CN112472798A - 胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用 - Google Patents
胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明属于医药技术领域，特别涉及胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用。本发明的实验表明，腹腔注射IGF2重组蛋白可有效降低溃疡性结肠炎小鼠结肠炎症因子IL1β、TNFα水平，并恢复结肠中胰岛素样生长因子1型受体、胰岛素样生长因子2结合蛋白2以及紧密连接蛋白ZO1、Occludin的表达水平。此外，IGF2重组蛋白给药可使DSS模型小鼠的结肠长度明显恢复。结果显示，IGF2重组蛋白可通过上调IGF1R、IGF2BP2的表达，降低结肠中炎症因子水平，并提高紧密连接蛋白的表达水平，从而有效防止溃疡性结肠炎小鼠的结肠损伤，可用于制备治疗溃疡性结肠炎的药物制剂。
Description
技术领域
本发明属于医药技术领域，特别涉及胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用。
背景技术
溃疡性结肠炎(UC)是溃疡性结肠炎属非特异性炎症性肠病，世界卫生组织称为特发性结肠炎。是一种病因不明的局限于结肠粘膜及粘膜下层的慢性非特异性炎症过程，病变多位于乙状结肠和直肠，也可延伸至降结肠，甚至整个结肠。其主要特点是结直肠黏膜呈现连续弥漫性炎症，临床表现为腹泻、粘液脓血便、腹痛等，其具体发病机制尚未完全阐明。UC的治愈难度大，长期迁延不愈，有潜在恶变的危险，病因不明，且有病程较长、反复发作的特点，并且发病率呈逐年升高的趋势。因此，寻找有效治疗溃疡性结肠炎的药物成为亟待解决的临床难题之一。
目前，溃疡性结肠炎的药物治疗主要依赖于美沙拉嗪皮质类固醇和硫嘌呤。皮质类固醇依赖性和耐药性是临床上的重要问题。免疫抑制剂，如环孢菌素和他克莫司，以及抗肿瘤坏死因子-α药物，如英夫利昔单抗和阿达木单抗，可用于具有中度至重度的溃疡性结肠炎；然而，由于免疫抑制，先前对这些药物的反应丧失或不耐受以及感染，正日益成为溃疡性结肠炎临床治疗中显现的问题。因此，探索溃疡性结肠炎替代疗法的研究很有意义。
胰岛素样生长因子2(IGF2)，又称为生长调节素A，它在细胞的增殖分化及胚胎的生长发育等过程中起重要作用。0.5％-1％的IGF2以游离形式存在，其余IGF2主要与IGFmRNA结合蛋白(IGF2BP1，IGF2BP2以及IGF2BP3)以结合形式存在于生物体内。IGF2可分别与胰岛素样生长因子1型受体(IGF1R)、胰岛素样生长因子2型受体(IGF2R)以及胰岛素受体结合，从而发挥其在RNA/DNA合成、葡萄糖代谢、氨基酸转运等方面的促进作用。
目前，关于IGF2重组蛋白在治疗溃疡性结肠炎方面的应用未见报道。
发明内容
本发明的目的在于提供一种胰岛素样生长因子2(IGF2)重组蛋白在制备治疗溃疡性结肠炎药物中的应用。
为了实现上述发明目的，本发明提供以下技术方案：
胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用。
作为优选，所述治疗溃疡性结肠炎药物还包括药学上可接受的辅料。
作为优选，所述药学上可接受的辅料选自填充剂、粘合剂、崩解剂、增溶剂、溶剂中的一种或几种。
作为优选，所述治疗溃疡性结肠炎药物的剂型为注射型。
作为优选，所述治疗溃疡性结肠炎药物的输注方式为腹膜内输注。
作为优选，所述治疗溃疡性结肠炎药物可有效抑制DSS模型中结肠组织炎症因子IL1β、TNFα的释放。
作为优选，所述治疗溃疡性结肠炎药物可有效抑制结肠组织紧密连接蛋白ZO1、Occludin表达的下调。
作为优选，所述治疗溃疡性结肠炎药物可有效抑制结肠组织中IGF1R、IGF2BP2表达的下调。
本发明还提供了所述治疗溃疡性结肠炎药物在治疗溃疡性结肠炎药物中的应用。
本发明还提供了所述治疗溃疡性结肠炎药物在治疗溃疡性结肠炎小鼠的结肠损伤并恢复结肠长度的药物中的应用。
本发明观察IGF2重组蛋白对DSS诱导的溃疡性结肠炎的治疗效果，发现IGF2重组蛋白具有明显的结肠黏膜层保护作用，并有效降低结肠炎症因子IL1β、TNFα水平，恢复紧密连接蛋白ZO1、Occludin的表达，并上调IGF1R、IGF2BP2的表达水平。
与现有技术相比，本发明的创新性为：
本发明第一次公开了IGF2重组蛋白可通过上调IGF1R及IGF2BP2水平，降低结肠中炎症因子水平，增强结肠中紧密连接蛋白的表达，从而有效防止溃疡性结肠炎的发生，可用于治疗炎症因素诱导的溃疡性结肠损伤。为溃疡性结肠损伤的治疗提供了新的可能性。
附图说明
