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Stress Damages and Expressions of Hsp60and Hsf-1in the Heat Stressed Myocardial Cells of Neonatal Rats in Vitro

作　者: Rehana Buriro
导　师: 鲍恩东
学　校: 南京农业大学
专　业: 基础兽医
关键词: Heat stress Rat Hsp60 Hsf-1 mRNA Caspase Cytochrome-c
分类号: S852.3
类　型: 博士论文
年　份: 2012年
下　载: 1次
引　用: 0次
阅　读: 论文下载

内容摘要

Heat stress causes harmful changes in the cells, and prolonged duration leads to loss of function in cells, either by necrosis or by apoptosis. Heat shock proteins (Hsps) play an important role to maintain the homeostasis of the cell and help it to survive even during stressful period. Hsp60is one the main protein of Heat shock proteins’s family and it is mainly called as mitochondrial protein. The main purpose of this study was to investigate the expression and transcription of Hsp60and HSF-1and their corresponding mRNA in primary myocardial cells of neonatal rats in vitro, to correlate the relationship of Hsp60and HSF-1at various durations of heat stress, to observe the cytoprotective mechanism of Hsp60in response to heat stress.For this purpose the primary myocardial cells were cultured and divided into9groups including control group which is kept at37℃at5%CO2and95%humidity. Rest of groups was heat stressed for various durations from10,20,40,60,120,240,360and480min respectively at42℃at5%CO2and95%humidity.The heat stress model of primary myocardial cells of neonatal rats was developed through detection of myocardial enzyme elevation including AST, LDH, CK, CK-MB, as these are considered as myocardial markers and by observing the cytopathological lesions in control as well as heat stressed myocardial cells. The enzymes showed varying degrees of elevations. Level of AST was found to be significantly high throughout the stress period as compared to control group, whereas LDH displayed the significant elevation at20and60min of heat stress. CK was found significantly higher at120min and CK-MB activity was observed60and120min of heat stress. The cytopathological lesions were observed at the beginning of heat stress i.e10min with granular and vacuolar degeneration and were found persistent throughout the heat stress period, with obvious pyknotic nucleus after120min of heat stress treatment. These findings revealed that myocardial cells of neonatal rats are damaged by heat stress.The western blot results showed that Hsp60expression was decreased significantly after20and40min and then increased after120min of heat stress and was found stable till240min of heat stress. Meanwhile the immunocytochemical results showed the most prominent signals with thick Hsp60density after240min of heat stress at42℃as compared to that of60min. This change in Hsp60density indicated that due to consumption of Hsp60, the cells started to produce sufficient Hsp60after240min of heat stress. Whereas, the cytopathological lesions showed the vacuolar degeneration after60 min of heat stress and pyknosis occurred at240min of heat stress. These findings suggest that myocardial cells were protected to certain extent from heat stress until and unless Hsp60was produced to help myocardial cellsThe in vitro relationship between Hsf-1and Hsp60and their corresponding mRNA was observed to find whether Hsf-1production influence Hsp60formation or not. Hsp60expression showed fluctuations during heat stress and observed significantly low after20min of heat stress and then increased at120min and remained elevated up to240min of heat stress. After that point, Hsp60expression was reduced as compared to control group. However, hsp60mRNA transcription levels increased significantly after20min and followed the same pattern up to240min of heat stress and then decreased significantly after360min of heat stress. The levels of Hsf-1expression were declined till120min of heat stress but increased significantly after that point and remained elevated till480min of heat stress, whereas Hsf-1mRNA levels increased significantly after10min and sustained same pattern up to the end of heat treatment that is480min. These results indicate that Hsf-1is not the only factor involved in regulating the hsp60mRNA transcription and is involved in the formation of other Hsps too.It was also observed that Hsp60production could not prevent the apoptosis, if the heat stress duration is too long because the levels of Hsp60expression decreased after20min and then increased at120min and then decreased at360min. But the expression of Caspase-3and Cytochrome-c increased significantly after40min of heat stress, suggesting that myocardial cells were unable to cope with the persistent heat stress and going to apoptosis. The changes in levels of Cytochrome-c are similar to that of cleaved Caspase-3, suggesting the two proteins are closely related during heat stress. However, Caspase8and9found relatively stable during the stress period.

