The effect of recombinant mud carp insulin-like growth factor-Ⅰ (IGF-Ⅰ) on expression of growth hormone(GH)in mud carp, Cirrhinus molitorella
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摘要:
使用半定量RT-PCR方法检测了鲮各组织中GH mRNA的表达,并分析重组鲮IGF-Ⅰ处理后鲮GH表达变化情况。结果表明:鲮的GH mRNA表达具有明显的组织特异性,在鲮的肝、肾、脾、肌肉、肠、鳃、性腺、心脏和皮肤中都未检测到GH mRNA表达,只在鲮脑中检测到GH mRNA表达;经重组鲮IGF-Ⅰ处理后,鲮脑中GH mRNA稍有升高,但在肝和肌肉中仍未检测到GH mRNA表达;重组鲮IGF-Ⅰ处理前后,鲮血清中GH浓度变化不明显。
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关键词:
- 鲮 /
- 生长激素 /
- 胰岛素样生长因子-Ⅰ /
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Abstract:The study was conducted to examine the expression of GH mRNA in different tissues of mud carp by semi-quantitative RT-PCR and the effects of recombinant mud carp insulin-like growth factor-Ⅰ(rmcIGF-Ⅰ) on expression of GH in mud carp. GH mRNA detection result of brain of mud carp was positive, and the other tissues detections results were negative, including liver, kidney, spleen, muscle, intestine, gill, gonad, heart and skin. GH mRNA levels increased a little in brain in response to rmcIGF-Ⅰ, but the treated fish exhibited no obvious change in sera GH concentration following rmcIGF-Ⅰ injection.
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Keywords:
- mud carp /
- Cirrhinus molitorella /
- GH /
- IGF-Ⅰ
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在脊椎动物中,生长激素(growth hormone,GH)和胰岛素样生长因子-Ⅰ(insulin-like growth factor-Ⅰ,IGF-Ⅰ)是2种重要的生长因子,它们在动物的生长、发育、生殖和代谢调控中起重要作用。GH是一种由单一肽链构成的非糖多肽激素,由垂体前叶的生长激素细胞合成和分泌。GH分泌活动受到脑特别是下丘脑产生的生长释放激素(growth hormone releasing hormone,GHRH)和生长激素释放抑制激素(growth hormone release inhibiting hormone,GHIH)这2种激素的调控[1]。鱼类胰岛素样生长因子-Ⅰ(IGF-Ⅰ)是一种由70个氨基酸组成的蛋白质,其生物活性受IGF-Ⅰ受体及IGF-Ⅰ结合蛋白(IGFBPs)调节。肝脏是鱼类IGF-ⅠmRNA的主要分泌表达位点,但大部分肝外组织也有IGF-Ⅰ的表达,以旁分泌和自分泌的模式发挥作用[2-4]。近年来国内外对鱼类的GH/IGF-Ⅰ的分子调控及信号转导的机理进行了广泛的研究,并取得了一定进展[5-6],而有关鱼类自身GH和IGF-Ⅰ的相互影响研究甚少。
鲮(Cirrhinus molitorella)是我国南方主要淡水养殖鱼类之一,在我国台湾、越南、菲律宾等国家和地区亦有养殖。为深入研究鲮GH的表达调控机理及GH/IGF-Ⅰ的相互作用,本研究在克隆鲮GH和IGF-ⅠcDNA序列的基础上[3, 6],首先检测了鲮GH mRNA组织表达,并研究重组鲮IGF-Ⅰ对其GH mRNA表达影响。
1. 材料与方法
1.1 重组鲮IGF-Ⅰ
重组鲮IGF-Ⅰ由本实验室参照ZHANG等[7]的研究进行表达和纯化,纯化后重组鲮IGF-Ⅰ充分透析除盐,冷冻干燥,溶解在0.7%的生理盐水中待用。
1.2 实验动物处理以及组织的提取
