Effects of different long-chain fatty acids on lipid uptake and CD36 palmitoylation in small intestinal epithelial cells of mouse
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摘要:目的
探究不同长链脂肪酸对小鼠小肠上皮细胞脂肪酸摄取和分化簇36(Cluster of differentiation 36, CD36)棕榈酰化的影响。
方法在细胞水平，用长链饱和脂肪酸棕榈酸(Palmitic acid, PA)、长链多不饱和脂肪酸亚油酸(Linoleic acid, LA)、长链单不饱和脂肪酸油酸(Oleic acid, OA)处理小鼠小肠上皮细胞ModeK，通过BODIPY染色比较细胞的脂质摄取情况，采用免疫荧光和Western blot检测CD36的膜定位及棕榈酰化水平，利用棕榈酰化抑制剂评估CD36的棕榈酰化对于脂肪酸摄取的必要性。在活体试验中，对小鼠急性灌胃不同油脂，即猪油、豆油和橄榄油(分别富含PA、LA和OA)，研究小鼠空肠的脂质摄取、血清甘油三酯(Triglyceride, TG)水平及CD36棕榈酰化水平。
结果BODIPY染色结果显示，添加LA和OA后，ModeK细胞内的平均荧光强度显著高于PA处理的细胞(P<
0.0001 )，说明LA和OA促进了小肠上皮细胞对脂质的摄取；免疫荧光结果显示，与PA相比，LA和OA可使CD36更多地定位于质膜上；棕榈酰化检测结果表明，LA和OA可显著升高质膜上CD36的棕榈酰化水平(P<0.05, P<0.001)；此外，棕榈酰化抑制剂2−溴棕榈酸酯(2-Bromopalmitate, 2BP)可显著逆转LA和OA对小肠上皮细胞脂质摄取的促进作用(P<0.001, P<0.0001 )。活体急性灌胃结果显示，与灌胃猪油相比，灌胃豆油和橄榄油显著促进空肠上皮细胞脂质摄取(P<0.05, P<0.0001 )，提升血液TG水平以及空肠组织质膜组分中CD36棕榈酰化水平(P<0.05)。结论相比于长链饱和脂肪酸PA，长链不饱和脂肪酸LA和OA可显著增加小肠上皮细胞的脂质摄取，OA效果更明显，这可能与长链不饱和脂肪酸促进CD36在质膜上的棕榈酰化水平及定位有关。
Abstract:ObjectiveTo investigate the impacts of various long-chain fatty acids on fatty acid uptake and the palmitoylation of cluster of differentiation 36 (CD36) in mouse small intestinal epithelial cells.
MethodIn vitro, ModeK, the mouse intestinal epithelial cells, were subjected to treatment with various long-chain fatty acids, including saturated fatty acid palmitic acid (PA), polyunsaturated linoleic acid (LA) and monounsaturated oleic acid (OA). A comparison of the cellular lipid uptake was conducted by BODIPY staining. Additionally, immunofluorescence and Western blot techniques were employed to evaluate the membrane localization of CD36 and its palmitoylation status, and the palmitoylation inhibitor was utilized to appraise the necessity of CD36 palmitoylation for fatty acid uptake. In vivo experiments involved the acute oral administration to mice with different oils, including lard, soy oil and olive oil, which were rich in PA, LA and OA respectively. It aimed to examine lipid uptake in the mouse jejunum, blood triglyceride (TG) level, and the palmitoylation level of CD36.
ResultThe BODIPY staining results indicated that the average fluorescence intensity within ModeK cells subjected to treatment with LA and OA were significantly higher than that in cells treated with PA (P<
0.0001 ). This finding suggested that LA and OA enhanced lipid uptake by intestinal epithelial cells. The immunofluorescence results demonstrated that, in comparison to PA, LA and OA resulted in a greater localization of CD36 on the plasma membrane. Synchronously, palmitoylation assay results further indicated that LA and OA significantly increased the palmitoylation level of CD36 on plasma membrane (P<0.05, P<0.001). Moreover, the palmitoylation inhibitor 2-bromopalmitate (2BP) reversed the promoting effect of LA and OA on lipid uptake by intestinal epithelial cells (P<0.001, P<0.0001 ). And the results of acute gavage in vivo showed that gavage of soy oil and olive oil significantly promoted lipid uptake by jejunal epithelial cells compared to gavage of lard (P<0.05, P<0.0001 ), as well as elevated blood triglyceride (TG) level and the palmitoylation level of CD36 in the plasma membrane fraction of jejunal tissue (P<0.05).ConclusionIn comparison to PA, a long-chain saturated fatty acid, the long-chain unsaturated fatty acids LA and OA exhibit the ability to enhance lipid uptake in small intestinal epithelial cells, with OA demonstrating a more pronounced effect. And this enhancement may be attributed to the promotive palmitoylation and localization of CD36 on the plasma membrane, which is facilitated by long-chain unsaturated fatty acids.