图1为Western Blot法检测溃疡性结肠炎小鼠结肠的IGF2蛋白水平；
图2为Western Blot法检测IGF2重组蛋白对溃疡性结肠炎小鼠结肠组织IGF1R、IGF2BP2、ZO1以及Occludin蛋白水平的影响；
图3为HE染色显示IGF2重组蛋白对溃疡性结肠炎小鼠结肠黏膜层的影响；
图4为IGF2重组蛋白给药后溃疡性结肠炎小鼠结肠的外观形态拍照。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
本实验所选动物为8周C57BL/6雄性小鼠，体重约25克，购自湖南斯莱克景达，许可证号：SCXK(湘)2019-0004，小鼠放至人工昼夜12小时循环照明环境中饲养，自由摄食及饮水。C57BL/6雄性小鼠被随机分为灭菌水+BSA对照组(8只)、DSS+BSA造模组(8只)、DSS+IGF2重组蛋白给药组(8只)。对照组每天腹腔注射1次BSA(15μg/kg)，灭菌水自由饮；造模组每天腹腔注射1次BSA(15μg/kg)，5％硫酸葡聚糖(DSS)自由饮；给药组每天腹腔注射1次IGF2重组蛋白(15μg/kg)，5％DSS自由饮。给药7天后，使用4％水合氯醛腹腔注射麻醉小鼠，麻醉完全后取结肠组织，保存于-80℃，用于Western Blot检测。
结肠组织Western Blot步骤如下：
(1)结肠组织中加入RIPA蛋白裂解液后，在组织匀浆机中匀浆1分钟。
(2)4℃，12000rpm离心20分钟，吸取蛋白上清液。
(3)使用碧云天牌BCA蛋白定量试剂盒检测结肠组织蛋白浓度，用蛋白裂解液将蛋白样本稀释至统一浓度后，分别使用各蛋白样本将5×蛋白上样缓冲液稀释成1×，并煮沸10分钟进行蛋白变性。
(4)将变性后的蛋白样本加入到十二烷基硫酸钠聚丙烯酰胺凝胶的浓缩胶梳孔中，将蛋白在100V恒压情况下进行电泳1.5h。
(5)电泳后的凝胶置于200mA恒流情况下转膜2h，将蛋白转印至PVDF膜上。
(6)10％脱脂奶粉常温封闭含蛋白的PVDF膜2h后，一抗4℃孵育12h。
(7)PBST清洗后，使用对应抗性的二抗室温孵育1h，PBST再次清洗。
(8)将PVDF膜置于凝胶成像仪中，滴加化学发光显影液进行显影拍照。
实验结果如图1和图2所示。实验结果显示：溃疡性结肠炎小鼠结肠组织蛋白中IGF2的蛋白条带明显弱于对照组，IGF2在溃疡性结肠炎小鼠结肠中蛋白水平降低；溃疡性结肠炎小鼠结肠组织蛋白中IGF1R、IGF2BP2、ZO1以及Occludin的蛋白条带明显弱于对照组，而IGF2重组蛋白给药后的溃疡性结肠炎小鼠结肠中IGF1R、IGF2BP2、ZO1以及Occludin的蛋白条带强于溃疡性结肠炎小鼠造模组，说明IGF2重组蛋白可明显恢复溃疡性结肠炎小鼠结肠组织中IGF2受体-IGF1R、IGF2 mRNA结合蛋白-IGF2BP2以及紧密连接蛋白ZO1、Occludin的蛋白表达水平。
实施例2
实验动物来源、饲养条件以及给药方案均参照实施例1。在麻醉小鼠后，取结肠组织，分别拍照后，置于4％多聚甲醛固定24h，用于组织HE染色。
结肠组织HE染色步骤：
(1)将固定后的结肠组织按照30％酒精-50％酒精70％酒精-80％酒精-90％酒精-95％酒精-100％酒精-100％酒精(各1h)进行梯度酒精脱水后，使用二甲苯浸泡2h透明，然后石蜡液浸蜡1h，使用石蜡包埋机进行石蜡包埋制作石蜡块。
(2)使用石蜡切片机将石蜡块切成5μm的石蜡切片。
(3)石蜡切片用二甲苯浸泡脱蜡1h后，按照100％酒精-100％酒精-90％酒精-80％酒精-70％酒精-蒸馏水(各1min)进行梯度水化。
(4)石蜡切片上滴加苏木素染液染色5min后，用1％盐酸酒精分化2-3秒。
(5)自来水冲洗，伊红染液染色2-5min。
(6)切片按照70％酒精(10sec)-80％酒精(10sec)-90％酒精(1min)-100％酒精(1min)-100％酒精(1min)脱水后，二甲苯浸泡2-3min透明晾干后，滴加中性树脂封片，显微镜下拍照。
实验结果见图3，图4以及表1所示。
表1.IGF2重组蛋白干预对DSS模型小鼠结肠长度的影响
***P﹤0.001vs灭菌水+BSA组；###P﹤0.001vs DSS+BSA组。采用One-way ANOVA法进行统计分析。
图3和表1显示：DSS模型组小鼠结肠长度(4.84±0.113cm)明显小于对照组(7.76±0.146cm)，而IGF2重组蛋白给药后，小鼠结肠长度(6.04±0.065cm)明显恢复。IGF2重组蛋白可明显恢复DSS导致的小鼠结肠长度减少现象。
图4实验结果显示：相对于正常对照组，DSS模型组小鼠结肠黏膜层杯状细胞明显减少，大量炎性细胞浸润，而IGF2重组蛋白干预后，结肠黏膜层杯状细胞数目明显增多，炎性细胞浸润情况有所缓解。IGF2重组蛋白可明显抑制DSS导致的小鼠结肠黏膜层损伤。
