c-J un在肉雞腹水綜合征過程中肺動(dòng)脈壁的表達(dá)變化及維拉帕米的干預(yù)研究

王建琳,喬健

(1.青島農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,山東青島266109;2.中國農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院,北京海淀100193)

c-J un在肉雞腹水綜合征過程中肺動(dòng)脈壁的表達(dá)變化及維拉帕米的干預(yù)研究

王建琳1,喬健2

(1.青島農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,山東青島266109;2.中國農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院,北京海淀100193)

本試驗(yàn)研究低溫誘發(fā)肉雞腹水綜合征過程中肺動(dòng)脈壁c-Jun的表達(dá),及阻斷鈣信號(hào)對(duì)肺動(dòng)脈壁c-Jun表達(dá)的影響和對(duì)肉雞腹水綜合征發(fā)生的干預(yù)情況,為肉雞腹水綜合征發(fā)生機(jī)制的研究提供基礎(chǔ).240只15日齡AA肉雞分為對(duì)照組、低溫組和維拉帕米組,15～50日齡每組隨機(jī)取6只測(cè)定右心肥大指數(shù),取肺組織做石蠟組織切片進(jìn)行c-Jun免疫組化染色分析.結(jié)果顯示,29～50日齡低溫組肉雞右心肥大指數(shù)較對(duì)照組和維拉帕米組明顯升高(P<0.05),低溫組肉雞20～50 μm、50～100 μm和100～200 μm肺小動(dòng)脈c-Jun陽性指數(shù)分別從22、29日齡和29日齡開始到50日齡極顯著高于對(duì)照組和維拉帕米組(P<0.01).研究結(jié)果認(rèn)為,低溫誘發(fā)肉雞腹水綜合征過程中不同直徑大小肺小動(dòng)脈壁c-Jun表達(dá)明顯增強(qiáng),阻斷鈣信號(hào)可明顯抑制肺小動(dòng)脈壁c-Jun的表達(dá)和肉雞腹水綜合征的發(fā)生.

肉雞;腹水綜合征;肺小動(dòng)脈;c-Jun;鈣信號(hào)

肉雞腹水綜合征(Ascites Syndrome,AS)是世界公認(rèn)的肉雞三大疾病之一,給肉雞養(yǎng)殖業(yè)造成了巨大的經(jīng)濟(jì)損失.Julian(1993)提出了肉雞AS的發(fā)生假說,即誘發(fā)因素-肺動(dòng)脈高壓-右心肥大、衰竭-后腔靜脈回流受阻-肝臟淤血性營養(yǎng)不良-腹水發(fā)生[1].其中右心肥大是機(jī)體對(duì)肺動(dòng)脈高壓的結(jié)構(gòu)性代償,用右心肥大指數(shù)(右心室/全心室的重量,RV:TV)作為評(píng)價(jià)指標(biāo),來反映肺動(dòng)脈高壓的形成、右心衰竭和肉雞AS的形成[2].

肉雞肺血管重塑是肺動(dòng)脈高壓的形態(tài)學(xué)基礎(chǔ),是肉雞AS的促發(fā)因素,以中膜增厚為其主要特征[3].肺動(dòng)脈平滑肌細(xì)胞增殖是中膜增厚的直接原因,與原癌基因c-myc等的表達(dá)及鈣信號(hào)等密切相關(guān)[4-6],而有關(guān)肉雞AS發(fā)生過程中肺小動(dòng)脈c-Jun蛋白的表達(dá)情況及鈣信號(hào)對(duì)其表達(dá)的影響等還未見報(bào)道.故本研究擬用免疫組織化學(xué)染色技術(shù)研究肉雞AS發(fā)生過程中肺動(dòng)脈c-Jun蛋白表達(dá)的動(dòng)態(tài)變化,及用鈣通道拮抗劑-維拉帕米阻斷鈣信號(hào)后肺動(dòng)脈c-Jun蛋白的表達(dá)情況和右心肥大指數(shù)的變化情況,為肉雞AS發(fā)生機(jī)制的研究提供基礎(chǔ).

