BMSCs通过miR-34a调节PNUTS在慢性缺氧大鼠心室重构中的作用

发布时间：2018-06-08 01:00

本文选题：慢性缺氧 + 心室重构　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]慢性缺氧是导致心室重构的主要因素之一。心室重构是导致各种心脏病发生心衰、心律失常的重要的病理机制,也是心脏老化的标志。近年来国内外研究包括本课题组前期实验研究发现BMSCs (骨髓间充质干细胞)移植可有效改善心肌细胞凋亡及心肌纤维化,BMSCs移植治疗可望成为改善心室重构的可行性治疗手段之一。但BMSCs改善心室重构的作用机制尚不甚清楚。PNUTS(Serine/threonine protein phosphatase 1 regulation 10; PPPIR10;丝氨酸/苏氨酸蛋白磷酸酶1调节亚基10)是蛋白磷酸酶1 ( PP1)的调节亚基,其主要有抗细胞凋亡、促进急性心肌梗死后心功能恢复的作用,近来有研究表明BMSCs中可表达PNUTS,但是BMSCs是否通过PNUTS改善慢性缺氧导致的心室重构以及PNUTS和慢心缺氧导致的心室重构的相关性未见报道。故本课题建立SD大鼠慢性缺氧心室重构模型,并用BMSCs治疗各缺氧大鼠模型。检测慢性缺氧心室重构模型及BMSCs治疗后大鼠的心室miR-34a、pnuts mRNA表达量、PNUTS浓度,以及血清和心肌组织中的PⅠCP、PⅢNP浓度。探讨PNUTS在慢性缺氧所致的心室重构及BMSCs改善慢心缺氧导致的心室重构中的作用。[方法]45只大鼠随机分为9组,每组5只,分别为:A组(对照组)、B组(缺氧7天)、C组(缺氧14天)、D组(缺氧21天)、E组(缺氧28天)、B'组(缺氧7天后腹腔注射浓度为5×106/mL的干细胞1mL,治疗4周)、C'组(缺氧14天后腹腔注射浓度为5×106/mL的干细胞1mL,治疗4周)、D'组(缺氧21天后腹腔注射浓度为5×106/mL的干细胞1mL,治疗4周)、E'组(缺氧28天后腹腔注射浓度为5×106/mL的干细胞1mL,治疗4周),运用全自动缺氧实验装置建立缺氧组模型,运用干细胞治疗4周后称取大鼠体重,按0.3mL/100g的量腹腔注射10%水合氯醛麻醉大鼠并进行解剖,打开腹腔,于腹主动脉采血,离心后取血清;打开胸腔取心脏制作病理切片,HE染色观察各缺氧组及治疗组大鼠心肌组织的细胞形态、水肿、肥大、变性、坏死及炎性细胞浸润情况等病理性改变;Masson染色观察各缺氧组及治疗组大鼠心肌组织的心肌胶原分布及含量,计算胶原容积分数(CVF);荧光Tunel检测各缺氧组及治疗组大鼠心肌组织中心肌细胞凋亡情况,计算细胞凋亡率;运用qPCR检测各缺氧组、治疗组大鼠心肌组织及BMSCs中miR-34a表达量、pnuts mRNA表达量;运用ELISA检测各缺氧组和治疗组大鼠血清中PⅠCP、PⅢNP浓度,心肌组织中PⅠCP、PⅢNP浓度,PNUTS浓度,BMSCs中PNUTS浓度;分析miR-34a表达量、pnuts mRNA表达量、PNUTS浓度、PⅠCP、PⅢNP浓度、胶原容积分数及细胞凋亡率之间的相互关系。[结果]1、与对照组比较,各缺氧组大鼠血清和心肌组织中PⅠCP、PⅢNP含量显著增加(P＜0.01);各缺氧组间两两分析比较显示,血清及心肌组织中的PⅠCP、PⅢNP含量差异均有显著性(P＜0.01),并随着缺氧时间的增加而增加。2、与对照组比较,HE染色显示各缺氧组大鼠心肌细胞均有不同程度的水肿、空泡变性、肥大、炎症细胞浸润及纤维化;Masson染色显示心肌细胞间隙胶原蛋白增加,胶原沉积增加,缺氧组的CVF显著增多(P0.01);病理切片荧光Tunel显示心肌细胞凋亡增加,心肌细胞凋亡率显著增加(P0.01)。各缺氧组间两两分析比较显示:心肌病理改变、纤维化程度、CVF、心肌细胞凋亡率差异有显著性(P＜0.01),并且随着时间的增加而增加。3、与对照组比较,各缺氧组心肌组织miR-34a表达量显著增加(P0.01),pnuts mRNA表达量显著减少(P0.01);各缺氧组间两两比较显示:miR-34a表达量差异显著(P＜0.01),且随着缺氧时间的增加而增加,pnuts mRNA表达量差异显著(P＜0.01),且随着缺氧时间的增加而减少。4、相关性分析显示:大鼠血清和心肌组织的PⅠCP、PⅢNP浓度与CVF呈显著正相关(P0.01) ; PⅠCP、PⅢNP浓度与心肌细胞凋亡率凋亡呈显著正相关(P＜0.01)。5、干细胞中miR-34a表达量显著低于对照组(P0.01),干细胞中pnutsmRNA表达量、PNUTS浓度显著高于对照组(P＜0.01)。6、干细胞治疗组(B'组、C'组、D'组、E'组)血清及心肌组织中的PⅠCP、PⅢNP浓度较相应的各缺氧组显著下降(P0.01)。7、干细胞治疗组(B'组、C'组、D'组、E'组)的心肌组织异常病理改变、纤维化程度较相应的各缺氧组显著改善,心肌组织中的CVF及心肌细胞凋亡率较相应的各缺氧组显著下降(P0.01)。8、干细胞治疗组(B'组、C'组、D'组)心肌组织miR-34a表达量较相应的各缺氧组显著减少(P0.01),pnuts mRNA表达量显著增多(P＜0.01),PNUTS浓度显著增多(P0.01);干细胞治疗组E'组心肌组织miR-34a表达量、pnuts mRNA表达量及PNUTS浓度较缺氧组E组改变不显著(P0.01)。9、相关性分析显示:除E组(缺氧28天组),余各缺氧组和治疗组心肌组织miR-34a表达量与pnuts mRNA表达量呈显著负相关(P0.01),缺氧组及治疗组PNUTS的含量与PⅠCP、PⅢNP含量、CVF、细胞凋亡率呈显著负相关(P0.01)。线性回归分析提示:缺氧组及治疗组miR-34a与PNUTS含量显著相关(R2=0.856、R2=0.862),回归系数分别为-0.132 (P＜0.05) ; -0.162 (P0.05);PNUTS含量与缺氧组PⅠCP、PⅢNP含量显著相关(R2=0.986、R2=0.949)回归系数分别为-22.518(P0.05)、-11.188(P0.05);PNUTS含量与治疗组PⅠCP、PⅢNP含量显著相关(R2=0.992、R2=0.981)回归系数分别为-7.673 (P＜0.05)、-5.478 (P0.05) ; PNUTS含量与缺氧组及治疗组CVF显著相关(R2=0.995、R2=0.992)回归系数分别为-0.168 (P0.05)、-0.090 (P0.05) ; PNUTS 含量与缺氧组及治疗组心肌细胞凋亡率显著相关(R2=0.999、R2=0.997)回归系数分别为-0.643 (P＜0.05)、-0.113 (P0.05)。[结论]1、慢性缺氧心室重构模型建立成功,在缺氧21天到缺氧28天时心室发生重构最严重;2、慢性缺氧心室重构过程中,心肌纤维化、心肌细胞凋亡出现时间基本一致,两者共同参与心室重构的发展过程,且在缺氧21天到缺氧28天时改变更明显;3、BMSCs对慢性缺氧大鼠的心室重构有明显改善作用,但不能完全逆转心室重构。4、miR-34a可以抑制pnuts在心肌中的表达;5、miR-34a在大鼠骨髓间充质干细胞中低表达,pnuts在大鼠骨髓间充质干细胞中高表达;6、PNUTS与慢性缺氧导致的心室重构具有显著相关性;7、BMSCs可通过miR-34a调控的PNUTS改善慢性缺氧导致的心室重构,但是对于缺氧时间较长的慢性缺氧模型,BMSCs并不通过miR-34a调控的PNUTS发挥作用。