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Keywords:
- Long-chain fatty acid /
- Mouse /
- Intestinal epithelial cell /
- Lipid intake /
- CD36 /
- Palmitoylation
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禽白血病是由禽白血病病毒(Avian leukemia virus，ALV)引起的一类禽免疫抑制性肿瘤性传染病。可自然感染鸡群的禽白血病病毒包括ALV-A~E、J 和K 7个亚群，其中ALV-A是引起我国鸡群发生禽白血病的主要病原[1-2]。ALV-A可使感染鸡群产生免疫抑制、生产性能下降，乃至发生特征性肿瘤而死亡，给养禽业造成巨大的经济损失[3-4]。目前，该病鲜有商品化疫苗和有效的治疗方法，主要通过净化种群和生物安全措施进行预防[5]。然而，近年来禽白血病的流行病学调查研究发现，ALV-A在我国地方鸡种[6-7]、商品肉鸡[8]、蛋鸡[9]及野生鸟类[10]中普遍存在。可见，现有措施并不能完全控制A亚群禽白血病在中国的发生与流行，该病已成为威胁我国养鸡业(尤其是种鸡业)可持续健康发展的重大疾病之一。因此，研发更适合防控我国A亚群禽白血病的新策略已迫在眉睫。国外已有研究证实，提高宿主对A亚群禽白血病的遗传抗性，开展A亚群禽白血病的抗病育种可成为防控该病的有效策略[11-13]。
ALV-A由tva受体基因编码的细胞表面特异性受体Tva介导侵入宿主细胞，继而发生感染[14]。tva受体基因的遗传突变会导致Tva受体蛋白的完全缺失或表达一个不适宜作为ALV-A受体的缺陷型Tva受体蛋白，从而引起宿主细胞对ALV-A的感染产生遗传抗性[15]。在国外白来航近交品系tva受体基因中已经鉴定了tvar1、tvar2、tvar3和tvar4 4个ALV-A遗传抗性位点[16-17]。Chen等[18]首次从中国鸡种中成功鉴定了tvar5和tvar6 2个ALV-A遗传抗性位点。另外，我们前期研究(未发表)发现中国鸡种tva受体基因存在新的自然突变位点：tva基因编码区第3位碱基由G突变为A，推测该突变引起tva基因起始密码子序列由ATG突变为ATA，将该tva基因自然突变命名为tva c.3G>A，但其对宿主感染A亚群禽白血病病毒的影响尚不清楚。因此，本研究拟通过ALV-A体外感染试验和体内攻毒试验，从体外、体内2个层面验证tva c.3G>A突变是否引起宿主对ALV-A的感染产生遗传抗性，以期鉴定新的ALV-A遗传抗性位点。
1. 材料与方法
1.1 试验动物、病毒、细胞株及质粒
黄羽肉鸡品系CB01~CB15[19]抗凝血样采自于温氏食品集团股份有限公司，每个品系随机采取36~60份血样，共670份血液样品。1日龄无特定病原(Specific pathogen free，SPF)鸡苗购自广东大华农有限公司。ALV-A GD08株、DF-1细胞系为广东省畜禽健康养殖与环境控制重点实验室保存。RCASBP(A)-GFP重组质粒为广东省畜禽健康养殖与环境控制重点实验室前期构建并保存[18]。
1.2 主要试剂
血液/细胞/组织基因组DNA提取试剂盒购自天根生化科技(北京)有限公司；质粒小量提取试剂盒、无内毒素质粒大量提取试剂盒、凝胶 DNA回收试剂盒均购自OMEGA公司；ReverTra Ace®qPCR RT Kit、KOD-FX购自Toyobo公司；FastStart SYBR Green Master(Rox)购自Roche公司；反转录试剂盒、pMD19-T、PrimeScript® One Step RT-PCR Kit购自Takara公司；Opti-DMEM无血清培养基、DMEM细胞培养基、胎牛血清、胰蛋白酶、青链霉素均购自Gibco公司；TRIZOL reagent、Lipofectamine 3000转染试剂购自Invitrogen公司。
1.3 黄羽肉鸡品系tva基因的遗传变异分析
参考NCBI数据库中鸡tva基因的DNA序列(GenBank登录号：AY531262.1)，应用 Primer 5.0软件设计3对引物，分3个片段(1、2和3段)PCR扩增tva基因全长序列3 607 bp，引物信息见表1。提取黄羽肉鸡品系CB01~CB15血液样品的基因组DNA，用该3对引物PCR扩增tva基因全长序列。将PCR扩增产物送生工生物工程(上海)股份有限公司测序，应用DNAstar和Mutation Surveyor基因序列分析软件比对tva基因序列和测序序列，分析黄羽肉鸡品系tva基因的遗传变异，对tva c.3G>A突变位点进行基因分型。
