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他汀类药物对SD乳鼠心肌成纤维细胞增殖的抑制作用和凋亡的诱导作用及其信号转导通路研究
The Effects of Atorvastatin on Aldosterone Induced Proliferation and Collagen Synthesis in Cultured Neonatal Cardiac Fibroblasts and Atorvastatin Induced Fibroblasts Apoptosis
【作者】 王倩；
【导师】 崔炜；
【作者基本信息】 河北医科大学 ， 内科学， 2010， 博士
【摘要】 慢性充血性心力衰竭（CHF）是各种心脏疾病发展的终末阶段,其发病率和病死率随着全球老龄化的不断加剧逐年增多。目前虽有多种药物可改善心衰患者症状,但真正能够阻断甚至逆转心衰进程的疗法或药物尚未问世。因此,关于心衰的发病机制和治疗的研究仍在不断深入。他汀类药物是临床上应用广泛的降脂药,有研究表明,他汀对心衰患者可能有益。本研究以培养的新生SD大鼠心肌成纤维细胞（CFs）为研究对象,采用比色法、流式细胞术、Western Blot和细胞免疫荧光染色,共聚焦显微镜扫描技术等实验方法,观察了醛固酮（Ald）诱导的心脏成纤维细胞增殖、DNA合成、胶原合成及细胞周期变化,及可能的分子机制;同时观察HMG-CoA还原酶抑制剂阿托伐他汀（Ato）对Ald诱导的心脏成纤维细胞增殖、DNA合成、胶原合成及细胞周期变化的影响及其可能的分子机制;还应用比色法和形态学观察、流式细胞术等凋亡检测方法,分别测定了Ato和瑞舒伐他汀（Ros）对CFs增殖和凋亡的影响;并对脂溶性的Ato和水溶性的Ros对心肌成纤维的抑制作用进行了比较。为阐明心肌纤维化的发生机制,防治心肌纤维化提供实验依据。实验内容主要包括以下五部分:第一部分醛固酮对SD乳鼠心肌成纤维细胞增殖和胶原合成的促进作用及其信号转导通路目的:研究Ald对SD乳鼠CFs增殖和胶原合成的促进作用及其作用机制。方法:用胰酶消化法分离并培养新生SD大鼠的CFs,以Ald诱导其增殖和胶原合成,并加入螺内酯（Spi）干预,采用MTT法检测CFs的增殖,Brdu法检测CFs的DNA合成,流式细胞术检测CFs细胞周期,羟脯氨酸试剂盒检测CFs胶原合成,免疫荧光染色、激光共聚焦显微镜扫描半定量p-ERK1/2、p-AKT、Cyclin D1、Cyclin E2的表达变化。结果:1. Ald和Spi对CFs增殖的影响:MTT结果表明,与对照组比较,10-9 mol/L～10-7 mol/L Ald组的平均吸光度值均显著升高（P <0.05,P <0.05,P <0.01）。10-10 mol/L Ald组的平均吸光度值与对照组比较没有显著差异。10-9 mol/L～10-7mol/L Ald组同时给予10(-6 mol/L Spi,平均吸光度值均明显低于相应Ald组（P <0.05,P <0.05,P <0.01）,而与对照组比较没有显著差异。10-10 mol/L与10-9 mol/L、10-9 mol/L与10-8 mol/L、10-8 mol/L与10-7 mol/L之间均无显著差异。10-10 mol/L Ald组同时给予10-6 mol/L Spi,平均吸光度值无显著变化,与对照组比较也无显著差异。2. Ald和Spi对CFs DNA合成的影响:Brdu结果表明,与对照组比较,10-9 mol/L～10-7 mol/L Ald组的平均吸光度值均显著升高（P <0.01,P <0.01,P <0.01）。10(-9mol/L～10-7 mol/L Ald组同时给予10-6mol/L Spi,平均吸光度值均明显低于相应Ald组（P <0.01,P <0.01,P <0.01）,而与对照组比较没有显著差异。10-7 mol/L Ald组的平均吸光度值明显高于10-8 mol/L Ald组（P <0.05）,10-8 mol/L与10-9 mol/L Ald组间吸光度值没有显著差异。3. Ald和Spi对CFs细胞周期的影响:采用流式细胞术对CFs细胞周期进行分析,结果表明:1G1期细胞百分比:与对照组比较,10-7 mol/L Ald作用24 h、48 h、72 h的G1期细胞百分比均显著减少（ P <0.01,P <0.01,P <0.01）。同时给予10-6 mol/L Spi组的G1期细胞百分比均明显高于相应Ald组（P <0.01,P <0.01,P <0.01）。10(-7 mol/LAld作用48 h组的G1期细胞百分比明显低于24 h组（P <0.01）,10-7mol/LAld作用72 h组的G1期细胞百分比明显低于48 h组（P <0.01）。2S期细胞百分比:与对照组比较, 10-7 mol/L Ald作用24 h、48 h、72 h的S期细胞百分比均显著增加（P <0.01, P <0.01, P <0.01）。同时给予10-6 mol/L Spi组的S期细胞百分比均明显低于相应Ald组（P <0.01, P <0.01, P <0.01）。10(-7mol/LAld作用48 h组的S期细胞百分比明显高于24h组（P <0.01）,10-7mol/LAld作用72 h组的S期细胞百分比明显高于48 h组（P <0.01）。3PI:与对照组比较, 10-7 mol/L Ald作用24 h、48 h、72 h的PI均显著增加（P <0.01,P <0.01,P <0.01）。同时给予10-6 mol/L Spi组的PI均明显低于相应Ald组（P <0.01,P <0.01,P <0.01）。10-7 mol/LAld作用48 h组的PI明显高于24 h组（P <0.01）,10(-7mol/LAld作用72 h组的PI明显高于48 h组（P <0.01）。4. Ald和Spi对CFs胶原合成的影响:羟脯氨酸的量能反映胶原代谢情况。羟脯氨酸含量测定表明:与对照组比较,相应时间点的10-7 mol/L Ald组的平均吸光度值均显著升高（P <0.01,P <0.01,P <0.01）。与10-7 mol/L Ald刺激组比较,相应时间点的10-7 mol /L Ald+ 10-6 mol/L Spi组的平均吸光度值均显著降低（P <0.01,P <0.01,P <0.01）。对照组与相应时间点的10-7 mol/L Ald+10-6 mol/L Spi组比较均有显著差异（P <0.01,P <0.01,P <0.01）,即10-6 mol/L Spi不能完全逆转10-7 mol /L Ald的促胶原合成作用。10-7 mol/L Ald 48 h组与10(-7mol/L Ald 24 h组比较,10-7 mol/L Ald 72 h组与10-7 mol/L Ald 48 h组比较,平均吸光度值均显著升高（P <0.01, P <0.01）,即随时间延长,Ald刺激的胶原合成显著增加。5. Ald对CFs周期蛋白Cycin D1表达的影响:免疫荧光染色,共聚焦显微镜扫描检测蛋白表达结果表明,与对照组比较,10(-7mol /L Ald刺激CFs 24 h、48 h均可明显上调CFs胞核内的Cycin D1表达（P <0.01,P <0.05）,且Ald作用24 h时Cycin D1表达上调较48 h更为显著（P <0.01）。6. Ald对CFs周期蛋白Cyclin E2表达的影响:免疫荧光染色,共聚焦显微镜扫描检测蛋白表达结果表明,与对照组比较,10(-7mol /L Ald刺激CFs 24 h、48 h、72 h均可明显上调CFs胞核内的Cycin E2表达（P <0.01,P <0.05,P <0.05）,且Ald作用24 h时Cycin E2表达上调较48 h、72 h更为显著（P <0.01,P <0.01）。7. Ald对CFs周期蛋白P-ERK1/2表达的影响:免疫荧光染色,共聚焦显微镜扫描检测蛋白表达结果表明,与对照组比较,10(-7mol /L Ald刺激CFs 5 min、15 min、30 min、45min、60 min、2 h、4 h、8 h、24 h均可明显上调P-ERK1/2表达（P <0.01,P <0.01,P <0.01,P <0.01,P <0.01,P <0.01,P <0.01,P < 0.05）,且在Ald刺激30 min和4 h时P-ERK1/2表达量2次达峰。8. Ald对CFs周期蛋白p-AKT表达的影响:免疫荧光染色,共聚焦显微镜扫描检测蛋白表达结果表明,与对照组比较,10(-7mol /L Ald刺激CFs 45 min、60 min、2 h均可明显上调P-AKT表达（P <0.01,P <0.01,P <0.01）,但无明显峰值。（见Fig. 1-6）结论:外源性Ald可诱导CFs增殖、DNA合成和胶原合成,发挥其致心肌纤维化作用,此作用与其通过ERK1/2和Akt通路上调周期蛋白Cycin D1和Cyclin E2表达有关。第二部分阿托伐他汀对醛固酮诱导的SD乳鼠心肌成纤维细胞增殖和胶原合成的抑制作用及其机制探讨目的:研究Ato对Ald诱导的SD乳鼠CFs增殖和胶原合成的抑制作用及其作用的分子机制。方法:用胰酶消化法分离并培养新生SD大鼠的CFs,以Ald诱导其增殖和胶原合成,并加入Ato干预,采用MTT法检测CFs的增殖,Brdu法检测CFs的DNA合成,流式细胞术检测CFs周期,羟脯氨酸试剂盒检测CFs胶原合成,免疫荧光染色,激光共聚焦显微镜扫描和Western Blot半定量p-ERK1/2、p-AKT、Cyclin D1、Cyclin E2的表达变化。结果:1. Ato对Ald诱导的CFs增殖的影响:与对照组比较, 10(-7 mol/L Ald组的平均吸光度值显著升高（ P <0.01）,10-7 mol/L Ald+10(-6 mol/L Spi、10-8 mol/L、10-7 mol/L Ato组无显著差异。10-7 mol/L Ald +10-6 mol/L、10-5 mol/L Ato组的平均吸光度值均显著低于对照组（P <0.01,P <0.01）。与10-7 mol/L Ald组比较,同时给予10-6 mol/L Spi、10-8 mol/L～10-5 mol/L Ato组的平均吸光度值均显著降低（均P <0.01）。2. Ato对Ald诱导的CFs DNA合成的影响:与对照组比较,10-7 mol/L Ald组的平均吸光度值显著升高（P <0.01）,10-7 mol/L Ald+10-6mol/L Spi、10-8 mol/L、10-7 mol/L Ato组无显著差异。10-7 mol/L Ald +10-6 mol/L、10-5 mol/L Ato组的平均吸光度值均显著低于对照组（P <0.01,P <0.01）。与10-7 mol/L Ald组比较,同时给予10-6 mol/L Spi、10-8 mol/L～10-5 mol/L Ato组的平均吸光度值均显著降低（均P <0.01）。3. Ato对Ald诱导的CFs细胞周期的影响:1对各组各时间段的G1期细胞百分比,进行统计学分析,结果表明:与对照组比较,10-7 mol/L Ald作用24 h、48 h、72 h均能明显降低G1期细胞百分比（均P <0.01）。