The effect of recombinant growth hormone (GH)on insulin-like growth factor-Ⅰ (IGF-Ⅰ) expression in mud carp, Cirrhinus molitorella
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摘要:
使用半定量RT-PCR方法，研究了鲮(Cirrhinus molitorella)各组织中IGF-ⅠmRNA组织表达，并分析重组鲮GH处理后鲮IGF-Ⅰ表达变化情况。结果表明，鲮IGF-ⅠmRNA在肝组织中表达最高，其次是肾和脑，另外在肠、鳃、脾、性腺和心脏组织中也有表达，而在肌肉、皮肤中没有检测到鲮IGF-ⅠmRNA的表达；按120 μg·g-1鱼体重的剂量经腹腔注射重组鲮GH，观察12 h后鲮IGF-Ⅰ表达变化情况，发现经重组鲮GH处理后，鲮肝组织IGF-ⅠmRNA表达水平显著升高，脑组织IGF-ⅠmRNA表达水平也稍有升高，而在肌肉中仍未检测到IGF-ⅠmRNA的表达；重组鲮GH处理前后，对照组中鲮血清中IGF-Ⅰ的含量为145.59±21.84 ng·mL-1，试验组中鲮血清中IGF-Ⅰ的含量为247.71±2.83 ng·mL-1，经t检验，P=0.043 < 0.05，表明重组鲮GH处理前后鲮血清中IGF-Ⅰ的含量存在显著性差异。
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关键词:
- 鲮 /
- 胰岛素样生长因子-Ⅰ /
- 生长激素
Abstract:Expression patterns of IGF-ⅠmRNA in different tissues of mud carp (Cirrhinus molitorella)were studied using semi-quantitative RT-PCR method and the effects of recombinant mud carp growth hormone(rmcGH)on expression of IGF-Ⅰin mud carp were investigated. The IGF-ⅠmRNA was detected successfully in liver, kidney, brain, intestine, gill, spleen, gonad, and heart, and no IGF-ⅠmRNA was detected in skin and muscle. The IGF-Ⅰ mRNA detected was higher in liver than in other tissues. Mud carp were received either intra-peritoneal injections of rmcGH (120 μg·g-1 body weight) or vehicle, tissue samples and blood were collected 12 hour later. Total RNA was isolated and assayed for IGF-ⅠmRNA using semi-quantitative RT-PCR. Blood was extracted to determine the levels of IGF-Ⅰ in serum. IGF-Ⅰlevels increased from 145.59±21.84 to 247.71±2.83 ng·mL-1 (P=0.043) in the serum after rmcGH injection.
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Keywords:
- mud carp /
- Cirrhinus molitorella /
- insulin-like growth factor-Ⅰ /
- growth hormone
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鳀鱼(Engraulis japonicas)属鳀科(Engranlidae)、鳀属(Engraulis)，是一种小型中上层游泳鱼类[1]。广泛分布于北起库页岛，南至台湾海峡的太平洋西北部水域。我国主要分布在渤海、黄海和东海海域[2]。作为一种小型中上层鱼类，鳀鱼的数量变动容易受到各种环境因素的影响。
亲体-补充量关系(stock and recruitment relationship，SRR)是渔业资源评估和管理的基础[3-4]之一。依据SRR模型也可以计算和确定许多重要的渔业管理目标值，如最大持续产量(maximal sustainable yield，MSY)，和维持MSY所需的亲体量等。然而，由于补充量的剧烈波动、亲体量的测量误差以及时间序列的相关性和空间分布上的变动性等常常使参数的估计及模型的选择存在很大的不确定性[4]。SRR的估计是渔业资源评估中最困难的任务之一。
渔业生物的亲体数量对种群的补充量大小具有重要的影响，BEVERTON-HOLT[5]和RICKER[6]先后确立了SRR，建立了相应的鱼类繁殖模型，这些模型长期为渔业生物学家在不同类型的渔业种群中应用[7]。此文选用对环境因子响应比较显著的Ricker模型对资源数据进行拟合[8]。
