PUMA介导缺氧复氧大鼠心肌细胞凋亡的实验研究

发布时间：2018-01-11 12:30

本文关键词：PUMA介导缺氧复氧大鼠心肌细胞凋亡的实验研究　出处：《南昌大学》2010年硕士论文　论文类型：学位论文

【摘要】： 凋亡引起的心肌细胞损失是缺血/再灌注损伤(ischemia/reperfusion injury,IRI)的重要病理特征。阐明对心肌IRI中心肌细胞凋亡起关键性作用的蛋白质及其信号转导机制,对揭示心肌IRI的本质寻找并确立药物靶点,有效防治心肌IRI具有重要的现实意义。 p53上调凋亡调制物(p53 upregulated modulator of apoptosis, PUMA)是近年发现促凋亡作用最强的BH3-only蛋白质家族成员之一,是Bax/Bak上游的主要促凋亡蛋白质,能隔离所有的Bcl-2抗凋亡蛋白质作用。PUMA在多种病理性因素的刺激下通过p53依赖和非依赖途径激活介导细胞凋亡,在凋亡通路上发挥着举足轻重的作用。本实验采用原代大鼠心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)模型模拟在体心肌IRI,探讨促凋亡蛋白PUMA是否介导H/R诱导心肌细胞凋亡的发生?遏制PUMA表达是否可以下调心肌细胞凋亡而达到减轻H/R损伤的作用? 第一部分:PUMA在大鼠心肌细胞H/R损伤中的作用及意义目的: 应用乳鼠原代心肌细胞H/R损伤模型,探讨PUMA在大鼠心肌细胞H/R损伤中的作用及意义。方法: 原代培养的心肌细胞被随机分为5组,实验重复3次。①正常对照(control)组;②H/R 2h组;③H/R 4h组;④H/R 6h组;⑤H/R 12h组。采用生化自动分析仪测定LDH活性、MTT法测定细胞存活率、Annexin V-FITC和PI联合染色的流式细胞术检测凋亡率、RT-PCR和Western blot方法检测PUMA及凋亡相关蛋白Bax、Bcl-2的mRNA及蛋白的表达变化及分光光度法检测Caspase3活性变化。结果: 1、H/R诱导心肌细胞凋亡 H/R处理后心肌细胞凋亡率迅速上升,其中以H/R 6h最为明显,细胞凋亡率(23.44±4.16)%显著高于Control组(3.12±0.46)% (P0.05);H/R 12h Caspase3活性达峰值为Control组的(4.57±0.48)倍(P0.05)。 2、H/R诱导PUMA及凋亡相关蛋白Bax、Bcl-2表达变化 RT-PCR图像分析显示:PUMA mRNA H/R 2h开始升高(0.45±0.05),6h达峰值(0.76±0.06),持续到12h(0.71±0.07)仍高于Control组(0.29±0.02)(P0.05);Bax mRNA在H/R 2h开始升高(0.63±0.07),6h达峰值(0.96±0.09),持续到12h(0.79±0.09)仍高于Control组(0.28±0.05)(P0.05);与Control组(0.97±0.08)相比,Bcl-2 mRNA H/R 2h开始下降(0.82±0.07),12h降至最低(0.47±0.05)(P0.05)。 Western Blot图像分析显示:Control组中PUMA蛋白的表达量很低(0.08±0.01),H/R4h出现明显上调(0.33±0.04),6h显著升高(0.68±0.07),12h达峰值(0.72±0.07)(P0.05);与Control组(0.28±0.04)相比,Bax蛋白H/R 2h开始升高(0.49±0.05),6h显著升高(0.92±0.08),12h达峰值(0.97±0.10)(P0.05);与Control组(0.68±0.07)相比,Bcl-2蛋白H/R 2h开始降低(0.56±0.06),12h降至最低(0.26±0.02)(P0.05)。结论: H/R诱导心肌细胞凋亡。PUMA在正常培养的心肌细胞中表达很低,心肌细胞H/R后迅速上调,其表达变化与心肌细胞凋亡率、Caspase3活性变化及凋亡相关蛋白Bax表达变化正相关,与Bcl-2表达变化负相关,提示促凋亡蛋白PUMA可能通过上调Bax表达及下调Bcl-2表达,导致Caspase3活性增加介导H/R诱导的心肌细胞凋亡。第二部分: PUMA特异性siRNA对大鼠心肌细胞H/R损伤的影响目的: 应用脂质体转染的方法将PUMA特异性siRNA(si-PUMA)转染心肌细胞,探讨si-PUMA对心肌细胞H/R损伤的影响。方法: 原代培养的心肌细胞被随机分为4组,实验重复3次。①正常对照(control)组;②H/R 6h组;③转染错义siRNA+ H/R 6h组(si-SCR);④转染si-PUMA +H/R 6h组(si-PUMA)。siRNA干扰心肌细胞PUMA表达36 h后,制备心肌细胞H/R损伤模型,在H/R 6h即PUMA表达最显著时间点,观察下调PUMA表达对心肌细胞H/R损伤的作用。观察指标和方法同前。结果: 1、si-PUMA特异性下调心肌细胞PUMA表达。si-PUMA组PUMA mRNA(0.10±0.002)显著低于H/R 6h组(1.06±0.08)(P0.05);si-PUMA组PUMA蛋白(0.33±0.04)显著低于H/R 6h组(0.84±0.09)(P0.05)。 2、与H/R 6h组(60.7±6.84)%相比,si-PUMA组细胞存活率(75.3±4.29)%明显上调(P0.05);与H/R 6h组(1237±107 ) U/L相比,si-PUMA组LDH活性(802±55) U/L显著降低(P0.05);与H/R 6h组(23.96±5.02)%相比,si-PUMA组细胞凋亡率(14.16±4.02)%明显下降(P0.05)。 3、与H/R 6h组(1.06±0.07)相比,si-PUMA组Bax蛋白(0.64±0.05)表达下调(P0.05);与H/R 6h组(0.52±0.06)相比,si-PUMA组Bcl-2蛋白(0.68±0.08)表达上调(P0.05);与H/R 6h组(4.62±0.34)相比,si-PUMA组Caspase3活性(2.97±0.25)活性下降(P0.05)。结论: 化学合成的si-PUMA可以通过脂质体方法高效转染心肌细胞。特异性干扰PUMA表达后,心肌细胞存活率升高、LDH溢出减少,凋亡率明显下调,其主要机制可能是si-PUMA干扰PUMA的促凋亡作用,导致Bax表达下调和Bcl-2表达上调而使Caspase-3的活化程度降低,造成心肌细胞凋亡率下降。提示si-PUMA对心肌细胞H/R损伤具有较好的保护作用。本文结论: 1、促凋亡蛋白PUMA介导了H/R心肌细胞损伤,是H/R诱导心肌细胞凋亡的关键分子。 2、提示PUMA可做为心肌IRI的治疗靶点,si-PUMA可用于心肌IRI分子治疗药物的筛选。可以通过基因治疗或是药物干预的方式,在适当程度上遏制PUMA的表达,减轻心肌IRI。
