【作者】 李晓岩；

【导师】 冉丕鑫； 王健；

【作者基本信息】 广州医学院 ， 呼吸内科， 2010， 博士

【摘要】 目的:探讨骨形态形成蛋白4（BMP4）对大鼠远端肺动脉平滑肌细胞内钙离子浓度的影响及其机制,为进一步探究其在低氧性肺动脉高压（HPH）发病过程的可能作用与机制打下基础。研究内容和方法:一、BMP4对大鼠远端肺动脉平滑肌细胞内钙离子浓度的影响将200-300g雄性Wistar大鼠麻醉后,取出心肺组织,置于显微镜下,用显微镊和显微剪刀于显微镜下小心分离出远端（4-5级）肺动脉,剥离内外膜后,采用胶原酶消化法获得远端肺动脉平滑肌细胞,将细胞种于放置在35mm培养皿内的圆形载玻片上,培养至第三天细胞密度达到50%,将细胞随机分为三个组:（1）未处理组,未给予刺激物; （2）对照组,用等量的BMP4溶解缓冲液处理PASMCs 60h; （3）BMP4组,用BMP4（50ng/ml）处理PASMCs60h。细胞用Fura-2在细胞培养箱内孵育1h,使Fura-2进入细胞内,用荧光显微镜检测,根据标准曲线,推算细胞内Ca²⁺浓度。二、BMP4影响大鼠远端肺动脉平滑肌细胞TRPC1,6 mRNA和蛋白质表达及其机理研究1、BMP4对PSMC表达TRPC1、6 mRNA和蛋白质的影响:实验采用原代培养大鼠远端肺动脉平滑肌细胞,培养至第五天细胞密度达到80-90%,将细胞随机分为三个组:（1）未处理组,未给予刺激物;（2）对照组,用等量的BMP4溶解缓冲液处理PASMCs 60h;（3）BMP4组,用BMP4（50ng/ml）处理PASMCs60h。收集细胞,用iQ SYBR Green supermix试剂实时定量细胞中的TRPC1,6 mRNA的表达;用Western Blotting检测TRPC1,6蛋白质的表达。2、BMP4对Smad1/5/8 ,ERK1/2, p38MAPK信号通路的影响:原代培养大鼠远端肺动脉平滑肌细胞,培养至第五天细胞密度达到80-90%,将细胞随机分为七个组:（1）对照组,用等量的BMP4溶解缓冲液处理PASMCs15min;（2）BMP4（50ng/ml）处理组:分别用BMP4（50ng/ml）处理PASMCs15min、30min、1h、4h、8h、60h。收集细胞,用Western Blotting检测BMP4对Smad1/5/8 ,ERK1/2, p38MAPK信号通路的影响。3、BMPRⅡSiRNA、SB及PD对BMP4/Smad1/5/8 ,ERK1/2, p38MAPK信号通路的影响:用Western Blotting检测BMPRⅡ-SiRNA、PD和SB对BMP4刺激Smad1/5/8、ERK1/2和p38MAPK信号通路的影响。4、SB及PD对BMP4促进大鼠远端PSMCs表达TPRC1,6mRNA和蛋白质的干预作用:原代培养大鼠远端肺动脉平滑肌细胞,培养至第五天细胞密度达到80-90%,将细胞随机分为四个组:（1）空白对照组,细胞未给予任何刺激物;（2） DMS0对照组, DMSO预处理细胞30min, BMP4（50ng/ml）处理PASMCs60h; （3）SB组,SB（5μM）预处理细胞30min, BMP4（50ng/ml）处理PASMCs60h;（4）PD组,PD（10μM）预处理细胞30min, BMP4（50ng/ml）处理PASMCs60h;收集细胞,用iQ SYBR Green supermix试剂实时定量细胞中的TRPC1,6 mRNA的表达,用Western Blotting检测TRPC1,6蛋白质的表达。5、SB或PD对BMP4增加PSMCs内钙离子浓度的影响的研究:原代培养大鼠远端肺动脉平滑肌细胞,培养至第三天细胞密度达到50%,将细胞随机分为四个组:1)空白对照组,细胞未给予任何刺激物;（2） DMS0对照组, DMSO预处理细胞30min, BMP4（50ng/ml）处理PASMCs60h;（3）SB组,SB（5μM）预处理细胞30min, BMP4（50ng/ml）处理PASMCs60h （4）PD组,PD（10μM）预处理细胞30min, BMP4（50ng/ml）处理PASMCs60h。细胞用Fura-2孵育,使Fura-2进入细胞内,用荧光显微镜检测,根据标准曲线,推算细胞内Ca²⁺浓度。三、慢性低氧对小鼠肺组织内BMP内源性拮抗蛋白表达的影响实验选用雄性的BMP4基因敲除的同种系BMP4+/+ C57BL/6J纯合子小鼠和BMP4+/- C57BL/6J杂合子小鼠,将BMP4+/+ C57BL/6J小鼠随机分为5组（n=3或4）:即正常对照组、低氧1天组、低氧3天组、低氧7天组和低氧21天组。6只BMP4+/- C57BL/6J小鼠随机分成低氧1天和对照2组。将低氧小鼠置于低氧装置内,调节箱内氧浓度为10%,每天24小时持续低氧。对照组小鼠除吸入空气外,其它饲养条件与实验组相同。低氧完成后,麻醉小鼠取出心肺组织,左肺储存于-80。C冰箱备用,右肺组织提取总RNA;用实时定量PCR检测BMP内源性拮抗蛋白的mRNA表达水平。结果:一、BMP4能够增加大鼠远端PASMCs内钙离子浓度,未处理组、仅以溶解液处理对照组和50ng/ml BMP4处理组,[Ca²⁺]i分别为204.07±1.22nM; 170.64±0.68nM; 415.52±3.94nM,实验组PASMC内钙离子浓度明显高于对照组,P值〈0.05。