在以前对黄海鳀鱼的亲体-补充量(stock and recruitment，SR)研究当中，研究者都将各种环境因素综合到一个误差项中进行研究。此文尝试将各个环境因子独立出来，再通过多元回归方法进行分析，探讨其对于SRR的影响程度。
1. 材料与方法
1.1 数据来源
1.1.1 黄海鳀鱼SR数据
以ZHAO等[8]研究中1987~2002年的渔业声学调查数据和渔获量统计数据及所估算的自然死亡系数和成时间序列的SR数据作为此文的数据来源(图 1)。选取1990~2001年间的12组SR数据作为观测数据。
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图 1 1987~2001年黄海鳀鱼亲体-补充量数据散点图图中连线连接相邻年份的补充数据Figure 1. Scatter plot of the stock and recruitment data for the anchovy (E.japonicas) stock in the Yellow SeaThe line joins recruitment values with the year indicated.以1龄鱼的个体数量作为补充量的量度指标。用前一年度冬季鳀鱼声学调查测得的越冬群体生物量与前一年度鳀鱼1/2的年渔获量之差作为亲体量的度量指标。其中前一年度1月份鳀鱼越冬群体的生物量则是在相应生物学评估结果基础上，经过以VPA返算的补充量对其1龄鳀鱼的生物量进行调整而得[8]。假设雌雄个体比例为1 : 1，每个成熟个体的生殖力与体重呈简单的正比函数关系。
1.1.2 水温、营养盐数据
黄海水文状况以国家海洋局黄海千里岩海洋环境监测站的观测数据为指标。采用1990~2001年间黄海千里岩海区的表层水温(sea surface temperature，SST)(图 2)和磷酸盐浓度(phosphate concentration，PC)数据(图 3)作为此研究的原始数据。
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图 2 黄海千里岩1990~2001年4~7月份平均日表层水温数据(折线图)与黄海鳀鱼补充量数据(柱状图)变化趋势图Figure 2. Variations of mean SST from April to July (line) and recruitment (bar) observed at the Qianliyan Marine Environment Monitoring Station in the Yellow Sea in the period of 1990~2001![]()
图 3 黄海千里岩1990~2001年平均磷酸盐浓度数据(折线图)与黄海鳀鱼补充量数据(柱状图)变化趋势图Figure 3. Variations of mean phosphates concentration (line) and recruitment (bar) observed at the Qianliyan Marine Environment Monitoring Station in the Yellow Sea in the period of 1990~20011.2 方法
1.2.1 SRR模型
选用通常使用的研究亲体数量(spawning stock biomass，SSB)和补充量(recruitment，R)之间关系的Ricker模型[8]，并使用多元回归的分析方法筛选了黄海千里岩海域逐月的SST、年平均SST、年平均PC等环境数据。
Ricker的SRR模型表达式为：R=αSe-βS
其中α、β是模型中的待定参数。
用环境条件指数$\alpha_i=\alpha_0+\sum\limits_{i=1}^n \alpha_i X_i$取代模型中的参数α，Ricker模型的表达形式变为：
$$ R=\left(\alpha_0+\sum\limits_{i=1}^n \alpha_i X_i\right) S_t \exp \left(-\beta S_t\right) $$ α0，α1，……，αi为环境条件指数回归方程中的参数，X1，……，Xi的含义如表 1。
表 1 黄海千里岩水域环境因子与黄海鳀鱼补充量数据的相关系数Table 1. The correlation of environmental factors and recruitment data observed at the Qianliyan Marine Environment Monitoring Station in the Yellow Sea环境因子
environmental factorsXi 相关系数
correlation coefficient年平均磷酸盐浓度
mean phosphates concentrationX1 0.4091 平均表层水温
average sea surface temperature1月 X2 0.2656 2月 X3 0.3707 3月 X4 -0.0761 4月 X5 -0.6213 5月 X6 -0.6614 6月 X7 -0.4987 7月 X8 -0.5290 8月 X9 -0.0451 9月 X10 -0.4223 10月 X11 -0.3174 11月 X12 -0.1593 12月 X13 -0.1838 全年 X14 -0.4679 4~7月 X15 -0.8096 1.2.2 AIC(akaike information criterion)与BIC(bayesian information criterion)
