimental group were all increased than in the controlled group (P<0.05).We observed that ultrasound stimulation increases the intracellular calcium concentration in vascular smooth muscle cells in 0.5W/cm2×5min group,including extracellular calcium influx and intracellular calcium release.These findings suggest that ultrasound induction of apoptosis of cell is associated with calcium signaling pathways that are activated in rat vascular smooth muscle cells cultured in vitro.
方法：不同剂量、频率为650KHz的超声波辐照体外培养的大鼠血管平滑肌细胞。照射后24h将细胞收集、涂片并固定，用 TUNEL标记法及电镜观察凋亡细胞，用免疫组织化学方法检测B细胞淋巴瘤-2伴x蛋白(Bax蛋白)表达：用荧光指示剂Fura-2/AM于单波长荧光分光光度仪上测定细胞内钙离子浓度。
结果:
1 TUNEL检测：0.25W/cm2×2min组超声辐照血管平滑肌细胞后24h，TUNEL阳性细胞率为5.4%，对照组为4.7%，无显著差异（P>0.05）；1.0W/cm2×5min组及0.5W/cm2×5min组超声辐照细胞后24h，TUNEL阳性细胞率明显升高，分别为20.2%和15.6%，显著高于0.25W/cm2×2min组及对照组（P<0.05）。
2 电镜观察：1.0W/cm2×5min组及0.5W/cm2×5min组超声在辐照后24h，均有细胞质基质电子密度深，有较多小空泡和髓样结构及凋亡小体形成，以1.0W/cm2×5min组更为明显。
3 Bax蛋白表达：在超声辐照细胞后24h，0.25W/cm2×2min组细胞Bax蛋白表达率与对照组无显著差异（P>0.05）；1.0W/cm2×5min组及0.5W/cm2×5min组Bax蛋白表达率明显升高，显著高于0.25W/cm2×2min组及对照组（P<0.05）。
4 细胞内钙离子浓度测定：0.25W/cm2×2min组，在无钙测定液条件下，
细胞内Ca2+为138.5±18.7nmol/L，与静息态无性差异（P>0.05）；在有钙测定液条件下，细胞内Ca2+为281.4±32.2nmol/L，与静息态有显著性差异（P<0.05）。
本课题受国家自然基金主任基金（30040030）资助
0.5W/cm2×5min 组：不论测定液是否有钙，细胞内Ca2+均显著升高（P<0.05），分别升为523.7±70.9nmol/L和265.1±49nmol/ L ，且在有钙测定液条件下明显高于无钙测定液条件下测定的细胞内Ca2+（P<0.05）。
结论：声强为0.5W/cm2 及1.0W/cm2超声辐照培养的大鼠血管平滑肌细胞，5min能诱导其凋亡，1.0W/cm2×5min组及0.5W/cm2×5min组Bax蛋白表达率明显升高；0.5W/cm2超声辐照5min 能引起细胞外钙内流及细胞内钙的释放，而使细胞胞浆内游离钙离子浓度升高，提示，0.5W/cm2超声诱导细胞凋亡可能与钙信号介导有关。
Objective: To study the induction of apoptosis of rat vascular smooth muscle cell cultured in vitro irradiated with various intensity and exposure time ultrasound ,and to provide experimental bases for its clinical application in future.we observe the effects of ultrasound exposure on intracellular free calcium of rat vascular smooth muscle cell cultured in vitro,trying to seek if cell apoptosis by ultrasound induction mediates by intracellular calcium signaling.
Methods: Rat vascular smooth muscle cells were cultured in vitro with DMEM. Cells were irradiated by ultrasound with dosage series and a frequency of 650 KHz . Apoptosis was detected 24 hours after irradiation by TUNEL marking method and electronic microscope respectively, Immunohistochemical method was used to evaluate the expressions of Bax;Concentrations of intracellular calcium in cultured smooth muscle cells were measured by calcium sensitive fluorescence indicator Fura-2-/AM technique.
