Effects of feed restriction on mammary gland development of pubertal mice and the involved mechanisms
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摘要:目的
研究限饲对初情期小鼠乳腺发育的影响及机制，为动物乳腺发育的营养调控提供科学依据。
方法选用24只4周龄C57BL/6雌性小鼠，分为对照组和限饲组。试验为期4周，每周称体质量并统计采食量和饮水量，在试验结束前对小鼠进行体成像和体组成检测。试验结束后采集小鼠乳腺并称质量。采集血液，测定血清中胰岛素样生长因子(IGF-1)和雌二醇(E2)的水平。采用Whole-mount染色观察小鼠乳腺发育情况，统计小鼠乳腺终末乳芽(TEB)和导管分支数量。利用免疫荧光染色检测乳腺组织中增殖细胞核抗原(PCNA)的表达。
结果限饲组小鼠体质量、体增质量、平均日采食量、平均日能量摄入均极显著低于对照组(P<0.001)，但其饮水量显著高于对照组(P<0.05)。限饲对小鼠肌肉含量和脂肪含量无显著影响。限饲使小鼠的乳腺质量降低了29%(P<0.05)，对乳腺指数无显著影响。Whole-mount染色结果表明，限饲显著抑制初情期小鼠的乳腺发育，其乳腺组织的TEB数量和导管分支数量均显著低于对照组(P<0.05)。此外，限饲对血清中E2水平无显著影响，但使血清中IGF-1水平降低了34%(P<0.05)。限饲显著抑制了小鼠乳腺组织中PCNA蛋白的表达。
结论限饲可显著抑制初情期小鼠乳腺发育，这可能与限饲可降低血清中IGF-1水平和乳腺组织中增殖相关蛋白PCNA表达有关。
Abstract:ObjectiveTo investigate the effect of feed restriction on mammary gland development of pubertal mice and explore the involved mechanisms, and provide a scientific basis for nutritional regulation during mammary gland development of animals.
MethodTwenty-four 4-week-old C57BL/6J female mice were randomly divided into the control group and feed restriction group. The trial lasted for four weeks. The body weight, feed intake and water intake were recorded weekly, and the body imaging and body composition of mice were examined before the end of the trial. At the end of the trial, the mouse mammary gland was collected and weighed. The blood was collected and the levels of insulin-like growth factor (IGF-1) and estradiol (E2) in serum were measured. The mammary gland development was observed and assessed by using whole-mount staining and the numbers of mammary gland terminal end bud (TEB) and duct branches were analyzed. Immunofluorescence staining was used to detect the expression of proliferating cell nuclear antigen (PCNA) in the mammary gland tissue of pubertal mice.
ResultThe body weight, body weight gain, average daily feed intake and average daily energy intake of mice in feed restriction group were significantly lower than those of mice in control group (P<0.001), while the water intake of feed restriction group was significantly higher than that of control group (P<0.05). Feed restriction had no effect on muscle and fat contents. In addition, feed restriction significantly decreased the mammary gland weight by 29%(P<0.05), with no effect on mammary gland index. The results of whole-mount staining showed that feed restriction significantly inhibited the development of mammary gland, with significant lower number of TEB and duct branches (P<0.05). Furthermore, feed restriction significantly reduced the serum level of IGF-1 by 34%(P<0.05), with no influence on serum E2 level. Feed restriction significantly inhibited PCNA protein expression in the mammary gland of pubertal mice.
ConclusionFeed restriction significantly inhibits mammary gland development of pubertal mice, which might be associated with the decreased level of IGF-1 in serum and expression of proliferation-associated protein PCNA in mammary gland tissue.