AIC与BIC在统计学上，作为模型选择的标准已经被广泛应用到很多领域[9]。此文采用AIC和BIC标准对SRR模型进行比较。
AIC=-2ln (L)+2m
BIC=-2ln(L)+mln(n)
其中m是模型中参数的个数，n是观测数据的个数，L表示最大似然值。取得最小AIC或BIC的模型被选为最适模型。
2. 结果
根据图 1所示的1990~2001年黄海鳀鱼亲体数量(×106 t)与补充量(×1011尾)资料，用Ricker模型进行拟合，求得各项参数值为α=1.5931，β=0.3058。AIC与BIC值分别为37.2062，38.176。
表 1显示了黄海鳀鱼补充量与黄海千里岩水域逐月平均、全年平均、4~7月平均SST及PC数据的相关系数。
这里，设$\alpha_t=\frac{R_t}{S_t} \exp \left(\beta S_t\right)$是一个对黄海鳀鱼补充量有重要综合影响的可变环境因子[10-11]，其与补充量的关系如图 4所示。为了对各项环境数据进行统计计算，设定Xi代表各项环境因子，其对应关系如表 1所示。
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图 4 黄海千里岩环境条件指数αt和黄海鳀鱼补充量指数R变化趋势图Figure 4. Variations of environmental conditions index and the recruitment observed at the Qianliyan Marine Environment Monitoring Station in the Yellow Sea有$\alpha_i=\alpha_0+\sum\limits_{i=1}^n \alpha_i X_i$[10-11]，根据表 1中显示的相关系数，采用多元线性回归方法拟合对αt有重要影响的环境因子，年平均PC、9月平均和4~7月平均SST，即有αt=α0+α1X1+α10X10+α15X15。求得各项参数值α0=7.974；α1=-0.0144；α10=0.216；α15=-0.713。
Rt=(7.974-0.0144Xt+0.216X10-0.713X15)Stexp (-0.3058St)，AIC与BIC的值分别为9.3159和11.7404。将由此求得的Rt的计算值与补充量R的观测值进行作图对比(图 5)，较之单纯使用Ricker模型，加入环境指数的模型拟合的效果更好。
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图 5 黄海鳀鱼补充量观测值和计算值比较图Figure 5. Comparison of observed and calculated recruitment of anchovy (E.japonicas) stock in the Yellow Sea3. 讨论
3.1 环境因子与补充量的相关性
逐月分析黄海千里岩海域平均月SST与补充量的关系，可以看出，4~7月份的SST对黄海鳀鱼的补充量有着显著的影响，因为黄海鳀鱼性腺发育、生殖和仔稚鱼育肥的主要时期是每年的4~10月份[8]，由表 1也可以看出4~7月的SST与黄海鳀鱼补充量的相关系数最大。
3.2 环境条件指数
从图 4中可以看到，1996年(包括1996年)之前的环境条件指数与补充量的变化趋势以及波动程度都极其相似，1996年之后的环境因子与补充量的变化趋势虽然比较相似，但是变化程度有了明显的不同。由ZHAO等[8]研究的年捕捞量数据可知，1997~2000年间的黄海鳀鱼产量逐年明显高于其它年，捕捞力量的加剧可能导致了这一现象的出现。
黄海鳀鱼SRR的主要影响因素在1997年之后发生了变化，1990~1997年间，黄海鳀鱼补充量在波动中保持相对的稳定，1999年之后黄海鳀鱼资源大幅衰退，补充量随之显著降低。由此可见，只有在亲体量变化水平相当的情况下，环境因子的影响才相对显著。
3.3 模型结果比较分析
表 2是黄海鳀鱼SRR模型参数的估计值，以及AIC、BIC的计算值。从中可以看出，加入环境条件因子的Ricker模型对补充量的估计效果更好。其AIC与BIC的值都远远小于单纯用Ricker模型进行拟合的结果。说明环境条件对黄海鳀鱼SRR的影响是比较明显的，对环境条件因子的研究还是有一定意义的。
表 2 黄海鳀鱼SRR模型参数的估计值，AIC、BIC计算值Table 2. Estimated parameters and AIC, BIC in stock recruitment models (SRR)模型
model参数 estimated parameters AIC BIC α β α0 α1 α10 α15 Ricker模型
Ricker model1.5931 0.3058 - - - - 37.2062 38.176 加入αt的Ricker模型
Ricker model with αt- 0.3058 7.974 -0.0144 0.216 -0.713 9.3159 11.7404 鳀鱼资源的衰退，将对鳀鱼渔业产生巨大的负面影响。从图 1可以看出，黄海鳀鱼种群正在迅速向原点位置衰退，为了保证黄海鳀鱼渔业的可持续发展和利用，预防性管理措施的实施是极为必要的。
