中国农业科学
中國農業科學
중국농업과학
SCIENTIA AGRICULTURA SINICA
2015年
7期
1437-1444
,共8页
王继英%王彦平%郭建凤%王怀中%林松%张印%武英
王繼英%王彥平%郭建鳳%王懷中%林鬆%張印%武英
왕계영%왕언평%곽건봉%왕부중%림송%장인%무영
细胞因子%模式识别受体%基因表达%大蒲莲%长白%仔猪
細胞因子%模式識彆受體%基因錶達%大蒲蓮%長白%仔豬
세포인자%모식식별수체%기인표체%대포련%장백%자저
cytokines%pattern recognition receptors%gene expression%Dapulian%Landrace%piglets
【目的】中国地方猪种在抗病性和免疫力方面优于国外猪种，但是对中外猪种抗病力差异的遗传基础和免疫机理却了解甚少。有推测不同品种猪对疾病的抵抗力不同可能在细胞因子和受体的表达水平上有所反映。本研究以抗病力强的大蒲莲和抗病力相对较弱的长白猪的断奶仔猪为研究对象，对6个具有重要功能的细胞因子（促炎症细胞因子（IL8和 TNFα），抗炎症细胞因子（IL10），干扰素调节因子（IRF3）和干扰素（IFNA 和 IFNG））和1个最具代表性的模式识别受体（TLR4）基因在大蒲莲和长白仔猪群体外周血液中的基因表达量进行检测，并对各基因进行品种和母猪效应分析，为了解中外不同猪种的免疫水平奠定基础。【方法】采集大蒲莲（104头）和长白（171头）35日龄仔猪的外周血，提取总 RNA，逆转录为 cDNA 后，利用荧光定量 PCR 法，首先对6个常用的候选内参基因（ACTB、GAPDH、B2M、TBP、RPL4和 PPIA）利用 geNorm 软件进行稳定性评价分析，筛选仔猪外周血 RNA 基因定量分析中稳定表达的内参基因，然后利用该内参基因进行校正，对上述细胞因子和模式识别受体基因在大蒲莲和长白仔猪群体外周血中的表达量进行定量，并利用最小二乘法对细胞因子和受体在大蒲莲和长白仔猪群体中的表达量进行品种和母猪效应分析。【结果】研究结果归纳为4个方面：1.外周血样品中内参基因稳定性值 M 从大到小依次为：ACTB（0.595）＞GAPDH（0.571）＞PPIA（0.469）＞RPL4（0.466）＞TBP（0.461）＞B2M（0.440），选择表达稳定性最高的基因 B2M 作为内参基因对细胞因子和受体基因的表达量进行校正。2.检测的6个细胞因子的基因表达量存在很大的差异，各细胞因子基因表达量ΔCt 最大值和最小值之间差值分别为9.23—14.25和5.83—8.73；受体 TLR4的ΔCt 值的最大和最小值之间相差相对较小，大蒲莲和长白仔猪分别为5.03和3.82。3.大蒲莲和长白两品种间相比较，细胞因子在长白猪群体中的表达量平均值均高于大蒲莲群体，而受体 TLR4的基因表达量在长白猪群体中的表达量低于大蒲莲群体；而且，大蒲莲群体内个体间细胞因子和受体表达量变异系数（平均31.43%）要大于长白群体（平均19.10%），这与大蒲莲选育程度低，群体内个体间的差异程度要大相一致。4.品种效应分析表明除 IL10外，其他6个目的基因的ΔCt 值品种间差异达显著（P＜0.05）或极显著（P＜0.01）水平。母猪效应分析表明母猪对各细胞因子和受体的影响均达到了极显著水平（P＜0.01）。【结论】成功筛选了外周血基因表达分析中稳定表达的内参基因（B2M），对常规饲养管理下大蒲莲和长白仔猪外周血中的主要细胞因子和受体基因的表达进行群体水平分析。本研究结果为了解中外不同猪种的种质特性和抗病品种的选育提供了基础数据。
【目的】中國地方豬種在抗病性和免疫力方麵優于國外豬種，但是對中外豬種抗病力差異的遺傳基礎和免疫機理卻瞭解甚少。有推測不同品種豬對疾病的牴抗力不同可能在細胞因子和受體的錶達水平上有所反映。本研究以抗病力彊的大蒲蓮和抗病力相對較弱的長白豬的斷奶仔豬為研究對象，對6箇具有重要功能的細胞因子（促炎癥細胞因子（IL8和 TNFα），抗炎癥細胞因子（IL10），榦擾素調節因子（IRF3）和榦擾素（IFNA 和 IFNG））和1箇最具代錶性的模式識彆受體（TLR4）基因在大蒲蓮和長白仔豬群體外週血液中的基因錶達量進行檢測，併對各基因進行品種和母豬效應分析，為瞭解中外不同豬種的免疫水平奠定基礎。【方法】採集大蒲蓮（104頭）和長白（171頭）35日齡仔豬的外週血，提取總 RNA，逆轉錄為 cDNA 後，利用熒光定量 PCR 法，首先對6箇常用的候選內參基因（ACTB、GAPDH、B2M、TBP、RPL4和 PPIA）利用 geNorm 軟件進行穩定性評價分析，篩選仔豬外週血 RNA 基因定量分析中穩定錶達的內參基因，然後利用該內參基因進行校正，對上述細胞因子和模式識彆受體基因在大蒲蓮和長白仔豬群體外週血中的錶達量進行定量，併利用最小二乘法對細胞因子和受體在大蒲蓮和長白仔豬群體中的錶達量進行品種和母豬效應分析。【結果】研究結果歸納為4箇方麵：1.外週血樣品中內參基因穩定性值 M 從大到小依次為：ACTB（0.595）＞GAPDH（0.571）＞PPIA（0.469）＞RPL4（0.466）＞TBP（0.461）＞B2M（0.440），選擇錶達穩定性最高的基因 B2M 作為內參基因對細胞因子和受體基因的錶達量進行校正。2.檢測的6箇細胞因子的基因錶達量存在很大的差異，各細胞因子基因錶達量ΔCt 最大值和最小值之間差值分彆為9.23—14.25和5.83—8.73；受體 TLR4的ΔCt 值的最大和最小值之間相差相對較小，大蒲蓮和長白仔豬分彆為5.03和3.82。3.大蒲蓮和長白兩品種間相比較，細胞因子在長白豬群體中的錶達量平均值均高于大蒲蓮群體，而受體 TLR4的基因錶達量在長白豬群體中的錶達量低于大蒲蓮群體；而且，大蒲蓮群體內箇體間細胞因子和受體錶達量變異繫數（平均31.43%）要大于長白群體（平均19.10%），這與大蒲蓮選育程度低，群體內箇體間的差異程度要大相一緻。4.品種效應分析錶明除 IL10外，其他6箇目的基因的ΔCt 值品種間差異達顯著（P＜0.05）或極顯著（P＜0.01）水平。母豬效應分析錶明母豬對各細胞因子和受體的影響均達到瞭極顯著水平（P＜0.01）。【結論】成功篩選瞭外週血基因錶達分析中穩定錶達的內參基因（B2M），對常規飼養管理下大蒲蓮和長白仔豬外週血中的主要細胞因子和受體基因的錶達進行群體水平分析。本研究結果為瞭解中外不同豬種的種質特性和抗病品種的選育提供瞭基礎數據。
