本文選題：心肌細(xì)胞 + 內(nèi)毒素　； 參考：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】：革蘭氏陰性菌感染時,細(xì)菌的細(xì)胞壁成分脂多糖(LPS)大量釋放入血,可以造成內(nèi)毒素血癥,常引起機(jī)體廣泛的失控性炎癥和免疫反應(yīng),導(dǎo)致機(jī)體最后出現(xiàn)休克、全身炎癥反應(yīng)綜合癥、多器官功能衰竭等嚴(yán)重?fù)p害。對體內(nèi)重要器官——心臟而言,LPS可以導(dǎo)致心肌收縮力減弱、心率減慢,嚴(yán)重影響心臟功能。本實(shí)驗(yàn)室前期的研究也發(fā)現(xiàn)： (1)在整體水平,LPS導(dǎo)致大鼠心肌收縮力減弱,心率減慢,嚴(yán)重降低了心臟收縮功能； (2)在組織水平,LPS導(dǎo)致心肌組織明顯破壞,肌纖維溶解斷裂,纖維間質(zhì)充血嚴(yán)重； (3)在細(xì)胞骨架方面,LPS引起心肌細(xì)胞皮質(zhì)部分纖維狀肌動蛋白(F-Actin)染色密度降低,細(xì)胞內(nèi)排列異常,形成應(yīng)力纖維；LPS刺激體外培養(yǎng)的新生鼠心肌細(xì)胞8 h,心肌細(xì)胞內(nèi)Desmin免疫熒光染色密度顯著降低,Desmin分布異常； (4)在基因表達(dá)水平,LPS可以引起心肌細(xì)胞骨架蛋白Actin和Tubulin基因表達(dá)發(fā)生顯著性變化,而Desmin基因表達(dá)變化不明顯。 以上結(jié)果提示,LPS引起心臟功能和形態(tài)結(jié)構(gòu)的嚴(yán)重?fù)p害是毋庸置疑的,但是LPS對心肌細(xì)胞骨架結(jié)構(gòu)的影響如何,目前仍所知甚少。特別是心肌細(xì)胞骨架蛋白在LPS長時間作用后形態(tài)學(xué)上的具體表現(xiàn)及其機(jī)制目前仍然不是十分清楚。 原子力顯微鏡(AFM),是一種新興的表面分析儀器,與傳統(tǒng)的電子顯微鏡相比,AFM具有非常高的橫向分辨率和縱向分辨率,并且AFM樣品制備簡單,不需要包埋、覆蓋、染色等一系列處理,在形態(tài)學(xué)研究方面,不僅可以掃描樣品表面的形貌,提供樣品空間三維圖像,以及長、寬、高、表面粗糙程度等大量信息,而且還可以滿足人們對細(xì)胞連接、細(xì)胞內(nèi)骨架等細(xì)微結(jié)構(gòu)進(jìn)行研究的要求,現(xiàn)在已經(jīng)開始在生物學(xué)領(lǐng)域發(fā)揮越來越重要的作用。 因此,本實(shí)驗(yàn)擬采用AFM掃描心肌細(xì)胞膜骨架和細(xì)胞內(nèi)骨架,以進(jìn)一步明確LPS對心肌細(xì)胞造成的損害,并對其機(jī)制進(jìn)行初步探討。實(shí)驗(yàn)分為如下三部分進(jìn)行： 第一部分,LPS對細(xì)胞膜骨架表面形貌的影響：原代培養(yǎng)新生鼠心肌細(xì)胞,LPS分別刺激1 h、4 h和8 h,然后應(yīng)用AFM對細(xì)胞進(jìn)行掃描,觀察LPS對細(xì)胞膜骨架表面形貌、細(xì)胞投影面積、表面積和體積等指標(biāo)的影響； 第二部分,LPS對心肌細(xì)胞內(nèi)骨架的影響：LPS刺激離體培養(yǎng)的新生鼠心肌細(xì)胞,低濃度非離子去垢劑Triton X-100處理細(xì)胞,去除細(xì)胞膜和可溶性蛋白后,應(yīng)用AFM掃描細(xì)胞內(nèi)骨架,并對細(xì)胞內(nèi)骨架進(jìn)行定量分析,觀察細(xì)胞內(nèi)骨架排列以及含量的變化；同時應(yīng)用免疫化學(xué)方法,對細(xì)胞內(nèi)F-Actin進(jìn)行熒光染色,以探索LPS對細(xì)胞內(nèi)骨架的影響； 第三部分,LPS引起離體心肌細(xì)胞急性肥大的機(jī)制探討：Na+-K+-ATP酶在細(xì)胞正常生理代謝及細(xì)胞內(nèi)外離子平衡中起著非常重要的作用。因此我們測定了LPS刺激后離體培養(yǎng)的新生鼠心肌細(xì)胞膜上Na+-K+-ATP酶的活性,并用Na+-K+-ATP酶抑制劑哇巴因(Ouabain)刺激心肌細(xì)胞,測定哇巴因刺激后心肌細(xì)胞面積、直徑以及細(xì)胞總蛋白含量的變化,觀察哇巴因能否引起心肌細(xì)胞肥大；同時應(yīng)用Ca2+螯合劑EGTA加以干預(yù),以探討Ca2+在LPS引起離體心肌細(xì)胞急性肥大中的作用。 