表 1 tva受体基因全长序列PCR扩增引物信息Table 1. Primers used to amplify the whole sequence of tva receptor gene片段 Fragment 引物名称 Primer name 引物序列(5′→3′) Primer sequence 退火温度/℃ Annealing temperature 片段大小/bp Segment size 1 P1-F GTTCAGCAGATCCTCATCTCCCG 62 1308 P1-R GGCCATTGTGCGATCTAAGAGGG 2 P2-F AGCCCTCTTAGATCGCACAA 60 1253 P2-R GTGACACCGAGCACAAAATG 3 P3-F GTTGGAGCTGGATGAGCACT 60 1132 P3-R TGAGGGAATTCCTGTCACCT 1.4 tva基因mRNA的RT-PCR扩增
采用Trizol法提取tva c.3G>A突变位点野生型tva c.3G/G和纯合突变型tva c.3A/A种鸡血液的总RNA，参考反转录试剂盒说明书反转录成cDNA。参考文献[18]中的引物序列和PCR反应体系、扩增条件，利用KOD-FX高保真酶RT-PCR扩增tva基因整个编码序列。回收、纯化的RT-PCR产物克隆入pMD19-T，送生工生物工程(上海)股份有限公司测序，测序结果应用Lasergene 7.1软件进行分析。
1.5 鸡胚成纤维细胞分离与培养
采集10日龄鸡胚消毒后，用镊子打开气室和壳膜，夹起鸡胚，用眼科剪刀和镊子剔除头、爪、骨头和内脏，把鸡胚组织块置于平皿中，用PBS液漂洗去除血污。将漂洗好的组织块放入5 mL离心管，用眼科剪刀剪至肉糜状，用0.25%(w)的胰酶在37 ℃水浴箱中消化15~20 min，加入适量的小牛血清终止消化。使用200目细胞滤膜过滤后，1000 r/min离心5 min，弃掉上清液，用完全培养基对细胞进行重悬，分装入无菌培养皿中，获取纯化的鸡胚成纤维细胞 (Chicken embryo fibroblast，CEF)，细胞置于CO2体积分数为5%、37 ℃的培养箱内培养。
1.6 流式细胞术检测RCASBP(A)-GFP报告病毒感染
将重组质粒RCASBP(A)-EGFP转染入DF-1细胞中，转染后第7天，拯救并收集DF-1细胞上清液中表达绿色荧光蛋白的重组病毒RCASBP(A)-GFP，测定病毒感染单位(IU)后，分装保存于−80 ℃备用。分离与培养tva c.3G>A突变位点野生型tva c.3G/G、杂合突变型tva c.3G/A和纯合突变型tva c.3A/A CEF，分别接种于24孔板中，每孔接种5×104个细胞，培养24 h后，每孔接种5×105 IU/mL的RCASBP(A)-GFP病毒液。孵育2 h后，弃掉病毒液，用含1%(φ) 胎牛血清的维持液继续培养。感染后第1、2、3、7天，使用 Cytomics FC 500 分析仪(Beckman Coulter，USA)通过荧光激活细胞分选(Fluorescence-activated cell sorting，FACS)定量 tva c.3G>A突变位点不同基因型CEF的GFP 阳性细胞百分比。
1.7 ALV-A体内感染试验
前期研究发现tva c.3G>A突变主要存在于CB06品系。从CB06品系随机挑选75只1日龄雏鸡随机分为3组，每组25只商品鸡和3只SPF鸡作为对照，饲养于3个隔离器，并随意提供饲料和水。1日龄时，每只雏鸡腹腔接种ALV-A GD08株病毒液(S/P=2.1)0.2 mL，5日龄时，再攻毒1次。攻毒后1周，采集每只雏鸡抗凝血样，抽取全基因组DNA，通过直接测序方法对每只雏鸡tva c.3G>A突变位点进行基因分型。攻毒后1个月，采集雏鸡的血样，利用Trizol试剂盒抽提血样总RNA，利用ALV-A GD08株特异性检测引物，RT-PCR检测每只雏鸡的病毒血症情况，确定tva c.3G>A突变不同基因型雏鸡对ALV-A GD08株的感染状态[18]。
1.8 统计分析
所有数据均使用GraphPad Prism 7.0软件进行绘图和数据统计分析，2组数据比较采用独立样本t检验分析，数据结果以平均值±标准差表示。利用Popgene软件分析tva c.3G>A突变位点在我国黄羽肉鸡品系中的基因型频率分布。
2. 结果与分析
2.1 黄羽肉鸡品系中tva c.3G>A突变位点的鉴定