【목적】중국지방저충재항병성화면역력방면우우국외저충，단시대중외저충항병력차이적유전기출화면역궤리각료해심소。유추측불동품충저대질병적저항력불동가능재세포인자화수체적표체수평상유소반영。본연구이항병력강적대포련화항병력상대교약적장백저적단내자저위연구대상，대6개구유중요공능적세포인자（촉염증세포인자（IL8화 TNFα），항염증세포인자（IL10），간우소조절인자（IRF3）화간우소（IFNA 화 IFNG））화1개최구대표성적모식식별수체（TLR4）기인재대포련화장백자저군체외주혈액중적기인표체량진행검측，병대각기인진행품충화모저효응분석，위료해중외불동저충적면역수평전정기출。【방법】채집대포련（104두）화장백（171두）35일령자저적외주혈，제취총 RNA，역전록위 cDNA 후，이용형광정량 PCR 법，수선대6개상용적후선내삼기인（ACTB、GAPDH、B2M、TBP、RPL4화 PPIA）이용 geNorm 연건진행은정성평개분석，사선자저외주혈 RNA 기인정량분석중은정표체적내삼기인，연후이용해내삼기인진행교정，대상술세포인자화모식식별수체기인재대포련화장백자저군체외주혈중적표체량진행정량，병이용최소이승법대세포인자화수체재대포련화장백자저군체중적표체량진행품충화모저효응분석。【결과】연구결과귀납위4개방면：1.외주혈양품중내삼기인은정성치 M 종대도소의차위：ACTB（0.595）＞GAPDH（0.571）＞PPIA（0.469）＞RPL4（0.466）＞TBP（0.461）＞B2M（0.440），선택표체은정성최고적기인 B2M 작위내삼기인대세포인자화수체기인적표체량진행교정。2.검측적6개세포인자적기인표체량존재흔대적차이，각세포인자기인표체량ΔCt 최대치화최소치지간차치분별위9.23—14.25화5.83—8.73；수체 TLR4적ΔCt 치적최대화최소치지간상차상대교소，대포련화장백자저분별위5.03화3.82。3.대포련화장백량품충간상비교，세포인자재장백저군체중적표체량평균치균고우대포련군체，이수체 TLR4적기인표체량재장백저군체중적표체량저우대포련군체；이차，대포련군체내개체간세포인자화수체표체량변이계수（평균31.43%）요대우장백군체（평균19.10%），저여대포련선육정도저，군체내개체간적차이정도요대상일치。4.품충효응분석표명제 IL10외，기타6개목적기인적ΔCt 치품충간차이체현저（P＜0.05）혹겁현저（P＜0.01）수평。모저효응분석표명모저대각세포인자화수체적영향균체도료겁현저수평（P＜0.01）。【결론】성공사선료외주혈기인표체분석중은정표체적내삼기인（B2M），대상규사양관리하대포련화장백자저외주혈중적주요세포인자화수체기인적표체진행군체수평분석。본연구결과위료해중외불동저충적충질특성화항병품충적선육제공료기출수거。
Objective] Though Chinese indigenous breeds have stronger disease resistance and immunity than imported commercial breeds, little is known about the genetic basis and immune mechanism of their difference. It is supposed that the gene expression level of cytokines and receptors may be the reasons leading to the different disease resistances among breeds. In the present study, the mRNA expression levels of seven genes in piglets of Dapulian and Landrace were determined, including two pro-inflammatory cytokines (IL8 and TNFα), one anti-inflammatory cytokine (IL10), one interferon regulatory factor (IRF3), two interferon (IFNA and IFNG) and one pattern recognition receptor (TLR4), and their breed and sow effects in the two breeds were analyzed, which would benefit the understanding of the immunity levels of Chinese indigenous breeds and the commercial breeds.