与10-7 mol/L Ald组比较,10-7～10-5 mol/L Ato、10-6 mol/L Spi+10-7 mol/L Ald 24 h、48 h、72 h,G1期细胞均明显增多（均P <0.01）。2对各组各时间段的S期细胞百分比,进行统计学分析,结果表明:与对照组比较,10-7 mol/L Ald作用24 h、48 h、72 h均能明显增加S期细胞百分比（均P <0.01）。与10-7 mol/L Ald组比较,10-7～10-5 mol/L Ato、10-6 mol/L Spi+10-7 mol/L Ald 24 h、48 h、72 h,S期细胞均明显较少（均P <0.01）。3对各组各时间段的PI进行统计学分析,结果表明:与对照组比较,10-7 mol/L Ald作用24 h、48 h、72 h均能明显增加PI（均P <0.01）。与10-7 mol/L Ald组比较,10-7～10-5 mol/L Ato、10-6 mol/L Spi +10-7 mol/L Ald 24 h、48 h、72 h, PI均明显降低（均P <0.01）。4. Ato和Ald对CFs凋亡的影响:流式细胞术检测亚二倍体峰,结果显示,与对照组比较,10(-6、10-5 mol/L Ato+10-7 mol/L Ald 24 h、48 h、72 h组凋亡细胞比例显著增加（均P <0.01）。10(-6、10(-5mol/L Ato+10-7 mol/L Ald 48 h组凋亡细胞比例分别较对应的10(-6、10-5 mol/L Ato+10-7 mol/L Ald 24 h组显著增加（P <0.01,P <0.01）,10(-6、10-5 mol/L Ato+10-7 mol/L Ald 72 h组凋亡细胞比例分别较对应的10-6、10-5 mol/L Ato+10-7 mol/L Ald 48 h组显著增加（P <0.01,P <0.01）。5. Ato对Ald诱导的CFs胶原合成的影响:对各组各时间段培养基中的羟脯氨酸含量进行测定,结果表明:与对照组比较,10-7 mol/L Ald作用24 h、48 h、72 h,平均吸光度值均显著升高（均P <0.01）。与10-7 mol/L Ald组比较,同时给予10-6 mol/L Spi、10-8～10-5 mol/L Ato组的平均吸光度值均显著降低（均P <0.01）。6. Ato对Ald诱导的CFs周期蛋白Cyclin D1表达的影响:10-5 mol/L Ato可明显抑制10-7 mol/L Ald上调的Cyclin D1的表达（P <0.01）。7. Ato对Ald诱导的CFs周期蛋白Cyclin E2表达的影响:10-5 mol/L Ato可明显抑制10-7 mol/L Ald上调的Cyclin E2的表达（P <0.01）。8. Ato对Ald诱导的CFs p-ERK1/2表达的影响:110-7 mol/L Ald刺激CFs 30 min同时给予10-7～10-5 mol/L Ato进行干预,免疫荧光染色,共聚焦显微镜扫描结果表明,各浓度Ato均不能抑制P-ERK1/2表达。210-7 mol/L Ald刺激CFs 4 h同时给予10-7～10-5mol/L Ato进行干预,免疫荧光染色,共聚焦显微镜扫描结果表明,10-7～10-5 mol/L Ato均能抑制P-ERK1/2表达（均P <0.01）。10-6 mol/L较10-7 mol/L Ato对P-ERK1/2表达的抑制作用更强（P <0.01）。310-7 mol/L Ald刺激CFs 4 h同时给予不同浓度Ato进行干预,Western结果表明,10-7～10-5 mol/L Ato均能抑制P-ERK1/2表达（均P <0.01）。9. Ato对Ald诱导的CFs P-AKT表达的的影响:110-7 mol/L Ald刺激CFs 1 h同时给予不同浓度Ato进行干预,免疫荧光染色,共聚焦显微镜扫描结果表明,10-7 10-5 mol/L Ato均能抑制P-AKT表达（均P <0.01）。10-6 mol/L与10-7 mol/L Ato比较,10-5 mol/L与10-6 mol/L Ato比较,对P-AKT表达的抑制作用均有显著差异（P <0.01,P <0.01）。210-7 mol/L Ald刺激CFs 1 h同时给予不同浓度Ato进行干预,Western结果表明,10-7～10-5 mol/L Ato均能抑制P-AKT表达（均P <0.01）。结论:Ato可明显抑制Ald诱导的CFs增殖、DNA合成和胶原合成,抑制Ald的致心肌纤维化作用,此作用与其通过抑制ERK1/2和Akt通路,进而抑制周期蛋白Cycin D1和Cyclin E2表达有关。第三部分阿托伐他汀对SD乳鼠心肌成纤维细胞增殖的抑制作用和对凋亡的诱导作用目的:研究Ato对SD乳鼠CFs增殖的抑制作用和凋亡的诱导作用。方法:用胰酶消化法分离并培养新生SD大鼠的CFs,并加入Ato干预,采用MTT法检测CFs的增殖,Brdu法检测CFs的DNA合成,倒置生物显微镜和荧光显微镜检测凋亡细胞形态,流式细胞术检测CFs凋亡率。结果:1. Ato对CFs增殖的影响:与对照组比较,10-7 mol/L～10-5 mol/L Ato组的平均吸光度值均显著降低（均P <0.01）,10-5 mol/L FPP +10-5 mol/L Ato组的平均吸光度值显著降低（P <0.01）,即10-5 mol/L FPP不能完全拮抗10-5 mol/L Ato对CFs增殖的抑制作用。与Ato单独作用组比较,同时给予10-5mol/L FPP,10-7 mol/L～10-5 mol/L Ato组的平均吸光度值均显著升高（均P <0.01）。2. Ato对CFs DNA合成的影响:与对照组比较,10-7 mol/L～10-5 mol/L Ato组的平均吸光度值均显著降低（均P <0.01）,10-5 mol/L FPP +10-5 mol/L Ato组的平均吸光度值均显著降低（P <0.01）,即10-5 mol/L FPP不能完全拮抗10-5 mol/L Ato对CFs DNA合成的抑制作用。与Ato单独作用组比较,同时给予10-5 mol/L FPP,10-7 mol/L～10-5 mol/L Ato组的平均吸光度值均显著升高（均P <0.01）。3. Ato对CFs的形态学影响:1倒置生物显微镜观察不同浓度Ato对CFs的影响,可见未加Ato处理组的CFs细胞间隙小且贴壁良好,几乎没有脱壁漂浮的圆形死细胞。Ato处理后,部分细胞胞质回缩,细胞间间隙增大,细胞贴壁能力不同程度减弱,随Ato浓度增大,处理时间延长,细胞形态改变越来越明显。2采用瑞氏—吉姆萨染色法,对空白组和各处理组细胞进行染色后,倒置生物显微镜下观察,可见未加Ato处理组的CFs呈梭形、三角形、多边形,胞浆着色均匀,淡粉红色,胞核较大呈深粉色或淡紫色,细胞贴壁良好,细胞间隙很小。Ato处理后,CFs出现了不同程度的胞质回缩、变形,核固缩、深染,细胞间隙增大等一系列改变。随Ato浓度增大,处理时间延长,细胞的变化越发显著。3Hochest33258染色后,荧光显微镜下进行观察,可见未加Ato处理组的CFs胞核形状规则,呈椭圆形,着色较浅,呈淡蓝色荧光。Ato处理后,胞核出现不同程度固缩、变形、甚至崩解为大小不一的碎片,着色较深,呈深蓝色。4. AnexinV/PI双染,流式细胞术检测Ato对CFs凋亡的影响:1对不同浓度Ato处理组各时间段早期凋亡细胞百分比,进行统计学分析,结果表明:与相应对照组比较,10-7～10-5 mol/L Ato作用24 h、48 h、72 h均能明显增加早期凋亡细胞百分比（均P <0.01）。对相同浓度Ato作用不同时间,和相同时间点不同浓度Ato作用后的早期凋亡细胞百分比分别进行比较,结果显示除10-7 mol/L与10-6 mol/L Ato间作用24 h和72 h无显著差异外,Ato可时间、剂量依赖性的增加早期凋亡CFs的百分比（均P <0.05）。2对不同浓度Ato处理组各时间段晚期凋亡细胞百分比,进行统计学分析,结果表明:与相应对照组比较,10-7～10-5 mol/L Ato作用24 h、48 h、72 h均能明显增加晚期凋亡细胞百分比（均P <0.01）。对相同浓度Ato作用不同时间,和相同时间点不同浓度Ato作用后的晚期凋亡细胞百分比分别进行比较,结果显示除10-7 mol/L Ato作用24 h和48 h之间、10-6 mol/L Ato作用24 h和48 h之间无显著差异外, Ato可时间、剂量依赖性的增加晚期凋亡CFs的百分比（均P <0.05）。3对不同浓度Ato处理组各时间段总凋亡细胞百分比,进行统计学分析,结果表明:与相应对照组比较,10-7～10-5 mol/L Ato作用24 h、48 h、72 h均能明显增加总凋亡细胞百分比（均P <0.01）。对相同浓度Ato作用不同时间,和相同时间点不同浓度Ato作用后的凋亡细胞百分比分别进行比较,结果显示Ato可时间、剂量依赖性的增加凋亡CFs的百分比（均P <0.05）。5. PI单染,流式细胞术检测亚二倍体峰:与相应对照组比较,10-7～10-5 mol/L Ato作用24 h、48 h、72 h均能明显增加凋亡细胞百分比（均P <0.01）。与10-5 mol/L Ato单独作用组比较,同时给予10-5 mol/L FPP在24 h、48 h和72 h均能部分逆转Ato对CFs的凋亡诱导作用（均P <0.01）。结论:10-7～10-5mol/L Ato不仅可抑制CFs增殖和DNA合成,还可时间和剂量依赖性的诱导CFs凋亡。第四部分瑞舒伐他汀对SD乳鼠心肌成纤维细胞增殖的抑制作用和对凋亡的诱导作用目的:研究Ros对SD乳鼠CFs增殖的抑制作用和凋亡的诱导作用。方法:用胰酶消化法分离并培养新生SD大鼠的CFs,并加入Ros干预,采用MTT法检测CFs的增殖,Brdu法检测CFs的DNA合成,倒置生物显微镜和荧光显微镜检测凋亡细胞形态,流式细胞术检测CFs凋亡率。结果:1. Ros对CFs增殖的影响:与对照组比较,10-8 mol/L～10-5 mol/L Ros组的平均吸光度值均显著降低（均P <0.01）,10-5 mol/L FPP +10-5 mol/L Ros组的平均吸光度值显著降低（P <0.01）,即10-5 mol/L FPP不能完全拮抗10-5 mol/L Ros对CFs增殖的抑制作用。与Ros单独作用组比较,同时给予10-5 mol/L FPP,10-8 mol/L～10-5 mol/L Ato组的平均吸光度值均显著升高（P <0.05,P <0.01,P <0.01, P <0.01）。2. Ros对CFs DNA合成的影响:与对照组比较,10-7 mol/L～10-5 mol/L Ros组的平均吸光度值均显著降低（均P <0.01）,10-5 mol/L FPP +10-5 mol/L Ros组的平均吸光度值显著降低（P <0.01）,即10-5 mol/L FPP不能完全拮抗10-5 mol/L Ros对CFs DNA合成的抑制作用。与Ros单独作用组比较,同时给予10-5 mol/L FPP, 10-7 mol/L～10-5 mol/L Ros组的平均吸光度值均显著升高（均P <0.01）。