Results:
1 Apoptosis was detected 24 hours after irradiation by TUNEL marking method.The ratio of positive cells marked cell by TUNEL was 5.4%( 0.25W/cm2×
2min) no more than in the controlled group 4.7% (P>0.05). The ratio of positive cells marked cell by TUNEL was 20.2%(1.0W/cm2×5min)and 15.6%(0.5W/cm2×5min) respectively higher than in the controlled group statistically(P<0.05).
2 Electron-microscopical observations:In experimental gorups(1.0W/cm2×5min and 0.5W/cm2×5min),many apoptotic phenomena were observed:electronic density of cytoplasm was high, showing many vesiculated and the form of apoptotic body,especially in 1.0W/cm2×5min group.
3 Bax protein expression:Except 0.25W/cm2×2min group,the positive rates of bax protein in experimental group were all increased than in the controlled group statistically (P<0.05).
4 In 0.25W/cm2×2min group,ultrasound increased [Ca2+]i in vascular smooth muscle cell in presence of calcium in medium ,but did not in absence of calcium in medium. In 0.5W/cm2×5min group,ultrasound increased [Ca2+]i in vascular smooth muscle cells in either presence or absence of calcium in medium,but [Ca2+]i was significantly higher (523.7±70.9nmol/L) presence of calcium than that (265.1±49.3nmol/L) in absence of calcium statistically (P<0.05).
Conclusions: We found that ultrasound can induce apoptosis of rat vascular smooth muscle cells cultured in vitro in 1.0W/cm2×5min and 0.5W/cm2×5min group. In 1.0W/cm2×5min and 0.5W/cm2×5min group, the positive rates of bax protein in experimental group were all increased than in the controlled group (P<0.05).We observed that ultrasound stimulation increases the intracellular calcium concentration in vascular smooth muscle cells in 0.5W/cm2×5min group,including extracellular calcium influx and intracellular calcium release.These findings suggest that ultrasound induction of apoptosis of cell is associated with calcium signaling pathways that are activated in rat vascular smooth muscle cells cultured in vitro.
引文
1 Ross R.The pathogenesis of atherosclerosis:a perspecttive for the 1990s. Nature Sci ,1990,362(4):801-809
2 Kitazume H,Kubo I,Iwama T,et al.Long-term agniographic follow-up of lesions patent 6 months after percutaneous coronary angioplasty.Am Heart J,1995,129(2):441-444
3 Wiedermann JG,Marboe C.Intracoroary irradiation markedly reduces restenosis after ballon angioplasty in a porcine model.J Am Coll Cardiol,1994, 23(10):1491-1498
4 Candado JA,Waksman R,Gurdiel O,et al.Long-term angiographic and outcome after percutaneous transluminal coronary angioplasty and intracoronary radiation therapy in humans. Circulation, 1997,96(5):722-732
5 Teitein PS,Massull OV,Jani S.Catheterbased radiotherapy to inhibit restenosis after coronary stenting.N Engl J Med,1997,336(11):1697-1703
6 Hamon M,Lecluse E,Monassier JP,et al.Pharmacological approaches to the prevention of restenosis after coronary angiopasty.Drugs Aging,1998,13(4): 291-301
7 Versavi F,gaspardone A,Fabrizio Tomai MP,et al.A comparsion of coronary-artery stenting with angioplasty for isolated stenosis of the proximal left anterion descending coronary artery.N Engl J Med,1997,336(5):817-822
8 Bauters C,Meurice T,Hamon M,et al.Methanisms and prevention of restenosis :from experimental models to clinical practice.Cardiovascuar Res,1996, 31(3):835-846
9 Mercator E.Effect of high dose angiotensin-convering enzyme inhibition on restenosis:final results of the MARCATOR study,a multicenter,double-blind, place-controlled trial of cilazapril.J AM Coll Cardiol,1995,24(2):362-369
Deutsch E.The emerging role of low-molecular-weight heparin and antiplateled therapies in the cardiac catheterization labortory.Am Heart J,1999,138(6):
10 577-585
11 Vykhodtseva N.,Mcdannold N,Martin H,et al.Apoptosis in ultrasound produced threshold lesions in the rabbit brain. Ultrasound Med Biol.,2001,27(1):111-117.
