CN112790154B - 一种高温应激诱导小黄鱼肝脏细胞凋亡模型的构建方法 - Google Patents
一种高温应激诱导小黄鱼肝脏细胞凋亡模型的构建方法 Download PDFInfo
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Abstract
本发明属于氧化应激模型技术领域,具体涉及一种高温应激诱导小黄鱼肝脏细胞凋亡模型的构建方法,具体为在养殖水温为20℃左右时,按照2℃/h的升温速率处理小黄鱼,水温上升至32±0.5℃,此温度处理6‑12h,使其发生氧化应激反应诱导细胞凋亡。本发明明确了高温应激前的基础水温、升温速率及需要达到的高温及其处理时间,提出了模型建立的评判指标。通过高温应激建立的小黄鱼肝脏细胞凋亡模型,可以用于研究小黄鱼肝脏氧化损伤的发病机制,对于揭示小黄鱼肝脏氧化损伤机制及研发肝脏高效保护药物也具有重要的理论和实际意义。
Description
技术领域
本发明属于氧化应激模型构建技术领域,具体涉及一种高温应激诱导小黄鱼肝脏细胞凋亡模型的构建方法。
背景技术
小黄鱼作为我国“四大海产”之一,具有非常重要的经济价值。随着规模化人工繁殖及养殖技术的日趋成熟,小黄鱼养殖业将得到快速发展。在养殖过程中,水体温度直接关系到养殖对象的成活率、生长速度等。已有研究表明水温32℃会导致小黄鱼产生氧化应激反应,严重时将导致其大量死亡。因此,阐明小黄鱼高温应激响应的分子机制、提供有效的缓解高温应激的技术手段,对于小黄鱼的推广、健康养殖和科学管理至关重要。
高温诱导机体发生氧化应激反应,造成体内含氧化合物——ROS异常增多,破坏细胞正常结构,引起细胞凋亡等组织损伤。衡量机体氧化应激反应的指标包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)等抗氧化酶,以及丙二醛(MDA)。热休克蛋白GRP94基因表达量可从分子层面反应机体发生应激反应情况。同时,通过测定细胞凋亡比例可直接评估高温应激诱导产生细胞凋亡水平。
目前,研究高温对鱼类肝脏细胞凋亡的影响的在体实验还没有一个统一的技术规范,研究结果差异很大,难以标准化,导致研究结果相互借鉴作用微乎其微。因此,我们以小黄鱼为对象,建立了一个标准的高温诱导的氧化应激实验模型,以便更好地开展小黄鱼响应高温应激的调控机制研究。
发明内容
针对现有技术中存在的问题,本发明设计的目的在于提供一种高温应激诱导小黄鱼肝脏细胞凋亡模型的构建方法。
本发明通过 以下技术方案加以实现:
本发明提供了一种高温应激诱导小黄鱼肝脏细胞凋亡模型的构建方法,在正常养殖水温为20℃左右时,水体溶氧>6.0mg/L以上,按照2℃/h的升温速率对养殖小黄鱼进行温度处理,温度上升至32±0.5℃时停止升温,维持此温度6h以上,为研究高温应激诱导小黄鱼肝损伤的调节机制提供载体。
上述小黄鱼肝脏高温应激模型的构建过程具体为:选取体质健壮、体表无损、6月龄左右全同胞小黄鱼,自然养殖水温在20℃左右时,将实验鱼放入0.3m3实验桶中,每个桶放养数量30-40尾,暂养2周,暂养期间,通过温度调控设备将养殖水温控制在20±0.5℃,溶氧>6.0mg/L,pH=7.7-7.9,每天早晚两次投喂人工配合饲料,饲料颗粒直径3mm左右,进行饱食投喂,投喂后半小时每个桶进行清污,换水70%。实验开始后,利用温度控制仪进行缓慢升温处理,升温速率严格控制为2℃/h,上升至32±0.5℃时,维持此温度,高温处理过程中需增加充氧量,防止温度升高水体溶氧降低,确保溶氧>6.0mg/L,实验过程中小黄鱼停止投喂。分别于达到32℃的0、6、12h,采集肝脏,监测肝脏组织各项指标的变化,所述小黄鱼肝脏氧化应激模型判定标准为:肝脏中SOD、GSH-PX活性先显著升高后显著降低;MDA显著升高;热休克蛋白GRP94基因的表达量显著增加。当小黄鱼肝脏组织各项指标达到氧化应激模型建立标准,并且肝脏细胞凋亡比例明显增加,可判定高温诱导的小黄鱼肝脏细胞凋亡模型构建成功。
