1. 华南农业大学 动物科学学院，广东 广州 510642;
2. 华南农业大学 海洋学院，广东 广州 510642
收稿日期：2018-11-12；接收日期：2019-02-27；网络出版时间：2019-03-19
基金项目：国家自然科学基金(No.31601911)资助
摘要：神经生长因子(Nerve growth factor，NGF)是一种能促进神经元发育、分化、再生的蛋白。为高效生产药效更佳的人源NGF (hNGF)药物，最近，笔者实验室构建出唾液腺特异表达hNGF的转基因小鼠，并从该转基因小鼠唾液中纯化获得具有高生物学活性的hNGF蛋白。为了选择性别和日龄最适宜的转hNGF基因小鼠用于收集纯化hNGF蛋白，文中比较了28日龄(性成熟前)雄性、雌性，63日龄(性成熟后)雄性、雌性转hNGF基因小鼠，共4组转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液鼠源NGF (mNGF)蛋白量和唾液hNGF蛋白量等指标。结果显示，63日龄的转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液mNGF蛋白量和唾液hNGF蛋白量显著高于28日龄同一性别的转hNGF基因小鼠，且63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF蛋白量显著高于同一日龄的雌性转hNGF基因小鼠；在4组小鼠中，63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF含量最高，比28日龄雌性转hNGF基因小鼠高出约46倍，最适宜用于收集唾液并从中纯化hNGF。
关键词：转基因小鼠性别日龄人神经生长因子唾液
Effects of gender and age on protein secretion in saliva of transgenic mice expressing human nerve growth factor
Sha Liao1, Qingchun Zhu1, Zhenfang Wu1, Zicong Li1, Fang Zeng1,2
1. College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China;
2. College of Marine Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
Received: November 12, 2018; Accepted: February 27, 2019; Published: March 19, 2019
Supported by: National Natural Science Foundation of China (No. 31601911)
Corresponding author: Fang Zeng. Tel: +86-20-85284985; Fax: +86-20-85280369; E-mail: zengf8210@163.com.
Abstract: Nerve growth factor (NGF) can promote the development, differentiation and regeneration of neurons. Recently, in order to efficiently produce human NGF (hNGF) drugs with better efficacy, we created transgenic mice expressing hNGF specifically in their salivary glands, and purified highly active hNGF protein from their saliva. Some studies reported that the NGF secretion in mouse saliva is affected by gender and age. Here, in order to select hNGF transgenic mice with high NGF secretion for saliva collection and hNGF purification, we divided transgenic mice into 4 groups, including 28-day-old young males and females, 63-day-old adult males and females. We compared their saliva volume, total salivary protein amount, salivary mNGF protein amount and salivary hNGF protein amount. The results showed that the saliva volume as well as amounts of total salivary protein, salivary mNGF protein and salivary hNGF protein secreted by 63-day-old transgenic mice were significantly higher than those secreted by sex-match 28-day-old transgenic mice, and the salivary hNGF protein amount secreted by male transgenic mice at the age of 63 days was significantly higher than that of female transgenic mice at the same age; Among 4 groups of mice, 63-day-old male transgenic mice secreted the highest salivary hNGF content, which was about 46 times higher than that secreted by the 28-day-old female transgenic mice. Therefore, 63-day-old male transgenic mice should be selected for saliva collection and hNGF purification.
