应用AFM探讨LPS对离体心肌细胞骨架形貌结构的影响

发布时间：2018-06-10 19:00

本文选题：心肌细胞 + 内毒素　；参考：《南方医科大学》2010年硕士论文

【摘要】：革兰氏阴性菌感染时,细菌的细胞壁成分脂多糖(LPS)大量释放入血,可以造成内毒素血症,常引起机体广泛的失控性炎症和免疫反应,导致机体最后出现休克、全身炎症反应综合症、多器官功能衰竭等严重损害。对体内重要器官——心脏而言,LPS可以导致心肌收缩力减弱、心率减慢,严重影响心脏功能。本实验室前期的研究也发现： (1)在整体水平,LPS导致大鼠心肌收缩力减弱,心率减慢,严重降低了心脏收缩功能； (2)在组织水平,LPS导致心肌组织明显破坏,肌纤维溶解断裂,纤维间质充血严重； (3)在细胞骨架方面,LPS引起心肌细胞皮质部分纤维状肌动蛋白(F-Actin)染色密度降低,细胞内排列异常,形成应力纤维；LPS刺激体外培养的新生鼠心肌细胞8 h,心肌细胞内Desmin免疫荧光染色密度显著降低,Desmin分布异常； (4)在基因表达水平,LPS可以引起心肌细胞骨架蛋白Actin和Tubulin基因表达发生显著性变化,而Desmin基因表达变化不明显。以上结果提示,LPS引起心脏功能和形态结构的严重损害是毋庸置疑的,但是LPS对心肌细胞骨架结构的影响如何,目前仍所知甚少。特别是心肌细胞骨架蛋白在LPS长时间作用后形态学上的具体表现及其机制目前仍然不是十分清楚。原子力显微镜(AFM),是一种新兴的表面分析仪器,与传统的电子显微镜相比,AFM具有非常高的横向分辨率和纵向分辨率,并且AFM样品制备简单,不需要包埋、覆盖、染色等一系列处理,在形态学研究方面,不仅可以扫描样品表面的形貌,提供样品空间三维图像,以及长、宽、高、表面粗糙程度等大量信息,而且还可以满足人们对细胞连接、细胞内骨架等细微结构进行研究的要求,现在已经开始在生物学领域发挥越来越重要的作用。因此,本实验拟采用AFM扫描心肌细胞膜骨架和细胞内骨架,以进一步明确LPS对心肌细胞造成的损害,并对其机制进行初步探讨。实验分为如下三部分进行：第一部分,LPS对细胞膜骨架表面形貌的影响：原代培养新生鼠心肌细胞,LPS分别刺激1 h、4 h和8 h,然后应用AFM对细胞进行扫描,观察LPS对细胞膜骨架表面形貌、细胞投影面积、表面积和体积等指标的影响；第二部分,LPS对心肌细胞内骨架的影响：LPS刺激离体培养的新生鼠心肌细胞,低浓度非离子去垢剂Triton X-100处理细胞,去除细胞膜和可溶性蛋白后,应用AFM扫描细胞内骨架,并对细胞内骨架进行定量分析,观察细胞内骨架排列以及含量的变化；同时应用免疫化学方法,对细胞内F-Actin进行荧光染色,以探索LPS对细胞内骨架的影响；第三部分,LPS引起离体心肌细胞急性肥大的机制探讨：Na+-K+-ATP酶在细胞正常生理代谢及细胞内外离子平衡中起着非常重要的作用。因此我们测定了LPS刺激后离体培养的新生鼠心肌细胞膜上Na+-K+-ATP酶的活性,并用Na+-K+-ATP酶抑制剂哇巴因(Ouabain)刺激心肌细胞,测定哇巴因刺激后心肌细胞面积、直径以及细胞总蛋白含量的变化,观察哇巴因能否引起心肌细胞肥大；同时应用Ca2+螯合剂EGTA加以干预,以探讨Ca2+在LPS引起离体心肌细胞急性肥大中的作用。结果如下：第一部分： (1)LPS对细胞膜骨架表面形貌的影响：AFM扫描获得细胞膜骨架表面形貌图像,并进行三维成像,结果显示,细胞核和细胞质边界清晰,细胞膜骨架明显,并向细胞四周延伸出数量不等,长短不一的伪足状突起；无论是正常心肌细胞还是LPS作用后心肌细胞,细胞膜骨架表面形貌都有许多山峰状突起和孔洞样凹陷存在；在细胞不同部位获得细胞膜骨架表面形貌的高度曲线,可见曲线高低不同,曲线疏密程度也不同,这表明细胞膜表面骨架排列复杂,细胞膜骨架表面凹凸不平,高低不一并且骨架排列疏密程度也不均一；但是仅仅从扫描图像上来看,无法看出LPS作用后,心肌细胞膜骨架表面形貌是否发生变化,就此我们对细胞膜表面的粗糙程度进行了定量分析,结果发现,正常对照组、LPS 1 h组、LPS 4 h组和LPS 8 h组,四个组之间细胞膜骨架表面粗糙程度无显著性差异(F=2.020,P=0.113)； (2)LPS对单个心肌细胞投影面积、表面积和体积的影响：正常对照组、LPS 1 h组、LPS4h组和LPS 8 h组,四个组之间单个心肌细胞投影面积呈显著性差异(F=5.683, P=0.001)。