1　材料與方法

240只14日齡AA肉仔雞隨機(jī)分為3組,為對(duì)照組(21±2)℃、低溫組(11±2)℃和維拉帕米組(11±2)℃(0.01%維拉帕米飲水),每組80只,每組每周隨機(jī)取6只翅靜脈注射戊巴比妥鈉處死測(cè)定右心肥大指數(shù)[7].

取右側(cè)肺組織下1/3固定、制備石蠟組織切片,應(yīng)用多克隆抗體即用型免疫組化試劑盒(北京中杉金橋生物技術(shù)有限公司)進(jìn)行c-Jun免疫組織化學(xué)染色,并用蘇木素進(jìn)行復(fù)染,顯微鏡下觀察結(jié)果.著色為棕色或黃色的為陽性細(xì)胞,其余為陰性細(xì)胞.結(jié)合圖像處理系統(tǒng)進(jìn)行分析計(jì)算直徑為20～50 μm、50～100 μm和100～200 μm肺小動(dòng)脈陽性細(xì)胞數(shù)所占總細(xì)胞數(shù)的百分比,作為c-Jun陽性指數(shù)(PI)[8].

用SPSS11.5軟件分析數(shù)據(jù),數(shù)據(jù)表示為:平均數(shù)±標(biāo)準(zhǔn)差(Mean±SD),用Independenr-samples T Test方法分析組間差異性,P<0.05為差異顯著,P< 0.01為差異極顯著.

表1　各組肉雞右心肥大指數(shù)(RV:TV)的動(dòng)態(tài)變化(n=6)

2　結(jié)果

2.1各組肉雞右心肥大指數(shù)(RV:TV)的動(dòng)態(tài)變化低溫組肉雞RV:TV從29日齡開始到43日齡極顯著高于對(duì)照組(P<0.01),50日齡時(shí)顯著高于對(duì)照組(P<0.05),見表1.

2.2各組肉雞20～50 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-Jun)的變化環(huán)境低溫誘發(fā)肉雞肺血管重塑過程中,肺動(dòng)脈血管平滑肌細(xì)胞c-Jun蛋白表達(dá)明顯增加.如表2所示,低溫組肉雞20-50 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-Jun)從22日齡開始到50日齡極顯著高于對(duì)照組(P<0.01).

表2　各組肉雞20～50 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-J un)的變化(n=6)

2.3各組肉雞50～100 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-Jun)的變化低溫組肉雞50～100 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-Jun)從29日齡開始到50日齡極顯著高于對(duì)照組(P<0.01),見表3.

2.4各組肉雞100～200 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-Ju)的變化低溫組肉雞100～200 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-Jun)從29日齡開始到50日齡極顯著高于對(duì)照組(P<0.01),見表4.

表3　各組肉雞50～100 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-J un)的變化(n=6)

表4　各組肉雞100～200 μm肺小動(dòng)脈血管平滑肌細(xì)胞PI(c-J un)變化(n=6)

3　討論

肺動(dòng)脈平滑肌細(xì)胞增殖導(dǎo)致肺動(dòng)脈中膜增厚,其增殖過程受到部分原癌基因的調(diào)控,研究報(bào)道c-fos和c-jun等原癌基因參與了人類和哺乳動(dòng)物血管平滑肌細(xì)胞的增殖[9-10].增殖的血管平滑肌細(xì)胞胞漿內(nèi)鈣離子濃度明顯升高,表明胞漿內(nèi)鈣離子內(nèi)流的增加是血管平滑肌細(xì)胞增殖所必須的[11].而胞漿內(nèi)鈣離子濃度的升高也涉及到了原癌基因c-fos的激活與表達(dá)[12].本研究整個(gè)過程中,正常組肉雞不同直徑的肺小動(dòng)脈c-Jun蛋白表達(dá)比較平穩(wěn),低溫組肉雞20～50 μm,50～100 μm,100～ 200 μm肺小動(dòng)脈c-Jun蛋白分別于低溫處理一周(22日齡)開始增加,并且大部分一直處于持續(xù)增加狀態(tài).與對(duì)照組相比,低溫組肉雞肺動(dòng)脈c-Jun蛋白的表達(dá)于22日齡或29日齡極顯著升高,表明環(huán)境低溫明顯誘發(fā)了肉雞肺小動(dòng)脈c-Jun蛋白的表達(dá).維拉帕米組肉雞肺小動(dòng)脈c-Jun蛋白的表達(dá)從22日齡或29日齡較低溫組極顯著降低,表明維拉帕米可明顯抑制低溫誘發(fā)的肉雞肺小動(dòng)脈c-Jun蛋白的表達(dá).結(jié)合前期維拉帕米可明顯抑制低溫誘發(fā)的肉雞肺動(dòng)脈平滑肌細(xì)胞增殖的研究結(jié)果[6],認(rèn)為鈣信號(hào)參與了低溫誘發(fā)的肉雞肺小動(dòng)脈c-Jun蛋白的表達(dá),參與了肺血管重塑.