[Abstract]:Loss of cardiomyocytes apoptosis induced by the ischemia / reperfusion injury (ischemia/reperfusion, injury, IRI) the important pathological features. Clarify the protein and the signal transduction mechanism of critical function on apoptosis of myocardial IRI in heart muscle cells, to reveal the essence of myocardial IRI to find and establish drug targets, has important practical significance for the effective prevention and treatment of myocardial IRI.
P53 regulation of apoptosis modulator (p53 upregulated modulator of apoptosis, PUMA) is one of the recently discovered BH3-only protein family members of apoptosis is the strongest, the main Pro apoptotic protein Bax/Bak upstream, can isolate all the anti apoptotic protein.PUMA Bcl-2 in a variety of pathological factors stimulated by p53 dependent and non dependent pathway the activation of apoptosis, plays an important role in the apoptosis pathway.
This experiment adopts the primary rat myocardial cells with hypoxia / reoxygenation (hypoxia/reoxygenation, H/R) model to simulate the in vivo myocardial IRI, to investigate whether apoptosis protein PUMA mediates H/R induced apoptosis of myocardial cells contain? Whether PUMA expression can down regulate cardiomyocyte apoptosis and reduce H/R damage?
Part 1: the role and significance of PUMA in H/R injury of rat cardiac myocytes
Objective:
To explore the role and significance of PUMA in H/R injury of rat cardiac myocytes by using the H/R damage model of rat primary cardiomyocytes.
Method:
The primary cultured myocardial cells were randomly divided into 5 groups, the experiment was repeated 3 times. The normal control group (control); H/R 2H group; the H/R group 4H; the H/R 6h group; the H/R 12h group. LDH activity was determined by automatic biochemical analyzer, cell viability was measured by MTT assay, cytometry the flow rate of Annexin V-FITC and apoptosis PI staining, RT-PCR and Western blot for the detection of PUMA and apoptosis related protein Bax, mRNA and protein levels of Bcl-2 and Caspase3 activity was determined by spectrophotometry.
Result:
1, H/R induced cardiomyocyte apoptosis
The apoptotic rate of cardiomyocytes increased rapidly after H/R treatment, especially H/R 6h. The apoptotic rate (23.44 + 4.16)% was significantly higher than that of Control group (3.12 + 0.46)% (P0.05), and H/R 12h Caspase3 activity reached the peak value of Control (4.57 + 0.48) times (P0.05).