結(jié)果如下： 第一部分： (1)LPS對細(xì)胞膜骨架表面形貌的影響：AFM掃描獲得細(xì)胞膜骨架表面形貌圖像,并進(jìn)行三維成像,結(jié)果顯示,細(xì)胞核和細(xì)胞質(zhì)邊界清晰,細(xì)胞膜骨架明顯,并向細(xì)胞四周延伸出數(shù)量不等,長短不一的偽足狀突起；無論是正常心肌細(xì)胞還是LPS作用后心肌細(xì)胞,細(xì)胞膜骨架表面形貌都有許多山峰狀突起和孔洞樣凹陷存在；在細(xì)胞不同部位獲得細(xì)胞膜骨架表面形貌的高度曲線,可見曲線高低不同,曲線疏密程度也不同,這表明細(xì)胞膜表面骨架排列復(fù)雜,細(xì)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；但是僅僅從掃描圖像上來看,無法看出LPS作用后,心肌細(xì)胞膜骨架表面形貌是否發(fā)生變化,就此我們對細(xì)胞膜表面的粗糙程度進(jìn)行了定量分析,結(jié)果發(fā)現(xiàn),正常對照組、LPS 1 h組、LPS 4 h組和LPS 8 h組,四個組之間細(xì)胞膜骨架表面粗糙程度無顯著性差異(F=2.020,P=0.113)； (2)LPS對單個心肌細(xì)胞投影面積、表面積和體積的影響：正常對照組、LPS 1 h組、LPS4h組和LPS 8 h組,四個組之間單個心肌細(xì)胞投影面積呈顯著性差異(F=5.683, P=0.001)。LPS 8 h組與正常對照組相比顯著性增大(P=0.004)；而LPS 1h組和LPS4h組與正常對照組相比,細(xì)胞投影面積無顯著性差異(P值分別為1.000、0.322)； 四個組之間單個心肌細(xì)胞表面積呈顯著性差異(F=5.748, P=0.001)。LPS 8h組與正常對照組相比顯著性增大(P=0.004)：而LPS 1h組和LPS 4h組與正常對照組相比,無顯著性差異(P值分別為1.000、0.336)； 四個組之間單個心肌細(xì)胞體積呈顯著性差異(F=15.961, P=0.000)。LPS 8h組與正常對照組相比,單個細(xì)胞體積顯著性增大(P=0.000)；而LPS 1h組和LPS 4h組與正常對照組相比,無顯著性差異(P值分別為1.000、0.316)； 第二部分： (1)LPS對細(xì)胞內(nèi)骨架排列的影響：正常對照組、LPS 1h組、LPS 4h組、LPS 8 h組,四個組之間細(xì)胞內(nèi)骨架表面纖維密度指數(shù)(SFDI)呈顯著性差異(F=39.735, P=0.000)。LPS1 h組、LPS 4h組和LPS 8h組,細(xì)胞內(nèi)骨架SFDI與正常對照組相比均顯著性增大(P值均為0.000)。這表明隨著LPS刺激時間的延長,細(xì)胞內(nèi)骨架排列越來越疏松； (2)LPS對單位面積上細(xì)胞骨架體積(Volume/area)的影響：正常對照組、LPS 1h組、LPS 4h組、LPS 8h組,四個組之間細(xì)胞內(nèi)骨架Volume/area呈顯著性差異(F=26.658,P=0.000)。LPS 8 h組與正常對照組相比,細(xì)胞內(nèi)骨架Volume/area顯著性增大(P=0.000)；而LPS 1 h組和LPS 4h組與正常對照組相比,細(xì)胞內(nèi)骨架Volume/area均無顯著性差異(P值分別為0.870、0.081)。這表明LPS作用較長時間才能引起細(xì)胞內(nèi)骨架含量的增多； (3)LPS對細(xì)胞內(nèi)骨架單根纖維寬度的影響：正常對照組、LPS 1 h組、LPS 4h組、LPS 8h組,四個組之間細(xì)胞內(nèi)骨架單根纖維的寬度呈顯著性差異(F=16.128,P=0.000)。LPS作用后纖維寬度逐漸增大,LPS 1 h組、LPS4h組、LPS 8 h組單根纖維寬度與正常對照組相比均顯著性增大(P值分別為0.033、0.000、0.000)。這表明隨著LPS作用時間的延長,細(xì)胞內(nèi)骨架單根纖維的寬度逐漸增大； (4)LPS對F-Actin分布的影響：LPS作用后,F-Actin排列變得疏松,LPS作用8h后應(yīng)力纖維形成明顯； (5)LPS對F-Actin熒光強(qiáng)度的影響：正常對照組、LPS 1h組、LPS4h組和LPS8h組,四個組之間細(xì)胞內(nèi)F-Actin熒光強(qiáng)度呈顯著性差異(F=7.216,P=0.000)。