当归多糖对大鼠乙酸性结肠炎的保护作用

摘要

目的: 研究当归多糖对大鼠乙酸性结肠炎损伤的保护作用及其初步机制.

方法: 建立大鼠乙酸性结肠炎模型. 实验设正常对照组, 模型对照组; 阳性药物对照组(5氨基水杨酸100 mg/kg); 当归多糖给药组(250, 500, 1 000 mg/kg), 每天灌肠给药一次, 用药7 d. 评价大鼠结肠黏膜损伤指数(CMDI)及粪便隐血实验(OBT), 检测结肠组织髓过氧化物酶(MPO), SOD活性, MDA、NO含量和TGF-β、EGF表达水平, 并作病理组织学观察.

结果: ASP灌肠明显降低模型组大鼠结肠显著升高的CMDI、OBT评分, MPO活性、MDA、NO含量(CMDI: 2.1±0.8, 1.8±0.6, 1.4±0.7 vs 2.9±0.6; OBT: 3.1±1.3, 2.7±1.1, 2.2±1.2 vs 3.8±0.8; MPO: 77.2±23.6, 72.5±16.8, 61.3±19.2 vs 98.1±26.9; MDA: 44.26±10.25, 38.72±14.84, 31.59±12.68 vs 31.59±12.68; NO: 0.252±0.041, 0.223±0.037, 0.217±0.032 vs 0.331±0.092, P<0.05或P<0.01), 明显升高模型组大鼠结肠显著降低的SOD活性与TGF-β表达水平 (SOD: 30.16±2.88, 31.27±2.73, 33.52±2.81 vs 28.33±1.17; TGF-β: 0.136±0.031, 0.153±0.036, 0.169±0.029 vs 0.105±0.021, P<0.05或P<0.01), 亦显著上调EGF: 表达(EGF: 0.178±0.021, 0.195±0.031, 0.191±0.022 vs 0.151±0.026, P<0.05或P<0.01). ASP用药呈一定量效关系. SF灌肠亦改善模型组大鼠结肠病理组织学表现.

结论: ASP灌肠明显缓解乙酸性结肠炎大鼠结肠损伤, 机制与促生长因子、抗氧化和一定的抗炎作用相关.

关键词: N/A

Protective effects of Angelica sinensis polysaccharides on acetic acid-induced rat colitis

Shao-Ping Liu, Wei-Guo Dong, Bao-Ping Yu, He-Sheng Luo, Jie-Ping Yu, Dong-Fang Wu

Shao-Ping Liu, Wei-Guo Dong, Bao-Ping Yu, He-Sheng Luo, Jie-Ping Yu, Department of Gastroenterology, Wuhan University Renmin Hospital, Wuhan 430060, Hubei Province, China

Dong-Fang Wu, Department of Pharmacy, Wuhan University Renmin Hospital, 238 Jiefang Road, Wuhan 430060, Hubei Province, China

Supported by: Science and Technology Committee of Hubei Province, No. 2001AA308B.

Correspondence to: Wei-Guo Dong, Wuhan University Renmin Hospital, 238 Jiefang Road, Wuhan 430060, Hubei Province, China. dongwg@public.wh.hb.cn

Received: May 14, 2003
Revised: May 20, 2003
Accepted: June 2, 2003
Published online: February 15, 2004

Abstract

AIM: To study the protective effects of Angelica sinensis polysaccharides (ASP) on colon injury in acetic acid-induced rat colitis and its mechanism.

METHODS: The colitis model of rats was produced by intracolon enema with acetic acid. The experimental animals were divided into 6 groups: normal, model, 5-ASA (100 mg/kg), and ASP (250, 500, 1 000 mg/kg), and treated intracolonically with saline, 5-ASA, and ASP respectively once a day for 7 days. The colon mucosa damage index(CMDI) and occult blood test (OBT) were evaluated. The activities of MPO and SOD, the contents of MDA and NO, the expression levels of TGF-β and EGF in the colon tissue were detected. H-E stained section was also observed.

