ERK1/2 siRNA干扰质粒对IGF-1诱导结肠平滑肌细胞产生干细胞因子的影响

摘要

目的: 探讨IGF-1诱导的大鼠结肠平滑肌细胞(SMC)产生干细胞因子(SCF)的信号途径.

方法: 以不同时间(0, 5, 15, 30, 45, 60 min)、不同浓度(0, 50, 100, 150 μg/L)的IGF-1诱导结肠SMC; 以ERK1/2 siRNA干扰质粒转染大鼠结肠SMC, RT-PCR及Western blot检测IGF-1诱导下结肠SMC产生ERK1/2及SCF的变化.

结果: 在15 min时IGF-1诱导结肠SMC表达磷酸化ERK1/2最强(0.417±0.036 vs 0.101±0.015, P<0.05). 在100 μg/L作用15 min时磷酸化ERK1/2和SCF表达最强(0.790±0.051 vs 0.336±0.013; 0.765±0.061 vs 0.289±0.021, 均P<0.05). 在IGF-1诱导下, 转染入ERK1/2 siRNA的结肠SMC产生的p-ERK1/2、t-ERK1/2及SCF较对照组明显降低(0.284±0.021 vs 0.732±0.005; 0.256±0.015 vs 0.712±0.023; 0.219±0.020 vs 0.673±0.013; 0.621±0.027 vs 1.725±0.012; 0.821±0.019 vs 1.751±0.043; 0.275±0.061 vs 0.531±0.047; 均P<0.05).

结论: IGF-1可以使结肠SMC表达磷酸化ERK1/2和SCF增加. ERK1/2 siRNA可使ERK1/2和结肠SMC产生的SCF明显降低, 从而证实IGF-1促进大鼠结肠SMC产生SCF是通过ERKMAPK通路.

关键词: ERK1/2; 转染; 胰岛素样生长因子1; 结肠平滑肌细胞; 干细胞因子

Transfection with siRNA against ERK1/2 inhibits IGF-1-induced stem cell factor expression in colonic smooth muscle cells

Yu-Feng Yuan, Ying-Juan Yu, Lin Lin

Yu-Feng Yuan, Ying-Juan Yu, Lin Lin, Department of Gastroenterology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China

Supported by: National Natural Science Foundation of China, No. 30971354; and the Natural Science Foundation of Jiangsu Province, No. BK2008466.

Correspondence to: Professor Lin Lin, Department of Gastroenterology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China. lin9100@yahoo.com.cn

Received: November 24, 2010
Revised: January 4, 2011
Accepted: January 11, 2011
Published online: February 28, 2011

Abstract

AIM: To investigate how insulin-like growth factor-1 (IGF-1) regulates the expression of stem cell factor (SCF) in colonic smooth muscle cells (SMCs).

METHODS: After rat colonic SMCs were treated with different concentrations of IGF-1 (0, 50, 100, 150 μg/L) for different durations (0, 5, 15, 30, 45, 60 min), the levels of phosphorylated ERK1/2 and SCF were determined by RT-PCR and Western blot. Rat colonic SMCs were then transfected with siRNA against ERK1/2 to examine the impact of ERK1/2 down-regulation on IGF-1-induced SCF expression.

RESULTS: After treatment with IGF-1, the level of phosphorylated ERK1/2 in colonic SMCs reached a peak at about 15 min (0.417 ± 0.036 vs 0.101 ± 0.015; P < 0.05). The optimal concentration of IGF-1 to induce the expression of phosphorylated ERK1/2 and SCF was 100 μg/L (0.790 ± 0.051 vs 0.336 ± 0.013; 0.765 ± 0.061 vs 0.289 ± 0.021, both P < 0.05). After treatment with IGF-1, the expression levels of phosphorylated ERK1/2, total ERK1/2, and SCF in colonic SMCs transfected with siRNA against ERK1/2 were lower than those in the control group (0.284 ± 0.021 vs 0.732 ± 0.005; 0.256 ± 0.015 vs 0.712 ± 0.023; 0.219 ± 0.020 vs 0.673 ± 0.013; 0.621 ± 0.027 vs 1.725 ± 0.012; 0.821 ± 0.019 vs 1.751 ± 0.043; 0.275 ± 0.061 vs 0.531 ± 0.047; all P < 0.05).