LPS 8 h组与正常对照组相比显著性增大(P=0.004)；而LPS 1h组和LPS4h组与正常对照组相比,细胞投影面积无显著性差异(P值分别为1.000、0.322)；四个组之间单个心肌细胞表面积呈显著性差异(F=5.748, P=0.001)。LPS 8h组与正常对照组相比显著性增大(P=0.004)：而LPS 1h组和LPS 4h组与正常对照组相比,无显著性差异(P值分别为1.000、0.336)；四个组之间单个心肌细胞体积呈显著性差异(F=15.961, P=0.000)。LPS 8h组与正常对照组相比,单个细胞体积显著性增大(P=0.000)；而LPS 1h组和LPS 4h组与正常对照组相比,无显著性差异(P值分别为1.000、0.316)；第二部分： (1)LPS对细胞内骨架排列的影响：正常对照组、LPS 1h组、LPS 4h组、LPS 8 h组,四个组之间细胞内骨架表面纤维密度指数(SFDI)呈显著性差异(F=39.735, P=0.000)。LPS1 h组、LPS 4h组和LPS 8h组,细胞内骨架SFDI与正常对照组相比均显著性增大(P值均为0.000)。这表明随着LPS刺激时间的延长,细胞内骨架排列越来越疏松； (2)LPS对单位面积上细胞骨架体积(Volume/area)的影响：正常对照组、LPS 1h组、LPS 4h组、LPS 8h组,四个组之间细胞内骨架Volume/area呈显著性差异(F=26.658,P=0.000)。LPS 8 h组与正常对照组相比,细胞内骨架Volume/area显著性增大(P=0.000)；而LPS 1 h组和LPS 4h组与正常对照组相比,细胞内骨架Volume/area均无显著性差异(P值分别为0.870、0.081)。这表明LPS作用较长时间才能引起细胞内骨架含量的增多； (3)LPS对细胞内骨架单根纤维宽度的影响：正常对照组、LPS 1 h组、LPS 4h组、LPS 8h组,四个组之间细胞内骨架单根纤维的宽度呈显著性差异(F=16.128,P=0.000)。LPS作用后纤维宽度逐渐增大,LPS 1 h组、LPS4h组、LPS 8 h组单根纤维宽度与正常对照组相比均显著性增大(P值分别为0.033、0.000、0.000)。这表明随着LPS作用时间的延长,细胞内骨架单根纤维的宽度逐渐增大； (4)LPS对F-Actin分布的影响：LPS作用后,F-Actin排列变得疏松,LPS作用8h后应力纤维形成明显； (5)LPS对F-Actin荧光强度的影响：正常对照组、LPS 1h组、LPS4h组和LPS8h组,四个组之间细胞内F-Actin荧光强度呈显著性差异(F=7.216,P=0.000)。LPS8h组与正常对照组相比,细胞内F-Actin荧光强度显著性增大(P=0.001);而LPS 1h组和LPS 4h组与正常对照组相比,F-Actin荧光强度均无显著性差异(P值分别为1.000、0.279)。第三部分： (1)LPS对心肌细胞总蛋白含量的影响：正常对照组、LPS 1 h组、LPS 4h组、LPS8h组,四个组之间心肌细胞总蛋白含量呈显著性差异(F=530.828,P=0.000)。LPS 8 h组与正常对照组相比,细胞总蛋白含量显著性增大(P=0.000); (2)LPS对Na+-K+-ATP酶活性的影响：正常对照组、LPS1h组、LPS4h组、LPS 8 h组,四个组之间心肌细胞膜Na+-K+-ATP酶活性呈显著性差异(F=79.710,P=0.000)。