RESULTS: Intracolon enema with ASP decreased the significanctly elevated extents of CMDI, OBT and levels of MPO, MDA, and NO in the model group (CMDI: 2.1±0.8, 1.8±0.6, 1.4±0.7 vs 2.9±0.6; OBT: 3.1±1.3, 2.7±1.1, 2.2±1.2 vs 3.8±0.8; MPO: 77.2±23.6 , 72.5±16.8, 61.3±19.2 vs 98.1±26.9; MDA: 44.26±10.25, 38.72±14.84, 31.59±12.68 vs 31.59±12.68; NO: 0.252±0.041, 0.223±0.037, 0.217±0.032 vs 0.331±0.092, P < 0.05 or P < 0.01), increased the significantly reduced level of expression of TGF-β and the activity of SOD in the model group (SOD: 30.16±2.88, 31.27±2.73, 33.52±2.81 vs 28.33±1.17; TGF-β: 0.136±0.031, 0.153±0.036, 0.169±0.029 vs 0.105±0.021, P < 0.05 or P < 0.01), and also increased significantly the expression of EGF (EGF: 0.178±0.021, 0.195±0.031, 0.191±0.022 vs 0.151±0.026, P < 0.05 or P < 0.01). The histological changes were also alleviated with ASP treatment.

CONCLUSION: Enteroclysis with ASP markedly relieve the colon injury in acetic acid-induced rats colitis, which is related with promoting growth factors, decreasing oxygen free radicals and some anti-inflammation effects.

Key Words: N/A

0 引言

当归多糖(angelica sinensis polysaccharides, ASP)具有调节免疫、抗肿瘤、抗感染、促进造血等广泛药理作用[1]. 近年研究发现, ASP体外通过促生长因子合成, 上调c-myc蛋白表达、升高鸟氨酸脱羧酶活性等作用, 促进大鼠胃上皮细胞增生与迁移, 加速其损伤模型的修复; ASP灌胃明显缓解大鼠实验性胃溃疡损伤, 促进溃疡愈合; ASP可拮抗大鼠胃肠道大量中性粒细胞浸润引起的炎症损伤; ASP亦能抑制 MDA, NO生成, 减少谷光甘肽的清除, 表现出抗氧化特性, 拮抗CCI4诱导的大鼠氧化性肝损伤[2-7]. 为探讨ASP对IBD肠组织损伤是否亦具有缓解保护作用机制, 我们建立大鼠乙酸性结肠炎模型, 首次观察ASP灌肠给药对其结肠损伤程度及氧自由基、生长因子水平的影响.

1 材料和方法

1.1 材料

健康Sprague-Dawley大鼠, 雌雄兼用, 体质量250±30 g, 由湖北预防医学科学院实验动物中心提供; ASP由武汉大学人民医院药学部参照文献[5]方法提取、纯化, 所用当归产于甘肃岷县, 使用前溶于生理盐水, 高压消毒; 5-ASA原料药, 国怡药业有限公司提供, 批号0209477; 分析纯乙酸, 上海生化试剂厂产品; MPO, SOD, MDA, NO检测试剂盒购于南京建成生物工程研究所; OB试纸, Tonyar Biotech公司产品; TGF-β, EGF多抗分别购自美国Santa Cruz和Zymed公司; SP试剂盒由北京中山生物技术有限公司提供; 其余试剂均为国产分析纯.

