CN116115738A - 细胞因子Igfbp7在制备治疗小鼠老年性肌肉萎缩的药物中的应用 - Google Patents
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Abstract
本发明公开了一种细胞因子Igfbp7在制备治疗小鼠老年性肌肉萎缩的药物中的应用，所述细胞因子Igfbp7用于干预小鼠老年性肌肉萎缩的效果好，具有干预人肌丢失相关疾病的前景。
Description
技术领域
本发明涉及骨骼肌丢失的治疗技术领域，尤其涉及一种细胞因子Igfbp7在制备治疗小鼠老年性肌肉萎缩的药物中的应用。
背景技术
骨骼肌功能的维持依赖于骨骼肌的结构基础，其质量的减少称为肌丢失，会导致骨骼肌功能退化乃至丧失，最终严重危害人体健康。目前肌丢失相关疾病主要有两大类：1)原发性肌肉疾病，主要是指进行性肌营养不良，这是一类遗传性肌丢失相关疾病，临床表现为肌肉无力和萎缩。2)继发性骨骼肌丢失相关疾病，主要是指肌肉衰减综合征(Sarcopenia)。Sarcopenia是随着老龄化过程出现的一种以骨骼肌质量和力量渐进性和全身性丧失为特征的综合症。正常人在30岁以后，每年骨骼肌质量减少0.1％到0.5％，65岁之后肌丢失会急剧加速。为了深入研究肌丢失相关疾病的发病机理及干预手段，研究人员开发了一系列重要的研究模型，其中最主要的是小鼠骨骼肌丢失模型，包括基因修饰小鼠、骨骼肌损伤小鼠模型及老年小鼠肌肉萎缩模型。但是，目前涉及干预小鼠骨骼肌丢失的相关技术手段有限，制约了相关研究的进展。
胰岛素样生长因子结合蛋白(Insulin-like growth factor binding proteins,Igfbps)是一类可以结合胰岛素样生长因子(Insulin-like growth factors，IGF)的细胞因子。Igfbps主要包括两类：IGF结合蛋白(Igfbp1-6)以及IGF结合蛋白相关蛋白(Igfbp-rp1-10)。Igfbp-rp1也称为Igfbp7，研究发现Igfbp7是一种分泌型糖蛋白，在肝脏、骨骼肌、胰腺及大脑都有表达。Igfbp7不同于其它Igfbps及Igfbp-rps，其与IGF-1的结合能力很弱。研究发现Igfbp7具有一些独特的功能，包括参与血管发育(被认为是肺动脉高压发生的早期标志物)和肿瘤发生发展(被发现具有抑制白血病干细胞的作用)，但细胞因子Igfbp7对骨骼肌丢失，特别是老年性肌肉萎缩的干预效果尚不清楚。
发明内容
本发明的目的是提供一种细胞因子Igfbp7在制备治疗小鼠老年性肌肉萎缩的药物中的应用，所述细胞因子Igfbp7用于干预小鼠老年性肌肉萎缩的效果好，具有干预人肌丢失相关疾病的前景。
本发明的技术方案是：
细胞因子Igfbp7在制备治疗老年性肌肉萎缩的药物中的应用。
所述细胞因子Igfbp7促进老年性骨骼肌增生。
所述骨骼肌为胫骨前肌和腓肠肌。
所述Igfbp7的用量为：0.1μg/g/次。
所述Igfbp7采用腹腔注射给药。
Igfbp7的氨基酸序列如SEQ ID NO:1所示：
细胞因子Igfbp7是一种肌细胞因子，由于衰老而导致肌细胞因子Igfbp7表达减少。申请人的实验表明，给老年性小鼠补充Igfbp7具有提高胫骨前肌和腓肠肌质量和肌力的作用，并证实Igfbp7具有促进老年性小鼠骨骼肌(胫骨前肌和腓肠肌)增生的作用，在后期干预人肌丢失相关疾病方面具有重要前景。
本发明提供了一种新的干预小鼠老年性肌肉萎缩的方法。
附图说明