Keywords: transgenic micegenderagehuman nerve growth factorsaliva
神经生长因子(Nerve growth factor，NGF)是一种具有神经元营养和促进突起生长的神经细胞生长调节因子。它对中枢及周围神经元的各种生理过程具有重要的调控作用。NGF在1952年被首次发现[1]，后续研究证明，NGF对治疗各种神经损伤或退行性疾病具有明显的效果[2]。由于对NGF的发现和研究作出了巨大的贡献，生物学家Cohen和Levi-Montalcini获得了1986年的诺贝尔医学生理奖[3-4]。从2002年至今，中国政府已批准4种NGF药物(商品名分别为恩经复、苏肽生、金路捷和丽康乐)上市用于治疗人类神经相关疾病，该4种药物均提取自小鼠唾液腺，属于鼠源NGF (mNGF)，且该类药物目前的价格非常昂贵，约1万元/mg。虽然mNGF与人NGF (hNGF)同源性高[5]，且对人类神经相关疾病具有确实疗效，但mNGF用于人类疾病治疗仍然存在免疫原性问题[6]。且已有研究表明，mNGF活性比hNGF低[7]，所以非常有必要研发hNGF药物。
迄今为止，有不少研究尝试利用各种方法来制备hNGF，如利用大肠杆菌表达系统[8-9]、酵母表达系统[10]、昆虫[11-12]或哺乳动物细胞[13-14]表达系统等，但因为上述表达系统存在翻译后修饰缺陷、产量低、成本高等问题而达不到理想效果。还有研究利用转基因兔乳腺作生物反应器制备hNGF[15]。但乳腺生物反应器存在性别和表达时期的限制，即必须在雌性动物的泌乳期才能表达目的蛋白，且乳中蛋白量及种类繁多，不利于纯化目的蛋白。许多动物在唾液腺中高水平表达高活性的内源NGF，这说明，动物唾液腺是NGF蛋白合成的理想场所。此外，以动物唾液腺作生物反应器表达外源蛋白还不受性别和表达时期的限制，且唾液中蛋白量及种类较少，有利于外源蛋白纯化。基于动物唾液腺的以上优点，本实验室曾芳等[16]已生产出唾液腺特异表达hNGF的转基因小鼠，并从其唾液中纯化获得了比mNGF有更高生物活性的hNGF蛋白，但该转基因小鼠的hNGF蛋白平均产量仍有待提高。
已有研究表明，小鼠唾液中的NGF蛋白分泌量存在明显的性别差异，雄性小鼠唾液腺中NGF的mRNA表达量显著高于雌性小鼠，且雄性小鼠唾液中的NGF蛋白量显著高于雌性小鼠，同时研究数据显示，成年(56日龄以上)雄鼠下颌唾液中NGF浓度显著高于幼年(30日龄左右)雄性小鼠[17-18]。另有研究表明，小鼠子宫组织中的NGF表达量随日龄增加而增加，在30日龄达到峰值，成年后维持在一定水平[19]；陆璐等[20]研究发现新生小鼠颌下腺不表达NGF，雄性小鼠2周龄左右才检测到NGF的表达信号，此后随日龄的增加而增加，1月龄左右时雄性小鼠颌下腺中NGF的mRNA迅速增多，到2月龄时NGF的mRNA持续保持较高水平转录。这些研究说明性别和日龄会影响小鼠NGF的表达量。
为了选择合适日龄及合适性别的转hNGF基因小鼠用于高效收集唾液并纯化hNGF蛋白，文中研究了性别和日龄对转hNGF基因小鼠唾液蛋白分泌的影响。为后续从转hNGF基因小鼠唾液中高效生产hNGF药物提供有利参考。
1 材料与方法1.1 转基因小鼠的配种扩繁8周龄的C57BL-6品系野生型雌性小鼠购自广东省医学实验动物中心，饲养5 d度过适应期后，每只注射7.5 IU的PMSG激素(购自宁波第二激素厂)，48 h后每只注射7.5 IU的HCG激素(购自宁波第二激素厂)[21]，注射后与10周龄的C57BL-6品系的转hNGF基因杂合子雄性小鼠[16]按2:1合笼，次日清晨检查阴道栓，将有栓雌鼠挑出分笼待产。
1.2 转基因小鼠的绿色荧光表达鉴定新生小鼠在出生后第3日龄进行绿色荧光表达鉴定。在黑暗环境中用荧光灯(BLS公司，中国)照射小鼠，通过滤光眼镜观察，在荧光灯照射下发出绿色荧光的则为转基因小鼠。将转基因小鼠和野生型小鼠放在一起，在自然光下和蓝色荧光灯照射下分别用不加滤光片和加了滤光片的相机拍摄照片。
1.3 转基因小鼠的PCR鉴定小鼠在25日龄打耳标后剪取长度约为5 mm的尾巴组织置于去DNA酶的1.5 mL离心管中，使用Omega公司的组织DNA提取试剂盒，按照其说明书进行小鼠尾样DNA抽提，抽提完成后，用微量核酸浓度仪(Nanodrop 2000，Thermo，美国)测定DNA样品浓度，放于?20 ℃冰箱保存。用PCR对转基因小鼠进行基因型检测(PCR仪为美国Life公司)。hNGF基因扩增引物为5′-AGC TGTGGAAGCTGGTGTTCCTG-3′、5′-CAGCTTC ACGGGGAGGCTG-3′，内参基因GAPDH的扩增引物为5′-CTCCCACTCTTCCACCTTCG-3′、5′-C CACCACCCTGTTGCTGTAG-3′ (设计软件：Primer Premier 6.0，由北京华大基因公司合成)，产物长度分别为290 bp和110 bp。转基因小鼠DNA可以扩增出内参基因片段和hNGF基因片段，而野生型小鼠DNA只能扩增出内参基因片段。PCR试剂购自广州东盛生物科技公司。
