利用Trim-Away技术降低人胰岛淀粉样多肽在大鼠胰岛素瘤细胞中的毒性

doi:10.16288/j.yczz.20-061

遗传 ›› 2020, Vol. 42 ›› Issue (6): 586-598.doi: 10.16288/j.yczz.20-061

利用Trim-Away技术降低人胰岛淀粉样多肽在大鼠胰岛素瘤细胞中的毒性

龚葭薇, 孔德麟, 杨琳, 聂玉哲, 梁洋, 滕春波()

东北林业大学生命科学学院,哈尔滨 150040

收稿日期:2020-03-07 修回日期:2020-06-06 出版日期:2020-06-20 发布日期:2020-06-15
通讯作者: 滕春波 E-mail:chunboteng@nefu.edu.cn
作者简介:龚葭薇,在读硕士研究生,专业方向：发育生物学。E-mail:869268764@qq.com
基金资助:
国家自然科学基金项目编号(31472159);黑龙江省自然科学基金重点项目资助编号(ZD2017001)

Toxicity reduction of human islet amyloid polypeptide by Trim-Away technique in insulinoma cells

Jiawei Gong, Delin Kong, Lin Yang, Yuzhe Nie, Yang Liang, Chun-Bo Teng()

College of Life Sciences, Northeast Forestry University, Harbin 150040, China

Received:2020-03-07 Revised:2020-06-06 Online:2020-06-20 Published:2020-06-15
Contact: Teng Chun-Bo E-mail:chunboteng@nefu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China No(31472159);the Natural Science Foundation of Heilongjiang Province No(ZD2017001)

摘要/Abstract

摘要：

人胰岛淀粉样多肽(human islet amyloid polypeptide, hIAPP)又称胰淀素(Amylin),是胰岛β细胞中胰岛素的共分泌蛋白,与胰岛素共同包裹在囊泡中被分泌出细胞。正常生理条件下,hIAPP有助于胰岛素分泌并调节机体血糖平衡;但其蛋白错误折叠或过量积累则会对细胞造成毒性,进而影响β细胞功能,导致机体罹患2型糖尿病(type 2 diabetes mellitus, T2DM)。为了清除细胞内过度积累的hIAPP,且不影响其正常的合成功能,本研究选用一种新的蛋白质降解技术——Trim-Away,该技术可以在短时间内降解目标蛋白质,且不会对靶蛋白的mRNA转录、翻译等功能产生影响。首先在大鼠(Rattus norvegicus)胰岛素瘤细胞(insulinoma cells, INS1)中过表达hIAPP模拟其过度累积的情况,并通过乳酸脱氢酶(lactate dehydrogenase, LDH)的释放、CCK8 (cell counting kit-8)的活性以及PI-Annexin V流式检测的阳性比例变化,证明hIAPP的过度积累造成β细胞的凋亡;通过实时定量PCR及ELISA检测发现胰岛素的合成和分泌都受到了阻碍;最后利用Trim-Away技术在hIAPP过表达的INS1细胞中特异性清除了过度积累的hIAPP蛋白。细胞活性实验证实清除hIAPP蛋白可以减少细胞的死亡,ELISA实验证实INS1细胞恢复了胰岛素的分泌能力。本研究验证了hIAPP过度积累对INS1细胞的毒性作用,并且证明Trim-Away技术在清除胰腺β细胞中hIAPP毒性具有效果,为利用Trim-Away治疗糖尿病提供了新的策略。

关键词: Trim-Away, 人胰岛淀粉样多肽, β细胞;, 胰岛素分泌

Abstract:

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a co-secreting protein of insulin in human pancreatic β-cells. It is encapsulated in vesicles and secreted out of the cells with insulin. hIAPP can promote insulin secretion and regulate blood glucose homeostasis in the body under the normal physiological conditions. However, hIAPP misfolding or excessive accumulation can cause toxic effects on the β cells, which in turn affect cell function, resulting in type 2 diabetes mellitus (T2DM) for the affected individuals. In order to eliminate the excessive accumulation of hIAPP in the cell and to maintain its normal synthetic function, we have adopted a new protein degradation technology called Trim-Away, which can degrade the target protein in a short time without affecting the mRNA transcription and translation synthesis function of the target protein. First, we overexpressed hIAPP in the rat insulinoma cells (INS1) to simulate its excessive accumulation and analyzed its effect in INS1 cells by measuring the release of LDH (lactate dehydrogenase), CCK8 activity and PI-Annexin V positive ratio. Results showed that excessive accumulation of hIAPP caused β cell apoptosis. Second, real-time quantitative PCR analysis and ELISA detection showed that the synthesis and secretion of insulin were hindered. We used Trim-Way technology to specifically eliminate the excessive accumulation of hIAPP protein in hIAPP overexpressing INS1 cells. Cell activity experiments confirmed that clearance of hIAPP reduced the cell death phenotype. Further ELISA experiments confirmed that INS1 cells restored insulin secretion ability. This study examined the toxic effect of hIAPP excessive accumulation in INS1 cells and demonstrated the cytotoxicity clearance effect of Trim-Way technology in pancreatic β-cells. Our research has provided a new strategy for using Trim-Away technology for treatment of diabetes.

Key words: Trim-Away, human islet amyloid polypeptide (hIAPP), β-cell;, insulin secretion

龚葭薇, 孔德麟, 杨琳, 聂玉哲, 梁洋, 滕春波. 利用Trim-Away技术降低人胰岛淀粉样多肽在大鼠胰岛素瘤细胞中的毒性[J]. 遗传, 2020, 42(6): 586-598.

Jiawei Gong, Delin Kong, Lin Yang, Yuzhe Nie, Yang Liang, Chun-Bo Teng. Toxicity reduction of human islet amyloid polypeptide by Trim-Away technique in insulinoma cells[J]. Hereditas(Beijing), 2020, 42(6): 586-598.

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目的	引物名称	序列(5'→3')
PCR	lv3-hIAPP	F: acctccatagaagattctagaATGGGCATCCTGAAGCTGC
	lv3-hIAPP	R: ctctccatggtggcgtctagaTAAAGGGGCAAGTAATTCAGTGG
	pcDNA3.1-hIAPP	F: tagtccagtgtggtggaattcATGGGCATCCTGAAGCTGC
	pcDNA3.1-hIAPP	R: tgctggatatctgcagaattcTAAAGGGGCAAGTAATTCAGTGG
	pcDNA3.1-humanTim21	F: tagtccagtgtggtggaattcATGGCTTCAGCAGCACGC
	pcDNA3.1-humanTim21	R: tgctggatatctgcagaattcATATAGTCAGTGGATCCTTGTGATCC
	pcDNA3.1-ratTrim21	F: tagtccagtgtggtggaattcATGTCACACTCTACAACCTCAAAAATG
	pcDNA3.1-ratTrim21	R: tgctggatatctgcagaattcGGTATCTTTAGTGGACAGAGCTTTAGAG
qPCR	Bax	F: TGTTACAGGGTTTCATCCAGG
	Bax	R: GTGTCCACGTCAGCAATCA
	Bcl2	F: AAGCACATCCAATAAAAGCGC
	Bcl2	R: GTTATCATACCCTGTTCTCCCG
	Ins1	F: CTAAGTGACCAGCTACAATCA
	Ins1	R: CCACTTCACGACGGGACTTG
	Ins2	F: TACAGTCGGAAACCATC
	Ins2	R: GAAGGTCACCTGCCCCCG
	PDX1	F: CCTACCCCGGGCTGCCGGCC
	PDX1	R: CCGGGGGACTTCCCTGTTCC
	NeuroD1	F: CTCTTTCAAACACGAACCATCC
	NeuroD1	R: GAATGGTGAAACTGACGTGC
	Nkx6.1	F: AGCACAAATCCAGCGGAG
	Nkx6.1	R: CTCCTTTCCTCGCCATCAC
	SLC2a2	F: AGATTGCTCCAACCACACTC
	SLC2a2	R: GCTTTTCTTTGCCCTGACTTC
	Actin	F: CAAGCAGGAGTACGATGAGTC
	Actin	R: AACGCAGCTCAGTAACAGTC

利用Trim-Away技术降低人胰岛淀粉样多肽在大鼠胰岛素瘤细胞中的毒性

Toxicity reduction of human islet amyloid polypeptide by Trim-Away technique in insulinoma cells

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