[Abstract]:[Objective] chronic hypoxia is one of the main factors leading to ventricular remodeling. Ventricular remodeling is an important pathological mechanism that causes heart failure and arrhythmia in all kinds of heart diseases. It is also a sign of heart aging. In recent years, domestic and foreign studies have found that BMSCs (bone marrow mesenchymal stem cells) transplantation can improve the heart effectively. Myocyte apoptosis and myocardial fibrosis, BMSCs transplantation is expected to be one of the feasible treatment methods to improve ventricular remodeling. However, the mechanism of BMSCs improvement of ventricular remodeling is not clear.PNUTS (Serine/threonine protein phosphatase 1 regulation 10; PPPIR10; serine / threonine protein phosphatase 1 regulation subunit 10) is protein phosphorus The regulatory subunit of acid enzyme 1 (PP1), which mainly has anti apoptosis and promotes cardiac function recovery after acute myocardial infarction, has recently shown that PNUTS can be expressed in BMSCs, but the correlation of BMSCs through PNUTS to improve ventricular remodeling caused by chronic hypoxia and the correlation of ventricular remodeling caused by PNUTS and slow cardiac hypoxia has not been reported. The chronic hypoxic ventricular remodeling model of SD rats was established and the model of hypoxia rats was treated with BMSCs. The chronic hypoxia ventricular remodeling model and the ventricular miR-34a, pnuts mRNA expression, PNUTS concentration, and P I CP, P III NP concentration in serum and myocardial tissues after BMSCs treatment were detected. The ventricular remodeling caused by chronic hypoxia was discussed. And BMSCs to improve the role of BMSCs in ventricular remodeling. [method]45 rats were randomly divided into 9 groups, 5 rats in each group, group A (control group), group B (7 days of hypoxia), group C (14 days of hypoxia), group D (21 days of hypoxia), group E (28 days of hypoxia), B'group (7 days after 7 days of hypoxia, 5 x 106/mL 1mL, 4 weeks), C' group 14 days (14 days of hypoxia) The posterior intraperitoneal injection of 1mL, 5 x 106/mL, was treated for 4 weeks. In group D'(21 days after hypoxia, 5 x 106/mL of stem cells 1mL, 4 weeks of treatment), E' group (28 days after hypoxia, 5 * 106/mL of stem cell 1mL, for 4 weeks), an anoxic group model was established with an automatic anoxic experimental device for 4 weeks. After being weighed, the rats were injected into 10% hydrochloral anaesthetized rats by intraperitoneal injection of 0.3mL/100g and dissected, open the abdominal cavity, take the blood from the abdominal aorta, and take the serum after centrifugation; open the chest and take the heart to make pathological sections. HE staining was used to observe the morphology, edema, hypertrophy, degeneration, necrosis and inflammation