本发明通过明确高温应激前的基础水温、升温速率及需要达到的高温及其处理时间,提出了模型建立的评判指标。通过高温应激建立的小黄鱼肝脏细胞凋亡模型,可以用于研究小黄鱼肝脏氧化损伤的发病机制,对于揭示小黄鱼肝脏氧化损伤机制及研发肝脏高效保护药物也具有重要的理论和实际意义。
附图说明
图1为高温应激对小黄鱼肝脏氧化应激指标的影响;
图2为不同处理组GRP94基因表达量测定结果;
图3为Tunel法检测细胞凋亡结果(DAPI 染出来的细胞核在紫外的激发下为蓝色,阳性凋亡细胞核为绿色)。
具体实施方式
以下结合具体实施例对本发明的技术方案进行详细的描述,以便更好地理解本技术方案。
实施例1:实验处理及样品采集
取体质健壮、体表无伤的6月龄小黄鱼全同胞家系,分为对照组C和实验组T,每组3个平行,每个平行放养实验鱼30尾,养殖于0.3m3的养殖桶中,暂养两周,暂养期间水温控制在20±0.5℃,溶氧>6.0mg/L,pH=7.7-7.9,每天早晚两次(07:00和16:00)投喂人工配合饲料,饲料颗粒直径3mm左右,进行饱食投喂,投喂后半小时每个桶进行清污,换水70%。实验开始时,停止投喂,对照组养殖条件不变,实验组通过加热棒,按照2℃/h的恒定速率进行匀速升温,温度达到32℃时,维持此温度,分别于达到此温度的0、6、12h,依次命名为T_0、T_6和T_12组,从实验组和对照组分别取样9尾,每桶取样3尾,麻醉后迅速解剖取肝脏,每份肝脏一式两份,一份用多聚甲醛保存,一份液氮速冻后保存在-80℃,备用。
实施例2:氧化应激指标测定与分析
低温保存的样本,分别按照试剂盒说明书检测SOD、CAT 和GSH-PX活性及MDA 含量。结果显示,32℃处理0h,4个氧化应激指标与对照组无显著差异(P>0.05),此时属于机体接触高温应激早期,还未引起应激反应。32℃处理6h后,抗氧化酶系统开始发挥作用,表现为抗氧化酶SOD和GSH-PX活性显著升高(P<0.05)。由于抗氧化酶的作用,使得脂质过氧化反应的产物 MDA 的含量虽然有所增加,但是与对照组和T_0组之间的差异并不显著。随着高温持续处理12h,机体抗氧化酶系统遭到一定程度破坏,表现为肝脏SOD和GSH-PX活性显著下降,此时,CAT活性和MDA含量则明显升高,见图1,表现出氧化应激反应。
实施例3:内质网关键基因GRP94基因表达量测定与分析
(1)RNA提取和cDNA合成
根据Trizol法提取肝脏样本组织总RNA,并用2.0 %琼脂糖凝胶电泳检测其完整性,使用NanoDrop One超微量分光光度计测量提取的RNA浓度和质量。根据Hifair® III1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)试剂盒说明书合成cDNA第一链,得到的cDNA溶液于-20℃冰箱保存、备用。
(2)基因序列片段的克隆与分析
根据小黄鱼转录组数据,通过生物信息学分析得到小黄鱼GRP94基因CDS参考序列,据此设计引物件表1。
表1 相关引物及其序列
以小黄鱼肝脏cDNA为模板进行GRP94片段的PCR扩增。PCR反应体系如下:模板DNA1μL、上下引物各1uL、2×Mix 12.5μL、ddH2O 9.5μL。hsp90b1基因的PCR反应条件为: 94 ℃预变性3min,94℃变性30s,56℃退火30s,72℃延伸2min30s,35个循环;最后72℃延伸10min。扩增后的产物经2%的琼脂糖凝胶电泳分离后割胶回收目的片段,纯化后的产物与pMD19-T载体连接,用DH5α感受态细胞进行转化,经固体培养基(含氨苄抗生素)培养后,筛选阳性克隆送到生工生物工程(上海)股份有限公司进行测序获得CDS序列。根据获得的CDS序列设计qRT-PCR的引物,见表1。