为了剖析中国黄羽肉鸡品系tva受体基因的遗传变异，分3个片段PCR扩增每只鸡tva受体基因的基因组区域。如图1所示，PCR扩增出tva基因的1、2和3片段的目的条带，片段大小与预期结果相符。将PCR产物进行Sanger测序，发现中国黄羽肉鸡品系tva基因DNA序列第260位碱基存在由G突变为A的自然突变(图2)，进一步分析推测该突变致使tva基因编码区第3位碱基由G突变为A，引起tva基因起始密码子序列由ATG突变为ATA，将该tva基因自然突变命名为tva c.3G>A。为了证实纯合突变型tva c.3A/A转录本存在G>A突变，RT-PCR扩增野生型tva c.3G/G和纯合突变型tva c.3A/A血液tva基因整个编码序列。结果如图3所示，tva c.3G/G和tva c.3A/A基因型血样均扩增出566和420 bp的条带，表明tva c.3G>A突变位点在不同基因型血样均可扩增出tva基因长、短2个转录本。RT-PCR产物的克隆测序结果表明，与野生型tva c.3G/G相比，纯合突变型tva c.3A/A基因型2个tva cDNA序列第3位碱基均由G突变为A，引起Tva受体蛋白氨基酸序列第1个氨基酸由甲硫氨酸(M)改变为异亮氨酸(I)。
![图 1 tva基因3个片段的PCR扩增结果]()
图 1 tva基因3个片段的PCR扩增结果M：DL2000 marker；1~3：tva基因的1、2和3片段Figure 1. PCR amplified results of three fragments of tva geneM: DL2000 marker; 1−3: Fragment 1, 2 and 3 of tva gene![图 2 tva c.3G>A突变位点不同基因型测序图]()
图 2 tva c.3G>A突变位点不同基因型测序图红色加粗标注为tva基因DNA序列第260位碱基由G突变为AFigure 2. Sequence traces for tva c.3G>A mutation sites of different genotypesThe positions of the mutates from G to A at 260th base of tva gene are underlined in red bold![图 3 tva c.3G>A突变位点不同基因型血样全长tva编码序列的RT-PCR扩增结果]()
图 3 tva c.3G>A突变位点不同基因型血样全长tva编码序列的RT-PCR扩增结果M：DL2000 marker；1：野生型tva c.3G/G；2：纯合突变型tva c.3A/AFigure 3. RT-PCR amplified results of the full-lengthtva coding sequences for blood samples from different genotypes of tva c.3G>A mutation siteM: DL2000 marker; 1: Wild type tva c.3G/G; 2: Homozygous mutant tva c.3A/A2.2 tva c.3G>A突变致宿主体外抗ALV-A感染
RCASBP(A)-EGFP重组质粒转染DF-1细胞48 h后，用倒置荧光显微镜观察到GFP荧光标记蛋白表达，表明成功拯救了RCASBP(A)-GFP荧光报告病毒(图4)。为探究tva c.3G>A突变对宿主细胞体外感染ALV-A的影响，利用拯救的RCASBP(A)-GFP荧光报告病毒分别感染tva c.3G/G、tva c.3G/A和tva c.3A/A CEF，感染后不同时间点利用流式细胞术检测RCASBP(A)-GFP对tva c.3G>A突变位点不同基因型CEF的感染情况，结果如图5和6所示。野生型tva c.3G/G CEF和杂合突变型tva c.3G/A CEF对RCASBP(A)-GFP病毒易感，而纯合突变型tva c.3A/A CEF抗RCASBP(A)-GFP的感染，表明tva c.3G>A突变导致宿主体外抗RCASBP(A)-GFP的感染。
![图 4 RCASBP(A)-EGFP质粒转染DF-1细胞48 h后的绿色荧光蛋白表达]()
图 4 RCASBP(A)-EGFP质粒转染DF-1细胞48 h后的绿色荧光蛋白表达绿色荧光为表达 GFP 的细胞；标尺=250 μmFigure 4. Expression of green fluorescent protein in DF-1 cells after transfection with RCASBP(A)-EGFP plasmid for 48 hGreen fluorescence represent cells expressing GFP; Scale =250 μm![图 5 tva c.3G/G、tva c.3G/A和tva c.3A/A CEF感染RCASBP(A)-EGFP的过程]()