12 Feril LB, Kondo T, Zhao QL,et al.Enhancement of hyperthmia- induced apoptosis by non-thermal effects of ultrasound. Cancer Lett, 2002,178(1):63-70.
13 Ashush H,Rozenszajn LA,Blass M,et al.Apoptosis induction of human myeloid leukemic cells by ultrasound exposure.Cancer Res, 2000, 60(4):1014-1020.
14 Stanton MT, Ettarh R, Arango D,et al. Diagnostic ultrasound induces change within numbers of cryptal mitotic and apoptotic cells in small intestine. Life Sci, 2001，68(13):1471-1475.
15 Alter A,Rozenszain LA,Miller HI,et al.ultrasound inhibits the adhesion and migration of smooth muscle cells in vitro.Ultrasound Med,1998,23:711-721
16 Ramirez A,Schwane JA,McFarland C,et al. The effect of ultrasound on collagen synthesis and fibroblast proliferation in vitro.Med Sci Sports Exerc, 1997 , 29(3): 326-32.
17 Lejbkowicz F, Salzberg S,et al. Distinct sensitivity of normal and malignant cells to ultrasoumd in vitro.Environ Health Perspect , 1997 , 105 (Suppl 6):1575-8.
18 Hrazdira I,Skorpikove J,Dolnikova M,et al.Ultrasonicaiiy induced cells.Eur J Ultrasound, 1998, 8(1):43-9.
19 Fitzgerald PJ,Takagi A,Moore P,et al.Intravascular sonotherapy decreases neointimal hyperplasia after stent implantation in swine.Circulation,2001 103(14):1828-1831
20 王关嵩,扬晓静,钱桂生,等.大鼠血管平滑肌细胞分离培养的探讨.第三军医大学学报, 1998,20(4):348-350
21 王关嵩,钱桂生,扬晓静,等.2种酶消化法培养大鼠血管平滑肌细胞生长特性的比较.第三军医大学学报,1999,21(10):765-767
22 Martin FR,Mansur AD,Dagmar M,et al.Its significance in laryngeal squamous cell carcinoma is independent of bcl-2 and bax protein expression.Cancer Res, 1999,85(3):591-599
23 Parvizi J, Parpura V, Greenleaf JF,et al.Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes. J Orthop Res, 2002，20(1):51-57
24 Adinno MA,AI-Karmis AM,Stoltz PA,et al.Effect of free radical scanvergers on changes in ion condunctance during exposure ultrasound.Membrane Biolchem, 1993,10(3):237-247
25 杜联芳,张青萍,刘望彭.诊断超声与细胞凋亡.中华超声影像学杂志,2000，9(6):374-376
26 杜联芳,张青萍,刘望彭.诊断超声对人早孕绒毛Bcl-2蛋白表达的研究.中国超声医学杂志,2001,17(1):7-9
2 Kitazume H,Kubo I,Iwama T,et al.Long-term agniographic follow-up of lesions patent 6 months after percutaneous coronary angioplasty.Am Heart J,1995,129(2):441-444
3 Wiedermann JG,Marboe C.Intracoroary irradiation markedly reduces restenosis after ballon angioplasty in a porcine model.J Am Coll Cardiol,1994, 23(10):1491-1498
4 Candado JA,Waksman R,Gurdiel O,et al.Long-term angiographic and outcome after percutaneous transluminal coronary angioplasty and intracoronary radiation therapy in humans. Circulation, 1997,96(5):722-732
5 Teitein PS,Massull OV,Jani S.Catheterbased radiotherapy to inhibit restenosis after coronary stenting.N Engl J Med,1997,336(11):1697-1703
6 Hamon M,Lecluse E,Monassier JP,et al.Pharmacological approaches to the prevention of restenosis after coronary angiopasty.Drugs Aging,1998,13(4): 291-301
7 Versavi F,gaspardone A,Fabrizio Tomai MP,et al.A comparsion of coronary-artery stenting with angioplasty for isolated stenosis of the proximal left anterion descending coronary artery.N Engl J Med,1997,336(5):817-822
8 Bauters C,Meurice T,Hamon M,et al.Methanisms and prevention of restenosis :from experimental models to clinical practice.Cardiovascuar Res,1996, 31(3):835-846
9 Mercator E.Effect of high dose angiotensin-convering enzyme inhibition on restenosis:final results of the MARCATOR study,a multicenter,double-blind, place-controlled trial of cilazapril.J AM Coll Cardiol,1995,24(2):362-369
Deutsch E.The emerging role of low-molecular-weight heparin and antiplateled therapies in the cardiac catheterization labortory.Am Heart J,1999,138(6):
10 577-585
11 Vykhodtseva N.,Mcdannold N,Martin H,et al.Apoptosis in ultrasound produced threshold lesions in the rabbit brain. Ultrasound Med Biol.,2001,27(1):111-117.