3. Ros对CFs的形态学影响:1倒置生物显微镜观察不同浓度Ros对CFs的影响,可见未加Ros处理组的CFs细胞间隙小且贴壁良好,几乎没有脱壁漂浮的圆形死细胞。Ros处理后,部分细胞胞质回缩,细胞间间隙增大,细胞贴壁能力不同程度减弱,随Ros浓度增大,处理时间延长,细胞形态改变越来越明显。2采用瑞氏—吉姆萨染色法,对空白组和各处理组细胞进行染色后,倒置生物显微镜下观察,可见未加Ros处理组的CFs呈梭形、三角形、多边形,胞浆着色均匀,淡粉红色,胞核较大呈深粉色或淡紫色,细胞贴壁良好,细胞间隙很小。Ros处理后,CFs出现了不同程度的胞质回缩、变形,核固缩、深染,细胞间隙增大等一系列改变。随Ros浓度增大,处理时间延长,细胞的变化越发显著。3Hochest33258染色后,荧光显微镜下进行观察,可见未加Ros处理组的CFs胞核形状规则,呈椭圆形,着色较浅,呈淡蓝色荧光。Ros处理后,胞核出现不同程度固缩、变形、甚至崩解为大小不一的碎片,着色较深,呈深蓝色。4. AnexinV/PI双染,流式细胞术检测Ros对CFs凋亡的影响:1对不同浓度Ros处理组各时间段早期凋亡细胞百分比,进行统计学分析,结果表明:与相应对照组比较,除10-7 mol/L Ros作用24 h外,10-7 mol/L Ros作用48 h、72 h ,10-6、10-5 mol/L Ros作用24 h、48 h、72 h均能明显增加早期凋亡细胞百分比（均P <0.05）。对相同浓度Ros作用不同时间,和相同时间点不同浓度Ros作用后的早期凋亡细胞百分比分别进行比较,结果显示Ros可时间、剂量依赖性的增加早期凋亡CFs的百分比（均P <0.05）。2对不同浓度Ros处理组各时间段晚期凋亡细胞百分比,进行统计学分析,结果表明:与相应对照组比较,除10-7 mol/L Ros作用24 h外,10-7 mol/L Ros作用48 h、72 h ,10-6、10-5 mol/L Ros作用24 h、48 h、72 h均能明显增加晚期凋亡细胞百分比（均P <0.05）。对相同浓度Ros作用不同时间,和相同时间点不同浓度Ros作用后的晚期凋亡细胞百分比分别进行比较,Ros可时间、剂量依赖性的增加晚期凋亡CFs的百分比（均P <0.05）。3对不同浓度Ros处理组各时间段总凋亡细胞百分比,进行统计学分析,结果表明:与相应对照组比较,10(-7～10-5 mol/L Ros作用24 h、48 h、72 h均能明显增加总凋亡细胞百分比（均P <0.05）。对相同浓度Ros作用不同时间,和相同时间点不同浓度Ros作用后的凋亡细胞百分比分别进行比较,结果显示Ros可时间、剂量依赖性的增加凋亡CFs的百分比（均P <0.05）。5. PI单染,流式细胞术检测亚二倍体峰:与相应对照组比较,10(-7～10-5 mol/L Ros作用24 h、48 h、72h均能明显增加凋亡细胞百分比（均P <0.01）。与10-5 mol/L Ato单独作用组比较,同时给予10(-5 mol/L FPP在24 h、48 h和72 h均能部分逆转Ros对CFs的凋亡诱导作用（均P <0.01）。结论:10(-7～10(-5mol/L Ros不仅可抑制CFs增殖和DNA合成,还可以时间和剂量依赖性的诱导CFs凋亡。第五部分阿托伐他汀和瑞舒伐他汀对SD乳鼠心肌成纤维细胞增殖抑制作用比较目的:比较Ato和Ros对SD乳鼠CFs增殖的抑制作用和凋亡的诱导作用。方法:用胰酶消化法分离并培养新生SD大鼠的CFs,并加入Ato或Ros干预,采用MTT法检测CFs的增殖,Brdu法检测CFs的DNA合成,流式细胞术检测CFs细胞周期,羟脯氨酸试剂盒检测CFs胶原合成,流式细胞术检测CFs凋亡。结果:1. CFs增殖测定:对MTT测定结果进行统计分析显示,与空白对照组相比,Ros和Ato 10(-7～10-5 mol/L均可抑制新生大鼠CFs增殖（均P < 0.01）。Ros与Ato各相同剂量组相比,对CFs增殖的抑制效果无显著差异。2. CFs DNA合成测定:对Brdu测定结果进行统计分析显示,与空白对照组相比,Ros和Ato 10(-7～10-5 mol/L组均可抑制新生大鼠CFs DNA合成（均P < 0.01）。Ros与Ato各相同剂量组相比,对CFs DNA合成的抑制效果无显著差异。3. CFs细胞周期测定:流式细胞术检测细胞周期,结果显示,与空白对照组比较,Ros和Ato 10(-7～10-5 mol/L组作用24 h均可增加CFs处于G0/G1期的百分率,降低CFs处于S期的百分率,并降低细胞增殖指数（PI）（均P < 0.05）。Ros和Ato各相同剂量组间,处于G0/G1期、G2/M期和S期的CFs比例及PI均无显著差异,即2药对细胞周期的阻滞作用相似。4.流式细胞术检测亚二倍体峰:流式细胞术检测亚二倍体峰,可反应每个处理组中晚期凋亡细胞所占比率,结果显示,10(-7～10(-5mol/L Ato和Ros均可诱导CFs凋亡（均P < 0.01）,且2药均有明显的剂量依赖,随药物浓度增加,凋亡细胞比率随之增加（均P < 0.01）。Ros与Ato各相同剂量组相比,对CFs凋亡的促进效果无显著差异。5. CFs胶原分泌测定:羟脯氨酸测定结果显示,Ros和Ato 10(-7～10-5 mol/L组均可抑制新生大鼠CFs胶原分泌（均P < 0.01）。Ros对CFs胶原分泌的抑制作用呈明显的剂量依赖,随Ros浓度增加,作用逐渐增强（均P < 0.01）。10(-7与10-6 mol/L Ato间对胶原分泌的抑制作用无显著差异,10(-6与10-5 mol/L Ato间在各时间点对胶原分泌的抑制作用差异显著（均P < 0.01）。Ros与Ato各相同剂量组相比,对CFs胶原合成的抑制效果无显著差异。结论:Ros与Ato对SD乳鼠CFs增殖和DNA合成及胶原合成的抑制作用无明显差异。Ros与Ato对乳鼠CFs凋亡的诱导作用无明显差异。
【Abstract】 Chronic congestive heart failure was the final stage of various heart disease. The morbility and mortality of CHF increased year by year accopanied by the aging of the world population. Although there were many drugs and treatments which could relieve the symptoms of CHF, but drugs or treatments which could really block or reverse the course of CHF were still in need. Therefore study on the pathogenesis and therapeusis of CHF will go on.Statins were hypolipemia drugs applied widely in clinical practice. Some recent studies have indicated statin may be benificial to CHF patients. The present study investigated the effects of aldosterone on proliferation, DNA synthsis, cell cycle change and cllagen synthsis in cultured neonatal rat cardiac fibroblasts with chromatometry, flow cytometry, Western blot, and cell immunofluorescence technique, and the potential molecular mechanism. Meanwhile, the current study investigated the effects of HMG-CoA reducase inhibitor atorvastatin on proliferation, DNA synthesis, cell cycle change, and collagen synthsis in cultured neonatal cardiac fibroblasts induced by aldosterone, and the potential molecular mechanisms. Thereafter, the present study investigated the effects of atorvastatin and rosuvastatin on proliferation, DNA synthsis and apoptosis of cultured neonatal rat cardiac fibroblasts with chromatometry, flow cytometry and invert microscope, fluorescence microscope. In the last section, the proliferation promotion and apoptosis induction effects of atorvastatin was compared with rosuvastatin. The study provided new ideas and experimental evidences for anti-myocardial fibrosis therapy. The present study included five parts as following:Part 1 The effects of aldosterone on proliferation and collagen synthesis in cultured neonatal cardiac fibroblastsObjective: To investigate the effects of Ald on proliferation and collagen synthesis in cultured neonatal cardiac fibroblasts.