图 5 tva c.3G/G、tva c.3G/A和tva c.3A/A CEF感染RCASBP(A)-EGFP的过程Figure 5. Time course of infection of tva c.3G/G, tva c.3G/A and tva c.3A/A CEFs with RCASBP(A)-EGFP![图 6 流式细胞术检测tva c.3G>A突变位点不同基因型CEF感染RCASBP(A)-GFP 7 d后的GFP阳性细胞率]()
图 6 流式细胞术检测tva c.3G>A突变位点不同基因型CEF感染RCASBP(A)-GFP 7 d后的GFP阳性细胞率Figure 6. GFP positive cell rates for CEFs of different genotypes of tva c.3G>A mutation site seven days after infection with RCASBP(A)-GFP detected by flow cytometry2.3 tva c.3G>A突变致宿主体内抗ALV-A感染
为探究tva c.3G>A突变对宿主体内感染ALV-A的影响，利用ALV-A野毒感染tva c.3G>A突变野生型、杂合突变型、纯合突变型雏鸡。作为阳性对照，9只SPF雏鸡攻ALV-A野毒后均为ALV-A阳性，说明ALV-A体内攻毒试验成立。ALV-A攻毒试验结果显示，野生型tva c.3G/G雏鸡(25只)攻ALV-A野毒后均为ALV-A阳性，杂合突变型tva c.3G/A雏鸡(28只)攻ALV-A野毒后病毒血症阳性率为75%，而纯合突变型tva c.3A/A雏鸡(22只)攻ALV-A野毒后均为ALV-A阴性(表2)。结果表明，tva c.3G>A突变导致宿主体内抗ALV-A的感染，ALV-A体内攻毒试验结果与ALV-A体外感染试验结果一致，证实tva c.3G>A突变位点为ALV-A的遗传抗性位点。
表 2 ALV-A攻毒后雏鸡病毒血症阳性率Table 2. Positive infection rate of viremia in chicks infected by ALV-A雏鸡 Chick 基因型 Genotype 阳性样品数/总样品数 No. of positive samples/Total No. of samples 阳性感染率/% Positive infection rate SPF tva c.3G/G 9/9 100 CB06 tva c.3G/G 25/25 100 tva c.3G/A 21/28 75 tva c.3A/A 0/22 0 2.4 tva c.3G>A抗性位点在不同黄羽肉鸡品系的基因型频率分布
不同黄羽肉鸡品系tva c.3G>A抗性位点的基因分型结果如表3所示。CB01、CB08、CB10和CB15品系中检测到tva c.3G>A抗性位点的杂合基因型tva c.3G/A，其频率分别为0.05、0.05、0.10和0.14，在CB01、CB08、CB10和CB15品系中检测到纯合抗性基因型tva c.3A/A，其频率分别为0.10、0.15、0.23和0.08，其余黄羽肉鸡品系所检样品均为野生型tva c.3G/G。
表 3 我国黄羽肉鸡品系 tva c.3G>A抗性位点的基因型频率分布Table 3. Genotypic frequency of tva c.3G>A resistance locus in Chinese yellow-feathered broiler lines品系 Line 样品数/只 No. of samples 基因型 Genotype tva c.3G/G tva c.3G/A tva c.3A/A CB01 60 0.85 0.05 0.10 CB02 50 1 0 0 CB03 36 1 0 0 CB04 30 1 0 0 CB05 48 1 0 0 CB06 60 1 0 0 CB07 30 1 0 0 CB08 60 0.80 0.05 0.15 CB09 30 1 0 0 CB10 60 0.67 0.10 0.23 CB11 35 1 0 0 CB12 45 1 0 0 CB13 60 1 0 0 CB14 30 1 0 0 CB15 36 0.78 0.14 0.08 3. 讨论与结论
本研究鉴定了中国黄羽肉鸡品系tva受体基因中存在1种自然突变位点，即tva基因编码区第3位碱基由G突变为A，引起tva基因起始密码子序列由ATG突变为ATA，致使宿主对ALV-A体外、体内的感染产生遗传抗性，证实tva c.3G>A突变位点为ALV-A的遗传抗性位点。据我们所知，本研究首次报道了受体基因起始密码子序列突变可以引起宿主遗传性抗特定ALV亚群的感染，这增强了我们对ALV-A宿主共同进化的理解。
