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学科主题: 儿科学
题名:
二氧化硫通过ERK途径抑制血管紧张素Ⅱ诱导的高血压小鼠血管平滑肌细胞增殖
作者: 武辉娟
答辩日期: 2016-11-17
导师: 杜军保
专业: 儿科学
授予单位: 北京大学
授予地点: 北京大学第一临床医学院
学位: 硕士
关键词: 二氧化硫,血管紧张素Ⅱ,高血压,血管平滑肌细胞增殖
其他题名: Sulfur dioxide inhibits vascular smooth muscle cell proliferation in angiotensin II-induced hypertensive mice via ERK pathway
分类号: R541.4
摘要:

【目的】血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖是动脉粥样硬化、高血压和血管再狭窄等疾病共同的细胞病理基础之一。阐明VSMC增殖的调控机制对于防治血管重构性疾病和逆转增生性血管病变有着非常重要的临床意义。既往本课题组研究发现气体信号分子二氧化硫(sulfur dioxide, SO2)可抑制VSMC增殖,但其具体分子机制尚未完全明了。本研究的目的在于阐明SO2对血管紧张素Ⅱ(angiotensin II, Ang II)诱导的高血压小鼠主动脉平滑肌细胞增殖的抑制作用及分子机制。

【方法】 36只雄性C57小鼠按随机字数表法随机分为对照组、AngⅡ组和AngⅡ+SO2组。对AngⅡ组和AngⅡ+SO2组小鼠背部皮下植入胶囊式AngⅡ泵,剂量为1000ng/kg.min,对AngⅡ+SO2组小鼠每天腹腔注射SO2供体,对照组和AngⅡ组小鼠每天腹腔注射同剂量的生理盐水,持续2周。通过尾动脉法分别测定各组小鼠实验前和实验后1周及2周的动脉收缩压。实验2周后,采集小鼠血标本及胸主动脉组织标本。应用高效液相色谱荧光法(HPLC-FD)分别测定主动脉组织及血浆中SO2的含量。胸主动脉组织标本制成石蜡切片通过弹力纤维染色观察主动脉中膜厚度,通过免疫组织化学染色法及Western blot杂交检测主动脉组织中抗体增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)和磷酸化ERK(P-ERK)的表达。培养A7R5型血管平滑肌细胞,共分6组(对照组、AngⅡ组、AngⅡ+SO2组、PD98059组、AngⅡ+PD98059组及AngⅡ+SO2+PD98059组),当细胞数量长至70%~80%,进行同步化处理24h,然后分组在培养基中加药,加药浓度为SO2:100mol/L(SO2供体Na2SO3/NaHSO3摩尔比3:1),AngⅡ:1mol/L,PD98059:20mol/L,加药时间0.5h,提取细胞蛋白,western blot实验测定总ERK(T-ERK)及P-ERK的表达,加药时间24h,提取细胞蛋白,应用western blot实验测定增殖指标PCNA的表达。