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley （S-D） rats, the effects of Ald and Spi on proliferation were measured by MTT colorimetric assay, the effects of Ald and Spi on DNA synthesis were measured by Brdu colorimetric assay, the effects of Ald and Spi on cell cycle were measured by flow cytometry technique, synthesis of collagen was observed by the hydroxyproline concentration determined, p-ERK1/2, p-AKT, Cyclin D1, Cyclin E2 level were tested by immunofluorescence technique.Results: 1. Effects of Ald and Spi on proliferation of CFs: Compared with the control group, 10-9 mol/L 10-7 mol/L Ald significantly increased the proliferation of fibroblasts respectively （P <0.01, P <0.05, P < 0.05）, 10-10 mol/L Ald didn’t increased the proliferation of fibroblasts, and there were no difference among those four groups. Compared with 10-9 mol/L, 10-8 mol/L, 10-7 mol/L Ald groups, the proliferation of fibroblasts in 10-9 mol/L, 10-8 mol/L, 10-7 mol/L Ald +10-6 mol/L Spi groups were significantly decreased respectively （P <0.01, P<0.05, P<0.05） , which were not different from the control group.2. Effects of Ald and Spi on DNA synthesis of CFs: Compared with the control group, 10-9 mol/L 10-7 mol/L Ald significantly increased the DNA synthesis of fibroblasts respectively （All P <0.01）. DNA synthesis in 10-7 mol/L Ald group was more than 10-8 mol/L Ald group （P <0.01）, but there were no difference between 10-8 mol/L and 10-9 mol/L Ald groups. Compared with 10-9 mol/L, 10-8 mol/L, 10-7 mol/L Ald groups, DNA synthesis of fibroblasts in 10-9 mol/L, 10-8 mol/L, 10-7 mol/L Ald +10-6 mol/L Spi groups were significantly decreased respectively （All P <0.01） , which were not different from the control group.3. Effects of Ald and Spi on cell cycle change of CFs: 1) G1 phase cell proportion: Compared with the control group, 10-7mol/L Ald significantly decreased the proportion of fibroblasts in G1 phase at 24 h, 48 h, 72 h respectively （All P <0.01）. The proportion of fibroblasts in G1 phase in 10(-7mol/L Ald 48 h group was less than 10-7 mol/L Ald 24 h group, and 10(-7mol/L Ald 72 h group was less than 10-7 mol/L Ald 48 h group（P <0.01, P <0.01）. Compared with 10-7mol/L Ald groups, the proportion of fibroblasts in G1 phase in 10-7 mol/L Ald +10-6 mol/L Spi groups were significantly increased at 24 h, 48 h, 72 h respectively （All P <0.01）.2 S phase cell proportion: Compared with the control group, 10(-7mol/L Ald significantly increased the proportion of fibroblasts in S phase at 24 h, 48 h, 72 h respectively （All P <0.01）. The proportion of fibroblasts in S phase in 10-7 mol/L Ald 48 h group was more than 10(-7 mol/L Ald 24 h group, and 10-7 mol/L Ald 72 h group was more than 10(-7 mol/L Ald 48 h group（P <0.01, P <0.01）. Compared with 10(-7mol/L Ald groups, the proportion of fibroblasts in S phase in 10(-7mol/L Ald +10-6 mol/L Spi 24 h, 48 h, 72 h groups were significantly decreased respectively （All P <0.01）.3 PI: Compared with the control group, 10-7 mol/L Ald significantly increased PI of fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. PI of fibroblasts in 10-7 mol/L Ald 48 h group was more than 10-7 mol/L Ald 24 h group, and 10-7 mol/L Ald 72 h group was more than 10-7 mol/L Ald 48 h group（P <0.01, P <0.01）. Compared with 10-7 mol/L Ald groups, PI of fibroblasts in 10-7 mol/L Ald +10-6 mol/L Spi 24 h, 48 h, 72 h groups were significantly decreased respectively （All P <0.01）.4. Effects of Ald and Spi on collagen synthesis in CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the collagen synthesis in fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Collagen synthesis in 10-7 mol/L Ald 48 h group was more than 10(-7 mol/L Ald 24 h group, 10-7 mol/L Ald 72 h group was more than 10(-7 mol/L Ald 48 h group （P <0.01, P <0.01）. Compared with 10-7 mol/L Ald 24 h, 48 h, 72 h groups, collagen synthesis in fibroblasts in correspondent 10-7 mol/L Ald +10-6 mol/L Spi groups were significantly decreased respectively （All P <0.01） .5. Effects of Ald on expression of Cyclin D1 in CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the expression of Cyclin D1 in fibroblasts nucleus at 24 h, 48 h respectively （P <0.01, P <0.05）. The expression of Cyclin D1 in 10-7 mol/L Ald 24 h group was higher than 10-7 mol/L Ald 48 h group.6. Effects of Ald on expression of Cyclin E2 in CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the expression of Cyclin E2 in fibroblasts nucleus at 24 h, 48 h, 72 h respectively （P <0.01, P <0.05, P <0.05）. The expression of Cyclin E2 in 10-7 mol/L Ald 24 h group was higher than 10-7 mol/L Ald 48 h and 72 h groups.7. Effects of Ald on expression of P-ERK1/2 in CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the expression of P-ERK1/2 in fibroblasts at 5 min, 15 min, 30 min, 60 min, 2 h, 4 h, 8 h, 24 h respectively （All P <0.05）. The expression of P-ERK1/2 in 10-7 mol/L Ald 30 min and 4 h group were the highest among all groups.8. Effects of Ald on expression of p-AKT in CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the expression of p-AKT in fibroblasts at 45 min, 60 min, 2 h respectively （P <0.01, P <0.01, P <0.01）. But there was not significant peak amplitude.Conclusion: Exogenous aldosterone stimulated cardiac fibroblasts proliferation, DNA synthesis and collagen synthesis via activating P-ERK1/2 and p-AKT. Ald increased CFs proliferation by upregulating Cycin D1 and Cyclin E2 expression. Part 2 The effects of atorvastatin on aldosterone induced proliferation and collagen synthesis in cultured neonatal cardiac fibroblastsObjective: To investigate the effects of Ato on Ald induced proliferation and collagen synthesis in cultured neonatal cardiac fibroblasts.