1.4 小鼠唾液采集根据日龄、性别及基因型将小鼠分成4组，分别为28日龄转基因雄性、28日龄转基因雌性、63日龄转基因雄性、63日龄转基因雌性，每组4只。每只小鼠腹腔共注射氯胺酮(剂量按每克体重25 μg)和甲苯噻嗪(剂量按每克体重1.1 μg)进行麻醉[16]，麻醉后用移液枪吸取(每只小鼠吸取20 min)小鼠口腔分泌的唾液放入离心管中[22]，放?80 ℃冰箱保存备用。
1.5 小鼠唾液总蛋白检测将采集好的小鼠唾液适当稀释，使用康为世纪生物科技公司生产的BCA蛋白浓度测定试剂盒，按照其说明书对唾液中的总蛋白浓度进行测定。用酶标仪(美国伯腾仪器有限公司，型号：Synergy H1)在540–590 nm范围内测定BSA标准品和每个样品的吸光值，绘制标准曲线，计算唾液样品中的总蛋白浓度。
1.6 酶联免疫吸附测定法(ELISA)检测分析将采集好的小鼠唾液适当稀释后，使用小鼠β-NGF ELISA试剂盒和人β-NGF ELISA试剂盒(均购自武汉尹艾博科技有限公司)分别对转基因小鼠唾液中mNGF蛋白和hNGF蛋白浓度进行测定。用酶标仪(美国伯腾仪器有限公司，型号：Synergy H1)测定样品吸光值，绘制标准曲线，计算唾液样品中的mNGF蛋白和hNGF蛋白浓度。
1.7 统计分析在Excel表中计算出蛋白浓度及其标准差等数据，并绘制柱状图，利用IBM SPSS Statistics 21进行显著性分析，比较同一性别不同日龄以及相同日龄、不同性别转基因小鼠的差异显著性。
2 结果与分析2.1 转hNGF基因小鼠绿色荧光表达及PCR鉴定荧光灯照射检测显示，转hNGF基因小鼠有明显荧光表达，而野生型小鼠则无荧光表达(图 1A)；PCR检测显示，转hNGF基因小鼠基因组中携带hNGF基因，而野生型小鼠基因组中无hNGF基因(图 1B)。荧光灯照射检测结果与PCR检测结果完全一致。
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| 图 1 转hNGF基因小鼠鉴定(A：转hNGF基因小鼠荧光表达鉴定；B：转hNGF基因小鼠PCR鉴定) Fig. 1 Identification of hNGF transgenic mice. (A) Analysis of EGFP expression in transgenic mice. (B) PCR identification of transgenic founder mice. "TG" represents transgenic mice; "WT" represents wild-type; "M" represents molecular markers; "–" represents negative control using wild-type DNA as template; "+" represents positive control using plasmid as template; N represents negative control using water as template and GAPDH was used as an internal control gene. |
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2.2 性别及日龄对转hNGF基因小鼠唾液分泌量的影响28日龄的转hNGF基因雄鼠与雌鼠唾液分泌量差异不显著，且63日龄的转hNGF基因雄鼠与雌鼠唾液分泌量也无显著差异；63日龄的转hNGF基因雄鼠与雌鼠唾液分泌量都极显著高于28日龄的同一性别转hNGF基因小鼠(图 2)。