【结果】:1. SO2抑制AngⅡ诱导的小鼠血压升高。对照组、AngⅡ组和AngⅡ+SO2组小鼠分别在实验开始前、1周后和2周后测定尾动脉血压。结果显示:实验开始前对照组、AngⅡ组、AngⅡ+SO2组收缩压均值间无统计学差异[(100.9±1.9)mmHg vs (101.1±1.8) mmHg vs (99.2±1.9) mmHg](P>0.05)。实验开始1周后分别测定三组小鼠收缩压,结果显示,与对照组相比,AngⅡ组小鼠收缩压明显升高[(144.5±5.6)mmHg vs (103.7±5.3)mmHg](P<0.01),AngⅡ+SO2组小鼠收缩压低于AngⅡ组[(129.1±5.0)mmHg vs (144.5±5.6)mmHg](P<0.01)。实验开始2周后,与对照组相比,AngⅡ组小鼠收缩压明显升高[(153.8±6.8)mmHg vs (102.8±6.6)mmHg](P<0.01);而给予二氧化硫干预后, AngⅡ+SO2组小鼠收缩压低于AngⅡ组[(123.2±6.1)mmHg vs (153.8±6.8)mmHg](P<0.01)。2.AngⅡ抑制小鼠血清和主动脉组织中SO2的生成。实验2周后三组小鼠分别取血清和组织裂解上清液用HPLC法测定SO2含量,结果显示:与对照组相比,AngⅡ组小鼠血清中的SO2含量明显降低[(34.51±6.14µmol/L) vs (46.65±6.89µmol/L)](P<0.05),而给予SO2干预后的小鼠,与AngⅡ组相比,AngⅡ+SO2组小鼠的血清SO2含量明显升高[(46.59±6.39µmol/L) vs (34.51±6.14µmol/L)](P<0.01)。与对照组相比,AngⅡ组小鼠主动脉组织中的SO2含量明显降低[(4.79±0.88µmol/L) vs (9.47±0.84µmol/L)](P<0.05),而给予SO2干预后的小鼠,与AngⅡ组相比,AngⅡ+SO2组小鼠的主动脉组织中SO2含量明显升高[(8.29±0.87µmol/L) vs (4.79±0.88µmol/L)](P<0.01)。3. SO2抑制AngⅡ诱导的小鼠主动脉中膜的增厚。实验小鼠主动脉组织弹力纤维染色结果显示,与对照组相比,AngⅡ组的血管中膜厚度有增加趋势;AngⅡ+SO2组的血管中膜厚度较AngⅡ组有降低趋势。4. SO2抑制AngⅡ诱导的小鼠主动脉组织PCNA和P-ERK的高表达。实验小鼠主动脉组织免疫组织化学染色结果显示,AngⅡ组主动脉组织中PCNA表达较对照组有增高趋势,而AngⅡ+SO2组中PCNA表达较AngⅡ组有降低趋势。AngⅡ组主动脉细胞核中高表达P-ERK细胞数较对照组有增加趋势,而AngⅡ+SO2组主动脉细胞核中高表达P-ERK细胞数较AngⅡ组有降低趋势。实验小鼠的主动脉组织提取组织蛋白通过western blot法检测PCNA和P-ERK的表达,结果显示,与对照组相比,AngⅡ组主动脉组织蛋白的PCNA表达明显增高[PCNA/β-actin灰度值比 (0.61±0.17) vs (0.19±0.03)] (P<0.01),而与AngⅡ组相比,AngⅡ+SO2组的PCNA表达明显降低[PCNA/β-actin灰度值比 (0.23±0.04) vs (0.61±0.17)] (P<0.01);与对照组相比,AngⅡ组主动脉组织蛋白的P-ERK表达明显增高[P-ERK/T-ERK灰度值比 (0.83±0.05) vs (0.42±0.04)] (P<0.01),而与AngⅡ组相比,AngⅡ+SO2组的P-ERK表达明显降低[P-ERK/T-ERK灰度值比 (0.37±0.04) vs (0.83±0.05)](P<0.01)。5. ERK磷酸化抑制剂PD98059可以阻断SO2对VSMC增殖的抑制作用。VSMC细胞蛋白,通过western blot法检测PCNA、T-ERK(总ERK)和P-ERK(磷酸化ERK)的表达,结果显示,与对照组相比,AngⅡ组细胞PCNA表达明显增高[PCNA/β-actin灰度值比(0.98±0.11) vs (0.58±0.14)] (P<0.05);而与AngⅡ组相比,AngⅡ+SO2组的PCNA表达[PCNA/β-actin灰度值比(0.30±0.09) vs (0.98±0.11)]明显降低 (P<0.05)。与对照组相比,AngⅡ组细胞P-ERK/T-ERK表达明显增高[P-ERK/T-ERK灰度值比(0.92±0.01) vs (0.75±0.03)](P<0.05);而与AngⅡ组相比,AngⅡ+SO2组的P-ERK/T-ERK表达降低[P-ERK/T-ERK灰度值比(0.44±0.03) vs (0.92±0.01)](P<0.05)。然而在有PD98059存在的条件下,SO2则不能抑制由AngⅡ诱导的细胞PCNA表达增强[PCNA/β-actin灰度值比(0.97±0.03) vs (0.91±0.10)](P>0.05)。