1.2 方法

参照文献[Gut 1996; 39: 407-415]制备动物模型, 乙醚麻醉固定大鼠, 橡胶输液管由肛门轻缓插入结肠内约8 cm, 推入80 mL/L乙酸2 mL, 作用20 s后, 立即注入5 mL生理盐水, 冲洗2次. 实验设正常对照组、模型对照组、阳性药物对照组、ASP给药组（250, 500, 1 000 mg/kg）, 每组8只, 均灌肠给药, 1次/d, 给药时间从制备模型后24 h开始, 用药7 d. 正常对照组及模型对照组均给予等量生理盐水灌肠. 实验完成后用OB试纸检测粪便隐血实验(OBT), 显色评分标准按OB试纸说明进行. 处死动物, 参照文献[8]评价CMDI, 评分标准为: 0 = 无损伤; 1 = 轻度充血、水肿, 表面光滑, 无糜烂或溃疡; 2 = 充血水肿, 黏膜粗糙呈颗粒状, 有糜烂或肠粘连; 3 = 高度充血水肿, 黏膜表面有坏死及溃疡形成, 溃疡最大纵径小于1 cm, 肠壁增厚或表面有坏死及炎症; 4 = 在3分基础上溃疡最大纵径大于1 cm, 或全肠壁坏死. 在距肛门7-9 cm处取适量结肠新鲜标本, 快速置于液氮中速冻, 待测SOD, MDA, NO, MPO, 检测操作按相应试剂盒说明进行; 用40 mL/L多聚甲醛溶液固定结肠组织标本, 常规石蜡包埋、切片, HE染色并光镜观察. TGF-β、EGF免疫组化检测步骤严格按试剂盒说明进行, 均以非免疫性山羊血清封闭非特异性抗原, 一抗分别为1: 100, 1: 150的TGF-β与EGF兔IgG多抗, 二抗均为生物素化羊抗兔IgG工作液, 以PBS代替一抗做阴性对照, 以细胞核蓝色为阴性, 胞质内或核膜上呈棕黄色为阳性. 每张切片选取10个400倍视野, 采用全自动图像分析仪与HPIAS-2000图像分析软件, 分别扫描记录阳性细胞的吸光度A值, 取其平均值, 作为此切片TGF-β, EGF的相对含量.

统计学处理 数据以mean±SD表示, 采用SPSS医学统计软件处理, 两组间均数差异的比较采用t检验.

2 结果

2.1 结肠损伤程度和炎症反应

模型组大鼠CMDI, OBT与MPO活性较正常对照显著升高(P<0.01), 不同剂量ASP灌肠显著降低CMDI, 中、大剂量ASP亦使OBT评分, MPO活性明显降低(P<0.05或 P<001). 大剂量ASP改善CMDI, OBT作用与100 mg/kg 5-ASA相当(表1). 结肠标本HE染色亦显示, 模型组黏膜高度充血水肿, 上皮溃疡形成, 局部见坏死, 大量炎性细胞浸润, 腺体中杯状细胞显著减少而大剂量ASP用药组黏膜充血水肿, 上皮溃疡坏死明显减轻, 炎性细胞浸润亦有所缓解.

表1 ASP对结肠炎大鼠CMDI, OBT的影响 (mean±SD, n = 8).

分组	剂量(mg/kg)	CMDI(分)	OBT(分)	MPO(nkat/g)
正常对照	0	0.0±0.0	0.0±0.0	413±192
模型对照	0	2.9±0.6b	3.8±0.8b	1 635±448b
5-ASA	100	1.6±0.9d	2.6±1.1c	493±180d
ASP	250	2.1±0.8c	3.1±1.3	1 287±393
ASP	500	1.8±0.6d	2.7±1.1c	1 209±280c
ASP	1 000	1.4±0.7d	2.2±1.2d	1 022±320d

^bP<0.01 vs 正常对照组;

^cP<0.05,

^dP<0.01 vs 模型对照组.

2.2 结肠氧由基和生长因子

模型组大鼠MDA, NO含量显著升高, TGF-β表达、SOD活性显著降低(P<0.01), EGF表达无明显变化, ASP灌肠显著降低MDA, NO含量, 升高TGF-β, EGF表达水平, SOD活性(P<0.05或P<0.01), 而100 mg/kg 5-ASA对模型组TGF-β, EGF表达水平无明显影响. ASP用药呈一定量效关系(表2, 图1-4).