of the group of rats in the anoxic group and the treatment group. Pathological changes such as cell infiltration, Masson staining was used to observe the distribution and content of myocardial collagen in the hypoxia group and the treatment group, and calculate the collagen volume fraction (CVF); fluorescence Tunel was used to detect the apoptosis of cardiac muscle cells in the myocardial tissue of each anoxic group and the treatment group, and the apoptosis rate was calculated. QPCR was used to detect the hypoxia group. The concentration of P I CP, P III NP, P I CP, P III concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration of pnuts mRNA in the rats' myocardial tissue and the expression of pnuts mRNA in the BMSCs and the serum of the rats in the hypoxia group and the treatment group were detected by ELISA. The correlation between the number of integral and the rate of apoptosis. [results]1, compared with the control group, the content of P I CP and P III NP in the serum and myocardial tissues of the rats of each anoxic group increased significantly (P < 0.01). The differences in the content of P I CP and P III NP in the serum and myocardial tissues were statistically significant (P < 0.01), and with the anoxia. Increased.2, compared with the control group, HE staining showed that the myocardial cells in the hypoxia group had edema, vacuolar degeneration, hypertrophy, inflammatory cell infiltration and fibrosis; Masson staining showed the increase of collagen in the intercellular space, the increase of collagen deposition, and the increase of CVF in the hypoxia group (P0.01); pathological section fluorescence Tunel. The apoptosis rate of cardiac myocytes increased significantly (P0.01). The 22 analysis and comparison between the anoxic groups showed that the myocardial pathological changes, the degree of fibrosis, CVF, the apoptosis rate of myocardial cells were significantly different (P < 0.01), and.3 was added with the increase of time, and compared with the control group, the miR-34a expression of myocardial tissue in each anoxic group was compared with the control group. Significantly increased (P0.01) and pnuts mRNA expression decreased significantly (P0.01). The 22 comparison between the anoxic groups showed that the expression of miR-34a was significantly different (P < 0.01), and increased with the increase of the hypoxia time, and the expression of pnuts mRNA was significantly different (P < 0.01), and decreased with the increase of oxygen deficiency time. The correlation analysis showed that the serum and myocardium of rats were serum and myocardium. The concentration of P I CP and P III NP was significantly positively correlated with CVF (P0.01); P I CP, P III NP concentration was significantly positively correlated with apoptosis rate of cardiomyocytes (P < 0.01), and the expression amount in stem cells was significantly lower than that of the control group. The concentration of P I CP in serum and myocardial tissue in group C', group