【结论】:SO2可以降低Ang II引起的高血压,抑制Ang II诱导的血管重构、血管平滑肌的增殖,该效应与SO2抑制ERK磷酸化有关。

英文摘要:

Objective: Vascular smooth muscle cell (VSMC) proliferation is one of common cell pathological basis of atherosclerosis, hypertension, vascular restenosis and some other disease. Clarifying the mechanisms underlying VSMC proliferation has great clinical significance for the prevention and treatment of vascular remodeling and the reverse of vascular disease with hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the specific molecular mechanism has not been fully elucidated. The study was designed to test if SO2 inhibited VSMC proliferation in mice with hypertension induced by angiotensin II (AngII) and its molecular mechanisms.

Methods: Thirty-six male C57 mice were randomly divided into control group, AngII group and AngII+SO2 group. Mice in AngII group and AngII+SO2 group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng/kg/min. In addition, mice in AngII+SO2 received intraperitoneal injections of SO2 donor, while, mice in control group and AngII group received intraperitoneal injections of same does of normal saline, consecutively for 2 weeks. Through the tail artery manometry, systolic pressure of each mouse in every group was measured respectively before the experiment, 1 week later and 2 weeks later during the experiment. After 2 weeks, blood samples and thoracic aorta tissue specimens were collected. We measured the concentration of SO2 in aortic tissue homogenate and in plasma using high performance liquid omatography with fluorescence determination (HPLC-FD). Thoracic aorta tissue specimens were made with paraffin slice. The thickness of the aorta tunica media was analyzed by elastic fiber staining, and the expression of proliferating cell nuclear antigen (PCNA) and phosphorylated ERK (P-ERK) in aortic tissues were detected through immunohistochemistry and Western blot hybridization. VSMC of A7R5 cell lines was cultured and divided into six groups (control group, AngⅡ group, AngⅡ+SO2 group, PD98059 group, AngⅡ+PD98059 group, and AngⅡ+ SO2+PD98059 group). When increasing to 70%-80%, the cell lines were made synonized treatment for 24 h. Then, we added drug seperately to the culture medium of each group. Drug concentration was as follows SO2: 100 mol/L (SO2 donor: Na2SO3/NaHSO3 mole ratio 3:1), and AngⅡ: 1 mol/L,PD98059: 20 mol/L. After 0.5 h of drug treatment, proteins were isolated from the cells to measure the expression of total ERK (T-ERK) and P-ERK through western blot. After 24 h of drug treatment, proteins were isolated from the cells to measure the expression of PCNA through western blot.