二、BMP4能够促进大鼠远端PASMCs的TRPC1、6mRNA及蛋白的表达,未处理组和对照组比较无差异表达,与对照组比较BMP4组TRPC1,TRPC6mRNA表达量明显升高,分别上升1.69倍,2.08倍（P〈0.05）,BMP4组与对照组相比,BMP4分别使TRPC1/β-actin,TRPC6/β-actin的蛋白灰度比值增加1.61倍,1.41倍（P〈0.05）,结果具有统计学意义。三、BMP4作用于大鼠远端PASMCs,可以迅速激活Smad信号通路（15min）,而后随时间的延长逐渐减弱,也可以迅速激活P38MAPK及ERK1/2信号通路（15min）,并且在作用4h时出现第二个激活高峰。四、BMPRⅡsiRNA转染大鼠远端PASMCs,成功地沉默了BMPRⅡ蛋白表达的同时,减弱了BMP4对Smad磷酸化水平。五、P38MAPK信号通路的特异性抑制剂SB及ERK1/2信号通路的特异性抑制剂PD能够降低BMP4刺激所引起的TRPC1、6mRNA的表达,用ERK1/2的抑制剂PD处理组TRPC1和TPC6mRNA的相对表达量分别降低1.6倍和1.7倍（P〈0.05）;用P38MAPK的抑制剂SB处理组TRPC1和TPC6mRNA的相对表达量分别降低1.5（P〈0.05）。六、P38MAPK信号通路的特异性抑制剂SB及ERK1/2信号通路的特异性抑制剂PD能够降低BMP4刺激所引起的TRPC1、6蛋白表达增加,SB+B4、PD+B4与DMSO+B4组比较TRPC1蛋白表达TRPC1/β-actin的灰度比值分别下降2.3倍（P〈0.05）;SB+B4、PD+B4与DMSO+B4组比较TRPC6蛋白表达TRPC6/β-actin的灰度比值分别下降2.7倍（P〈0.05）。七、P38MAPK信号通路的特异性抑制剂SB及ERK1/2信号通路的特异性抑制剂PD均能够降低BMP4对大鼠远端PASMCs内基础Ca2+浓度增加的程度,用ERK1/2的抑制剂PD和P38MAPK的抑制剂SB处理的PASMCs内这种BMP4诱导的Ca2+浓度增加分别降低1.9倍和1.6倍（P〈0.05）。八、在正常的BMP4+/+ C57BL/6J小鼠肺组织中至少存在8种BMP内源性拮抗剂,分别是chordin、chordin-1、Noggin、Mgp、FSRP、CV-2、Fst和Gremlin。九、8种BMP内源性拮抗蛋白的mRNA的表达随小鼠缺氧的时间呈先上升后下降的趋势,低氧第一天表达增加尤其明显,其中FSN的表达趋势较其他几种BMP内源性拮抗蛋白明显,别增加3.64倍、1.23倍、2.0倍、2.0倍、1.9倍、2.8倍、1.4倍、28倍（P〈0.05）。十、常氧状态下,BMP4+/+ C57BL/6J纯合子小鼠和BMP4+/- C57BL/6J杂合子小鼠肺组织内大多数BMP内源性拮抗蛋白mRNA的表达无明显差异,两组对照, BMP4+/-杂合子小鼠肺组织内Noggin和CV2 mRNA的表达上升,分别为1.36倍、1.91倍（P〈0.05）, chordin mRNA的表达降低,下降0.59倍（P〈0.05）。十一、低氧状态下,BMP4+/+ C57BL/6J纯合子小鼠和BMP4+/- C57BL/6J杂合子小鼠肺组织内大多数BMP内源性拮抗蛋白mRNA的表达无明显差异,两组对照,两组对照,BMP4+/+ C57BL/6J纯合子小鼠肺组织内Noggin、Gremlin和FSN的mRNA表达明显高于BMP4+/- C57BL/6J杂合子小鼠,分别为4倍、2.69倍和28倍（P〈0.05）。十二、正常氧和低氧一天条件下相比较,BMP4+/- C57BL/6J杂合子小鼠肺组织内大多数BMP内源性拮抗蛋白的mRNA的表达,无明显差异,在低氧状态下BMP4+/-杂合子小鼠肺组织内CV2 mRNA的表达下降2倍（P〈0.05）,提示低氧状态下CV-2的下调在BMP4基因突变基础上对HPH的发病起重要作用。结论:一、BMP4促进细胞内Ca²⁺浓度增加是低氧性肺动脉高压发病机制中新的观点。BMP4可以激活Smad、P38MAPK和ERK1/2信号通路。BMP4可以通过激活P38MAPK,ERK1/2途径,刺激大鼠远端PASMCs的TRPC1,6mRNA及蛋白的表达增加,通过TRPC1,6组成的Ca²⁺通道增加细胞内的Ca²⁺浓度,从而增加血管的紧张度及促进血管平滑肌增殖,最终导致肺动脉高压。二、低氧条件下,细胞首先表现出应激反应,增加BMP内源性拮抗蛋白的表达,随后出现与BMP不协调的表达,部分内源性拮抗剂与BMP4存在依赖关系,表明内源性拮抗剂在低氧性肺动脉高压的发生机制上存在一定的作用。更多还原

【Abstract】 OBJECTIVE:To investigate that machenism of [Ca2+] i change in peripheral PASMCs exposed to BMP4 and role of BMPs antagonists in pathogenesis of hypoxic pulmonary arterie hypertension.METHODS:1 Determine whether there was [Ca2+] i chang in distal PASMCs exposed to BMP4 （50 ng/ml）Distal PASMCs were treated with BMP4 （50 ng/ml）. Ca²⁺ in PASMCs