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley （S-D） rats, the effects of Ato on Ald induced proliferation were measured by MTT colorimetric assay, the effects of Ato on Ald induced DNA synthesis were measured by Brdu colorimetric assay, the effects of Ato on Ald induced cell cycle change were measured by flow cytometry technique, synthesis of collagen was observed by the hydroxyproline concentration determined, p-ERK1/2, p-AKT, Cyclin D1, Cyclin E2 level were tested by immunofluorescence technique and Western Blot.Results: 1. Effects of Ato on Ald induced proliferation of CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the proliferation of fibroblasts （P <0.01）. Compared with 10-7 mol/L Ald group, the proliferation of fibroblasts in 10-7 mol/L Ald +10-6 mol/L Spi, 10-8 mol/L 10-5 mol/L Ato groups were significantly decreased respectively （All P <0.01）. There were not difference between control group and 10-7 mol/L Ald +10-6 mol/L Spi, 10-8 mol/L, 10-7 mol/L Ato groups, however, the proliferation of fibroblasts in 10-7 mol/L Ald +10(-6 mol/L, 10-5 mol/L, Ato groups were lower than control group （P <0.01, P <0.01）. Trere were not difference between 10-7 mol/L Ald+10-8 mol/L Ato group and 10-7 mol/L Ald+10-7 mol/L Ato group, 10-7 mol/L Ald+10(-6 mol/L Ato group and 10-7 mol/L Ald+10-5 mol/L Ato group. The proliferation of fibroblasts in 10-7 mol/L Ald+10-6 mol/L Ato group was lower than 10-7 mol/L Ald+10-7 mol/L Ato group. Trere were no difference between 10-7 mol/L Ald+10-6 mol/L Spi group and 10-7 mol/L Ald+10-8 mol/L, 10-7 mol/L Ato groups. The proliferation of fibroblasts in 10-7 mol/L Ald+10-6 mol/L, 10-5 mol/L Ato groups was lower than 10(-7 mol/L Ald+10-6 mol/L Spi group.2. Effects of Ato on Ald induced DNA synthsis of CFs: Compared with the control group, 10-7 mol/L Ald significantly increased DNA synthsis of fibroblasts （P <0.01）. Compared with 10-7 mol/L Ald group, DNA synthesis of fibroblasts in 10-7mol/L Ald +10(-6mol/L Spi, 10-8 mol/L 10-5 mol/L Ato groups were significantly decreased respectively （All P <0.01）. Trere were not difference between control group and 10-7 mol/L Ald+10(-6mol/L Spi, 10-8 mol/L, 10-7 mol/L Ato groups, however, DNA synthesis of fibroblasts in 10(-7mol/L Ald +10-6 mol/L, 10-5 mol/L Ato groups were lower than control group （P <0.01, P <0.01）. DNA synthesis of fibroblasts in 10-7 mol/L Ald+10-7 mol/L Ato group was lower than 10(-7 mol/L Ald+10-8 mol/L Ato group, DNA synthesis of fibroblasts in 10(-7 mol/L Ald+10-6 mol/L Ato group was lower than 10-7 mol/L Ald+10(-7 mol/L Ato group, DNA synthesis of fibroblasts in 10-7 mol/L Ald+10(-5 mol/L Ato group was lower than 10-7mol/L Ald+10-6 mol/L Ato group（All P <0.01）.3. Effects of Ato on Ald induced cell cycle change of CFs: 1 S phase cell proportion: Compared with the control group, 10-7 mol/L Ald significantly increased the proportion of fibroblasts in S phase at 24 h, 48 h, 72 h respectively （All P <0.01）. Compared with 10-7 mol/L Ald group, the proportion of fibroblasts in S phase in 10-7 mol/L Ald+10-6 mol/L Spi, 10-7 10-5 mol/L Ato 24 h, 48 h, 72 h groups were significantly decreased respectively （All P <0.01）.2 PI: Compared with the control group, 10-7 mol/L Ald significantly increased PI of fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Compared with 10-7 mol/L Ald groups, PI of fibroblasts in 10-7 mol/L Ald +10-6 mol/L Spi, 10(-7 10-5 mol/L Ato 24 h, 48 h, 72 h groups were significantly decreased respectively （All P <0.01）.4. Effects of Ato on Ald induced collagen synthesis increase in CFs: Compared with the control group, 10-7 mol/L Ald significantly increased the collagen synthesis in fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Compared with 10-7 mol/L Ald 24 h, 48 h, 72 h groups, collagen synthesis in fibroblasts in 10-7 mol/L Ald+10-6 mol/L Spi, 10(-7 10-5 mol/L Ato 24 h, 48 h, 72 h groups were significantly decreased respectively （All P <0.01）.5. Effects of Ato on Ald induced expression of Cyclin D1 in CFs: Compared with 10-7mol/L Ald 24 h group, 10-7 mol/L Ald +10-5 mol/L Ato significantly inhibited the expression of Cyclin D1 in fibroblasts nucleus at 24 h （P <0.01）.6. Effects of Ato on Ald induced expression of Cyclin E2 in CFs: Compared with 10-7 mol/L Ald 24 h group, 10-7 mol/L Ald +10-5 mol/L Ato significantly