LPS作用心肌细胞4 h和8 h后,细胞膜Na+-K+-ATP酶活性与正常对照组相比均显著性下降(P值均为0.000)； (3)哇巴因对单个细胞面积、直径和总蛋白含量的影响：正常对照组、LPS组、哇巴因组,三个组之间心肌细胞面积呈显著性差异(F=68.098,P=0.000)。LPS组和哇巴因组与正常对照组相比,细胞面积均显著性增大(P值均为0.000)；并且哇巴因组与LPS组相比,也呈显著性差异(P=0.014)；三个组之间心肌细胞直径呈显著性差异(F=113.702,P=0.000)。LPS组和哇巴因组与正常对照组相比,细胞直径均显著性增大(P值均为0.000)；并且哇巴因组与LPS组相比,也呈呈著性差异(P=0.000)；三个组之间心肌细胞总蛋白含量呈显著性差异(F=273.387,P=0.000)。LPS组和哇巴因组与正常对照组相比,细胞总蛋白含量均显著性增大(P值均为0.000)；并且哇巴因组与LPS组相比,总蛋白含量也呈显著性差异(P=0.004)； (4)EGTA对LPS所致心肌细胞肥大的影响：正常对照组、EGTA组、LPS组、LPS+EGTA组,四个组之间细胞面积呈显著性差异(F=10.340,P=0.000)。EGTA组与正常对照组相比,细胞面积无显著性差异(P=1.000)；而LPS组与正常对照组相比,细胞面积显著性增大(P=0.000)；并且LPS+EGTA组与LPS组相比,细胞面积的减小也呈显著性差异(P=0.002)；四个组之间细胞直径呈显著性差异(F=28.507,P=0.000)。EGTA组与正常对照组相比,细胞直径无显著性差异(P=1.000)；而LPS组与正常对照组相比细胞直径显著性增大(P=0.000)；并且LPS+EGTA组与LPS组相比,细胞直径的减小也呈显著性差异(P=0.000)；四个组之间细胞总蛋白含量呈显著性差异(F=34.449,P=0.000)。LPS组与正常对照组和EGTA+LPS组分别相比,细胞总蛋白含量都呈显著性增多(P值分别为0.000、0.001)；而且LPS+EGTA组与正常对照组相比,细胞总蛋白含量的增多也呈显著性差异(P=0.032)。根据上述实验结果,本研究可得出以下初步结论： (1)LPS作用心肌细胞后,在较短时间内不会引起细胞膜骨架的明显变化; (2)LPS作用心肌细胞后,在较短时间内(1 h)会引起细胞内骨架排列变化,随着刺激时间的延长,细胞内骨架排列趋向疏松；LPS刺激较长时间(8h)会引起细胞内骨架含量的变化,细胞内骨架含量增多； (3)LPS作用于离体心肌细胞8 h后可以引起心肌细胞肥大； (4)心肌细胞膜上Na+-K+-ATP酶活性降低以及Ca2+内流增多引起胞浆内Ca2+浓度升高可能是LPS导致离体心肌细胞急性肥大的两个重要机制。
[Abstract]:When Gram-negative bacteria are infected, the cell wall component of the bacteria (LPS) is released into the blood, which can cause endotoxemia, often causing extensive uncontrolled inflammation and immune response in the body, which leads to the body's final shock, systemic inflammatory response syndrome, multiple organ failure and other serious damage. LPS can cause cardiac contractility to weaken, heart rate to slow down, and severe cardiac function.