CONCLUSION: IGF-1 treatment up-regulated the expression of phosphorylated ERK1/2 and SCF in colonic SMCs, while transfection with siRNA against ERK1/2 down-regulated IGF-1-induced expression of phosphorylated ERK1/2 and SCF, suggesting that the ERK/MAPK pathway may be involved in IGF-1-induced expression of phosphorylated ERK1/2 and SCF.

Key Words: ERK1/2; Transfection; Insulin-like growth factor-1; Colonic smooth muscle cell; Stem cell factor

0 引言

胃肠动力障碍性疾病与胃肠道Cajal间质细胞(interstitial cells of Cajal, ICC)的缺失或病变有关, 干细胞因子(stem cell factor, SCF)作为ICC生长、功能及表型维持的主要调控因子, 是影响ICC数量和超微结构改变的直接原因. 研究发现, 胰岛素样生长因子1(insulin-like growth factor 1, IGF-1)可调控胃平滑肌细胞(smooth muscle cell, SMC)合成SCF, 进而对胃ICC有保护作用[1]. 我们前期实验发现, IGF-1能促进体外培养的胃和结肠SMC合成SCF, 呈浓度和时间依赖性[2-4]; 且在细胞水平发现胃结肠SMC该作用是通过ERKMAPK通路完成的[3,4]. 本实验从基因水平进一步证实IGF-1诱导大鼠结肠SMC表达SCF的信号通路.

1 材料和方法

1.1 材料

ERK1/2 siRNA由Invitrogen公司合成, 序列参照文献[5], ERK1/2 siRNA序列为: 5'-GCCGCCGCCGCCGCCAT-3', 与MAPK mRNA翻译起始部位的17个碱基序列互补; 随机核苷酸序列: 5'-CGCGCGCTCGCGCACCC-3'. SD大鼠, 雌雄不拘, 体质量180-200 g(南京医科大学动物中心提供). DMEM培养液、胎牛血清(Gibco公司); 大豆胰蛋白酶抑制剂(Gibco, USA); Ⅱ型胶原酶(Sigma公司); 重组大鼠IGF-1(R&D, UK); SCF抗体(Santa Cruz, USA); phospho-p44/42 MAPK抗体、p44/42 MAPK抗体(CST, USA).

1.2 方法

1.2.1 结肠SMC的分离和培养: SD大鼠断椎处死, 快速取自肛门上2 cm至回盲部的全部结肠, 含抗生素的缓冲液反复漂洗、仔细剥离黏膜层和浆膜层. 将平滑肌组织减碎匀浆, 置入消化液(0.1%的Ⅱ型胶原酶和0.01%的大豆胰蛋白酶抑制剂)中, 30 ℃消化20 min, 1 000 r/min离心5 min、弃消化液, 反复2次, 加入含100 mL/L胎牛血清的DMEM培养液终止消化, 1 000 r/min离心5 min, DMEM培养液重悬细胞、过筛、50 mL/L CO₂、37 ℃孵育箱中培养, SMC长至致密单层时, 传代培养. 用3-5代SMC进行实验.

1.2.2 转染: 按FuGENE^®步骤进行操作: (1)将状态良好、处于对数生长期的SMC用2.5 g/L胰酶、完全培养基悬浮成单细胞悬液, 按照每孔3-6×10⁶个细胞接种于6孔板中; (2)补充完全培养液至2 mL, 细胞生长稳定且细胞贴壁覆盖率70%-80%左右时, 进行转染实验; (3)用100 μL无血清DMEM培养液稀释2 μg质粒; (4)震荡混悬FuGENE^®, 将6 μL FuGENE^®加入质粒稀释液; (5)充分混合, 室温孵育15 min; (6)将质粒复合物缓慢加入各培养皿中(加有2 mL不含抗生素的完全培养液)、混匀、置于37 ℃、50 mL/L的CO₂培养箱中培养; (7)第2天观察转染效率, 培养48 h.