Results: 1. Sulfur dioxide reduced hypertension caused by AngII. At the beginning of the experiment, after 1 week and after 2 weeks of the experiment, we measured the tail blood pressure of the mice in control group, AngII group and AngII+SO2 group. The result showed that before the experiment the mean systolic blood pressure in control group, AngII group and AngⅡ+SO2 group had no statistical difference (100.9±1.9)mmHg vs (101.1±1.8) mmHg vs (99.2±1.9) mmHg] (P>0.05). One week after the experiment, we measured the mean systolic blood pressure of the mice in three groups respectively. Compared with control group, the systolic blood pressure of mice in AngII group increased obviously [(144.5±5.6) mmHg vs (103.7±5.3) mmHg] (P<0.01), however, the systolic blood pressure of the mice in the AngII + SO2 group was lower than that in AngII group [(129.1±5.0) mmHg vs (144.5±5.6) mmHg ] (P<0.01). Two weeks after the experiment, compared with control group,the systolic blood pressure of the mice in AngII group increased obviously [(153.8±6.8)mmHg vs (102.8±6.6) mmHg] (P<0.01), however, with SO2 intervention, the systolic blood pressure of the mice in AngⅡ+SO2 group was lower than that in AngⅡ group [(123.2±6.1) mmHg vs (153.8±6.8)mmHg] (P<0.01). 2. Ang II reduced SO2 concentration in serum and aortic tissue of the mice. After 2 weeks of the experiment, we collected serum and aortic tissue homogenized supernatant of the mice in 3 groups respectively and measured the SO2 content by HPLC‑FD. We found that compared with control group, the SO2 concentration in serum of the mice in AngII group decreased obviously [(34.51±6.14 µmol/L) vs (46.65±6.89 µmol/L)] (P<0.05), but with sulfur dioxide intervention, the SO2 concentration in serum of the mice in AngII + SO2 group was higher than that in AngII group [(46.59±6.39 µmol/L) vs (34.51±6.14 µmol/L)] (P<0.01). Compared with control group the SO2 concentration in aortic tissue of the mice in AngII group decreased [(4.79±0.88 µmol/L) vs (9.47±0.84 µmol/L)] (P<0.05), but with sulfur dioxide intervention, the SO2 concentration in aortic tissue of the mice in AngII + SO2 group was higher than that in AngII group [(8.29±0.87 µmol/L) vs (4.79±0.88 µmol/L)] (P<0.01). 3. SO2 inhibited the thickening of the tunica media caused by AngII. Through elastic fiber staining methods for aortic tissue of the mice in experiment, we found that the thickness of the aortic tunica media in AngII group increased obviously compared to that in control group; however, the mean thickness of the in AngII+SO2 group was thinner than that in AngII group. 4. SO2 inhibited the high expression of PCNA and P-ERK caused by AngII in aortic tissues of the mice. The results of immunohistochemical staining showed that the PCNA expression in aortic tissues of the mice in AngII group was higher than that in control group. However, the PCNA expression in aortic tissues of the mice in AngII+SO2 group was lower than that in AngII group. The munber of high P-ERK expression cells in AngII group was higher than that in control group. However, the munber of high P-ERK expression cells in AngII+SO2 group was lower than that in AngII group. The result of measuring the expression of P-ERK(T-ERK) and P-ERK through western blot showed that the PCNA expression of aorta tissue protein in AngII group increased obviously compared with that in control group [the ratio of the grey value of PCNA/β-actin: (0.61±0.17) vs (0.19±0.03)] (P<0.01), however, the PCNA expression in AngII+SO2 group decreased obviously compared with that in AngII group [the ratio of the grey value of PCNA/β-actin: (0.23±0.04)vs (0.61±0.17)] (P<0.01); the P-ERK expression of aorta tissue protein in AngII group increased remarkably compared with that in control group [the ratio of the grey value of P-ERK/T-ERK: (0.83±0.05) vs (0.42±0.04)] (P<0.01), however, the P-ERK expression in AngII+SO2 group decreased obviously compared with that in AngII group [the ratio of the grey value of P-ERK/T-ERK: (0.83±0.05) vs (0.37±0.04)] (P<0.01). 5. PD98059, an ERK phosphorylation inhibitor, blocked the inhibitory effect of SO2 on VSMC proliferation. The results showed that, compared with control group, the PCNA expression in the AngII group increased [the ratio of the grey value of PCNA/β-actin (0.98±0.11) vs (0.58±0.14)] (P<0.05);compared with AngⅡgroup,the PCNA expression in the AngII + SO2 group decreased [the ratio of the grey value of PCNA/β-actin (0.30±0.09) vs (0.98±0.11)] (P<0.05)。Compared with control group, the P-ERK expression in the AngII group increased than that in control group [the ratio of the grey value of P-ERK/T-ERK (0.92±0.01) vs (0.75±0.03)](P<0.05);compared with AngⅡgroup,the P-ERK expression in the AngII + SO2 group decreased [the ratio of the grey value of P-ERK/T-ERK (0.44±0.03) vs (0.92±0.01)](P<0.05)。After adding PD98059, however, SO2 could not inhibit the PCNA expression induced by AngⅡ in cells [the ratio of the grey value of PCNA/β-actin (0.97±0.03) vs (0.91±0.10)] (P>0.05).

Conclusions: SO2 could reduce the hypertension caused by AngⅡ, and inhibit AngⅡ-induced VSMC proliferation via inhibiting ERK phosphorylation.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125270
Appears in Collections:北京大学第一临床医学院_学位论文

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作者单位: 北京大学第一临床医学院

Recommended Citation:
武辉娟. 二氧化硫通过ERK途径抑制血管紧张素Ⅱ诱导的高血压小鼠血管平滑肌细胞增殖[D]. 北京大学第一临床医学院. 北京大学. 2016.
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