(3)实时荧光定量PCR
采用Bio-Rad CFX Real-time PCR仪(CFX96,美国),按照Hieff UNICON® qPCRSYBR Green Master Mix(抗体法, No Rox)使用说明,以β-actin为内参引物,对GRP94进行实时定量PCR反应。反应体系如下:Hieff UNICON® qPCR SYBR Green Master Mix(抗体法,No Rox)10μL、上游引物 0.8μL、下游引物0.8μL、模板DNA 2μL、无菌超纯水6.4μL。反应条件为95℃预变性 30s,95℃变性10s,60℃ 退火30s,40个循环。每个样品设置技术重复三次以保证结果准确性,并在反应结束后检查扩增曲线和溶解曲线。实时荧光定量的结果,采用2-△△CT法计算其相对定量值。结果显示,在32℃处理的0h后,GRP94基因表达量有少量增加,但是与对照不显著;32℃持续处理6h后,GRP94基因表达量急剧增加,显著高于对照组和T_0组;温度处理12h后,GRP94基因表达量有所下降,显著低于T_6组,但是仍然显著高于对照组和T_0组,见图2。此研究结果表明,32℃高温处理6h有效诱导了小黄鱼肝脏中热休克蛋白GRP94基因的大量表达,说明了32℃高温处理诱导小黄鱼肝脏发生氧化应激反应。
实施例4:肝脏组织细胞凋亡检测与分析
多聚甲醛固定的样品经过梯度脱水、石蜡包埋、切片、烘干后,获得组织切片。按照细胞凋亡检测试剂盒(FITC)说明步骤进行染色后,于荧光显微镜下观察凋亡细胞(阳性凋亡细胞核为绿色)。结果显示,与对照组相比,温度达到32℃后的0h即可观察到肝脏组织中存在一定数量的凋亡细胞,随着热应激时间的延长,凋亡细胞比例明显增多,见图3,说明32℃高温处理有效诱导了肝脏组织发生细胞凋亡现象。结合前述肝脏组织发生明显的氧化应激反应,可判定高温应激成功诱导小黄鱼肝脏发生细胞凋亡。
Claims (5)
1.一种高温应激诱导小黄鱼肝脏细胞凋亡模型的建立方法,其特征在于包括以下步骤:
1)自然养殖水温在18-22℃时,选取体质健壮、体表无损、5-7月龄全同胞小黄鱼, 置于养殖桶中暂养2周;
2)暂养结束后,利用温度控制仪按照2℃/h的升温速率对养殖小黄鱼进行温度处理,同时增加充氧量,确保溶氧>6.0mg/L,
3)养殖桶温度上升至32±0.5℃时,维持此温度,分别于达到该温度条件下的0、6、12h,采集肝脏,监测肝脏组织各项指标的变化。
2.如权利要求1所述的一种高温应激诱导小黄鱼肝脏细胞凋亡模型的建立方法,其特征在于步骤1)中养殖桶体积为0.3m3,养殖桶放养数量为30-40尾。
3.如权利要求1所述的一种高温应激诱导小黄鱼肝脏细胞凋亡模型的建立方法,其特征在于步骤1)中暂养期间,通过温度调控设备将养殖水温控制在20±0.5℃,溶氧>6.0mg/L,pH=7.7-7.9。
4.如权利要求1所述的一种高温应激诱导小黄鱼肝脏细胞凋亡模型的建立方法,其特征在于步骤1)中暂养期间,每天早晚两次投喂人工配合饲料,进行饱食投喂,投喂后半小时养殖桶进行清污,换水70%。
5.如权利要求1所述的一种高温应激诱导小黄鱼肝脏细胞凋亡模型的建立方法,其特征在于步骤3)中肝脏采集后,每份肝脏一式两份,一份用多聚甲醛保存备用,一份液氮速冻,保存于-80℃的温度条件下备用。
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