图1 TGF-β在模型组大鼠结肠组中的表达SP×400.

表2 ASP对结肠炎大鼠结肠氧自由基与生长因子的影响(mean±SD, n = 8).

分别	剂量(mg/kg)	SOD(ukat/g)	MDA(umol/g)	NO(mmol/g)	TGF-β(A×10-3)	EGF(A×10-3)
正常对照	0	602±32	9.2±3.8	176±45	146±25	162±23
模型对照	0	472±20b	57.5±12.4b	331±92b	105±21b	151±26
5-ASA	100	548±49d	34.7±9.4d	194±35d	97±27	159±28
ASP	250	503±48	44.3±10.2c	252±41c	136±31c	178±21c
ASP	500	521±46c	38.7±14.8c	223±37d	153±36d	195±31d
ASP	1000	559±47d	31.6±12.7d	217±32d	169±29d	191±22d

^bP<0.01 vs 正常对照组;

^cP<0.05,

^dP<0.01 vs 模型对照组.

图2 TGF-β在ASP大剂量用药组大鼠结肠组中的表达SP×400.

图3 EGF在模型组大鼠结肠组中的表达SP×400.

图4 EGF在ASP大剂量用药组大鼠结肠组中的表达SP×400.

3 讨论

乙酸诱导的大鼠实验性结肠炎是成熟经典的IBD模型, 因其发病机制、病理表现与人类IBD相似, 常用于IBD治疗药物的筛选与评价[8-9]. 本研究显示, ASP灌肠显著减轻模型组大鼠结肠黏膜损伤程度, 明显缓解便血症状, 表现出明显的结肠损伤保护作用. MPO活性是间接反映IBD炎症程度的重要指标[10], 本研究亦显示中、大剂量ASP明显降低模型组MPO活性, 显示出一定的抗炎效果, 但其作用仍明显弱于100 mg/kg 5-ASA (P<0.01). ASP抗炎机制之一可能为其含有药理特性类似于肝素的成分, 可通过与一系列炎症前化学激动反应相互作用, 产生拮抗炎症效应[4]. IBD肠黏膜中OFR水平明显升高, 抗氧化系统存在缺陷, 大量OFR对自身组织产生攻击作用, 充当或激活炎性递质, 使结肠炎症损伤进一步加重[11-14]. 过量的弱氧自由基NO不仅趋化中性粒细胞和单核细胞, 还使血管扩张, 通透性增加, 有助于炎症的始动与发展, 且可与超氧阴离子反应生成具有高度细胞毒性的过氧亚硝酸盐, 损伤结肠上皮细胞的功能和结构, 破坏肠道黏膜屏障的完整性[15-18]. 抗氧化剂与抑制NO生成, 可有效缓解IBD结肠损伤[8,18-19]. 本研究显示模型组大鼠OFR与NO大量生成, ASP灌肠明显降低MDA、NO含量, 升高SOD活性, 表现出较强抗OFR作用, 从而缓解结肠氧化损伤与炎症反应. IBD肠黏膜中iNOS活性显著升高是NO大量病理性生成的主要原因[20]. 研究证实ASP可剂量依赖性抑制iNOS活性[5-6], 这可能是其减少炎症结肠组织NO含量的机制. ASP通过抗氧化、清除氧自由基作用, 从而抑制氧自由基对IBD重要炎症调控因子NF-κB的激活, 亦可能是其抗炎效应的机制[5,21-22]. TGF-β, EGF是对多种组织损伤修复和再生具有重要意义的细胞生长因子, 具有相互协同促进作用[23-28], 且均可阻止氧化剂诱导的肠黏膜屏障损伤与功能低下, 对肠道黏膜屏障的完整性及功能维持具有重要意义[29-32]. 研究证实TGF-β不足可加重IBD肠道损伤, 外源性给予TGF-β, EGF均可明显缓解IBD肠道损伤[33-37]. 本研究显示模型组大鼠结肠TGF-β水平显著降低, EGF无明显改变; ASP灌肠显著升高二者表达水平, 表明其明显促进TGF-β、EGF的合成分泌, 其促生长因子作用与新近研究报道相似[2-3], 而5-ASA对二者表达无明显影响. 结肠组织中显著增加的TGF-β、EGF可通过促多种组织细胞增生、分化、迁移作用, 直接加速结肠上皮细胞、黏膜屏障及血管损伤的修复与再生, 并发挥细胞保护作用, 这可能是ASP缓解模型组大鼠结肠损伤及肠道出血的主要机制.