尽管种鸡群净化策略和生物安全措施已用来控制禽白血病[20]，但这些传统方法并不能完全消除禽白血病在中国和东南亚国家的发生与流行[21-22]。ALV在鸡群中的流行，致使暴露于ALV的宿主可能会受到选择性压力而对ALV的感染产生完全抗性或者至少降低了对 ALV 感染的易感性。tva、tvb、tvc和chNEH1受体基因分别编码Tva、Tvb、Tvc和NHE1受体蛋白，分别介导宿主细胞对ALV-A、ALV-B/D/E、ALV-C和ALV-J的感染[14, 23-25]。受体基因的遗传突变会导致受体蛋白的完全缺失或表达一个缺陷型受体蛋白，从而引起宿主对ALV的感染产生遗传抗性。目前，已在国外某些白来航近交品系和中国肉鸡品系中鉴定了致使宿主细胞对ALV-A、ALV-B/D/E和ALV-C感染产生遗传抗性的受体基因遗传突变[16, 24,26]。Elleder等[16]在白来航品系C中发现tva基因第619碱基由C变为G(tvar1)，引起编码的氨基酸由Cys改变为Trp，大大降低ALV-A囊膜糖蛋白与Tva受体的亲和力，从而对ALV-A的感染产生遗传抗性。Elleder等[16]在白来航品系72中发现tva基因碱基序列第305—306位点插入CTCG 4个碱基(tvar2)，导致Tva受体蛋白缺失表达，从而产生对ALV-A的抗性。Chen等[18]研究发现中国鸡种存在tvar3、tvar4、tvar5和tvar6 ALV-A遗传抗性位点，其为位于tva受体基因内含子1破裂点信号保守区域的4种缺失突变，tvar3为tva基因第507—516碱基(序列为ACCCCGCCCC)缺失、tvar4为tva基因第507—511碱基(序列为ACCCC)缺失、tvar5为tva基因第502—511碱基(序列为CGCTCACCCC)缺失、tvar6为tva基因第502—516碱基(序列为CGCTCACCCCGCCCC)缺失，这4种tva受体基因遗传突变均影响tva基因mRNA的剪切，降低了ALV-A囊膜糖蛋白与Tva受体蛋白结合的亲和力，从而降低宿主细胞对ALV-A的易感性。本研究在我国黄羽肉鸡品系中发现tva受体基因始密码子序列由ATG突变为ATA，引起Tva受体蛋白氨基酸序列第1个氨基酸由甲硫氨酸(M)改变为异亮氨酸(I)，推测该突变导致Tva受体蛋白的表达完全缺失，从而引起宿主抗ALV-A的感染。
抗病育种是控制禽白血病的有效策略和重要途径，为评估不同黄羽肉鸡品系对ALV-A的遗传抗性，本研究对15个黄羽肉鸡品系tva c.3G>A抗性位点进行了基因分型，结果发现CB01、CB08、CB10和CB15品系存在tva c.3G>A抗性位点，其纯合抗性基因型tva c.3A/A的频率分别为0.10、0.15、0.23和0.08，提示这些黄羽肉鸡品系具有良好的抗 ALV-A 遗传改良潜力，可从这些鸡品系中筛选出培育抗ALV-A感染的育种素材，并运用于ALV-A 遗传抗性鸡品种的选育，为实现A亚群禽白血病抗病育种提供理论依据和技术支撑。
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图 1 长链脂肪酸对小肠上皮细胞脂肪酸摄取及CD36膜定位的影响
A：ModeK细胞的BODIPY染色结果图，比例尺为50 μm；B：BODIPY平均荧光强度统计图，****表示差异达到P<
0.0001 的显著水平(One-way ANOVA)；C：CD36膜定位免疫荧光染色图，比例尺为50 μm。Figure 1. Effects of long-chain fatty acids on lipid uptake by intestinal epithelial cells and the membrane localization of CD36
A: Image of BODIPY staining results of ModeK cells, the scale bar is 50 μm; B: BODIPY average fluorescence intensity statistical chart, **** indicates significant difference at P<
0.0001 level (One-way ANOVA); C: CD36 membrane localization immunofluorescence staining image, the scale bar is 50 μm.![]()
图 2 长链脂肪酸对小肠上皮细胞质膜上CD36棕榈酰化水平的影响
A：质膜上棕榈酰化CD36的蛋白条带，B：条带的灰度值统计图，相对密度代表Palm-CD36/Input-CD36的相对表达量，*和***分别表示差异达到P< 0.05和P<0.001的显著水平(One-way ANOVA)。