1.2.3 分组: SMC+IGF-1(100 μg/L)[3], 分别培养0, 5, 15, 30, 45, 60 min. 不同浓度组: SMC+IGF-1(0, 50, 100, 150 μg/L)培养15 min. 转染组: 将结肠SMC分为对照组、反义组(加入ERK1/2 siRNA)、随机组(加入随机核苷酸), 培养48 h, 再加入100 μg/L IGF-1培养16 h. 以上实验重复3次.

1.2.4 RP-PCR法检测总ERK1、ERK2和SCF mRNA: 按TRIzol试剂提取各组细胞的总mRNA, 以cDNA为模板进行PCR扩增. (1)SCF和内参GAPDH的循环条件: 94 ℃预变性5 min、94 ℃变性15 s、55 ℃退火30 s、72 ℃延伸15 s、共循环30次, 最后于72 ℃延伸8 min. SCF上游引物: 5'-TTC GCT TGT AAT TGG CTT TGC-3'; 下游引物: 5'-CAA CTG CCC TTG TAA GAC TTG CA-3'(76 bp). GAPDH上游引物: 5'-CCC CCA ATG TAT CCG TTG TG-3'; 下游引物: 5'-TAG CCC AGG ATG CCC TTT AGT-3'(118 bp). (2)ERK1/2循环条件: 94 ℃预变性5 min、94 ℃变性30 s、58 ℃退火30 s、72 ℃延伸30 s、共循环30次, 最后于72 ℃延伸7 min. ERK1上游引物: 5'-TAC ACG CAG TTG CAG TAC ATC G-3'; 下游引物: 5'-CGC AGG ATC TGG TAG AGG AAG T-3'(332 bp); ERK2上游引物5'-GGA GCT TGT GGA AAT ACC TTG G-3'; 下游引物: 5'-GAC GCA GTG TTC CTC TCT GCT A-3'(499 bp)[6]. PCR产物经2%琼脂糖凝胶电泳、观察、拍照.

1.2.5 Western bolt法检测SMC中的p-ERK1/2、t-ERK1/2和SCF蛋白: 蛋白裂解液提取各组细胞蛋白, BCA法测定蛋白浓度. 40 μg蛋白/泳道加样, 恒流30 mA电泳, 恒压100 V转膜60 min, 封闭2 h. 分别加入p-ERK1/2、t-ERK1/2和SCF一抗, 4 ℃过夜; 二抗1:5 000, 37 ℃孵育, 曝光、显影.

统计学处理 所有数据录入SPSS10.0软件包分析, 以mean±SD表示, 采用方差分析和成组t检验, P<0.05为有显著性差异.

2 结果

2.1 IGF-1诱导结肠SMC不同时间对p-ERK1/2和t-ERK1/2蛋白的影响

IGF-1(100 μg/L)作用于结肠SMC 5 min后p-ERK1/2表达开始增高, 15 min时表达最高(P<0.05), 之后逐渐降低, 但t-ERK1/2蛋白的表达无变化(P>0.05, 表1, 图1).

表1 IGF-1(100 μg/L)诱导结肠SMC对p-ERK1/2和t- ERK1/2蛋白的影响 (mean±SD).

分组	p-ERK1/2	t-ERK1/2
0 min	0.101±0.015c	0.132±0.021
5 min	0.214±0.019c	0.135±0.017
15 min	0.417±0.036a	0.134±0.026
30 min	0.306±0.023c	0.136±0.019
45 min	0.293±0.041c	0.134±0.013
60 min	0.209±0.053c	0.136±0.031

^aP<0.05 vs 0 min;

^cP<0.05 vs 15 min.

图1 100 μg/L的IGF-1作用于结肠SMC对p-ERK1/2蛋白的影响. 1: 0 min; 2: 5 min; 3: 15 min; 4: 30 min; 5: 45 min; 6: 60 min.

2.2 不同浓度IGF-1诱导结肠SMC对p-ERK1/2、t-ERK1/2和SCF蛋白的影响

IGF-1在50 μg/L时, p-ERK1/2和SCF表达开始增加(P<0.05), 在100 μg/L时表达最多(P<0.05), 之后逐渐下降, 但t-ERK1/2没有变化(P>0.05, 表2, 图2).