inhibited the expression of Cyclin E2 in fibroblasts nucleus at 24 h （P <0.01）.7. Effects of Ato on Ald induced expression of p-ERK1/2 in CFs: 1 There were not difference between 10-7 mol/L Ald 30 min group and 10-7 mol/L Ald +10-7 10-5 mol/L Ato 30 min groups on expression of p-ERK1/2 in CFs.2 Compared with 10-7 mol/L Ald 4 h group, 10-7 mol/L Ald +10-7 10-5 mol/L Ato significantly inhibited the expression of p-ERK1/2 in fibroblasts at 4 h （All P <0.01）. P-ERK1/2 expression in fibroblasts in 10-7 mol/L Ald +10-6 mol/L Ato 4 h group was lower than 10-7 mol/L Ald+10-7 mol/L Ato 4 h group （P <0.01）.3 Western Blot results show: compared with 10-7 mol/L Ald 4 h group, 10-7 mol/L Ald +10-7 10-5 mol/L Ato significantly inhibited the expression of p-ERK1/2 in fibroblasts at 4 h （P <0.01, P <0.01, P <0.01）.8. Effects of Ato on Ald induced expression of p-AKT in CFs: Compared with 10-7 mol/L Ald 1 h group, 10-7 mol/L Ald +10-7 10-5 mol/L Ato significantly inhibited the expression of p-AKT in fibroblasts at 1 h （All P <0.01）. p-AKT expression in fibroblasts in 10-7 mol/L Ald+10-6 mol/L Ato 1 h groups was lower than 10-7 mol/L Ald+10-7 mol/L Ato 1 h groups, p-AKT expression in fibroblasts in 10-7 mol/L Ald+10-5 the collagen synthesis in fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Compared with 10-7 mol/L Ald 24 h, 48 h, 72 h groups, collagen synthesis in fibroblasts in 10-7 mol/L Ald+10-6 mol/L Spi, 10-7～10-5 mol/L Ato 24 h, 48 h, 72 h groups were significantly decreased respectively （All P <0.01）.5. Effects of Ato on Ald induced expression of Cyclin D1 in CFs: Compared with 10-7mol/L Ald 24 h group, 10-7 mol/L Ald +10-5 mol/L Ato significantly inhibited the expression of Cyclin D1 in fibroblasts nucleus at 24 h （P <0.01）.6. Effects of Ato on Ald induced expression of Cyclin E2 in CFs: Compared with 10-7 mol/L Ald 24 h group, 10-7 mol/L Ald +10-5 mol/L Ato significantly inhibited the expression of Cyclin E2 in fibroblasts nucleus at 24 h （P <0.01）.7. Effects of Ato on Ald induced expression of p-ERK1/2 in CFs: 1 There were not difference between 10-7 mol/L Ald 30 min group and 10-7 mol/L Ald +10-7～10-5 mol/L Ato 30 min groups on expression of p-ERK1/2 in CFs.2 Compared with 10-7 mol/L Ald 4 h group, 10-7 mol/L Ald +10-7 10-5 mol/L Ato significantly inhibited the expression of p-ERK1/2 in fibroblasts at 4 h （All P <0.01）. P-ERK1/2 expression in fibroblasts in 10-7 mol/L Ald +10-6 mol/L Ato 4 h group was lower than 10-7 mol/L Ald+10-7 mol/L Ato 4 h group （P <0.01）.3 Western Blot results show: compared with 10-7 mol/L Ald 4 h group, 10-7 mol/L Ald +10-7～10-5 mol/L Ato significantly inhibited the expression of p-ERK1/2 in fibroblasts at 4 h （P <0.01, P <0.01, P <0.01）.8. Effects of Ato on Ald induced expression of p-AKT in CFs: Compared with 10-7 mol/L Ald 1 h group, 10-7 mol/L Ald +10-7～10-5 mol/L Ato significantly inhibited the expression of p-AKT in fibroblasts at 1 h （All P <0.01）. p-AKT expression in fibroblasts in 10-7 mol/L Ald+10-6 mol/L Ato 1 h groups was lower than 10-7 mol/L Ald+10-7 mol/L Ato 1 h groups, p-AKT expression in fibroblasts in 10-7 mol/L Ald+10-5 10-5 mol/L Ato +10-5 mol/L FPP groups were significantly increased respectively （All P <0.01）. 10-5 mol/L FPP could not reverse the inhibition effects of 10-5 mol/L Ato on CFs DNA synthesis completely. The inhibition effects of 10-7 mol/L Ato group was higher than 10-8mol/L Ato group （P <0.01）.3. Effects of Ato on morphology of CFs: 1 Observed with invert microscope, the shapes of fibroblasts without treated with Ato were regular, fusiform and polygon, intercellular space was small and anchoring growing. Fibroblasts in Ato group were shrunk and small. Intercellular space became large, and the ability of anchoring was reduced. The morphology of fibroblasts was changed more prominent, with the dose and time increase after Ato administration.2 Observed with invert microscope after Wright ’ s- Giemsa staining, the shapes of fibroblasts without treated with Ato were regular, fusiform and polygon, the endochylema were stained with uniform light pink, the nucleus were big and stained with deep pink or light purple, intercellular space was small and anchoring growing. Fibroblasts in Ato group were shrunk and small, with many pseudopodia. Intercellular space became large, nucleus became pyknosis and dark stained, and the ability of anchoring was reduced. The morphology of fibroblasts was changed more prominent, with the dose and time increase after Ato administration.3 Observed with fluorescence microscope after Hochest33258 staining, the nucleus without treated with Ato were regular, oval-shap and stained with light blue fluorescence. The nucleus in Ato group became pyknosis, disfiguration or disintegration, and dark stained. The morphology of nucleus was changed more prominent, with the