图1为小鼠多种组织细胞中Igfbp7的表达情况；
图2为不同年龄小鼠骨骼肌中Igfbp7的表达情况；
图3为Igfbp7处理对老年小鼠肌肉萎缩的干预效果。
具体实施方式
一.材料
C57BL/6J小鼠购自斯贝福(北京)生物技术有限公司；
Trizol购自Thermo公司；
反转录试剂及定量PCR相关试剂购自Takara公司；
Igfbp7抗体购自Proteintech公司；Tubulin抗体及Western blot相关试剂购自碧云天公司；
Igfbp7细胞因子购自Abcam公司；
石蜡切片及HE染色相关试剂购自碧云天公司。
其它试剂为市售的分析纯试剂。
二.实施例
实施例1：Igfbp7在小鼠多种组织细胞中的表达情况
1.1实验方法
选取3只8-10周龄C57BL/6J小鼠，处死后取多种组织或者细胞(小肠、肺、骨髓、脾脏、骨骼肌、肾脏、肝脏、胸腺、心脏、胃、脑和外周血)，利用Trizol裂解后提取RNA，反转录并开展定量PCR检测Igfbp7的表达，具体步骤包括：
1.利用Total RNA isolation Kit提取RNA；
2.使用PrimeScript RT reagent Kit反转录500ng RNA；
3.配制PCR反应体系(SYBR Premix Ex TaqII：12.5μL，PCR Forward Primer(10μM)：1.0μL，PCR Reverse Primer(10μM)：1.0μL，RT反应液(cDNA溶液)：2μL，dH2O：8.5μL)；
4.预变性：95℃，3min，变性：95℃，30s，退火：58℃，30s，延伸：72℃，30s(39cycles)，最后延伸：72℃，10min。
其中使用的引物序列包括：
Actin-F：5’-ACCTTCTACAATGAGCTGCG-3’(SEQ ID NO:2)
Actin-R：5’-CTGGATGGCTACGTACATGG-3’(SEQ ID NO:3)
Igfbp7-F：5’-CCTGTCCTCATCTGGAACAAG-3’(SEQ ID NO:4)
Igfbp7-R：5’-GCCCGTTACTTCATGCTTTTC-3’(SEQ ID NO:5)
此外，选取3只8-10周龄C57BL/6J小鼠，处死后取多种组织或者细胞，利用RIPA裂解后开展Western blot检测Igfbp7的表达，具体步骤包括：
1.细胞样品的裂解，利用RIPA裂解液裂解细胞后在沸水中煮5分钟，BCA法测定蛋白浓度；
2.样品电泳，样品加入5×loading buffer后，点样，先以60V电压电泳30分钟，随后110V电压电泳90分钟；
3.转膜，将胶中样品在电场条件下转至PVDF膜(200mA，2小时)；
4.5％脱脂奶粉封闭1小时；
5.一抗4℃过夜孵育；
6.洗膜后常温孵育二抗1小时；
7.洗膜后加入显影液，曝光拍照。
1.2实验结果
mRNA表达水平分析显示Igfbp7主要表达于骨骼肌、肾脏及脾脏中，其中以骨骼肌表达最高(参见图1A)。此外Western blot结果也证实骨骼肌细胞中Igfbp7的蛋白表达非常丰富(参见图1B)，这些结果说明Igfbp7是一种多组织表达的细胞因子，其中骨骼肌是重要分泌组织之一，Igfbp7是一种肌细胞因子。
实施例2：Igfbp7在不同年龄小鼠骨骼肌中表达情况
2.1实验方法
选取6对年轻及衰老小鼠(2月龄及16月龄)，处死后分离骨骼肌(包括胫骨前肌、腓肠肌、比目鱼肌、趾长伸肌)，利用Trizol裂解后提取RNA，反转录并开展定量PCR检测Igfbp7的表达，具体步骤参见实施例中的1.1。