(1) at the overall level, LPS induced myocardial contractility decreased, heart rate slowed down, and cardiac systolic function was seriously reduced.
(2) at the tissue level, LPS leads to obvious destruction of myocardial tissue, disintegration of muscle fibers and serious congestion of fibrous interstitium.
(3) in the cytoskeleton, LPS caused a decrease in the density of fibrous actin (F-Actin) in the cortex of cardiac myocytes, the abnormal arrangement in the cells and the formation of stress fibers. LPS stimulated 8 h of neonatal rat cardiomyocytes in vitro, and the density of Desmin immunofluorescence staining in myocardial cells decreased significantly, and the distribution of Desmin was abnormal.
(4) at the level of gene expression, LPS can induce significant changes in the expression of cardiac cytoskeletal proteins Actin and Tubulin, while Desmin gene expression is not changed significantly.
These results suggest that LPS causes serious impairment of cardiac function and morphologic structure, but the effect of LPS on the cytoskeleton structure of cardiac myocytes is still poorly understood. Especially, the specific morphological manifestation and mechanism of cardiac cytoskeleton protein after the long time effect of LPS is still not very clear.
Atomic force microscope (AFM) is a new type of surface analysis instrument. Compared with the traditional electron microscope, AFM has very high transverse resolution and vertical resolution, and the preparation of AFM sample is simple. It does not need a series of processing, such as embedding, covering, dyeing and so on. In the aspect of morphologic study, it can not only scan the surface of the sample, but also provide the appearance of the sample surface. The three-dimensional image of the sample space, as well as a large amount of information such as long, wide, high, and surface roughness, can also meet the requirements of the study of cell connections, the inner skeleton and other subtle structures, and now it has begun to play an increasingly important role in the field of Biology.
Therefore, this experiment is to use AFM to scan the cytoskeleton and the cytoskeleton of the cardiac myocytes to further clarify the damage caused by LPS to the cardiac myocytes, and to make a preliminary discussion on its mechanism. The experiment is divided into three parts:
The first part, the effect of LPS on the surface morphology of cell membrane skeleton: the primary culture of neonatal rat cardiomyocytes, LPS stimulation 1 h, 4 h and 8 h respectively, and then using AFM to scan the cells, observe the influence of LPS on the surface morphology of the membrane skeleton, the area of the cell projection, surface area and volume.
The second part, the effect of LPS on the cytoskeleton of myocardial cells: LPS stimulates the neonatal rat cardiomyocytes in vitro, and the low concentration nonionic detergent Triton X-100 is used to treat the cells, remove the cell membrane and soluble protein, and use AFM to scan the cytoskeleton, and analyze the cytoskeleton in the cell, and observe the arrangement and content of the cytoskeleton. Meanwhile, the F-Actin of cells was stained by immunofluorescence to explore the effect of LPS on the cytoskeleton.
The third part, the mechanism of acute hypertrophy induced by LPS in isolated cardiomyocytes: Na+-K+-ATP enzyme plays a very important role in the normal physiological metabolism of cells and the balance of intracellular and extracellular ion. Therefore, we measured the activity of Na+-K+-ATP enzyme on the membrane of neonatal rat cardiomyocytes cultured in vitro after LPS stimulation, and wow with the Na+-K+-ATP enzyme inhibitor. Ouabain stimulates cardiac myocytes to determine the changes in the area, diameter and total protein content of cardiac myocytes after ouabain stimulation, to observe whether ouabain can cause hypertrophy of cardiac myocytes, and the use of Ca2+ chelating agent EGTA to investigate the role of Ca2+ in the acute hypertrophy of isolated cardiomyocytes induced by LPS.