将鲮(500±10 g)从广州市番禺区鱼窝头镇养殖池塘中捕捞起后暂养于广东省鲮鱼原种场,待其状态稳定后,按鱼体重120 μg·g-1注射重组鲮IGF-Ⅰ或0.7%的生理盐水(对照),12 h后取鲮脑、肝和肌肉保存于液氮中,对照组取脑、肝、肌肉、肠、性腺、鳃、皮肤、脾脏、心脏和肾10种组织保存于液氮;并从每条鱼中抽取血液(大约3 mL)4℃冷凝30 min,4℃ 1 500 g离心15 min,取上清保存于-20℃,待检测GH和IGF-Ⅰ含量[8-9]。
1.3 总RNA的提取与RT-PCR
按Trizol Reagent(Invitrogen)操作说明提取各组织总RNA,用0.5×TBE配置1%琼脂糖凝胶,取2 μL总RNA与2 μL 2×DEPC处理后的加样缓冲液混合,65℃加热15 min后,立即于冰上放置2 min,加样电泳,鉴定所提取总RNA的质量。取2 μL总RNA测定光密度OD260和OD280值,计算总RNA浓度。
进行RT-PCR前必需测RNA浓度,逆转录体系对RNA量有一定要求,本研究按逆转录试剂盒TakaRa RNA LA PCRTM Kit(AMV)Ver 1.1说明书要求,所用总RNA量全部为500 ng。
按逆转录试剂盒TakaRa RNA LA PCRTM Kit(AMV)Ver.1.1方法进行逆转录:MgCl2 2 μL,10×RNA PCR缓冲液1 μL,DEPC处理H2O 4.25 μL,dNTP混合物(各10 mM)1 μL,RNA酶抑制剂0.25 μL,AMV逆转录酶0.5 μL,Oligo(dT)接头引物0.5 μL,总RNA 0.5 μL(500 ng)。50℃ 45 min,99℃ 5 min,5℃ 5 min,一个循环。
1.4 PCR循环数的确定及鲮GH mRNA组织表达
首先确定PCR扩增反应条件,即确定PCR扩增反应循环数[9]。用对照组肝cDNA作模板,引物:Actin-F,Actin-R(表 1)(该引物特异性强),94℃变性30 s,54℃退火30 s,72℃延伸45 s,每PCR循环数为15、20、25、30、35、40、45取4 μL,PCR产物在1.5%的琼脂糖凝胶电泳EB染色分析。循环数确定后利用看家基因(在鲮各组织中都表达)Actin做内标,PCR检测鲮GH mRNA各组织表达情况以及重组鲮IGF-Ⅰ处理后GH mRNA在脑、肝和肌肉的表达,所有PCR反应体系包括1 μL(0.15 μg)cDNA,0.25 μL rTaq酶(TaKaRa公司),正反向引物各1 μL,4 μL dNTP,5 μL 10×PCR缓冲液,双蒸水37.75 μL,总体积50 μL。PCR反应均94℃变性5 min,35个循环(94℃变性30 s,54℃退火30 s,72℃延伸45 s),最后72℃延伸10 min。GH mRNA检测引物序列见表 1。
表 1 扩增GH、Actin的引物序列Table 1. Oligonucleotide primers used to amplify cDNA for mud carp GH and Actin引物primer 序列sequence Actin-F: 5′-GTGTTGGCG/ATACAGGTCCTTACG-3′ Actin-R: 5′-CAGACTACCTC/GATGAAGATCCTGAC-3′ GH-F: 5′-ATGGAAAACCAGCGCCTCTTC-3′ GH-R: 5′-TGCATGTCCTTCTTGAAGCAAG-3′ 1.5 各组织mRNA表达检测结果分析
利用GeneTools软件分析,计算出步骤1.4中所有GH和Actin条带的相对质量,再将对应的GH与Actin质量相比,得出GH的相对表达量。
1.6 GH含量的检测
利用化学发光法(德普DPC)测定血清中GH浓度,利用放射性免疫法测定(放射免疫计数器SN679)血清中IGF-Ⅰ的含量[8]。
2. 结果
2.1 总RNA提取与RT-PCR
从100 mg鲮组织中提取RNA,经1%琼脂糖凝胶电泳检测,可见清晰的28S、18S条带(图 1),表明总RNA完整性很好。根据测定RNA的OD260和OD280值,计算其比值,比值均在1.9~2.0之间,也说明RNA纯度较高,可用于后续反应。
2.2 PCR循环数的确定
首先确定PCR反应条件,通过鲮的分子内标Actin基因的相对表达量确定PCR扩增反应循环数,用于检测鲮GH mRNA组织表达。由图 2结果可知,PCR循环数在25以下时,Actin的扩增量较低,电泳带不清晰;循环数在30以上时结果较好,循环数为35最为理想。
2.3 鲮GH mRNA组织表达与重组鲮IGF-Ⅰ对鲮GH mRNA组织表达的影响
运用半定量RT-PCR方法只在脑组织中检测到GH mRNA表达,而在鲮肝、肌肉、肾、皮肤、性腺、肠、鳃、心脏和脾组织都没有检测到GH mRNA表达;按照鱼体重120 μg·g-1注射重组鲮IGF-Ⅰ,12 h后,取肝脏、脑、肌肉,用Actin作内标,检测重组鲮IGF-Ⅰ处理后鲮肝、脑、肌肉组织GH mRNA表达变化。经重组鲮IGF-Ⅰ处理的鲮脑组织GH mRNA表达水平有所升高,在鲮肌肉和肝脏组织中仍未检测到GH表达(图 3)。
