干细胞因子对糖尿病小鼠结肠Cajal间质细胞的干预效应

摘要

目的: 探讨外源性干细胞因子(stem cell factor, SCF)能否改善糖尿病(diabetes mellitus, DM)小鼠结肠Cajal间质细胞(interstitial cells of Cajal, ICC)的异常病变.

方法: DM小鼠一次性ip链脲佐菌素(STZ, 150 mg/kg)造模, 将♂C57/BL6小鼠分为正常对照组(control组)、糖尿病组(DM组)、糖尿病+外源性SCF组(DM+SCF组); DM+SCF组ip SCF 0.2 µg/(kg•d), control组和DM组每天ip等量的磷酸盐缓冲液(pH = 7.4), 干预6 wk后处死所有小鼠, 以Western blot检测远端结肠组织中SCF的表达情况, 以免疫组化、透射电镜和Western blot观察远端结肠ICC的变化.

结果: DM小鼠远端结肠组织中SCF水平明显降低(178.97±13.51 vs 200.25±16.48, P<0.05), 且伴结肠组织中ICC数量减少(72±10 vs 102±12, P<0.05)、超微结构严重破坏. DM鼠给予外源性SCF干预后, 结肠组织中SCF表达(210.14±11.8)上调(P<0.05), 且ICC的数量(87±10, P<0.05)和超微结构显著改善.

结论: 外源性SCF对糖尿病相关的结肠ICC异常病变有一定改善或逆转作用.

关键词: 糖尿病; 干细胞因子; Cajal间质细胞; 结肠; 免疫印迹; 免疫组化

Effect of stem cell factor on colon interstitial cells of Cajal in murine with diabetes mellitus

Li-Ming Xu, Lin Lin, Yu-Rong Tang, Hong-Jie Zhang, Xue-Liang Li

Li-Ming Xu, Lin Lin, Yu-Rong Tang, Hong-Jie Zhang, Xue-Liang Li, Department of Gastroenterology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China

Supported by: the State Key Development Program for Basic Research of China, No. 2006CB503908.

Correspondence to: 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: December 24, 2007
Revised: March 12, 2008
Accepted: April 21, 2008
Published online: April 28, 2008

Abstract

AIM: To investigated whether exogenous stem cell factor (SCF) can improve the diabetes-associated depletion of interstitial cells of Cajal (ICC) in mice with diabetes mellitus (DM).

METHODS: DM mice were intraperitoneally injected with streptozocin (STZ) to induce an experimental model. Male C57/BL6 mice were randomly divided into control group, DM group and DM + SCF group. The mice in DM + SCF group were given exogenous SCF (0.2 µg/kg per day, ip) for 6 wk, and the mice in control group and DM group were given the same amount of phosphate buffer (pH = 7.4). All the mice were sacrificed after 6 wk. ICC changes in the distal colon were assessed by immunohistochemistry, transmission electron microscopy and Western blot, and SCF expression in the distal colon was analyzed by Western blot.

RESULTS: The expression of SCF in the distal colon was significantly reduced in DM group as compared with that in the control group (178.97 ± 13.51 vs 200.25 ± 16.48, P < 0.05), accompanied with the depletion (72 ± 10 vs 102 ± 12, P < 0.05) and microscopic lesions of ICC in the distal colon. The expression of SCF in the distal colon was increased in DM + SCF group (210.14 ± 11.8, P < 0.05), along with the dramatic improvement of ICC quantity (87 ± 10, P < 0.05) and ultrastructure in the distal colon as compared with those in DM group.

CONCLUSION: Exogenous SCF may improve the DM-associated depletion of colon ICC.

Key Words: Diabetes mellitus; Stem cell factor; Interstitial cells of Cajal; Colon; Western blot; Immunohistochemistry

0 引言

糖尿病(diabetes mellitus, DM)患者中, 胃肠动力障碍的发病率为25%-76%[1-2], 结肠动力障碍是DM常见并发症之一, 临床上可有便秘与腹泻等症状, 其中便秘在1型DM患者中发病率很高[3], 也是2型DM最常见的胃肠道症状[4], 严重影响患者的生活质量与血糖控制. Cajal间质细胞(interstitial cells of Cajal, ICC)是胃肠道起搏细胞, 且介导神经递质的产生和作用[5-6], 其数量和结构改变可能是DM结肠动力障碍重要原因[7-9]. ICC特异性表达的酪氨酸激酶受体c-Kit, 其配体干细胞因子[10](stem cell factor, SCF)已被证实对ICC具有重要调控作用[11-12]. 本研究将初步探讨外源性SCF对DM小鼠结肠ICC的作用或影响.

1 材料和方法

1.1 材料

4-6 wk♂C57/BL6小鼠(上海斯莱克公司), 链脲佐菌素(streptozocin, STZ, Sigma, USA), SCF抗体(R&D, UK), c-Kit抗体(Santa Cruz, USA), 二抗(Santa Cruz, USA), philips EM-400透射电镜等.