备注

编辑: N/A

参考文献

1.	Xie L, Yang LH, Li XH. Resarch on the pharmacologic effects of Angelica sinensis. Res Tradi Chin Med. 2000;16:56-58.  [PubMed]  [DOI]

2.	Ye YN, Koo MW, Li Y, Matsui H, Cho CH. Angelica sinensis modulates migration and proliferation of gastric epithelial cells. Life Sci. 2001;68:961-968.  [PubMed]  [DOI]

3.	Ye YN, Liu ES, Shin VY, Koo MW, Li Y, Wei EQ, Matsui H, Cho CH. A mechanistic study of proliferation induced by Angelica sinensis in a normal gastric epithelial cell line. Biochem Pharmacol. 2001;61:1439-1448.  [PubMed]  [DOI]

4.	Cho CH, Mei QB, Shang P, Lee SS, So HL, Guo X, Li Y. Study of the gastrointestinal protective effects of polysaccharides from Angelica sinensis in rats. Planta Med. 2000;66:348-351.  [PubMed]  [DOI]

5.	Ye YN, Liu ES, Li Y, So HL, Cho CC, Sheng HP, Lee SS, Cho CH. Protective effect of polysaccharides-enriched fraction from Angelica sinensis on hepatic injury. Life Sci. 2001;69:637-646.  [PubMed]  [DOI]

6.	Ding H, Peng R, Yu J. [Modulation of angelica sinensis polysaccharides on the expression of nitric oxide synthase and Bax, Bcl-2 in liver of immunological liver-injured mice]. Zhonghua Ganzangbing Zazhi. 2001;9 Suppl:50-52.  [PubMed]  [DOI]

7.	Ye YN, So HL, Liu ES, Shin VY, Cho CH. Effect of polysaccharides from Angelica sinensis on gastric ulcer healing. Life Sci. 2003;72:925-932.  [PubMed]  [DOI]

8.	Millar AD, Rampton DS, Chander CL, Claxson AW, Blades S, Coumbe A, Panetta J, Morris CJ, Blake DR. Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis. Gut. 1996;39:407-415.  [PubMed]  [DOI]

9.	Li L, Wang ZL, Ke JT, Zhang M, Shao JF, Zhong CN. Select animal models for experimental colitis model. Shijie Huaren Xiaohua Zazhi. 2001;9:584-585.  [PubMed]  [DOI]

10.	Li JH, Yu JP, He XF, Xu XM. Expression of NF-κB in rats with TNBS-induced ulcerative colitis. Shijie Huaren Xiaohua Zazhi. 2003;11:214-218.  [PubMed]  [DOI]

11.	Krieglstein CF, Cerwinka WH, Laroux FS, Salter JW, Russell JM, Schuermann G, Grisham MB, Ross CR, Granger DN. Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide and nitric oxide. J Exp Med. 2001;194:1207-1218.  [PubMed]  [DOI]

12.	Kirsner JB. Historical origins of current IBD concepts. World J Gastroenterol. 2001;7:175-184.  [PubMed]  [DOI]

13.	Pavlick KP, Laroux FS, Fuseler J, Wolf RE, Gray L, Hoffman J, Grisham MB. Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease. Free Radic Biol Med. 2002;33:311-322.  [PubMed]  [DOI]