图 3 半定量RT-PCR检测重组鲮IGF-Ⅰ处理前后鲮组织组织Actin和GH mRNA的组织表达B1. 半定量RT-PCR检测对照组中鲮组织Actin mRNA表达;B2. 半定量RT-PCR检测对照组中鲮组织GH mRNA表达;C1. 半定量RT-PCR检测重组鲮IGF-Ⅰ处理组中鲮组织Actin mRNA表达;C2. 半定量RT-PCR检测重组鲮IGF-Ⅰ处理组中鲮组织GH mRNA表达;D. 鲮组织GH mRNA的相对表达量;M. 100 bp DNA分子量标准;N. 负对照(不加模板);1~10.脑,肝,肌肉,肾,肠,鳃,脾,性腺,心脏,皮肤Figure 3. Semi-quantitative RT-PCR analysis of expression of Actin and GH mRNA in various tissues in mud carpB1. Actin mRNA detection in various tissues by semi-quantitative RT-PCR in control group; B2. GH mRNA detection in various tissues by semi-quantitative RT-PCR in control group; C1. Actin mRNA detection in various tissues by semi-quantitative RT-PCR in rmcIGF-Ⅰ in treated group; C2. GH mRNA detection in various tissues by semi-quantitative RT-PCR in rmcIGF-Ⅰin treated group; D. relative quantity of GH mRNA in various tissues in mud carp; M. 100 bp DNA molecular weight Marker; N. a negative control(no template); 1~10. brain, liver, muscle, kidney, intestine, gill, spleen, gonad, heart, skin2.4 血清中GH的含量
利用化学发光法测定血清中GH的含量,对照组中血清中GH的含量为0.037±0.0067 ng·mL-1,重组鲮IGF-Ⅰ处理组中血清中GH的含量为0.04±0.0058 ng·mL-1,经t检验,表明处理前后血清中GH含量不存在显著性差异;利用放射性免疫法测定血清中IGF-Ⅰ的含量,对照组中血清中IGF-Ⅰ的含量为145.59±21.84 ng·mL-1,重组鲮IGF-Ⅰ处理组中血清中IGF-Ⅰ的含量为302.16±29.24 ng·mL-1,经t检验,表明处理后血清中IGF-Ⅰ含量显著升高(表 2)。
表 2 对照组和重组鲮IGF-Ⅰ处理组鲮血清中GH和IGF-Ⅰ浓度Table 2. The GH and IGF-Ⅰconcentration in sera in control and rmcIGF-Ⅰtreated fish浓度±S.E.(n=3)/ ng·mL-1 concentration±S.E.(n=3) 对照组
control重组鲮IGF-Ⅰ处理组
rmcIGF-Ⅰtreated groupP值 生长激素含量
GH concentration0.037±0.0067 0.04±0.0058 P=1.000>0.05 胰岛素样生长因子含量
IGF-Ⅰconcentration145.59±21.84 302.16±29.24 P=0.013<0.053 3. 讨论
采用半定量RT-PCR方法对鲮GH mRNA的检测结果显示,只在鲮脑组织中检测到GH mRNA表达,而在鲮肝、肌肉、肾、皮肤、性腺、肠、鳃、心脏和脾组织中都未检测到GH mRNA表达;经重组mcIGF-Ⅰ处理,脑组织中鲮GH mRNA表达水平有所升高,而在鲮肌肉和肝脏中仍未检测到GH mRNA表达。由内标Actin在鲮各个组织中的表达量可以推断出,在鲮肝、肌肉、肾、皮肤、性腺、肠、鳃、心脏和脾脏组织中没有检测到GH mRNA以及处理前后鲮脑GH mRNA表达的变化,并不是因为从样品中抽提的RNA质量以及逆转录问题。重组mcIGF-Ⅰ处理前后,脑GH mRNA表达稍有升高,可能是因为重组mcIGF-Ⅰ在一定程度上刺激鲮脑分泌GH。