表2 IGF-1诱导结肠SMC对p-ERK1/2、t-ERK1/2和SCF蛋白的影响 (mean±SD).

IGF-1(μg/L)	p-ERK1/2	t-ERK1/2	SCF
0	0.336±0.013	0.409±0.021	0.289±0.021
50	0.554±0.046a	0.410±0.012	0.391±0.017a
100	0.790±0.051ce	0.411±0.013	0.654±0.061ce
150	0.591±0.029a	0.408±0.043	0.498±0.043a

^aP<0.05 vs IGF-1(0 μg/L);

^cP<0.05 vs IGF-1(50 μg/L);

^eP<0.05 vs IGF-1(150 μg/L).

图2 不同浓度IGF-1对结肠SMC中p-ERK1/2和SCF蛋白的影响. 1: 0 μg/L; 2: 50 μg/L; 3: 100 μg/L; 4: 150 μg/L.

2.3 IGF-1对于转染ERK1/2 siRNA的结肠SMC产生p-ERK1/2、t-ERK1/2的影响

与对照组相比, 反义组结肠SMC产生p-ERK1/2、t-ERK1/2显著降低(P<0.05), 随机组无显著变化(P>0.05, 表3, 图3), 说明ERK1/2 siRNA确实降低了ERK1/2的表达.

表3 IGF-1对转染ERK1/2 siRNA的结肠SMC产生p-ERK1/2、t-ERK1/2和SCF mRNA及蛋白的影响 (mean±SD).

分组	mRNA			蛋白
	ERK1	ERK2	SCF	p-ERK1/2	t-ERK1/2	SCF
对照组	0.732±0.005	0.712±0.023	0.673±0.013	1.725±0.012	1.751±0.043	0.531±0.047
反义组	0.284±0.021ac	0.256±0.015ac	0.219±0.020ac	0.621±0.027ac	0.821±0.019ac	0.275±0.061ac
随机组	0.721±0.037	0.725±0.037	0.669±0.027	1.754±0.041	1.847±0.011	0.542±0.037

^aP<0.05 vs 对照组;

^cP<0.05 vs 随机组.

图3 IGF-1对于转染ERK1/2 siRNA的结肠SMC产生p-ERK1/2和SCF的影响. A: mRNA; B: 蛋白; M: 100 bp DNA标记; 1: 正常组; 2: 反义组; 3: 随机组.

2.4 IGF-1对转染ERK1/2 siRNA的结肠SMC产生SCF的影响

与对照组相比, 反义组结肠SMC产生SCF显著降低(P<0.05), 随机组无显著变化(P>0.05, 表3, 图4).

图4 IGF-1对于转染ERK1/2 siRNA的结肠SMC产生SCF的影响. A: mRNA; B: 蛋白; M: 100 bp DNA标记; 1: 对照组; 2: 反义组; 3: 随机组.

3 讨论

胃肠动力障碍是消化科常见症候群(包括糖尿病胃肠动力障碍), 严重影响患者的生活质量, 其机制尚不清楚. ICC是一类起源于间充质的特殊细胞群, 以网状结构分布于消化系肠神经末梢和平滑肌细胞之间; ICC、肠神经细胞和SMC构成"功能元件", 共同调控胃肠道的各种生理功能[7,8]. 近年随着对胃肠道ICC结构、功能及其胃肠运动调控机制的深入研究, 已经证实ICC数量和结构异常与多种胃肠疾病相关[9-14].

SCF是一种多功能细胞生长因子, 包括可溶型干细胞因子(soluble stem cell factor, s-SCF)和膜结合型干细胞因子(membrane-bound stem cell factor, m-SCF), 两者都有生物学活性, 在多种细胞的增殖、分化和迁移过程中发挥重要的调控作用. 研究发现, 人体SCF可由多种细胞产生, 但维持胃肠ICC所需的SCF来源于胃肠SMC[1]. SCF与其天然配体c-Kit组成SCF-Kit系统, 直接参与ICC的分化、发育、增殖、表型维持等过程[15-18]. 我们的前期实验发现, SCF减少与胃肠ICC病变及胃肠运动障碍有直接关系, 给予外源性SCF可明显改善糖尿病模型鼠胃肠ICC及胃肠运动异常[16,19-20], 另外前期实验还提示, 体外培养胃和结肠SMC, 给予IGF-1可促进SCF表达[2-4].

