本文關(guān)鍵詞：PUMA介導(dǎo)缺氧復(fù)氧大鼠心肌細(xì)胞凋亡的實(shí)驗(yàn)研究　出處：《南昌大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： p53上調(diào)凋亡調(diào)制物(PUMA) 缺血再灌注損傷 缺氧復(fù)氧 心肌細(xì)胞 凋亡 RNA干擾

【摘要】： 凋亡引起的心肌細(xì)胞損失是缺血/再灌注損傷(ischemia/reperfusion injury,IRI)的重要病理特征。闡明對(duì)心肌IRI中心肌細(xì)胞凋亡起關(guān)鍵性作用的蛋白質(zhì)及其信號(hào)轉(zhuǎn)導(dǎo)機(jī)制,對(duì)揭示心肌IRI的本質(zhì)尋找并確立藥物靶點(diǎn),有效防治心肌IRI具有重要的現(xiàn)實(shí)意義。 p53上調(diào)凋亡調(diào)制物(p53 upregulated modulator of apoptosis, PUMA)是近年發(fā)現(xiàn)促凋亡作用最強(qiáng)的BH3-only蛋白質(zhì)家族成員之一,是Bax/Bak上游的主要促凋亡蛋白質(zhì),能隔離所有的Bcl-2抗凋亡蛋白質(zhì)作用。PUMA在多種病理性因素的刺激下通過(guò)p53依賴和非依賴途徑激活介導(dǎo)細(xì)胞凋亡,在凋亡通路上發(fā)揮著舉足輕重的作用。 本實(shí)驗(yàn)采用原代大鼠心肌細(xì)胞缺氧/復(fù)氧(hypoxia/reoxygenation,H/R)模型模擬在體心肌IRI,探討促凋亡蛋白PUMA是否介導(dǎo)H/R誘導(dǎo)心肌細(xì)胞凋亡的發(fā)生?遏制PUMA表達(dá)是否可以下調(diào)心肌細(xì)胞凋亡而達(dá)到減輕H/R損傷的作用? 第一部分:PUMA在大鼠心肌細(xì)胞H/R損傷中的作用及意義 目的: 應(yīng)用乳鼠原代心肌細(xì)胞H/R損傷模型,探討PUMA在大鼠心肌細(xì)胞H/R損傷中的作用及意義。 方法: 原代培養(yǎng)的心肌細(xì)胞被隨機(jī)分為5組,實(shí)驗(yàn)重復(fù)3次。①正常對(duì)照(control)組;②H/R 2h組;③H/R 4h組;④H/R 6h組;⑤H/R 12h組。采用生化自動(dòng)分析儀測(cè)定LDH活性、MTT法測(cè)定細(xì)胞存活率、Annexin V-FITC和PI聯(lián)合染色的流式細(xì)胞術(shù)檢測(cè)凋亡率、RT-PCR和Western blot方法檢測(cè)PUMA及凋亡相關(guān)蛋白Bax、Bcl-2的mRNA及蛋白的表達(dá)變化及分光光度法檢測(cè)Caspase3活性變化。 結(jié)果: 1、H/R誘導(dǎo)心肌細(xì)胞凋亡 H/R處理后心肌細(xì)胞凋亡率迅速上升,其中以H/R 6h最為明顯,細(xì)胞凋亡率(23.44±4.16)%顯著高于Control組(3.12±0.46)% (P0.05);H/R 12h Caspase3活性達(dá)峰值為Control組的(4.57±0.48)倍(P0.05)。 2、H/R誘導(dǎo)PUMA及凋亡相關(guān)蛋白Bax、Bcl-2表達(dá)變化 RT-PCR圖像分析顯示:PUMA mRNA H/R 2h開(kāi)始升高(0.45±0.05),6h達(dá)峰值(0.76±0.06),持續(xù)到12h(0.71±0.07)仍高于Control組(0.29±0.02)(P0.05);Bax mRNA在H/R 2h開(kāi)始升高(0.63±0.07),6h達(dá)峰值(0.96±0.09),持續(xù)到12h(0.79±0.09)仍高于Control組(0.28±0.05)(P0.05);與Control組(0.97±0.08)相比,Bcl-2 mRNA H/R 2h開(kāi)始下降(0.82±0.07),12h降至最低(0.47±0.05)(P0.05)。 Western Blot圖像分析顯示:Control組中PUMA蛋白的表達(dá)量很低(0.08±0.01),H/R4h出現(xiàn)明顯上調(diào)(0.33±0.04),6h顯著升高(0.68±0.07),12h達(dá)峰值(0.72±0.07)(P0.05);與Control組(0.28±0.04)相比,Bax蛋白H/R 2h開(kāi)始升高(0.49±0.05),6h顯著升高(0.92±0.08),12h達(dá)峰值(0.97±0.10)(P0.05);與Control組(0.68±0.07)相比,Bcl-2蛋白H/R 2h開(kāi)始降低(0.56±0.06),12h降至最低(0.26±0.02)(P0.05)。 