2.2实验结果
mRNA表达水平分析显示衰老小鼠较年轻小鼠骨骼肌细胞中Igfbp7的表达显著降低(参见图2)，提示Igfbp7减少可能参与老年性肌肉萎缩。
实施例3：Igfbp7处理对小鼠老年性肌肉萎缩的干预效果
3.1实验方法
选取老年小鼠(20月龄)，分为溶剂组和Igfbp7处理组，Igfbp7蛋白利用PBS溶解后，按照单次剂量0.1μg/g给处理中小鼠补充Igfbp7，腹腔注射，每周两次，持续8周，溶剂组注射100μL PBS，其余操作同Igfbp7处理组。8周后处死小鼠分离骨骼肌中的腓肠肌和胫骨前肌，称重。此外利用胫骨前肌石蜡切片及HE染色分析肌纤维，具体步骤包括：
1.从小鼠分离胫骨前肌后，PBS漂洗3次后，利用4％多聚甲醛固定6小时；
2.固定后的胫骨前肌置于10％蔗糖溶液脱水，沉底后转至20％蔗糖溶液脱水，再次沉底后转至30％蔗糖溶液，4℃过夜脱水；
3.将脱水后的胫骨前肌放于组织包埋机中，提前将融化的石蜡放入包埋框，于-20℃冻台冷却，石蜡凝固后从包埋框中取出修整形状；
4.将修整好的蜡块置于石蜡切片机上切片，切片厚度8μm。切片于40℃温水上漂浮，随后利用载玻片贴片切片组织，并放于60℃烘箱内烤片；
5.石蜡切片脱蜡水化：将载玻片分别浸于二甲苯脱蜡10min(2次)，无水乙醇2min(2次)，95％乙醇2min，80％乙醇2min，70％乙醇2min，蒸馏水2min；
6.苏木素染液染色5min，自来水冲洗10s；
7.分化液分化5s，自来水冲洗30s；
8.返蓝液返蓝30s，自来水冲洗30s；
9.伊红染色1min，自来水冲洗5s；10.脱水、透明及封片。
此外利用小鼠跑台分析小鼠力竭时间，具体步骤包括：小鼠在测试前3天每天在跑台上进行适应性运动(跑台速度：10m/min，时间：5min)。随后在测试当天小鼠在跑台上首先以10m/min速度运动5min，随后跑台速度慢慢增加至16m/min，记录小鼠力竭时(被持续电击10次时)的总运动时间。
3.2实验结果
Igfbp7处理组小鼠骨骼肌中腓肠肌和胫骨前肌质量较对照组显著增加(参见图3A和3B)，提示Igfbp7促进老龄小鼠胫骨前肌和腓肠肌增生。功能测试显示Igfbp7处理组小鼠具有更长的力竭时间，说明肌力增加(参见图3C)，胫骨前肌组织HE染色也显示Igfbp7处理组小鼠具有更大的肌纤维横截面积，说明胫骨前肌出现增生，有效改善了肌肉萎缩(参见图3D和3E)。这些结果说明Igfbp7有效改善了小鼠老年性肌肉萎缩，具有干预肌丢失的效果。
结论：Igfbp7是一种肌细胞因子，衰老导致肌细胞因子Igfbp7表达减少，给老年性小鼠补充Igfbp7具有提高胫骨前肌和腓肠肌质量和肌力的作用，Igfbp7具有干预老年性小鼠肌肉萎缩的作用。
Claims (5)
1.细胞因子Igfbp7在制备治疗老年性肌肉萎缩的药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述细胞因子Igfbp7促进老年性骨骼肌增生。
3.根据权利要求2所述的应用，其特征在于：所述骨骼肌包括胫骨前肌和腓肠肌。
4.根据权利要求1所述的应用，其特征在于：所述Igfbp7的用量为：0.1μg/g/次。
5.根据权利要求1所述的应用，其特征在于：所述Igfbp7采用腹腔注射给药。
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