dose and time increase after Ato administration.4. Effects of Ato on apoptosis of CFs detecting by flow cytometry technique with AnexinV/PI double staining: 1 Analysis of early apoptosis fibroblasts proportion: Compared with the correspondent control groups, 10-7 mol/L 10-5 mol/L Ato significantly increased the proportion of early apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Ato could increase early apoptosis fibroblasts proportion by time and dosage, except between 10-7 mol/L Ato 24h and 10-6 mol/L Ato 24h, 10-7 mol/L Ato 72h and 10-6 mol/L Ato 72h.2 Analysis of late apoptosis fibroblasts proportion: Compared with the correspondent control groups, 10-7 mol/L 10-5 mol/L Ato significantly increased the proportion of late apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Ato could increase late apoptosis fibroblasts proportion by time and dosage, except between 10-7 mol/L Ato 24 h and 10-7 mol/L Ato 48 h group, 10-6 mol/L Ato 24 h and 10-6 mol/L Ato 48 h group.3 Analysis of total apoptosis fibroblasts proportion: Compared with the correspondent control group, 10-7 mol/L 10-5 mol/L Ato significantly increased the proportion of total apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Ato could increase total apoptosis fibroblasts proportion by time and dosage.5. Effects of Ato on apoptosis of CFs detecting sub-G1 by flow cytometry technique with PI staining: Compared with the correspondent control groups, 10-7 mol/L 10-5 mol/L Ato significantly increased the proportion of apoptosis fibroblasts at 24 h, 48 h, 72 h （All P <0.01）. Compared with 10-5 mol/L Ato groups, the proportion of apoptosis fibroblasts in correspondent 10-5 mol/L Ato +10-5mol/L FPP 24 h, 48 h, 72 h groups were significantly increased respectively （All P <0.01）. Ato could increase apoptosis fibroblasts proportion by time and dosage （All P <0.05）.Conclusion: Ato could inhibit proliferation and DNA synthesis of CFs and induce CFs apoptosis. Part 4 The effects of rosuvastatin on proliferation inhibition and apoptosis induction of cultured neonatal cardiac fibroblastsObjective: To investigate the effects of Ros on proliferation inhibition and apoptosis induction of cultured neonatal cardiac fibroblasts.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley （S-D） rats, the effects of Ros on proliferation were measured by MTT colorimetric assay, the effects of Ros on DNA synthesis were measured by Brdu colorimetric assay, the effects of Ros on apoptosis were observed by inverted biological microscope and fluorescence microscope, and measured by flow cytometry technique.Results: 1. Effects of Ros on proliferation of CFs: Compared with the control group, 10-7 mol/L 10-5 mol/L Ros significantly inhibited the proliferation of fibroblasts respectively （All P <0.01）. Compared with 10-7 mol/L 10-5 mol/L Ros groups, the proliferation of fibroblasts in 10-7 mol/L 10-5 mol/L Ros +10-5 mol/L FPP groups were significantly increased respectively （All P <0.01）. 10-5 mol/L FPP could not reverse the inhibition effects of CFs proliferation in 10-5 mol/L Ros group completely. The inhibition effects of 10-7 mol/L Ros was higher than 10-8 mol/L Ros, and the inhibition effects of 10-6 mol/L Ros was higher than 10-7 mol/L Ros （P <0.01, P <0.01）.2. Effects of Ros on DNA synthesis of CFs: Compared with the control group, 10-7 mol/L 10-5 mol/L Ros significantly inhibited DNA synthesis of fibroblasts respectively （All P <0.01）. Compared with 10-7 mol/L 10-5 mol/L Ros groups, DNA synthesis of fibroblasts in 10-7 mol/L 10-5 mol/L Ros +10-5 mol/L FPP groups were significantly increased respectively （All P <0.01）. 10-5 mol/L FPP could not reverse the inhibition effects on CFs DNA synthesis in 10-5 mol/L Ros group completely. The inhibition effects of 10-7 mol/L Ros was higher than 10-8 mol/L Ros, and the inhibition effects of 10-5 mol/L Ros group was higher than 10-6 mol/L Ros （P <0.01, P <0.01）.3. Effects of Ros on morphology of CFs: 1 Observed with invert microscope, the shapes of fibroblasts without treated with Ros were regular, fusiform and polygon, intercellular space was small and anchoring growing. Fibroblasts in Ros group were shrunk and small. Intercellular space became large, and the ability of anchoring was reduced. The morphology of fibroblasts was changed more prominent, with the dose and time increase after Ros administration.2 Observed with invert microscope after Wright’s- Giemsa staining, the shapes of fibroblasts without treated with Ros were regular, fusiform and polygon, the endochylema were stained with uniform light pink, the nucleus were big and stained with deep pink or light purple, intercellular space was small and anchoring growing. Fibroblasts in Ros group were shrunk and small, with many pseudopodia. Intercellular space became large, nucleus became pyknosis