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作为哺乳动物的泌乳器官，良好的发育是乳腺充分发挥泌乳功能的前提，对动物子代的生长发育及畜牧业生产具有重要影响。因此，研究影响乳腺发育的因素及其机制对提高动物泌乳性能及畜牧业生产提质增效具有重要意义。
哺乳动物乳腺发育期包括初情期、妊娠期和泌乳期等不同阶段[1]。猪乳腺发育有3个关键阶段，分别是90日龄到性成熟期间、妊娠期后三分之一阶段和哺乳期[2]。乳腺的发育受到遗传、激素、营养和环境等不同因素的影响[1-3]，其中营养对乳腺的发育起着非常重要的作用[3-4]。前期研究发现，高脂日粮会抑制初情期小鼠乳腺的发育[5-6]；相反，限饲或者过瘦也会影响乳腺的发育，例如：母猪90日龄至性成熟期间，25%的限饲可显著降低乳腺实质质量、乳腺组织DNA和RNA含量[7]。此外，一定程度的能量限制是预防乳腺癌的潜在策略[8]，但是过量限饲也会抑制乳腺的正常发育。目前，对于限饲调控初情期哺乳动物乳腺发育的机制尚不清楚。
本试验以初情期小鼠为对象，在研究限饲对初情期小鼠乳腺导管发育的基础上，进一步探索限饲对初情期小鼠血清中胰岛素样生长因子1(Insulin-like growth factor 1,IGF-1)和雌二醇(Estradiol, E2)水平及乳腺组织中增殖相关蛋白增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)表达的影响，旨在揭示限饲对初情期小鼠乳腺发育的影响及其机制，为动物乳腺发育的营养调控提供科学依据。
1. 材料与方法
1.1 试验动物与试验设计
选用24只4周龄的C57BL/6J雌性小鼠(购于广东省医学实验动物中心)，预饲喂正常日粮3 d后，根据体质量随机分为2组：对照组(自由采食日粮)和限饲组(每天饲喂对照组小鼠前一天平均采食量的70%)，每组12只。将小鼠同室分笼饲养，自由采食饮水；每天光照与黑暗各12 h，环境温度控制在(25±1) ℃，湿度(60±5)%。每周进行小鼠采食量、饮水量和体质量的测定，采样前对小鼠进行体成像和体组成检测，饲养4周后，眼球采血静置离心分离得到血清，并对血清IGF-1和E2水平进行检测；通过颈部脱臼方式处死小鼠，采集小鼠乳腺组织、褐色脂肪和内脏脂肪组织并称质量，同时对乳腺进行染色和蛋白表达等检测。
1.2 试验材料
小鼠日粮购于广东省医学实验动物中心；百里酚、胭脂红和硫酸铝钾购于成都艾科达化学试剂有限公司；轻型小动物成分分析仪(上海纽迈电子科技有限公司)用于对小鼠进行体成像和体成分测定；IGF-1放射免疫试剂盒购于天津九鼎医学生物工程有限公司；PCNA抗体购于Cell Signaling Technology。
1.3 测定指标及方法
乳腺采集和Whole-mount染色：切下右侧乳腺腹腺体(第4对乳腺)并称质量，涂在玻璃载玻片上，进行Whole-mount染色[5]，显微镜下观察初情期小鼠乳腺组织结构，统计乳腺组织终末乳芽(Terminal end bud，TEB)数量和导管分支数量。
PCNA蛋白的免疫荧光染色：步骤为组织脱水、包埋、切片、抗原修复、血清封闭、孵一抗过夜、孵二抗、DAPI复染细胞核、封片，之后在免疫荧光显微镜下观察初情期小鼠乳腺组织荧光强弱并分析。
1.4 数据分析
数据结果用平均值±标准误表示，统计分析采用SigmaPlot 12.5软件分析，采用t检验比较对照组和限饲组的统计差异。
2. 结果与分析
2.1 限饲对初情期小鼠生长性状的影响
如图1a所示，限饲组小鼠的第1周体质量显著低于对照组(P<0.05)，从第2周开始，限饲组小鼠的体质量极显著低于对照组(P<0.001)；每周限饲组小鼠的体增质量均极显著低于对照组(图1b)；另外，限饲组小鼠平均日采食量和平均日能量摄入均极显著低于对照组(图1c、1d)；然而，限饲组小鼠平均日饮水量极显著高于对照组(图1e)。
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图 1 限饲对初情期小鼠生长性状的影响“*”、“**”和“***”分别表示与对照组相比差异达0.05、0.01、0.001的显著水平(t检验)Figure 1. Effects of feed restriction on growth traits of pubertal mice“*”,“**” and “***” indicate the difference with control group reaches 0.05, 0.01 and 0.001 significance levels respectively (t test)2.2 限饲对初情期小鼠体组成及不同部位脂肪的影响
体成像和体组成检测结果(图2、图3)表明，与对照组相比，限饲不会显著改变初情期小鼠的整体肌肉含量和脂肪含量。
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图 2 限饲对初情期小鼠体成像的影响红色部分代表脂肪Figure 2. Effect of feed restriction on the body imaging of pubertal miceRed part represents fat![]()
图 3 限饲对初情期小鼠体组成的影响Figure 3. Effects of feed restriction on the body composition of pubertal mice采样褐色脂肪和内脏脂肪的结果表明，与对照组相比，限饲对初情期小鼠的褐色脂肪指数(褐色脂肪质量占体质量百分比)无显著影响(图4A)，但可极显著降低内脏脂肪指数(内脏脂肪质量占体质量百分比)(图4B)。