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| 图 2 性别和日龄对转hNGF基因小鼠唾液分泌量的影响 Fig. 2 Effects of gender and age on saliva volume of hNGF transgenic mice. The amount of saliva collected in transgenic mice within 20 min after anesthesia. In the same gender but different ages, the marked "**" represents the difference is extremely significant (P < 0.01), "*" represents the difference is significant (P < 0.05). No superscripts mean the difference is not significant (P > 0.05). |
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2.3 性别及日龄对转hNGF基因小鼠唾液中总蛋白分泌的影响28日龄的转hNGF基因雄鼠与雌鼠唾液中总蛋白浓度之间差异不显著，63日龄转hNGF基因雄鼠与雌鼠唾液中总蛋白浓度之间差异也不显著，即同一日龄不同性别的转hNGF小鼠唾液中总蛋白浓度均无显著差异；63日龄的转hNGF基因雄鼠与雌鼠唾液中总蛋白浓度都极显著高于28日龄中同一性别的转hNGF基因小鼠(图 3A)。
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| 图 3 性别及日龄对转hNGF基因小鼠唾液中总蛋白分泌的影响(A：唾液中总蛋白浓度；B：唾液中总蛋白含量) Fig. 3 Effects of gender and age on total protein secretion in saliva of hNGF transgenic mice. (A) Concentration of total protein in saliva collected in transgenic mice within 20 min after anesthesia. (B) Content of total protein in saliva collected in transgenic mice within 20 min after anesthesia. In the same gender but different ages, the marked "**" represents the difference is extremely significant (P < 0.01), "*" represents the difference is significant (P < 0.05). No superscripts mean the difference is not significant (P > 0.05). |
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28日龄的转hNGF基因雄鼠与雌鼠唾液中总蛋白量之间差异不显著，63日龄的转hNGF基因雄鼠与雌鼠唾液中总蛋白量之间差异也不显著，即同一日龄的不同性别的转hNGF基因小鼠唾液中总蛋白量均无显著差异；63日龄的转hNGF基因雄鼠与雌鼠唾液中总蛋白量都极显著高于28日龄的同一性别转hNGF基因小鼠(图 3B)。
2.4 性别及日龄对转hNGF基因小鼠唾液中mNGF蛋白分泌的影响转hNGF基因小鼠唾液中mNGF蛋白的分泌情况与同日龄、同性别的野生型(WT)小鼠唾液中的mNGF蛋白分泌情况差异均不显著(图 4)。
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| 图 4 性别及日龄对转hNGF基因小鼠唾液中mNGF蛋白分泌的影响(A：转基因小鼠唾液中mNGF蛋白浓度；B：转基因小鼠唾液中mNGF蛋白相对浓度；C：转基因小鼠唾液中mNGF蛋白含量) Fig. 4 Effects of gender and age on mNGF secretion in saliva of hNGF transgenic mice. (A) Concentration of mNGF in saliva collected in transgenic mice within 20 min after anesthesia. (B) Relative concentration of mNGF in saliva collected in transgenic mice within 20 min after anesthesia. (C) Content of mNGF in saliva collected in transgenic mice within 20 min after anesthesia. In