was measured using fluorescent microscopy and Ca²⁺sensitive dye-Fura-2.2 Signaling Pathway between BMP4 and TRPC1、6 in distal pulmonary artery smooth muscle cells.1) BMP4 induced expression of TRPC1, 6 mRNA and ProteinThe PASMCs were treated with BMP4 （50 ng/ml） or vehicle for up to 60 hours. TRPC1, 6 mRNA extracted from treated cells were amplified by a standard PCR protocol using IQ SYBR Green supermix Real-Time PCR. Western Blotting was performed for identification of TRPC1, 6 proteins extracted from cells treated with BMP4 BMP4 （50 ng/ml）.2) Determine which signaling pathway was involved in BMP4-induced TRPC1, 6 expression up-regulatin.BMP4/Smad1/5/8 and ERK1/2, p38MAPK were examined in distal PASMCs treated with BMP4 using Western Blots. （Different time-point treatment） The PASMCs were pre-treated with inhibitor of ERK1/2 （PD098059 10μM）, ERK1/2 pathway was detected using Western Blot. The PASMCs were pre-treated with inhibitor of p38MAPK （SB2035805μM）, P38MAPK pathway was detected using Western Blot.3) Determine whether a kind of the inhibitors decreased [Ca2+]i in PASMCs exposed to BMP4The PASMCs exposed to BMP4 were pre-treated （30 min） with inhibitors of ERK1/2 （PD098059 10μM）, p38MAPK （SB203580 5μM）.Ca²⁺in PASMCs was measured using fluorescent microscopy and Ca²⁺sensitive dye-Fura-2. 3 Role of BMPs antagonists in pathogenesis of hypoxic pulmonary hypertension1) Determine whether there are some BMPs antagonists in lung tissue of mouse and how many kinds are there, so BMPs antagonists were established by polymerase chain reaction （PCR） and confirmed by gene sequencing.2) Identificat whether BMPs antagonists are depended-BMP4, and whether they are relative with hypoxia.They were detected by Real-time fluorescence quantitative PCR, the samples were lung tissue isolated from BMP4+/+ or BMP4+/- mice. （Wild-type or HET）.RESULTS:1 [Ca2+] i in peripheral PASMCs treated with BMP4 was increased. 2 BMP4 induces expression of TRPC1, 6 mRNA and Protein1) Incubation of peripheral PASMCs with BMP-4 （50ng/mL） led to phosphorylation of Smad1/5/8. Reprobing of blots for total Smad5 confirmed equal loading. Rapid phosphorylation of Smad1/5/8 was seen after 15 minutes of stimulation with BMP4.2) Incubation of peripheral PASMCs with BMP-4 （50 ng/mL） led to phosphorylation of p38MAPK