1.2 方法

1.2.1 建立DM小鼠模型、分组及干预: DM小鼠予一次性ip STZ(150 mg/kg)造模[13-14], 72 h后及1 wk后尾静脉采血, 两次血糖均≥12 mmol/L者为DM小鼠模型建立, 不符合条件者予以剔除. 所有实验小鼠分为正常对照组(control组, n = 6)、糖尿病组(DM组, n = 6)和糖尿病+外源性SCF干预组(DM+SCF组, n = 6). 干预(共6 wk): DM+SCF组给予SCF ip[0.2 µg/(kg•d)], control组和DM组每天ip等量的磷酸盐缓冲液(pH = 7.4), 6 wk结束后, 停止干预3 d后处死各组小鼠. 取小鼠距肛门1 cm左右的远端结肠组织约2 cm.

1.2.2 结肠组织SCF及c-Kit的表达(Western blot法): 取约0.5 g远端结肠研磨提取蛋白, BCA法测定蛋白浓度. 按50 µg蛋白/泳道加样, 恒流40 mA电泳, 恒压100 V转膜2 h, 50 mL/L牛奶封闭1.5 h. 检测SCF时, 加入SCF一抗(1:500), 37℃孵育1 h, 4℃过夜, 加入二抗(1:2000), 37℃孵育2 h, 曝光、显影. 检测c-Kit时, 一抗浓度为1:200, 二抗浓度为1:1500, 其余条件同前.

1.2.3 结肠组织c-Kit的表达(免疫组化, SP法): 取远端结肠约0.5 cm, 常规固定、包埋、切片, 脱蜡至水、抗原修复. 加c-Kit一抗(1:100)50 μL, 37℃孵育1 h后4℃过夜, 加生物素标记的二抗及链霉素抗生物素蛋白-过氧化酶各50 μL, 室温孵育各15 min, DAB显色, 复染、脱水、封片. 结果判断: 显微镜下胞质出现棕黄色片状或颗粒状物为阳性.

1.2.4 ICC超微结构观察(电镜): 取远端结肠约0.5 cm, 放入戊二醛固定, 经前固定、后固定、块染、脱水、浸渍后包埋、聚合, 半薄切片定位观察, 确定组织分层后, LKB-Ⅱ超薄切片机切片, 染色后透射电镜观察.

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为有统计学差异.

2 结果

2.1 远端结肠组织中SCF和c-Kit蛋白表达(Western blot)

干预6 wk后, DM组远端结肠中SCF和c-Kit蛋白分别较正常对照组显著降低(178.97±13.51 vs 200.25±16.48, 146.24±16.64 vs 196.73±8.48, P<0.05), DM+SCF组较DM组显著增加(210.14±11.8 vs 178.97±13.51, 169.36±15.8 vs 146.24±16.64, P<0.05). DM+SCF组与正常对照组中SCF蛋白表达无统计学差异(P>0.05), 而c-Kit蛋白有统计学差异 (P<0.05, 图1-2).

图1 Western blot检测远端结肠SCF和c-Kit蛋白表达. A: SCF; B: c-Kit. 1: 正常组; 2: DM组; 3: DM+SCF组.

图2 Western blot灰度扫描值. A: SCF; B: c-Kit. aP<0.05 vs 正常组; cP<0.05 vs DM组.

2.2 远端结肠组织中c-Kit表达(免疫组化)

干预6 wk后, DM组远端结肠c-Kit阳性细胞数较正常对照组明显减少(72±10 vs 102±12/5个高倍镜视野, P<0.05), DM+SCF组较DM组显著增加(87±10 vs 72±10/5个高倍镜视野, P<0.05), 但尚未达到正常对照组水平(87±10 vs 102±12/5个高倍镜视野, P<0.05, 图3).

图3 远端结肠c-Kit阳性细胞(×400). A: 正常组; B: DM组; C: DM+SCF组.

2.3 电镜结果

正常对照组小鼠远端结肠中ICC核膜完整, 胞质内富含细胞器, 如内质网、线粒体等, 细胞器结构清晰, 发育良好; ICC与周围的ICC、神经纤维以及平滑肌细胞间形成紧密连接. DM组小鼠远端结肠ICC出现基膜溶解, 细胞器破坏、数量减少, 内质网扩张, 线粒体肿胀甚至空泡样变性; ICC与周围细胞之间的紧密连接也被破坏. DM+SCF组结肠ICC的结构病变较DM组有所改善(图4).

图4 电镜结果. A: 正常组; B: DM组; C: DM+SCF组.