在哺乳动物中的研究表明[9-10],人IGF-Ⅰ对罗非鱼和虹鳟GH释放和表达都有抑制作用。LEEDOM等[11]利用重组牛生长激素(recombinant bovine somatotropin,rbST)(120 μg·g-1鱼体重)刺激罗非鱼,经刺激的罗非鱼血清GH不变;BIGA等[12]利用同剂量rbST刺激虹鳟,发现虹鳟性腺GH mRNA表达升高。MELAMED等[13]研究表明,大麻哈鱼促性腺激素释放激素(salmon gonadotropin-releasing hormone,sGnRH)对罗非鱼GH的释放有促进作用,但对其GH mRNA水平没有影响;LI等[1]研究发现,sGnRH既促进草鱼GH的释放,又刺激脑GH mRNA的表达。RILY等[14]发现经老鼠促生长激素释放激素(ghrelin,GHN)2周处理,罗非鱼垂体分泌的GH升高,但其脑垂体mRNA水平却没有变化;UNNIAPPAN和PETER[15]研究发现金鱼GHN可以促进其脑垂体GH mRNA的表达。由这些结论推断GH的表达和分泌可能与物种的特异性有关。PETEZ-SANCHEZ等[10]利用重组人IGF-Ⅰ处理虹鳟,发现重组人IGF-Ⅰ对虹鳟GH的释放有抑制作用。本研究中重组鲮IGF-Ⅰ对脑GH mRNA表达影响与文献报道的结论不尽一致,这表明GH与IGF-Ⅰ的表达、分泌机理及其相互作用远比我们目前所了解的情况复杂。
本实验中,外源IGF-Ⅰ的处理使鲮血清GH浓度变化不明显,并且重组鲮GH对鲮血清中GH浓度影响也不明显(数据另文发表)。这些结果与LEEDOM等[11]用重组牛生长激素处理罗非鱼,内源GH变化不明显的结论一致。
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图 3 半定量RT-PCR检测重组鲮IGF-Ⅰ处理前后鲮组织组织Actin和GH mRNA的组织表达
B1. 半定量RT-PCR检测对照组中鲮组织Actin mRNA表达;B2. 半定量RT-PCR检测对照组中鲮组织GH mRNA表达;C1. 半定量RT-PCR检测重组鲮IGF-Ⅰ处理组中鲮组织Actin mRNA表达;C2. 半定量RT-PCR检测重组鲮IGF-Ⅰ处理组中鲮组织GH mRNA表达;D. 鲮组织GH mRNA的相对表达量;M. 100 bp DNA分子量标准;N. 负对照(不加模板);1~10.脑,肝,肌肉,肾,肠,鳃,脾,性腺,心脏,皮肤
Figure 3. Semi-quantitative RT-PCR analysis of expression of Actin and GH mRNA in various tissues in mud carp
B1. Actin mRNA detection in various tissues by semi-quantitative RT-PCR in control group; B2. GH mRNA detection in various tissues by semi-quantitative RT-PCR in control group; C1. Actin mRNA detection in various tissues by semi-quantitative RT-PCR in rmcIGF-Ⅰ in treated group; C2. GH mRNA detection in various tissues by semi-quantitative RT-PCR in rmcIGF-Ⅰin treated group; D. relative quantity of GH mRNA in various tissues in mud carp; M. 100 bp DNA molecular weight Marker; N. a negative control(no template); 1~10. brain, liver, muscle, kidney, intestine, gill, spleen, gonad, heart, skin
表 1 扩增GH、Actin的引物序列
Table 1 Oligonucleotide primers used to amplify cDNA for mud carp GH and Actin
引物primer 序列sequence Actin-F: 5′-GTGTTGGCG/ATACAGGTCCTTACG-3′ Actin-R: 5′-CAGACTACCTC/GATGAAGATCCTGAC-3′ GH-F: 5′-ATGGAAAACCAGCGCCTCTTC-3′ GH-R: 5′-TGCATGTCCTTCTTGAAGCAAG-3′ 表 2 对照组和重组鲮IGF-Ⅰ处理组鲮血清中GH和IGF-Ⅰ浓度
Table 2 The GH and IGF-Ⅰconcentration in sera in control and rmcIGF-Ⅰtreated fish
浓度±S.E.(n=3)/ ng·mL-1 concentration±S.E.(n=3) 对照组
control重组鲮IGF-Ⅰ处理组
rmcIGF-Ⅰtreated groupP值 生长激素含量
GH concentration0.037±0.0067 0.04±0.0058 P=1.000>0.05 胰岛素样生长因子含量
IGF-Ⅰconcentration145.59±21.84 302.16±29.24 P=0.013<0.053 -
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