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图 1 鲮组织总RNA 1%琼脂糖凝胶电泳
1. 脑；2. 肝；3. 肾；4. 性腺；5. 肠；6. 鳃；7. 脾脏；8. 心脏；9. 皮肤；10. 肌肉
Figure 1. 1% agarose gel electrophoresis of total RNA from tissues of mud carp
1. brain; 2. liver; 3. kidney; 4. gonad; 5. intestine; 6. gill; 7. spleen; 8. heart; 9. skin; 10. muscle
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图 2 PCR循环数的确定
M. 100 bp DNA分子量标准；15~45. 循环数分别为15、20、25、30、35、40、45
Figure 2. Validation of PCR cycles
M. 100 bp DNA molecular weight marker; 15~45. Numbers on each lane represents the number of PCR cycles preformed.
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图 3 半定量RT-PCR检测重组鲮GH处理前后鲮actin和IGF-ⅠmRNA的组织表达
B1. 半定量RT-PCR检测对照组中鲮各组织中actin mRNA的表达；B2. 半定量RT-PCR检测对照组中鲮各组织中IGF-ⅠmRNA的表达；C1. 半定量RT-PCR检测处理组中鲮各组织中actin mRNA的表达；C2. 半定量RT-PCR检测处理组中鲮各组织中IGF-ⅠmRNA的表达；D. 处理前后鲮组织IGF-ⅠmRNA的相对表达；M. 100 bp DNA分子量标准；NC. 负对照；1~10. 肝、脑、肌肉、肾、肠、鳃、脾、性腺、心脏、皮肤
Figure 3. Semi-quantitative RT-PCR analysis of expression of Actin and IGF-Ⅰ mRNA in various tissues in mud carp
B1. Actin mRNA was detected in various tissues by semi-quantitative RT-PCR in control group; B2. IGF-ⅠmRNA was detected in various tissues by semi-quantitative RT-PCR in control group; C1. Actin mRNA was detected in various tissues by semi-quantitative RT-PCR in rmcGH in treated group; C2. IGF-Ⅰ mRNA was detected in various tissues by semi-quantitative RT-PCR in rmcGH in treated group; D. relative quantity of IGF-ⅠmRNA in various tissues in mud carp; M.100 bp DNA molecular weight marker; NC. a negative control (no template); 1~10. brain, liver, muscle, kidney, intestine, gill, spleen, gonad, heart, skin
表 1 扩增IGF-Ⅰ、Actin的引物序列
Table 1 Oligonucleotide primers used to amplify cDNA for mud carp IGF-Ⅰand Actin
引物 primer 序列 sequence Actin-F 5′-GTGTTGGCG/ATACAGGTCCTTACG-3′ Actin-R 5′-CAGACTACCTC/GATGAAGATCCTGAC-3′ IGF-Ⅰ-F 5′-ATGGAAAACCAGCGCCTCTTC-3′ IGF-Ⅰ-R 5′-TGCATGTCCTTCTTGAAGCAAG-3′ 
下载: 导出CSV
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 rmc IGF-Ⅰtreated fish
浓度±S.E.(n=3)/ng·mL-1 concentration±S.E. 对照组
control重组鲮IGF-Ⅰ处理组
rmcIGF-Ⅰtreated groupP值 胰岛素样生长因子-Ⅰ浓度
IGF-Ⅰconcentration145.59±21.84 247.71±2.83 P=0.043＜0.05 生长激素浓度
GH concentration0.037±0.0067 0.053±0.0067 P=0.508＞0.05
下载: 导出CSV
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