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图 4 限饲对初情期小鼠褐色脂肪和内脏脂肪的影响“***”表示与对照组相比差异达0.001的显著水平(t检验)Figure 4. Effects of feed restriction on the brown adipose tissue and visceral adipose tissue of pubertal mice“***” indicates the difference with control group reaches 0.001 significance level (t test)2.3 限饲对初情期小鼠乳腺发育的影响
小鼠乳腺Whole-mount染色如图5所示，与对照组小鼠相比，限饲组小鼠的乳腺组织在乳腺导管的密度和数量上有明显的降低；此外，与对照组小鼠相比，限饲组小鼠的乳腺质量显著降低29%，同时限饲组小鼠乳腺导管分支数量显著降低，导管的TEB数量极显著降低(表1)。
表 1 限饲对初情期小鼠乳腺(第4对右侧)发育性状的影响1)Table 1. Effects of feed restriction on developmental traits of mammary gland (the right one of the fourth pair) of pubertal mice处理
Treatment乳腺质量/g
Mammary gland weight乳腺指数/%
Mammary gland index终末乳芽数量
TEB number导管分支数量
Duct branch number对照 Control 0.059 5±0.004 7 0.298 1±0.023 7 38.666 7±0.666 7 27.666 7±1.201 9 限饲 Feed restriction 0.042 2±0.003 7* 0.258 5±0.019 2 21.666 7±0.666 7*** 21.666 7±1.201 9* 1) “*”和“***”分别表示与对照组相比差异达0.05和0.001的显著水平(t检验)
1)“*” and “***” indicate the difference with control group reaches 0.05 and 0.001 significance levels respectively (t test)![]()
图 5 限饲对初情期小鼠乳腺形态和导管发育的影响a、b分别为Whole-mount染色后对照组和限饲组小鼠乳腺组织的低倍显微镜照片；c、d分别为Whole-mount染色后对照组和限饲组小鼠乳腺组织的高倍显微镜照片；TEB：终末乳芽Figure 5. Effects of feed restriction on mammary gland morphology and duct development of pubertal micea and b are low-power microscope pictures of the breast tissue from mice in the control group and feed restriction group after whole-mount staining, respectively; c and d are high-power microscope pictures of the breast tissue from mice of the control group and feed restriction group after whole-mount staining, respectively; TEB: Terminal end bud2.4 限饲对初情期小鼠血清中IGF-1和E2水平的影响
如图6所示，与对照组相比，限饲可以使初情期小鼠血清中IGF-1水平降低34%，但是对小鼠血清中E2水平没有显著影响。
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图 6 限饲对初情期小鼠血清中IGF-1和E2水平的影响“*”表示与对照组相比差异达0.05的显著水平(t检验)Figure 6. Effects of feed restriction on the serum levels of IGF-1 and E2 of pubertal mice“*”indicates the difference with control group reaches 0.05 significance level (t test)2.5 限饲对初情期小鼠乳腺组织中增殖相关蛋白PCNA表达的影响
如图7所示，对照组乳腺组织导管免疫荧光染色(红色箭头部分)明亮清晰，而限饲组乳腺组织导管却暗淡模糊，由此得知，限饲可以显著降低乳腺导管增殖相关蛋白PCNA的表达。