[Method] Blood samples of clinically healthy Dapulian (n=104) and Landrace (n=171) piglets of 35 days old were collected, and their total RNA was extracted and reverse-transcribed into cDNA. Based on the quantitative real-time PCR, firstly, the expression stabilities of the six candidate genes (ACTB, GAPDH, B2M, TBP, RPL4 and PPIA) were evaluated by the geNorm program to select the most stable reference gene. Then, the selected reference gene was used for normalization, and the gene expression levels of the above mentioned cytokine and receptor genes were quantified in piglets of Dapulain and Landrace. Finally, the breed and sow effects of their expression levels in the two breeds were analyzed by Least Square Means. [Result] The expression stability values (M) of the six candidate reference genes in blood were as follows: ACTB (0.595) > GAPDH (0.571) > PPIA (0.469) > RPL4 (0.466)> TBP (0.461) > B2M (0.440). B2M, which had the most stable expression, was selected as the reference gene. In Dapulian and Landrace, the D-values between the maximum and minimum of ΔCt of the six cytokine genes were 9.23-14.25 and 5.83-8.73, respectively, indicating there were large difference among individuals of the two tested breeds. Compared with six cytokines, the D-values of TLR4 were smaller, 5.03 and 3.82 for Dapulian and Landrace respectively. Comparison between the two breeds, the expression levels of the six cytokines in Landrace were higher than that of Dapulian, while the expression levels of TLR4 in Dapulian was higher than that of Landrace. Additionally, the variation coefficients of all the cytokine and receptor genes in Dapulian (the average is 31.43%) were larger than those in Landrace (the average is 19.10%), which is consistent with the fact that Dapulian is one indigenous breed with less selecting strength compared with Landrace. The breed effect analyses indicated that, except IL10, breed effect had significant influence on all the other genes determined at 0.05 (P<0.05) level or 0.01 level (P<0.01). The sow effect analysis showed that it had significant influence on all the genes determined at 0.01 level (P<0.01). [Conclusion] In the present study, the stably expressed reference genes were successfully selected which are used for the gene expression research of porcine peripheral blood, seven genes’ mRNA expression levels of peripheral blood in normally raised Dapulian and Landrace piglets were determined at population level and the breed and sow effects on their expression were analyzed. Results of the study would provide important information concerning understanding of the variation between the indigenous and commercial breeds and mining the characteristics of indigenous breed.