肺血管重塑導(dǎo)致肺動(dòng)脈管腔明顯縮小,從而增加肺血管阻力,促進(jìn)了肺動(dòng)脈壓升高.肺動(dòng)脈壓升高將會(huì)使右心室壓力后負(fù)荷增加,故右心肥大是心臟對(duì)壓力負(fù)荷增加的形態(tài)學(xué)代償,故右心肥大指數(shù)可以直接反映右心肥大程度,也可以間接反映肺動(dòng)脈壓的高低[13].本研究結(jié)果發(fā)現(xiàn),肉雞低溫處理兩周后(29日齡)開始到試驗(yàn)結(jié)束(50日齡)右心肥大指數(shù)顯著或極顯著高于對(duì)照組,而維拉帕米組肉雞右心肥大指數(shù)在29日齡到50日齡時(shí)顯著或極顯著低于低溫組,表明環(huán)境低溫誘發(fā)了肉雞右心肥大和肺動(dòng)脈壓升高,維拉帕米明顯抑制了低溫誘發(fā)的肉雞右心肥大和肺動(dòng)脈壓升高.研究結(jié)果表明,鈣信號(hào)參與了肉雞右心肥大的發(fā)生和肺動(dòng)脈壓升高的過程.

綜合以上研究結(jié)果認(rèn)為,低溫誘發(fā)肉雞AS過程中肺小動(dòng)脈c-Jun蛋白表達(dá)明顯增強(qiáng),阻斷鈣信號(hào)可抑制低溫誘發(fā)的肉雞肺小動(dòng)脈c-Jun蛋白的過度表達(dá)和右心肥大的發(fā)生.

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Expression of c-J un in the Small Pulmonary Arterials of Broilers in the Development of Ascites Syndrome Induced by Low Ambient Tem perature and the Arrest Effect of Verapam il

WANG Jian-lin1,QIAO Jian2
(1.College of Animal science and Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China; 2.College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

Expression of c-Jun in pulmonary arterials of broilers in the development of ascites syndrome induced by low ambi?ent temperature and the effect of verapamil on the expression of c-Jun and occurrence of ascites syndrome were studied.15-day old broilers were divided into control group,low temperature group and verapamil group.The hearts and lungs in 6 broilers from ev?ery group were taken to study the index of right ventricular hypertrophy and the expression of c-Jun in pulmonary arterials respec?tively every week from 15 to 50 days old.The result showed that the index of right ventricular hypertrophy in low temperature groups was significantly higher than those in control group and verapamil group.The expression of c-Jun pulmonary arterials with 20～50 μm,50～100 μm and 100～200 μm diameters in broilers in low temperature group were significantly increased from 22 or 29 days to 50 days old compared with those in control group and verapamil group.The result suggested that the expression of c-Jun in pulmonary arterials with different diameters in broilers in the development of ascites syndrome were significantly increased, and blocking the calcium signal pathway could inhibit the high expression of c-Jun in pulmonary arterials and control the occur?rence of ascites syndrome in broilers.

broilers;ascites syndrom;pulmonary arterials;c-Jun;calcium signal

QIAO Jian

S858.31

A

0529-6005(2016)02-0009-03

2014-11-15

國家自然科學(xué)基金資助項(xiàng)目(30371063);山東省自然科學(xué)基金(ZR2013CL022)

王建琳(1976-),女,副教授,博士,主要從事禽病發(fā)生機(jī)制的研究,E-mail:bjsxxw@126.com

喬健,E-mail:qiaojian@cau.edu.cn