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图 7 限饲对初情期小鼠乳腺组织中PCNA蛋白表达的影响图中DAPI表示4′,6−二脒基−2−苯基吲哚荧光染色图片；PCNA表示增殖细胞核抗原荧光染色图片，红色箭头处为乳腺导管；Merge表示DAPI荧光染色和PCNA荧光染色合并图片；White light表示光学显微镜拍摄图片Figure 7. Effect of feed restriction on PCNA protein expression in mammary gland of pubertal miceIn the figure, DAPI represents 4′,6-diamidino-2-phenylindole fluorescence staining picture, red arrow points at mammary duct; PCNA represents the fluorescence staining image of PCNA; Merge represents the combined image of DAPI fluorescence staining and PCNA fluorescence staining; White light represents the image taken by an optical microscope3. 讨论与结论
3.1 限饲对初情期小鼠生长和体组成的影响
周期性能量限制能显著降低羔羊的生长性能[9]，对鸡和鹅的研究也观察到类似情况。叶耀辉等[10]的研究表明，限饲对猪胴体蛋白质日沉积量无显著影响，但能引起胴体脂肪沉积的减少，从而达到胴体瘦肉比例相对增加的目的。本研究发现，限饲同样也能显著降低小鼠的生长性能，体质量、体增质量、平均日采食量和平均日能量摄入均显著降低，但是平均日饮水量却显著升高，这可能是因为限饲组小鼠能量供给不足，代偿性通过增加饮水量来弥补能量的缺失。另外，限饲也能显著降低初情期小鼠内脏脂肪指数，但是对褐色脂肪指数、脂肪含量和肌肉含量均无显著影响。
3.2 限饲对初情期小鼠乳腺发育的影响
许多研究表明，青春期，妊娠期和哺乳期的乳腺快速增生期间，母猪的营养会影响乳腺发育。母猪90日龄至性成熟，相对于正常采食量25%的限饲可以显著降低乳腺实质质量、乳腺组织DNA和RNA含量，但是，在断奶后至90日龄对母猪进行34%的限饲不会影响乳腺的发育[7]。在本试验中，whole-mount染色结果表明，限饲可显著降低乳腺组织导管分支数量以及TEB的数量，提示限饲可显著抑制初情期小鼠乳腺的发育。Park等[11]的研究也发现，能量限制延迟了大鼠青春期的开始并延缓了母代和后代的生长，减少了乳腺中的脂肪沉积，但它并没有影响乳腺细胞性质。但是泌乳母猪饲料限制不会对乳汁组成和窝产仔数产生不利影响[12]。可见，不同阶段的限饲造成的影响也不相同。
3.3 限饲对初情期小鼠血清IGF-1和E2水平及乳腺组织中PCNA蛋白表达的影响
IGF-1是动物出生后生长调节的重要因子，有研究报道，IGF-1能增加乳腺上皮细胞存活率并刺激乳腺上皮细胞的增殖，还促进乳腺肿瘤发生[13]。Clemmons[14]的研究结果表明，血清中IGF- 1能够提高动物体内能量和蛋白的沉积，加快生长速度。E2是卵巢卵泡颗粒细胞分泌的一种自然雌激素，能增进和调节雌性器官及副性征的正常发育。有研究表明，17β−雌二醇能显著促进人乳腺癌细胞系MCF-7细胞的增殖[15]。本研究发现，限饲能显著降低初情期小鼠血清中IGF-1水平，这可能是导致限饲组小鼠生长发育受阻的重要原因。但是，限饲对初情期小鼠血清中E2水平无显著影响。
研究发现，IGF-1在初情期小鼠优先激活PI3K/Akt通路，增加细胞周期蛋白D1(Cyclin D1)表达[13]。本研究中乳腺组织的免疫荧光结果表明，限饲可以显著降低乳腺组织增殖相关蛋白PCNA的表达，提示限饲可能抑制血清IGF-1水平，从而降低乳腺组织增殖相关蛋白PCNA的表达来抑制乳腺发育。
综上所述，限饲可抑制初情期小鼠乳腺发育，其乳腺导管分支数量和TEB数量显著减少。限饲对初情期小鼠乳腺发育的抑制作用可能与其抑制增殖相关蛋白PCNA的表达和降低血清中IGF-1水平有关。本论文研究结果为动物和人类乳腺健康发育的营养调控提供了科学的参考依据。
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图 1 限饲对初情期小鼠生长性状的影响
“*”、“**”和“***”分别表示与对照组相比差异达0.05、0.01、0.001的显著水平(t检验)
Figure 1. Effects of feed restriction on growth traits of pubertal mice
“*”,“**” and “***” indicate the difference with control group reaches 0.05, 0.01 and 0.001 significance levels respectively (t test)
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图 2 限饲对初情期小鼠体成像的影响
红色部分代表脂肪
Figure 2. Effect of feed restriction on the body imaging of pubertal mice
Red part represents fat
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图 3 限饲对初情期小鼠体组成的影响
Figure 3. Effects of feed restriction on the body composition of pubertal mice
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图 4 限饲对初情期小鼠褐色脂肪和内脏脂肪的影响
“***”表示与对照组相比差异达0.001的显著水平(t检验)