the same gender but different ages, the marked "**" represents the difference is extremely significant (P < 0.01), "*" represents the difference is significant (P < 0.05), no superscripts mean the difference is not significant (P > 0.05). At the same age but different genders, if there are no superscripts, the difference is not significant (P > 0.05), if there are different lowercase letters in superscripts, the difference is significant (P < 0.05), if there are different uppercase letters in superscripts, the difference is extremely significant (P < 0.01). |
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28日龄的转hNGF基因雄鼠与雌鼠唾液中mNGF蛋白浓度差异不显著，但63日龄的转hNGF基因雄鼠唾液中mNGF蛋白浓度极显著高于63日龄的转hNGF基因雌鼠；63日龄的转hNGF基因雄鼠唾液中mNGF蛋白浓度极显著高于28日龄的转hNGF基因雄鼠，但63日龄与28日龄的转hNGF基因雌鼠唾液中mNGF蛋白浓度之间差异不显著；不同日龄且同一性别的野生型小鼠中，63日龄小鼠唾液mNGF蛋白浓度均高于28日龄，但雄性差异不显著，而雌性差异极显著；同一日龄不同性别的野生型小鼠中，雄鼠唾液mNGF蛋白浓度均高于雌鼠，但在63日龄时差异显著(图 4A)。
28日龄的转hNGF基因雄鼠与雌鼠唾液中mNGF蛋白相对浓度无显著差异，但63日龄的转hNGF基因雄鼠唾液中mNGF蛋白相对浓度极显著高于63日龄的转hNGF基因雌鼠；63日龄与28日龄同一性别的转hNGF基因小鼠唾液中mNGF蛋白相对浓度差异不显著；同日龄不同性别的野生型小鼠中，雄性唾液mNGF相对浓度均大于雌性，但在63日龄差异显著；而同一性别不同日龄的野生型小鼠唾液mNGF蛋白相对浓度差异均不显著(图 4B)。
28日龄的转hNGF基因雄鼠与雌鼠唾液中mNGF蛋白含量无显著差异，但63日龄的转hNGF基因雄鼠唾液中mNGF蛋白含量极显著高于63日龄的转hNGF基因雌鼠；63日龄的转hNGF基因雄鼠与雌鼠唾液中mNGF蛋白含量极显著高于28日龄的同一性别转hNGF基因小鼠；同一日龄不同性别的野生型小鼠中，雄性唾液mNGF蛋白含量均高于雌性，但在63日龄时差异显著；同一性别不同日龄的野生型小鼠中，63日龄小鼠唾液mNGF蛋白含量均极显著地高于28日龄小鼠唾液mNGF蛋白含量(图 4C)。
2.5 性别及日龄对转hNGF基因小鼠唾液中hNGF蛋白分泌的影响28日龄和63日龄的转hNGF基因雄鼠唾液中hNGF蛋白浓度都显著高于同一日龄的转hNGF基因雌鼠；63日龄的转hNGF基因雄鼠唾液中hNGF蛋白浓度显著高于28日龄的转hNGF基因雄鼠；28日龄和63日龄的转hNGF基因雌鼠唾液中hNGF蛋白浓度无显著差异(图 5A)。
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| 图 5 性别及日龄对转hNGF基因小鼠唾液中hNGF蛋白分泌的影响(A：转基因小鼠唾液中hNGF蛋白的浓度；B：转基因小鼠唾液中hNGF蛋白相对浓度；C：转基因小鼠唾液中hNGF蛋白的含量) Fig. 5 Effects of gender and age on hNGF secretion in saliva of hNGF transgenic mice. (A) Concentration of hNGF in saliva collected in transgenic mice within 20 min after anesthesia. (B) Relative concentration of hNGF in saliva collected in transgenic mice within 20 min after anesthesia. (C) Content of hNGF in saliva