实施例3
实验动物来源、饲养条件以及给药方案均参照实施例1。在麻醉小鼠后，取结肠组织保存于-80℃，用于Real-time PCR检测。
结肠组织Real-time PCR步骤：
(1)RNA提取及反转录：结肠组织中加入Trizol裂解液充分匀浆，加入氯仿萃取得到上层水相，加入等体积异丙醇混匀；离心弃上清，加入75％乙醇清洗RNA沉淀；风干后加入DEPC水溶解；按照反转录试剂盒(Thermo，货号：K1621)说明书将RNA反转录成cDNA。
(2)RT-PCR：10μl反应体系：TB反应液5μl，ROX染料0.2μl，上下游引物(10μM)各0.4μl；cDNA 1μl；去酶水3μl。
步骤1：95℃，5min
步骤2：95℃，15s；60℃，15s；72℃，45s，40个循环
步骤3：GAPDH作为内参基因，通过2-ΔΔCT计算目标基因的相对表达量。
所采用Q-PCR引物及序列如下表2所示：
表2Q-PCR引物及序列
实验结果见表3所示。
表3.IGF2重组蛋白干预对DSS模型小鼠结肠相关基因水平的影响
***P﹤0.001vs灭菌水+BSA组；#P﹤0.05vs DSS+BSA组。采用One-way ANOVA法进行统计分析。
实验结果显示：DSS模型小鼠结肠组织中IGF1R(0.57±0.040vs 1.00±0.048)、IGF2BP2(0.46±0.083vs 1.00±0.067)基因水平明显低于对照组，IL1β(46.60±15.590vs1.01±0.086)、TNFα(4.05±0.515vs 1.00±0.147)的基因水平高于对照组；给予IGF2重组蛋白后，IGF1R(0.84±0.010vs 0.57±0.040)、IGF2BP2(0.77±0.056vs 0.46±0.083)基因水平相对于造模组显著回升，而IL1β(13.48±2.891vs 46.60±15.590)、TNFα(2.65±0.277vs 4.05±0.515)的基因水平相对于造模组明显回落。IGF2重组蛋白给药可提高溃疡性结肠炎小鼠结肠组织中IGF1R、IGF2BP2基因水平，并下调炎症因子IL1β、TNFα的基因水平。
综上，IGF2重组蛋白可通过上调IGF1R及IGF2BP2水平，降低结肠中炎症因子水平，增强结肠中紧密连接蛋白的表达，从而有效防止溃疡性结肠炎的发生，可用于治疗炎症因素诱导的溃疡性结肠损伤。为溃疡性结肠损伤的治疗提供了新的可能性。
尽管已用具体实施例来说明和描述了本发明，然而应意识到，在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
SEQUENCE LISTING
<110> 南华大学附属第一医院
<120> 胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用
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Claims (10)
1.胰岛素样生长因子2重组蛋白在制备治疗溃疡性结肠炎药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物还包括药学上可接受的辅料。
3.根据权利要求2所述的应用，其特征在于，所述药学上可接受的辅料选自填充剂、粘合剂、崩解剂、增溶剂、溶剂中的一种或几种。
4.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物的剂型为注射型。
5.根据权利要求4所述的应用，其特征在于，所述治疗溃疡性结肠炎药物的输注方式为腹膜内输注。
6.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物可有效抑制DSS模型中结肠组织炎症因子IL1β、TNFα的释放。
7.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物可有效抑制结肠组织紧密连接蛋白ZO1、Occludin表达的下调。
8.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物可有效抑制结肠组织中IGF1R、IGF2BP2表达的下调。
9.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物在治疗溃疡性结肠炎药物中的应用。
10.根据权利要求1所述的应用，其特征在于，所述治疗溃疡性结肠炎药物在治疗溃疡性结肠炎小鼠的结肠损伤并恢复结肠长度的药物中的应用。
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