The results are as follows:
Part one:
(1) the effect of LPS on the surface morphology of the cytoskeleton: AFM scan obtained the image of the surface morphology of the cytoskeleton and carried out the three-dimensional imaging. The results showed that the nucleus and cytoplasm boundary were clear and the cytoskeleton was obvious, and the number of cells around the cells was different, and the normal myocytes and LPS were different. After the action, there are many mountain peaks and hole like sags in the surface morphology of the cell membrane skeleton, and the height curves of the surface morphology of the membrane skeleton in different parts of the cells are different and the degree of the density of the curve is different, which indicates that the skeleton of the membrane surface is complex and the surface of the cell membrane is concave and convex. The level of the skeleton arrangement was uneven. But only from the scanned image, it was impossible to see whether the surface morphology of the myocardial cell membrane changes after the LPS effect, and the roughness of the surface of the cell membrane was quantitatively analyzed. The results were found in the normal control group, the LPS 1 h group, the LPS 4 h group and the LPS. In 8 h group, there was no significant difference in the roughness of cell membrane skeleton between the four groups (F=2.020, P=0.113).
(2) the effect of LPS on the projection area, surface area and volume of single cardiac myocytes: the normal control group, the LPS 1 h group, the LPS4h group and the LPS 8 h group, and the significant difference in the projection area of the single cardiac myocytes between the four groups (F=5.683, P=0.001).LPS 8 h group was significantly increased (P=0.004) compared with the normal control group (P=0.004). Compared with the group, there was no significant difference in cell projection area (P = 1.000,0.322).
There was a significant difference in the surface area of single cardiac myocytes between the four groups (F=5.748, P=0.001).LPS 8h group was significantly increased compared with the normal control group (P=0.004), but there was no significant difference between the LPS 1H group and the LPS 4H group compared with the normal control group (P value was 1.000,0.336).
The volume of single cardiac myocytes was significantly different between the four groups (F=15.961, P=0.000).LPS 8h group, compared with the normal control group, the volume of single cell increased significantly (P=0.000), but there was no significant difference between the LPS 1H group and the LPS 4H group compared with the normal control group (P value was 1.000,0.316).
The second part:
(1) the effect of LPS on the cytoskeleton arrangement in the normal control group, the LPS 1H group, the LPS 4H group and the LPS 8 h group, the intracellular fibrous density index (SFDI) of the cytoskeleton between the four groups was significantly different (F=39.735, P=0.000).LPS1 H group, the intracellular skeleton and the normal control group were significantly increased (0). This indicates that with the prolongation of LPS stimulation time, the cytoskeletal arrangement of cells becomes more and more loose.
(2) the effect of LPS on cytoskeleton volume (Volume/area) per unit area: normal control group, LPS 1H group, LPS 4H group, LPS 8h group, and the intracellular cytoskeleton Volume/area (F=26.658, P=0.000) between the four groups (F=26.658, P=0.000). There was no significant difference in the cytoskeleton Volume/area between the 4H group and the normal control group (P value was 0.870,0.081, respectively). This showed that the longer time of LPS could cause the increase of the content of the cytoskeleton.
(3) the effect of LPS on the single fiber width of the cytoskeleton: normal control group, LPS 1 h group, LPS 4H group and LPS 8h group, the width of the single fiber in the cytoskeleton between the four groups was significantly different (F=16.128, P=0.000).LPS effect, the fiber width gradually increased, LPS 1 h group, 8 groups of single fiber width compared with the normal control group. All of them increased significantly (P = 0.033,0.000,0.000). This indicates that with the prolongation of LPS action time, the width of single fibrous skeleton in cells increases gradually.
(4) the effect of LPS on F-Actin distribution: after LPS, the F-Actin arrangement becomes loose, and the stress fibers become obvious after LPS acts on 8h.