3 讨论

DM结肠动力障碍的病理生理特点是: 结肠张力和收缩力低下、蠕动减慢、排空延迟[15], 其机制尚不完全清楚, 肠道ICC病变是重要原因之一[7], 临床缺乏有效的治疗手段. ICC是分布在胃肠神经末梢与平滑肌细胞之间的一类特殊细胞群, 他不仅是胃肠道起搏细胞, 产生并传导慢波, 还参与神经信号的传递, 控制胃肠平滑肌运动[6,16]. 研究证实ICC网络病变可导致胃肠起搏功能紊乱及电兴奋传导障碍, 胃肠平滑肌发生多种电节律紊乱(如慢波不规则或消失、收缩减弱或不能产生有效的推进性收缩), 临床上出现多种胃肠动力障碍症状; ICC的特异性标志是表达酪氨酸激酶受体c-Kit(CD117), 其胞外部分为SCF受体区, 胞内部分为酪氨酸激酶区, 很多研究已证明, 胃肠道ICC数量及结构的异常与多种胃肠疾病有关, 包括DM胃肠动力障碍、假性肠梗阻、先天性巨结肠、慢传输型便秘、食管失弛缓等[17-21]. 目前, 引起DM相关的ICC异常改变的病理生理学机制尚不清楚, 认为可能与胰岛素信号减弱、SCF减少或高糖等因素有关. 本实验结果发现DM 6 wk的小鼠远端结肠中ICC数量明显减少、ICC超微结构严重破坏, 与文献报道一致[9].

SCF是一种重要的多功能生长因子, 是c-Kit受体的天然配体, SCF与Kit结合组成Kit-SCF信号系统, 活化了酪氨酸激酶, 导致一系列磷酸化过程. Kit-SCF信号系统参与了机体所有Kit阳性细胞的发育、分化、增殖等过程[22-23], 同样也与ICC的增殖、分化和表型维持密切相关[11,24]. 小鼠SCF由10号染色体的steel(Sl)位点编码, 该编码序列缺失的成年(出生后20 d)Sl/Sld小鼠SCF合成严重受损, 胃肠道ICC难以识别[25]. Ward通过抑制3-磷脂酰醇激酶(PI3-K), 观察对新生及成年(出生后30 d)BALB/c小鼠空肠ICC的影响, 发现抑制PI3-K途径后新生小鼠和成年小鼠空肠ICC均减少, 认为PI3-K途径是SCF-Kit信号途径可能的作用环节[26]. 本实验中, DM小鼠远端结肠组织中SCF表达明显下调, 同时结肠ICC数量减少、超微结构被破坏; 而DM小鼠予以外源性SCF干预后, 结肠组织中SCF水平上调, 同时ICC数量及超微结构得到明显改善, 虽然未完全恢复到正常水平, 但该结果提示外源性SCF可以改善或逆转DM相关的结肠ICC病变; 异常的ICC未能完全恢复, 是否与干预时间、给药途径、剂量和/或信号通路等有关, 尚待验证.

SCF以两种形式存在, 即可溶型干细胞因子(soluble stem cell factor, s-SCF)(分子质量约24 kDa)和膜结合型干细胞因子(membrane-bound stem cell factor, m-SCF)(分子质量约27 kDa), 两者都有生物学活性. Rich et al[27]对BALB/c小鼠空肠ICC体外实验研究发现m-SCF对ICC有更大作用; 本实验采用的外源性SCF为重组小鼠SCF(分子质量约18 kDa), 对DM小鼠干预6 wk后, 测得结肠组织中SCF分子质量约30 kDa, 提示m-SCF表达增高, 同时使DM相关的结肠ICC病变得以改善, 提示外源性SCF可能为临床治疗DM胃肠动力障碍提供新的理论依据.

评论

背景资料

糖尿病胃肠动力障碍的发病机制目前仍不清楚, 临床上缺乏有效的治疗手段. 随着对Cajal间质细胞功能的研究日益增多, 人们对该疾病的认识不断深入, Cajal间质细胞有望成为治疗该病新的突破口.

同行评价者

关玉盘, 教授, 首都医科大学附属北京朝阳医院消化科.

研发前沿

近来, 干细胞因子在Cajal间质细胞以及胃肠动力障碍中的病理生理机制成为研究热点.

创新盘点

本研究首次在体内实验应用外源性干细胞因子对结肠Cajal间质细胞进行干预, 结果验证了干预的有效性及其对糖尿病小鼠Cajal间质细胞产生的显著影响.

应用要点

本研究结果可能为临床治疗糖尿病结肠功能障碍提供新的理论依据.

名词解释

c-Kit蛋白: 是已发现的Cajal间质细胞表面特异性标志物, 即酪氨酸激酶受体, 检测其含量可作为消化系Cajal间质细胞的定量指标, 干细胞因子是c-Kit的天然配基.

同行评价

本课题设计及研究方法合理, 结果与结论相符, 讨论条理清晰, 具有一定的科学性和可靠性, 也具有科研和临床实用价值.

备注

编辑:程剑侠 电编:郭海丽

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