結(jié)論: H/R誘導(dǎo)心肌細(xì)胞凋亡。PUMA在正常培養(yǎng)的心肌細(xì)胞中表達(dá)很低,心肌細(xì)胞H/R后迅速上調(diào),其表達(dá)變化與心肌細(xì)胞凋亡率、Caspase3活性變化及凋亡相關(guān)蛋白Bax表達(dá)變化正相關(guān),與Bcl-2表達(dá)變化負(fù)相關(guān),提示促凋亡蛋白PUMA可能通過(guò)上調(diào)Bax表達(dá)及下調(diào)Bcl-2表達(dá),導(dǎo)致Caspase3活性增加介導(dǎo)H/R誘導(dǎo)的心肌細(xì)胞凋亡。 第二部分: PUMA特異性siRNA對(duì)大鼠心肌細(xì)胞H/R損傷的影響 目的: 應(yīng)用脂質(zhì)體轉(zhuǎn)染的方法將PUMA特異性siRNA(si-PUMA)轉(zhuǎn)染心肌細(xì)胞,探討si-PUMA對(duì)心肌細(xì)胞H/R損傷的影響。 方法: 原代培養(yǎng)的心肌細(xì)胞被隨機(jī)分為4組,實(shí)驗(yàn)重復(fù)3次。①正常對(duì)照(control)組;②H/R 6h組;③轉(zhuǎn)染錯(cuò)義siRNA+ H/R 6h組(si-SCR);④轉(zhuǎn)染si-PUMA +H/R 6h組(si-PUMA)。siRNA干擾心肌細(xì)胞PUMA表達(dá)36 h后,制備心肌細(xì)胞H/R損傷模型,在H/R 6h即PUMA表達(dá)最顯著時(shí)間點(diǎn),觀察下調(diào)PUMA表達(dá)對(duì)心肌細(xì)胞H/R損傷的作用。觀察指標(biāo)和方法同前。 結(jié)果: 1、si-PUMA特異性下調(diào)心肌細(xì)胞PUMA表達(dá)。si-PUMA組PUMA mRNA(0.10±0.002)顯著低于H/R 6h組(1.06±0.08)(P0.05);si-PUMA組PUMA蛋白(0.33±0.04)顯著低于H/R 6h組(0.84±0.09)(P0.05)。 2、與H/R 6h組(60.7±6.84)%相比,si-PUMA組細(xì)胞存活率(75.3±4.29)%明顯上調(diào)(P0.05);與H/R 6h組(1237±107 ) U/L相比,si-PUMA組LDH活性(802±55) U/L顯著降低(P0.05);與H/R 6h組(23.96±5.02)%相比,si-PUMA組細(xì)胞凋亡率(14.16±4.02)%明顯下降(P0.05)。 3、與H/R 6h組(1.06±0.07)相比,si-PUMA組Bax蛋白(0.64±0.05)表達(dá)下調(diào)(P0.05);與H/R 6h組(0.52±0.06)相比,si-PUMA組Bcl-2蛋白(0.68±0.08)表達(dá)上調(diào)(P0.05);與H/R 6h組(4.62±0.34)相比,si-PUMA組Caspase3活性(2.97±0.25)活性下降(P0.05)。 結(jié)論: 化學(xué)合成的si-PUMA可以通過(guò)脂質(zhì)體方法高效轉(zhuǎn)染心肌細(xì)胞。特異性干擾PUMA表達(dá)后,心肌細(xì)胞存活率升高、LDH溢出減少,凋亡率明顯下調(diào),其主要機(jī)制可能是si-PUMA干擾PUMA的促凋亡作用,導(dǎo)致Bax表達(dá)下調(diào)和Bcl-2表達(dá)上調(diào)而使Caspase-3的活化程度降低,造成心肌細(xì)胞凋亡率下降。提示si-PUMA對(duì)心肌細(xì)胞H/R損傷具有較好的保護(hù)作用。 本文結(jié)論: 1、促凋亡蛋白PUMA介導(dǎo)了H/R心肌細(xì)胞損傷,是H/R誘導(dǎo)心肌細(xì)胞凋亡的關(guān)鍵分子。 2、提示PUMA可做為心肌IRI的治療靶點(diǎn),si-PUMA可用于心肌IRI分子治療藥物的篩選�？梢酝ㄟ^(guò)基因治療或是藥物干預(yù)的方式,在適當(dāng)程度上遏制PUMA的表達(dá),減輕心肌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.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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5 鄧宇s，

本文編號(hào)：1409544