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Protection Mechanism of Death-associated Protein(DAP5) on Cisplatin-induced Renal Tubular Epithelial Cell Apoptosis

Author: GaoJianJun
Tutor: CaiGuangYan
School: PLA Postgraduate Medical School
Course: Internal Medicine
Keywords: Cisplatin Tubular epithelial cells Apoptosis DAP5 Protein translation
CLC: R965
Type: Master's thesis
Year: 2011
Downloads: 35
Quote: 0
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Abstract


Background and Purpose: cisplatin (cisplatin, DDP) is currently one of the most commonly used drug in the tumor combined with chemotherapy. Limit its clinical application can cause an acute tubular injury, tubular epithelial apoptosis is one of the main mechanisms of cisplatin-induced acute tubular cell injury. Death-associated protein (Death Associated Protein, DAP5) as an important factor for the regulation of protein translation, in the case of stress or apoptosis classic protein translation pathway is inhibited, DAP5 by internal ribosome entry site (Internal Ribosome the Entry Site IRES) ways to promote the expression of some protein translation, in order to maintain normal cell survival. But DAP5 cisplatin-induced acute tubular injury specific mechanism is unclear. This study, in vivo and in vitro to establish cisplatin-induced renal tubular epithelial cell apoptosis model based on observation DAP5 expression changes in renal tubular epithelial cells during apoptosis induced by cisplatin study DAP5 apoptosis and related proteins, and to explore the specific regulatory mechanism DAP5 apoptosis in cisplatin-induced renal tubular epithelial cells. Then study DAP5 whether by an important signaling pathway that regulates protein translation (PI3K/Akt/mTOR), to provide a new method for clinical protection cisplatin-induced acute kidney injury. Method: 1, cisplatin-induced acute renal tubular epithelial cell apoptosis in vivo and in vitro models to establish: young Wistar rats after intraperitoneal injection of cisplatin 72h, the establishment of the tubular cell apoptosis in vivo models, the use of renal function and renal histopathology score to assess the degree of kidney damage and apoptosis TUNEL staining and Western blot detection of caspase-3, caspase-8 and caspase-9, Bax, Bcl-2 and other apoptosis-related indicators change; application of different concentration gradients cisplatin on human renal tubular epithelial cells (HKC) established after the stimulus 24h in vitro model, the application the nucleus Hochest staining and flow cytometry to detect apoptotic cells, western blot method to verify changes in the protein expression of the apoptosis-related indicators. HKC cell apoptosis induced by cisplatin DAP5 regulation: plasmid transfection and siRNA interference technology over expression and down the the HKC cells of DAP5 expression, based on observation of cisplatin induced both HKC cells wither The death of the impact of the application of flow cytometry degree of apoptosis, western blot detection cell of Bax expression of Bcl-2 protein levels, explore DAP5 in cisplatin-induced the HKC cell apoptosis in specific mechanism. Detection of cisplatin-induced renal tubular epithelial cell apoptosis process PI3K/Akt/mTOR signaling pathway changes, and triciribine (Akt inhibitor) and rapamycin (mTOR inhibitor) blocked the signaling pathway, with the western blot to detect changes in expression DAP5 relationship to explore PI3K/Akt/mTOR pathway DAP5,. The results: the rats by intraperitoneal injection of cisplatin 72h acute kidney injury, serum creatinine and blood urea nitrogen was significantly higher. Histopathological renal proximal tubular epithelial cell injury, appeared vacuolar degeneration and granular degeneration of proximal tubular dilatation, tubular cavity visible protein casts. Tubule brush border shedding cell derangement. Tubular epithelial cells by TUNEL staining positive rate increased significantly. Applications western blot of caspase-3, caspase-8 and caspase-9, Bcl-2, Bax apoptosis-related indicators for testing, and found that caspase-3, caspase-8 and caspase-9, Bax protein expression levels in the model group than The control group was significantly higher, Bcl-2 decreased significantly compared with the control group. The role of the in vitro with different concentrations of cisplatin (1-100μM) in HKC cells after 24h to adopt Hochest staining and flow cytometry found that apoptosis cisplatin concentration increases. Consistent with the change, and the results of in vivo experiments vitro apoptosis-related indicators, caspase-3, caspase-8 and caspase-9, Bax protein expression levels of cisplatin concentration increased, and Bcl-2 with the of decline. At the same time, in HKC cells during apoptosis induced by cisplatin, the visible DAP5 lysis to generate 86KDa fragment protein -DAP5/p86. Overexpression plasmid transfection after DAP5/p97 and DAP5/p86 may increase in the expression of Bcl-2, and reduce cisplatin-induced apoptosis; using siRNA the negative regulator DAP5 will enable the Bcl-2 expression decline, leading to increased apoptosis. The overexpression or negative regulation DAP5 are no effect on Bax. PI3K/Akt/mTOR signaling pathway and thus in the process of cisplatin-induced apoptosis detection, visible PI3K, p-Akt and p-mTOR downregulation, suggesting that this pathway is inhibited, the application of triciribine and the rapamycin blocking of PI3K / Akt / mTOR pathway seen after DAP5 decreased expression of Bcl-2 and Bax expression decreased. Thus PI3K/Akt/mTOR pathway in DAP5 positive regulator. Conclusion: This study successfully established cisplatin-induced apoptosis of renal tubular epithelial cells in vivo and in vitro models, the first time the mechanism of action of cisplatin-induced tubular epithelial cell apoptosis, DAP5 found in renal tubular epithelial cells apoptotic process DAP5 cleavage of 86KDa protein fragment DAP5/p86 in, after the full length DAP5/p97 and cracking DAP5/p86 can promote translation expression of anti-apoptotic gene Bcl-2, play a role in inhibition of apoptosis. Negative regulation DAP5 Bcl-2 expression can also declined. PI3K/Akt/mTOR pathway can positively regulate DAP5. In this study, by overexpression DAP5 mitigate cisplatin-induced renal tubular epithelial cell apoptosis, providing new ideas for the prevention and treatment of cisplatin renal toxicity.

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