12 Feril LB, Kondo T, Zhao QL,et al.Enhancement of hyperthmia- induced apoptosis by non-thermal effects of ultrasound. Cancer Lett, 2002,178(1):63-70.
13 Ashush H,Rozenszajn LA,Blass M,et al.Apoptosis induction of human myeloid leukemic cells by ultrasound exposure.Cancer Res, 2000, 60(4):1014-1020.
14 Stanton MT, Ettarh R, Arango D,et al. Diagnostic ultrasound induces change within numbers of cryptal mitotic and apoptotic cells in small intestine. Life Sci, 2001，68(13):1471-1475.
15 Alter A,Rozenszain LA,Miller HI,et al.ultrasound inhibits the adhesion and migration of smooth muscle cells in vitro.Ultrasound Med,1998,23:711-721
16 Ramirez A,Schwane JA,McFarland C,et al. The effect of ultrasound on collagen synthesis and fibroblast proliferation in vitro.Med Sci Sports Exerc, 1997 , 29(3): 326-32.
17 Lejbkowicz F, Salzberg S,et al. Distinct sensitivity of normal and malignant cells to ultrasoumd in vitro.Environ Health Perspect , 1997 , 105 (Suppl 6):1575-8.
18 Hrazdira I,Skorpikove J,Dolnikova M,et al.Ultrasonicaiiy induced cells.Eur J Ultrasound, 1998, 8(1):43-9.
19 Fitzgerald PJ,Takagi A,Moore P,et al.Intravascular sonotherapy decreases neointimal hyperplasia after stent implantation in swine.Circulation,2001 103(14):1828-1831
20 王关嵩,扬晓静,钱桂生,等.大鼠血管平滑肌细胞分离培养的探讨.第三军医大学学报, 1998,20(4):348-350
21 王关嵩,钱桂生,扬晓静,等.2种酶消化法培养大鼠血管平滑肌细胞生长特性的比较.第三军医大学学报,1999,21(10):765-767
22 Martin FR,Mansur AD,Dagmar M,et al.Its significance in laryngeal squamous cell carcinoma is independent of bcl-2 and bax protein expression.Cancer Res, 1999,85(3):591-599
23 Parvizi J, Parpura V, Greenleaf JF,et al.Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes. J Orthop Res, 2002，20(1):51-57
24 Adinno MA,AI-Karmis AM,Stoltz PA,et al.Effect of free radical scanvergers on changes in ion condunctance during exposure ultrasound.Membrane Biolchem, 1993,10(3):237-247
25 杜联芳,张青萍,刘望彭.诊断超声与细胞凋亡.中华超声影像学杂志,2000，9(6):374-376
26 杜联芳,张青萍,刘望彭.诊断超声对人早孕绒毛Bcl-2蛋白表达的研究.中国超声医学杂志,2001,17(1):7-9