全文目录

ABBREVIATIONS  9-10
ABSTRACT  10-12
CHAPTER 1 REVIEW  12-34
  1 STRESS  12-14
  2 HEAT SHOCK PROTEINS  14-17
    2.1. Highly conserved  17
    2.2. Wide spread availabilty in all organisms  17
    2.3. Non specificity in nature  17
    2.4. Other Features of HSPs  17
  3 REGULATION OF HSPs  17-19
  4 STRESS DAMAGES AND HSPs PROTECTION IN MYOCARDIAL CELLS  19-21
    4.1 Stress damage cause mortality in vivo  19
    4.2 Myocardial cell damage and enzyme elevations  19
    4.3 Regulation of Apoptosis  19-20
    4.4 Hsps and cell protection  20-21
  5 HSP60  21-24
  REFERENCES  24-34
CHAPTER 2 Stress damages of myocardial cells ofneonatal rats in vitro after heat stress  34-42
  Abstract  34
  1 Introduction  34-35
  2 Materials and Methods  35-37
    2.1 Cell culture and heat treatment  35
    2.2 Tests of the activities of LDH,AST,CK,and CK-MB  35
    2.3 Treatment of coverslips with polylysine solution  35-36
    2.4 Growth of cells on coverslip  36
    2.5 Fixation of cells  36
    2.6 Hematoxylin-Eosin (H&E)staining  36-37
    2.7 Statistical analysis  37
  3 Results  37-39
    3.1 The enzyme levels AST,LDH,CK and CK-MB in the cultured cellular supernatant  37-39
    3.2 Pathological Lesions of the heat stressed myocardial cells  39
  4 Discussion  39-42
CHAPTER 3 Localization and expression of Hsp60 in the heat stressed primarymyocardial cells of neonatal rats in vitro  42-52
  Abstract  42
  1 Introduction  42-44
  2 Materials and methods  44-47
    2.1 Cell culture and heat stress treatment  44
    2.2 Immunocytochemistry  44-45
    2.3 Western blotting  45
    2.4 Treatment of coverslips with polylysine solution  45-46
    2.5 Growth of cells on coverslip  46
    2.6 Fixation of cells  46
    2.7 Hematoxylin-Eosin (H&E) staining  46-47
    2.8 Statistical analysis  47
  3 Results  47-49
    3.1 The localization and distribution of Hsp60 in the myocardial cells in vitro  47
    3.2 Quantitative detection of Hsp60 expression in the heat-stressed myocardial cells ofneonatal rats in vitro  47
    3.3 Cytopathogical lesions in the heat stressed myocardial cells of rats in vitro  47-49
  4 Discussion  49-52
CHAPTER 4 Influences of Hsf-1 and its corresponding mRNA on the expression ofHsp60 and its corresponding mRNA in heat stressed myocardial cells of rats in vitro  52-62
  Abstract  52
  1 Introduction  52-54
  2 Materials and methods  54-56
    2.1 Cell culture and experimental treatment  54
    2.2 Western blotting  54
    2.3 Detection of hsp60 mRNA by lfuorescence quantitative real-time polymerase chainreaction (FQ RT-PCR)  54-56
    2.4 Statistical analysis  56
  3 Results  56-59
    3.1 Quantitative detection of Hsp60 expression in the heat-stressed myocardial cells of neonatal rats in vitro  56
    3.2 Quantitative detection of Hsf-1 expression in the heat-stressed myocardial cells of neonatal rats in vitro  56-57
    3.3 The transcription levels of hsp60 mRNA in the heat-stressed myocardial cells of neonatal rats in vitro  57
    3.4 The transcription levels of hsf-1 mRNA in the heat-stressed myocardial cells of neonatal rats in vitro  57-59
  4 Discussion  59-62
CHAPTER 5 Role of Hsp60 in preventing myocardial cells of neonatal rats againstapoptosis during heat stress  62-70
  1 Introduction  62-64
  2 Materials and Methods  64-65
    2.1 Cell culture and experimental treatment  64
    2.2 Western blotting  64
    2.3 Determination of Cytochrome-C,Caspase-3,Caspase-8 and Caspase-9  64-65
    2.4 Statistical analysis  65
  3 Results  65-68
    3.1 Semi-quantitative detection of Hsp60 expression in the heat-stressed myocardial cells of neonatal rats in vitro  65
    3.2 The expression of Cleaved Caspase-3 in the cultured myocardial cells in vitro  65-66
    3.3 The levels of Cytochrome-c in the cultured myocardial cells in vitro  66
    3.4 The levels of cleaved Caspase-8 in the cultured myocardial cells in vitro  66
    3.5 The levels of cleaved Caspase-9 in the cultured myocardial cells in vitro  66-68
  4 Discussion  68-70
REFERENCES  70-82
CONCLUSION  82-84
INNOVATION  84-86
ACKNOWLEDGEMENTS  86-88
PUBLICATIONS  88

Stress Damages and Expressions of Hsp60and Hsf-1in the Heat Stressed Myocardial Cells of Neonatal Rats in Vitro

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