(5) the effect of LPS on the fluorescence intensity of F-Actin: in the normal control group, the LPS 1H group, the LPS4h group and the LPS8h group, the intracellular F-Actin fluorescence intensity was significantly different between the four groups (F=7.216, P=0.000).LPS8h group, compared with the normal control group, the intracellular F-Actin fluorescence intensity was significantly increased (P=0.001), and the group and the normal control group were compared with the normal control group. There was no significant difference in F-Actin fluorescence intensity (P = 1.000,0.279).
The third part:
(1) the effect of LPS on the total protein content of cardiac myocytes: the total protein content of cardiac myocytes in the normal control group, the LPS 1 h group, the LPS 4H group and the LPS8h group showed significant difference (F=530.828, P=0.000).LPS 8 h group, compared with the normal control group, the total protein content of the cells increased significantly (P=0.000);
(2) the effect of LPS on the activity of Na+-K+-ATP enzyme: normal control group, LPS1h group, LPS4h group and LPS 8 h group, the activity of Na+-K+-ATP enzyme in myocardial cell membrane was significantly different between the four groups (F=79.710, P=0.000).LPS action of 4 h and 8 h, the activity of the cell membrane was significantly decreased compared with the normal control group (0);
(3) effect of ouabain on the area, diameter and total protein content of single cell: normal control group, LPS group and ouabain group, the area of cardiac myocytes in three groups was significantly different (F=68.098, P=0.000), and the cell area was significantly increased in group.LPS and ouabain group compared with normal control group (P value was 0); and ouabain group and LPS group There were also significant differences (P=0.014).
The diameter of the three groups was significantly different (F=113.702, P=0.000).LPS and ouabain group, compared with the normal control group, the cell diameter was significantly increased (P value was 0), and ouabain group compared with the LPS group, also showed a significant difference (P=0.000).
The total protein content of the three groups was significantly different (F=273.387, P=0.000).LPS and ouabain group compared with the normal control group, the total protein content was significantly increased (P value was 0), and ouabain group compared with the LPS group, the total protein content was also significant difference (P=0.004).
(4) the effect of EGTA on LPS induced cardiomyocyte hypertrophy: the cell area between the normal control group, the EGTA group, the LPS group, the LPS+EGTA group and the four groups was significantly different (F=10.340, P=0.000), and there was no significant difference in cell area (P=1.000) compared with the normal control group (P=1.000), while the cell area was significantly increased in the LPS group compared with the normal control group (P=0.000). There was also a significant difference in cell area between group LPS+EGTA and group LPS (P=0.002).
There was significant difference in cell diameter between the four groups (F=28.507, P=0.000), and there was no significant difference in cell diameter between the group.EGTA and the normal control group (P=1.000), while the cell diameter of the LPS group was significantly increased (P=0.000) compared with the normal control group (P=0.000), and the decrease of cell diameter in the LPS+EGTA group and LPS group was also significantly different (P=0.000).
The total protein content of the four groups was significantly different (F=34.449, P=0.000), and the total protein content in the.LPS group increased significantly compared with the normal control group and the EGTA+LPS group (P value was 0.000,0.001, respectively), and the increase of total protein content in the LPS+EGTA group was also significantly different from that in the normal control group (P=0.032).
Based on the above experimental results, the following conclusions can be drawn:
(1) LPS did not cause obvious changes in cell membrane skeleton in a short time after the action of cardiac myocytes.
(2) after the action of LPS on cardiac myocytes, the cytoskeleton arrangement changes in a short time (1 h), and the cytoskeleton arrangement tends to loose with the time of stimulation; LPS stimulation for a long time (8h) will cause the changes in the content of the cytoskeleton, and the content of the cytoskeleton increases.
(3) LPS can induce hypertrophy of cardiac myocytes after 8 h of isolated cardiomyocytes.
(4) the decrease of Na+-K+-ATP enzyme activity on the membrane of cardiac myocytes and the increase of intracellular Ca2+ in the cytoplasm by the increase of Ca2+ influx may be the two important mechanisms for the acute hypertrophy of isolated cardiomyocytes in vitro.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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