IGF-1是胰岛素样生长因子家族中的一员, 具有促进细胞增殖和分化, 调节细胞周期, 加速细胞物质代谢, 抑制细胞凋亡等功能[21]. IGF-I的信号转导途径主要为P13K途径和MAPK途径[22]. 文献报道, IGF-1可使SMC表面的IGF-1 R磷酸化, 进而激活细胞内下游的信号通路, 主要通过PI3K、MAPK信号转导通路, 以协同或分别或以相反的作用方式, 参与IGF-1介导的细胞增殖、分化、抗凋亡等效应[23]. 丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)是细胞内的一类丝氨酸/苏氨酸蛋白激酶, 该家族主要包括细胞外信号调节蛋白激酶(extracellular regulating kinase, ERK)、c-Jun氨基末端激酶和P38蛋白激酶[24,25], 他将细胞外刺激信号转导至细胞内及细胞核内, 引起细胞一系列生物学反应如细胞增殖、分化、转化及凋亡[26,27]. ERK通路是迄今研究较多、备受关注的重要MAPK信号通路. 在细胞中各种因素[28,29]激活ERK1/2后, 活化的ERK1/2激酶移位到细胞核, 调控信号转导途径的关键效应分子(如NF-κB、AP-1、c-Myc等)的表达, 在细胞的增殖中起重要作用[30,31]. 我们前期实验将ERKMAPK的抑制剂PD98059加入结肠SMC、使IGF-1诱导的SCF表达降低, 提示IGF-1是通过ERKMAPK通路诱导结肠SMC产生SCF[2-4]. 本实验向结肠SMC转染ERK siRNA使p-ERK1/2、总ERK1/2和SCF表达均降低(包括mRNA和蛋白), 说明干扰成功, 进而从基因水平证实IGF-1诱导结肠SMC产生SCF确实通过ERKMAPK通路.

总之, 本实验从基因水平证实IGF-1诱导结肠SMC产生SCF的信号通路, 为进一步探讨SCF的调控、合成、保护胃肠ICC及其信号途径和影响因素提供了可靠的实验依据, 有利于进一步探讨胃肠ICC在胃肠动力障碍性疾病中的作用、为开发新的治疗药物拓展了思路.

评论

背景资料

胃肠动力障碍性疾病与胃肠道Cajal间质细胞(ICC)的缺失或病变有关, 干细胞因子(SCF)作为ICC生长、功能及表型维持的主要调控因子, 是影响ICC数量和超微结构改变的直接原因.

同行评议者

王钦红, 副教授, 美国杜克大学医学院肿瘤生物系

研发前沿

尽管已知IGF-1在多种细胞的增殖分化过程中扮演着重要角色, 但在胃肠道SMC中的具体作用机制尚不清楚.

相关报道

Horvath等将糖尿病小鼠胃窦和胃体组织给予IGF-1, 可完全阻止ICC的减少, 提示IGF-1可能刺激平滑肌细胞表达干细胞因子, 进而对ICC起保护作用. 宁月季等在细胞水平用IGF-1刺激大鼠胃和结肠SMC, 发现IGF-1通过ERKMAPK通路刺激SMC产生SCF.

创新盘点

本研究首次应用RNA干扰技术对结肠SMC进行干预, 结果证实了IGF-1调控SMC产生SCF的信号通路.

应用要点

IGF-1通过ERKPAPK通路刺激结肠SMC表达SCF, 从而对ICC起调控作用, 从而为临床治疗胃肠动力障碍性疾病提供了新的理论依据.

同行评价

本文新颖性较好, 为更好地理解IGF-1调控SCF产生的机制提供实验理论基础.

备注

编辑: 李薇 电编:何基才

参考文献

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