2, H/R induced the changes of PUMA and apoptosis related protein Bax and Bcl-2 expression
RT-PCR image analysis showed that PUMA mRNA H/R 2H began to increase (0.45 + 0.05), and reached the peak at 6h (0.76 + 0.06), continued to 12h (0.71 + 0.07) is higher than that of group Control (0.29 + 0.02) (P0.05); Bax mRNA H/R 2H began to increase at (0.63 + 0.07), 6h (peak 0.96 + 0.09), continued to 12h (0.79 + 0.09) is higher than that of group Control (0.28 + 0.05) (P0.05); group Control (0.97 + 0.08) compared to Bcl-2 mRNA H/R 2H began to decline (0.82 + 0.07), 12h (0.47 + 0.05) to the lowest (P0.05).
Western Blot image analysis showed that the expression of PUMA protein in Control group was lower (0.08 + 0.01), H/R4h significantly increased (0.33 + 0.04), 6h (0.68 + 0.07) was significantly increased, and reached the peak at 12h (0.72 + 0.07) (P0.05); group Control (0.28 + 0.04) compared to Bax egg white H/R 2H began to increase (0.49 + 0.05), 6h (0.92 + 0.08) was significantly increased, and reached the peak at 12h (0.97 + 0.10) (P0.05); group Control (0.68 + 0.07) compared to Bcl-2 protein H/R 2H began to decrease (0.56 + 0.06), 12h (minimum 0.26 (+ 0.02) P0.05).
Conclusion:
H/R myocardial cell apoptosis induced by.PUMA in cultured myocardial cells in a low expression of H/R in myocardial cell after rapidly up-regulated, its expression changes and myocardial cell apoptosis rate, Caspase3 activity and apoptosis related protein Bax expression positive correlation, negative correlation with Bcl-2 expression, suggesting that the pro apoptotic protein PUMA and down-regulation of Bcl-2 expression by the upregulation of the expression of Bax, resulting in the increase of Caspase3 activity mediated myocardial cell apoptosis induced by H/R.
The second part: the effect of PUMA specific siRNA on H/R damage in rat cardiac myocytes
Objective:
PUMA specific siRNA (si-PUMA) was transfected into cardiomyocytes by liposome transfection, and the effect of si-PUMA on H/R damage in cardiac myocytes was investigated.
Method:
The primary cultured myocardial cells were randomly divided into 4 groups, the experiment was repeated 3 times. The normal control group (control); H/R 6h group; H/R group 6h transfection and missense siRNA+ (si-SCR); the si-PUMA +H/R transfection group 6h (si-PUMA).SiRNA interference PUMA expression of myocardial cells after 36 h, the preparation of the heart muscle cell damage model of H/R, H/R in 6h PUMA was the most significant point in time, to observe the down-regulation of PUMA expression on myocardial injury of H/R cells. With the index and method of observation.
Result:
1, si-PUMA specifically reduced the expression of PUMA in cardiac myocytes..si-PUMA group PUMA mRNA (0.10 + 0.002) was significantly lower than H/R 6h group (1.06 + 0.08) (P0.05); si-PUMA group PUMA protein (0.33 + 0.04) was significantly lower than that of H/R group (0.84 + 0.09) (P0.05).
2, H/R and 6h group (60.7 + 6.84)% compared with si-PUMA group, the cell survival rate (75.3 + 4.29)% was significantly increased (P0.05); group 6h and H/R (1237 + 107) U/L compared to si-PUMA group the activity of LDH (802 + 55) U/L decreased significantly (P0.05) and 6h group (H/R; 23.96 + 5.02)% compared to the apoptosis rate in si-PUMA group (14.16 + 4.02)% was significantly decreased (P0.05).
3, H/R and 6h group (1.06 + 0.07), si-PUMA group (0.64 + 0.05) Bax protein expression (P0.05 and H/R); group 6h (0.52 + 0.06), si-PUMA group (0.68 + 0.08) Bcl-2 protein expression (P0.05 and H/R); group 6h (4.62 + 0.34) compared si-PUMA group, the activity of Caspase3 (2.97 + 0.25) activity decreased (P0.05).
Conclusion:
Chemical synthesis of si-PUMA by liposome transfection efficiency of myocardial cells. The specific interference PUMA expression after myocardial cell survival rate increased, LDH reduced the overflow, the apoptosis rate was obviously reduced, the main mechanism may be pro apoptotic effect of si-PUMA PUMA interference, resulting in downregulation expression of Bcl-2 and Bax expression and the degree of activation of Caspase-3 decreased that cause myocardial cell apoptosis rate decreased. Suggesting that si-PUMA has a good protective effect on myocardial H/R injury.
The conclusion of this paper is as follows:
1, the apoptotic protein PUMA mediates the damage of H/R cardiomyocytes and is a key molecule in H/R induced cardiomyocyte apoptosis.
2, it suggests that PUMA can be used as a therapeutic target for myocardial IRI. Si-PUMA can be used to screen IRI molecular therapeutic drugs for myocardium. It can inhibit PUMA expression and reduce myocardial IRI. by appropriate gene therapy or drug intervention.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【共引文献】