Figure 4. Effects of feed restriction on the brown adipose tissue and visceral adipose tissue of pubertal mice
“***” indicates the difference with control group reaches 0.001 significance level (t test)
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图 5 限饲对初情期小鼠乳腺形态和导管发育的影响
a、b分别为Whole-mount染色后对照组和限饲组小鼠乳腺组织的低倍显微镜照片；c、d分别为Whole-mount染色后对照组和限饲组小鼠乳腺组织的高倍显微镜照片；TEB：终末乳芽
Figure 5. Effects of feed restriction on mammary gland morphology and duct development of pubertal mice
a and b are low-power microscope pictures of the breast tissue from mice in the control group and feed restriction group after whole-mount staining, respectively; c and d are high-power microscope pictures of the breast tissue from mice of the control group and feed restriction group after whole-mount staining, respectively; TEB: Terminal end bud
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图 6 限饲对初情期小鼠血清中IGF-1和E2水平的影响
“*”表示与对照组相比差异达0.05的显著水平(t检验)
Figure 6. Effects of feed restriction on the serum levels of IGF-1 and E2 of pubertal mice
“*”indicates the difference with control group reaches 0.05 significance level (t test)
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图 7 限饲对初情期小鼠乳腺组织中PCNA蛋白表达的影响
图中DAPI表示4′,6−二脒基−2−苯基吲哚荧光染色图片；PCNA表示增殖细胞核抗原荧光染色图片，红色箭头处为乳腺导管；Merge表示DAPI荧光染色和PCNA荧光染色合并图片；White light表示光学显微镜拍摄图片
Figure 7. Effect of feed restriction on PCNA protein expression in mammary gland of pubertal mice
In the figure, DAPI represents 4′,6-diamidino-2-phenylindole fluorescence staining picture, red arrow points at mammary duct; PCNA represents the fluorescence staining image of PCNA; Merge represents the combined image of DAPI fluorescence staining and PCNA fluorescence staining; White light represents the image taken by an optical microscope
表 1 限饲对初情期小鼠乳腺(第4对右侧)发育性状的影响1)
Table 1 Effects of feed restriction on developmental traits of mammary gland (the right one of the fourth pair) of pubertal mice
处理
Treatment乳腺质量/g
Mammary gland weight乳腺指数/%
Mammary gland index终末乳芽数量
TEB number导管分支数量
Duct branch number对照 Control 0.059 5±0.004 7 0.298 1±0.023 7 38.666 7±0.666 7 27.666 7±1.201 9 限饲 Feed restriction 0.042 2±0.003 7* 0.258 5±0.019 2 21.666 7±0.666 7*** 21.666 7±1.201 9* 1) “*”和“***”分别表示与对照组相比差异达0.05和0.001的显著水平(t检验)
1)“*” and “***” indicate the difference with control group reaches 0.05 and 0.001 significance levels respectively (t test)
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