LPS8h組與正常對照組相比,細(xì)胞內(nèi)F-Actin熒光強(qiáng)度顯著性增大(P=0.001);而LPS 1h組和LPS 4h組與正常對照組相比,F-Actin熒光強(qiáng)度均無顯著性差異(P值分別為1.000、0.279)。 第三部分： (1)LPS對心肌細(xì)胞總蛋白含量的影響：正常對照組、LPS 1 h組、LPS 4h組、LPS8h組,四個組之間心肌細(xì)胞總蛋白含量呈顯著性差異(F=530.828,P=0.000)。LPS 8 h組與正常對照組相比,細(xì)胞總蛋白含量顯著性增大(P=0.000); (2)LPS對Na+-K+-ATP酶活性的影響：正常對照組、LPS1h組、LPS4h組、LPS 8 h組,四個組之間心肌細(xì)胞膜Na+-K+-ATP酶活性呈顯著性差異(F=79.710,P=0.000)。LPS作用心肌細(xì)胞4 h和8 h后,細(xì)胞膜Na+-K+-ATP酶活性與正常對照組相比均顯著性下降(P值均為0.000)； (3)哇巴因?qū)蝹�細(xì)胞面積、直徑和總蛋白含量的影響：正常對照組、LPS組、哇巴因組,三個組之間心肌細(xì)胞面積呈顯著性差異(F=68.098,P=0.000)。LPS組和哇巴因組與正常對照組相比,細(xì)胞面積均顯著性增大(P值均為0.000)；并且哇巴因組與LPS組相比,也呈顯著性差異(P=0.014)； 三個組之間心肌細(xì)胞直徑呈顯著性差異(F=113.702,P=0.000)。LPS組和哇巴因組與正常對照組相比,細(xì)胞直徑均顯著性增大(P值均為0.000)；并且哇巴因組與LPS組相比,也呈呈著性差異(P=0.000)； 三個組之間心肌細(xì)胞總蛋白含量呈顯著性差異(F=273.387,P=0.000)。LPS組和哇巴因組與正常對照組相比,細(xì)胞總蛋白含量均顯著性增大(P值均為0.000)；并且哇巴因組與LPS組相比,總蛋白含量也呈顯著性差異(P=0.004)； (4)EGTA對LPS所致心肌細(xì)胞肥大的影響：正常對照組、EGTA組、LPS組、LPS+EGTA組,四個組之間細(xì)胞面積呈顯著性差異(F=10.340,P=0.000)。EGTA組與正常對照組相比,細(xì)胞面積無顯著性差異(P=1.000)；而LPS組與正常對照組相比,細(xì)胞面積顯著性增大(P=0.000)；并且LPS+EGTA組與LPS組相比,細(xì)胞面積的減小也呈顯著性差異(P=0.002)； 四個組之間細(xì)胞直徑呈顯著性差異(F=28.507,P=0.000)。EGTA組與正常對照組相比,細(xì)胞直徑無顯著性差異(P=1.000)；而LPS組與正常對照組相比細(xì)胞直徑顯著性增大(P=0.000)；并且LPS+EGTA組與LPS組相比,細(xì)胞直徑的減小也呈顯著性差異(P=0.000)； 四個組之間細(xì)胞總蛋白含量呈顯著性差異(F=34.449,P=0.000)。LPS組與正常對照組和EGTA+LPS組分別相比,細(xì)胞總蛋白含量都呈顯著性增多(P值分別為0.000、0.001)；而且LPS+EGTA組與正常對照組相比,細(xì)胞總蛋白含量的增多也呈顯著性差異(P=0.032)。 根據(jù)上述實(shí)驗(yàn)結(jié)果,本研究可得出以下初步結(jié)論： (1)LPS作用心肌細(xì)胞后,在較短時間內(nèi)不會引起細(xì)胞膜骨架的明顯變化; (2)LPS作用心肌細(xì)胞后,在較短時間內(nèi)(1 h)會引起細(xì)胞內(nèi)骨架排列變化,隨著刺激時間的延長,細(xì)胞內(nèi)骨架排列趨向疏松；LPS刺激較長時間(8h)會引起細(xì)胞內(nèi)骨架含量的變化,細(xì)胞內(nèi)骨架含量增多； (3)LPS作用于離體心肌細(xì)胞8 h后可以引起心肌細(xì)胞肥大； (4)心肌細(xì)胞膜上Na+-K+-ATP酶活性降低以及Ca2+內(nèi)流增多引起胞漿內(nèi)Ca2+濃度升高可能是LPS導(dǎo)致離體心肌細(xì)胞急性肥大的兩個重要機(jī)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

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