D' and E'group was significantly lower than that of the corresponding hypoxia group (P0.01).7. The myocardial tissue of the stem cell therapy group (B' group, C'group, D' group, group) was abnormal pathological changes, and the degree of fibrosis in the myocardial tissue was better than that of the corresponding hypoxia group. The hypoxia group decreased significantly (P0.01).8. The expression of miR-34a in myocardial tissue in the stem cell therapy group (group B', C' group, D'group) decreased significantly (P0.01), the mRNA expression of pnuts increased significantly (P < 0.01), PNUTS concentration increased significantly (P0.01). Compared with the hypoxia group, the changes in the E group were not significant (P0.01).9. The correlation analysis showed that the miR-34a expression of myocardial tissue in the hypoxia group and the treatment group was negatively correlated with the pnuts mRNA expression except the E group (28 days of hypoxia), and the content of PNUTS in the hypoxia group and the treatment group and P I CP, P III content, and the apoptosis rate showed significant negative correlation. The regression analysis showed that the content of miR-34a and PNUTS in the anoxic group and the treatment group were significantly correlated (R2=0.856, R2=0.862), the regression coefficients were -0.132 (P < 0.05), -0.162 (P0.05), PNUTS content and P I CP in the hypoxia group, and the regression coefficient of P III, respectively, and the treatment group. P I CP, P III NP content significantly correlated (R2=0.992, R2=0.981) regression coefficients were -7.673 (P < 0.05), -5.478 (P0.05), PNUTS content was significantly correlated with the hypoxia group and the treatment group. The regression coefficients of 2=0.999, R2=0.997) were -0.643 (P < 0.05) and -0.113 (P0.05). [conclusion]1, chronic hypoxia ventricular remodeling model was established successfully. The most serious remodeling of ventricle occurred during hypoxia 21 days to 28 days of hypoxia; 2. During the process of chronic hypoxia ventricular remodeling, myocardial fibrosis and myocardial cell apoptosis were basically the same, and both were involved in the heart. The development of ventricular remodeling is more obvious in 21 days of hypoxia to 28 days of hypoxia; 3, BMSCs can improve ventricular remodeling in rats with chronic hypoxia, but it can not completely reverse the ventricular remodeling.4, miR-34a can inhibit the expression of pnuts in the myocardium; 5, miR-34a is low expression in rat bone marrow mesenchymal stem cells, and pnuts in rat bone marrow High expression in mesenchymal stem cells; 6, PNUTS has a significant correlation with chronic hypoxia induced ventricular remodeling; 7, BMSCs can improve ventricular remodeling induced by chronic hypoxia by miR-34a regulated PNUTS, but BMSCs does not play a role in miR-34a regulated PNUTS in chronic hypoxia model with longer hypoxia.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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