and ERK1/2. Reprobing of blots for total p38MAPK and ERK1/2 confirmed equal loading. BMP4 stimulation led to rapid （15 min） transient activation of P38MAPK and ERK1/2, which returned to baseline by 30minuts, then phosphorylation, was evident at 4 hour. 3) The effect of MAPK inhibitors was examined at the 15-min time point. SB203580 and PD09859 can inhibit special signaling pathway compared with BMP4 alone.4) Immunoblotting analysis showed that three different concentrations of BMPRⅡsiRNA were silented BMPRⅡprotein expression almost 100% compared to no-targeting siRNA as a negative control. To confirm equal loading, blots were incubated withβ-actin antibody. In next experiments, 50nM concentration of BMPRⅡsiRNA was used.5) Real-time PCR demonstrated that induction of TRPC1 mRNA in response to BMP4 （50ng/ml） was decreased by the p38MAPK inhibitor, SB203580 or the ERK1/2 inhibitor, PD98059.6) Real-time PCR demonstrted that induction of TRPC6 mRNA in response to BMP4 （50ng/ml） was reduced by the p38MAPK inhibitor SB203580 or the ERK1/2 inhibitor, PD980509.7) The PASMCs transfected with no-targeting control siRNA showed no effect on BMP4-mediated P-Smad1/5/8 induction. However, with BMPRⅡsiRNA, P-Smad1/5/8 was reduced comparing with the no-targeting control level.8) In the PASMCs the pre-treated （30 min） with inhibitor of ERK1/2 （PD098059 10μM ）, p38MAPK （SB203580 5μm）, expressions of TRPC1, 6 protein were decreased.9) The increase of [Ca2+] i induced by BMP4 was attenuated by SB or PD.3 Eight BMPs antagonists were identificated by polymerase chain reaction （PCR） in lung tissue of mouse and confirmed by gene sequencingThe samples from lung tissue isolated from BMP4+/+ or BMP4+/- mice were detected by Real-time fluorescence quantitative PCR, and they were relative with hypoxia, most of the antagnists mRNA were increased after hypoxic 1 day and reduced with hypoxic time, which returned to basline by hypoxic 21 days. CONCLUSIONS:1. [Ca²⁺] i in peripheral PASMCs treated with BMP4 was increased; TRPC1, 6 are an important target of BMP signaling in PASMCs. Inhibition of p38MAPK and ERK1/2 parthway will decrease expression of TRPC1, 6 and represent a novel theory about PAH.2. There were eight BMP antagonists in lung tissue of mouse. Most of BMPs antagonists are depended-hypoxia.更多还原