Figure 2. Effects of long-chain fatty acids on the palmitoylation levels of CD36 on the plasma membrane of intestinal epithelial cells
A: Protein band of palmitoylated CD36 on the plasma membrane, B: Histogram of band gray value, relative density indicates the Palm-CD36/Input-CD36 expression ratio, * and *** indicate significant differences at P<0.05 and P<0.001 levels, respectively (One-way ANOVA).
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图 3 棕榈酰化阻断剂对肠上皮细胞摄取不同长链脂肪酸的影响
A：ModeK细胞的BODIPY染色结果图，比例尺为50 μm； B：BODIPY平均荧光强度统计图，***和****分别表示差异达到P<0.001和P<
0.0001 的显著水平(t检验)。Figure 3. Effects of palmitoylation inhibitor on the uptake of different long-chain fatty acids in intestinal epithelial cells
A: Image of BODIPY staining results of ModeK cells, the scale bar is 50 μm; B: BODIPY average fluorescence intensity statistical chart, *** and **** indicate significant differences at P<0.001 and P<
0.0001 levels, respectively (t-test).![]()
图 4 不同油脂对小肠脂质摄取及吸收的影响
A：空肠组织的BODIPY染色结果图，比例尺为100 μm，B：BODIPY平均荧光强度统计图，*和****分别表示差异达到P< 0.05和P<0.0001的显著水平(One-way ANOVA)； C：血清中甘油三酯(TG)浓度，*表示差异达到P<0.05的显著水平(t检验)。
Figure 4. Effect of different oils on lipid uptake and absorption in small intestinal epithelial cells
A: Image of BODIPY staining results in jejunal tissue, the scale bars is 100 μm. B: BODIPY average fluorescence intensity statistical chart, * and **** indicate significant differences at P< 0.05 and P<
0.0001 levels, respectively (One-way ANOVA); C: Triglyceride (TG) concentration in serum, * indicates significant difference at P<0.05 level (t-test).![]()
图 5 不同油脂对空肠组织质膜上CD36棕榈酰化水平的影响
A：质膜上棕榈酰化CD36的蛋白条带，B：条带的灰度值统计图，相对密度代表Palm-CD36/Input-CD36的相对表达量，*表示差异达到P<0.05的显著水平(One-way ANOVA)。
Figure 5. Effects of various oils on the palmitoylation levels of CD36 on the plasma membrane of jejunal tissue
A: Protein band of palmitoylated CD36 on the plasma membrane; B: Histogram of band gray value, relative density indicates the Palm-CD36/Input-CD36 expression ratio, * indicates significant difference at P<0.05 level (One-way ANOVA).
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