PUMA在大鼠胰腺移植缺血�苍俟嘧�損傷中的意義

【摘要】  目的：探討PUMA在移植胰腺缺血再灌注損傷(I/RI)中的早期促凋亡作用。方法：對(duì)封閉群大鼠建立大鼠腔靜脈內(nèi)分泌引流、腸道外分泌引流的動(dòng)物模型.再灌注后第0、1、2、3、4、6、9、12 h等時(shí)點(diǎn),每時(shí)點(diǎn)處死5只大鼠,切取移植胰腺,石蠟包埋切片,原位DNA缺口末端標(biāo)記(TUNEL)法測(cè)定胰腺組織細(xì)胞凋亡, Western blot方法測(cè)定移植胰腺組織PUMA,bcl��2、bax、caspase��3蛋白表達(dá)。結(jié)果：再灌注術(shù)后第0、1、2、3、4、6、9、12 h,胰腺細(xì)胞凋亡數(shù)分別為(29.42±4.93)、(47.65±6.43)、(74.80±9.73)、(106.35±16.80)、(148.71±19.50)、(123.96±15.54)、(97.32±10.60)和(57.42±9.56)個(gè)/高倍視野。各時(shí)點(diǎn)均顯著高于正常胰腺(23.48±4.26)。第4小時(shí)細(xì)胞凋亡數(shù)明顯增高,12 h降低到最低值,不同組細(xì)胞凋亡數(shù)的差異有統(tǒng)計(jì)學(xué)意義(P<0.01);PUMA以及其下游的bax和caspase��8蛋白表達(dá)在第4小時(shí)達(dá)到最高峰,12 h降低到最高值,bcl��2在第4小時(shí)達(dá)到最低值,蛋白表達(dá)差異均有統(tǒng)計(jì)學(xué)意義(P<0.05),以后逐漸升高,12 h降低到最低值,PUMA表達(dá)與各時(shí)點(diǎn)的細(xì)胞凋亡數(shù)有明顯的正相關(guān)(r=1.00,P<0.05)。結(jié)論：I/RI后細(xì)胞早期凋亡與PUMA活化有關(guān),PUMA是通過(guò)調(diào)節(jié)下游bax、caspase��8、bcl��2基因發(fā)揮凋亡促進(jìn)作用。

【關(guān)鍵詞】  胰腺移植; 缺血再灌注損傷;凋亡;PUMA蛋白;大鼠法律論文發(fā)表

　　[ABSTRACT] Objective: To study the pro�瞐poptosis effect of P53 up�瞨egulated modulator of apoptosis (PUMA) protein on ischemia�瞨eperfusion injury of rats stimulated during pancreatic transplantation.Methods: Rat models of pancreatic transplantation with venacava drainage and enteric drainage were established firstly. Then 5 animals in each group were sacrificed respectively at different time points including 0, 1, 2, 3, 4, 6, 9 and12 h after reperfusion, with pancreatic grafts being removed for apoptosic analysis by using DNA nick end labeling technique and protein expression analysis of PUMA, bcl��2, bax and caspase��3 by using western blot method. Results: Under high power field, the numbers of apoptosic cells in pancreatic grafts at each time point was 29.42 ±4.93, 47.65 ±6.43, 74.8 ±9.73, 106.35 ±16.8, 148.71 ±19.50, 123.96 ±15.54, 97.32 ±10.6 and 57.42 ±9.56, respectively which are all significantly higher than the number in normal pancreatic tissues (23.48 ±4.26). It indicated the numbers were obviously increased at 4h after profusion while decreased to the minimum at 12h with significant difference among each group (P<0.01). The expression of PUMA, bax and caspase��3 all started to up�瞨egulated at 1h after profusion and increased to maximum at 4h, then gradually decreased to minimum at 12h. While for bcl��2, the expression started to down�瞨egulated at 1h after profusion and decreased to the minimum at 4h, then gradually increased to maximum at 12h. There are significant differences among the protein expression (P<0.05). Besides, the PUMA expression is positively correlated to the number of apoptosic cells. (r=1.00, P<0.05). Conclusion: Cell apoptosis in ischemia�瞨eperfusion injury is highly correlated to the activation of PUMA. The pro�瞐poptosis effect of PUMA is generated by regulating bax, caspase��8 and bcl��2.法律論文發(fā)表

　　[KEY WORDS] Pancreas transplantation; Ischemia�瞨eperfusion injury; Apoptosis; PUMA protein; Wistar

　　近年來(lái)大量研究表明,細(xì)胞凋亡是器官移植缺血�苍俟嘧�損傷( ischemia�瞨eperfusioninjury, I/RI)的早期事件[1]，但細(xì)胞發(fā)生凋亡的機(jī)制不明。PUMA(p53 up�瞨egulated modulator of apoptosis)是2001年發(fā)現(xiàn)的p53下游促凋亡基因，是bcl��2家族BH��3亞家族中的成員，PUMA在受到p53缺氧、放射、化療等刺激信號(hào)下迅速被激活，從而導(dǎo)致PUMA下游bcl��2家族基因的活性改變，引起細(xì)胞色素C的釋放和Caspase酶的活化，最終導(dǎo)致細(xì)胞凋亡[2，3]。本研究應(yīng)用大鼠胰腺、十二指腸移植模型, 探討PUMA在大鼠胰腺、十二指腸移植I/RI中的早期凋亡促進(jìn)作用。

　　1 材料與方法法律論文發(fā)表

　　1.1 實(shí)驗(yàn)動(dòng)物

　　43只(對(duì)照組3只)清潔級(jí)封閉群SD大鼠(供體), 體質(zhì)量250～300 g ,雄性;清潔級(jí)封閉群Wistar大鼠, 40只，體質(zhì)量300～350 g, 雄性, 均由上海實(shí)驗(yàn)動(dòng)物中心提供。

　　1.2 主要試劑

　　鼠抗人PUMA多克隆抗體購(gòu)自Cell Signals公司，兔抗鼠bcl��2 、bax、 caspase��3、Actin多克隆抗體和Western bloting試劑購(gòu)自Santa Cru公司,細(xì)胞凋亡檢測(cè)試劑盒購(gòu)南京建成生物工程研究所。

　　1.3 大鼠胰腺移植動(dòng)物模型的制備法律論文發(fā)表

　　見(jiàn)文獻(xiàn)[4]。

　　1.4 移植模型的分組和處理

　　移植模型共分為8組，每組5只;各組大鼠于胰腺再