collected in transgenic mice within 20 min after anesthesia. In the same gender but different ages, the marked "**" represents the difference is extremely significant (P < 0.01), "*" represents the difference is significant (P < 0.05), no superscripts mean the difference is not significant (P > 0.05). At the same age but different genders, if there are no superscripts, the difference is not significant (P > 0.05), if there are different lowercase letters in superscripts, the difference is significant (P < 0.05), if there are different uppercase letters in superscripts, the difference is extremely significant (P < 0.01). |
| 图选项 |
28日龄和63日龄的转hNGF基因雄鼠唾液中hNGF蛋白相对浓度都高于同一日龄的转hNGF基因雌鼠，差异显著；28日龄与63日龄同一性别转hNGF基因小鼠唾液中hNGF蛋白相对浓度差异不显著(图 5B)。
28日龄和63日龄的转hNGF基因雄鼠唾液中hNGF蛋白含量都高于同一日龄的转hNGF基因雌鼠，差异显著；63日龄的转hNGF基因雄鼠和雌鼠唾液中hNGF蛋白含量都高于28日龄的同一性别的转hNGF基因小鼠，差异极显著(图 5C)。
3 讨论本研究结果显示，性别对转hNGF基因小鼠在28和63日龄分泌的唾液量、唾液总蛋白浓度、唾液总蛋白量无显著影响；对转hNGF基因小鼠在63日龄分泌的唾液mNGF蛋白浓度、相对浓度及总量均有显著影响，且雄性高于雌性；对转hNGF唾液hNGF蛋白浓度、相对浓度及总量都有显著影响，且雄性高于雌性。
本研究结果还显示，日龄对同一性别的转hNGF基因小鼠分泌的唾液量、唾液总蛋白浓度、唾液总蛋白量都有显著影响，且均为63日龄高于28日龄；对雄性转hNGF基因小鼠分泌的唾液mNGF蛋白浓度有显著影响，且63日龄高于28日龄；对同一性别的转hNGF基因小鼠分泌的唾液mNGF蛋白总量有显著影响，且63日龄高于28日龄；对雄性转hNGF基因小鼠分泌的唾液hNGF蛋白浓度有显著影响，且63日龄高于28日龄；对同一性别的转hNGF基因小鼠分泌的唾液hNGF蛋白总量有显著影响，且63日龄高于28日龄。
已有研究显示雄性小鼠唾液中NGF蛋白浓度显著高于雌性[16]，这与本研究结果相似。此外，还有研究显示雄性成年小鼠唾液腺中的NGF含量是雌性成年小鼠的10倍左右，而去势后的雄性成年小鼠唾液腺中的NGF含量则与雌性成年小鼠相当，用甲状腺激素和雄激素治疗后的去势雄性成年小鼠唾液腺中NGF含量分别增加6倍和20倍，且后者基本恢复至去势前的量[23]。刘玲爱等[24]也证明，通过注射外源雄激素可以有效提高小鼠唾液腺NGF的合成量。上述研究结果显示雄性激素在小鼠体内可以促进NGF蛋白的表达合成。这也提示我们，可以通过注射外源雄激素来提高转hNGF基因小鼠唾液中hNGF蛋白的分泌量，从而更高效生产hNGF蛋白。
本研究结果还显示，同一性别的转hNGF基因小鼠在63日龄分泌的唾液mNGF和hNGF蛋白总量均显著高于28日龄，说明hNGF基因小鼠分泌的唾液mNGF和hNGF蛋白总量随日龄增加而增加，这与另一研究报道的结果一致[20]。由于小鼠唾液蛋白的表达分泌主要受激素(例如性激素)调控，随着日龄的增加，小鼠体内的性激素含量也增加，可能就导致其唾液中NGF蛋白表达分泌增加[25-26]。
根据本研究结果，如果选择63日龄左右(性成熟后)的雄性转hNGF基因小鼠采集其唾液纯化hNGF蛋白，与选择28日龄(性成熟前)的雌性转hNGF基因小鼠相比，hNGF蛋白产量将提高约46倍。
综上所述，性别和日龄均对转hNGF基因小鼠唾液中的hNGF蛋白表达分泌量有显著影响。选择性成熟后的雄性转hNGF基因小鼠收集唾液纯化hNGF蛋白，将有利于提高该蛋白的制备效率。
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