and dark stained, and the ability of anchoring was reduced. The morphology of fibroblasts was changed more prominent, with the dose and time increase after Ros administration.3 Observed with fluorescence microscope after Hochest33258 staining, the nucleus without treated with Ros were regular, oval-shap and stained with light blue fluorescence. The nucleus in Ros group became pyknosis, disfiguration or disintegration, and dark stained. The morphology of nucleus was changed more prominent, with the dose and time increase after Ros administration.4. Effects of Ros on apoptosis of CFs detecting by flow cytometry technique with AnexinV/PI double staining: 1 Analysis of early apoptosis fibroblasts proportion: Compared with the correspondent control group, 10-7 mol/L 10-5 mol/L Ros significantly increased the proportion of early apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.05）, except 10-7 mol/L Ros 24 h group. Ros could increase early apoptosis fibroblasts proportion by time and dosage （All P <0.05）.2 Analysis of late apoptosis fibroblasts proportion: Compared with the correspondent control group, 10-7 mol/L 10-5 mol/L Ros significantly increased the proportion of late apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.05） , except 10-7 mol/L Ros 24 h group. Ros could increase late apoptosis fibroblasts proportion by time and dosage （All P <0.05）.3 Analysis of total apoptosis fibroblasts proportion: Compared with the correspondent control group, 10-7 mol/L 10-5 mol/L Ros significantly increased the proportion of total apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.05）. Ros could increase total apoptosis fibroblasts proportion by time and dosage.5. Effects of Ros on apoptosis of CFs detecting sub-G1 by flow cytometry technique with PI staining: Compared with the correspondent control group, 10-7 mol/L 10-5 mol/L Ros significantly increased the proportion of apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. Compared with 10-5 mol/L Ros groups, the proportion of apoptosis fibroblasts in correspondent 10-5 mol/L Ros +10-5 mol/L FPP 24 h, 48 h, 72 h groups were significantly increased respectively （All P <0.01）. Ros could increase apoptosis fibroblasts proportion by time and dosage （All P <0.05）.Conclusion: Ros could inhibit proliferation and DNA synthesis of CFs and induce CFs apoptosis.Part 5 Comparision of Effects on cardiac fibroblasts proliferation and apoptosis between Rosuvastatin and AtorvatatinObjective: To compare the effects of Ato and Ros on proliferation and apoptosis of cultured neonatal cardiac fibroblasts.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley （S-D） rats, the effects of Ato and Ros on proliferation were measured by MTT colorimetric assay, the effects of Ato and Ros on DNA synthesis were measured by Brdu colorimetric assay, the effects of Ato and Ros on cell cycle were measured by flow cytometry technique, synthesis of collagen was observed by the hydroxyproline concentration determined, and apoptosis were measured by flow cytometry technique.Results: 1. Effects of Ato and Ros on proliferation of CFs: Compared with the control group, 10-7 mol/L 10-5 mol/L Ato and Ros significantly inhibited the proliferation of fibroblasts respectively （All P <0.01）. The inhibition effects of Ato and Ros groups was equal to each other.2. Effects of Ato and Ros on DNA synthesis of CFs: Compared with the control group, 10-7 mol/L 10-5 mol/L Ato and Ros significantly inhibited the DNA synthesis in fibroblasts respectively （All P <0.01）. The inhibition effects of Ato and Ros groups was equal to each other.3. Effects of Ato and Ros on cell cycle of CFs: Compared with the control group, 10-7 mol/L 10-5 mol/L Ato and Ros significantly increased cell proportion in G0/G1 phase, and decreased cell proportion in S phase and PI （All P <0.01）. There were not difference between Ato and Ros groups on cell cycle inhibition.4. Effects of Ato and Ros on apoptosis of CFs detecting sub-G1 by flow cytometry technique with PI staining: Compared with the correspondent control group, 10-7 mol/L 10-5 mol/L Ato and Ros significantly increased the proportion of apoptosis fibroblasts at 24 h, 48 h, 72 h respectively （All P <0.01）. There were not difference between Ato and Ros groups on apoptosis induction.5. Effects of Ato and Ros on collagen synthesis in CFs: Compared with the control group, 10-7 mol/L 10-5 mol/L Ato and Ros significantly inhibited the collagen synthesis in fibroblasts respectively （All P <0.01）. The inhibition effects of Ato and Ros groups was equal to each other.Conclusion: Both Ato and Ro are effective antifibrotic agents. And their effects are equal to each other.
【Key words】 aldosterone； spironolactone； atorvastatin； rosuvastatin； SD rats； cardiac fibroblast； ERK； AKT/PKB； Cyclin； proliferation； apoptosis；