14.	Huycke MM, Abrams V, Moore DR. Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA. Carcinogenesis. 2002;23:529-536.  [PubMed]  [DOI]

15.	Feng ZJ, Yao XX. Regulating effect of NO-PGE2 on visceral angiectasis in portal hypertension. Shijie Huaren Xiaohua Zazhi. 2000;8:1154-1156.  [PubMed]  [DOI]

16.	Le QL, Wen XD. The role of nitric oxide in stress-induced gastric ulcers in rats. Shijie Huaren Xiaohua Zazhi. 2000;8:815-816.  [PubMed]  [DOI]

17.	Wang QG, He LY, Chen YW, Hu SL. Enzymohistochemical study on burn effect on rat intestinal NOS. World J Gastroenterol. 2000;6:421-423.  [PubMed]  [DOI]

18.	Bai AP, Shen ZX, Yu JP, Yu BP, Luo Y. Nitric oxide and the acute injury in colitis model. Shijie Huaren Xiaohua Zazhi. 1999;7:900-901.  [PubMed]  [DOI]

19.	Yavuz Y, Yüksel M, Yeğen BC, Alican I. The effect of antioxidant therapy on colonic inflammation in the rat. Res Exp Med (Berl). 1999;199:101-110.  [PubMed]  [DOI]

20.	Zhang K, Deng CS, Zhu YQ, Yang YP, Zhang YM. Signifisance of nuclear factor-κB, cyclooxygenase-2 and inducible nitric oxide synthase expression in human ulcerative colitis tissues. Shijie Huaren Xiaohua Zazhi. 2002;10:575-578.  [PubMed]  [DOI]

21.	Yamamoto Y, Gaynor RB. Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. J Clin Invest. 2001;107:135-142.  [PubMed]  [DOI]

22.	Liu SQ, Yu JP, Luo HS, Ran ZX. Effects of Ginkgo biloba extract on expression of NF-κB in rat liver fibrosis. Shijie Huaren Xiaohua Zazhi. 2002;10:922-926.  [PubMed]  [DOI]

23.	Sporn MB, Roberts AB. Transforming growth factor-beta: recent progress and new challenges. J Cell Biol. 1992;119:1017-1021.  [PubMed]  [DOI]

24.	Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991;251:936-939.  [PubMed]  [DOI]

25.	Fu XB, Yang YH, Sun TZ, Gu XM, Jiang LX, Sun XQ, Sheng ZY. Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor betain lung and its relation with lung repair. World J Gastroenterol. 2000;6:353-355.  [PubMed]  [DOI]

26.	Yuan YZ, Lou KX, Gong ZH, Tu SP, Zhai ZK, Xu JY. Effects and mechanisms of emodin on pancreatic tissue EGF expression in acute pancreatitis in rats. Shijie Huaren Xiaohua Zazhi. 2001;9:127-130.  [PubMed]  [DOI]

27.	Wan SM, Sun SH, Den MD, Ge QL, Yang YJ. TGF-β1 and PDGF-A expression in gastric cancer tissue and prognosis. Shijie Huaren Xiaohua Zazhi. 2002;10:36-39.  [PubMed]  [DOI]

28.	Zhuang ZH, Chen YL, Wang CD, Chen YG. Expression of TGF-β1 and TGF-β receptor in gastric carcinoma and precarcero-us lesions. Shijie Huaren Xiaohua Zazhi. 1999;7:507-509.  [PubMed]  [DOI]

29.	Banan A, Fields JZ, Talmage DA, Zhang L, Keshavarzian A. PKC-zeta is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury. Am J Physiol Gastrointest Liver Physiol. 2002;282:G794-G808.  [PubMed]  [DOI]

30.	Banan A, Fields JZ, Zhang Y, Keshavarzian A. Phospholipase C-gamma inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants. Am J Physiol Gastrointest Liver Physiol. 2001;281:G412-G423.  [PubMed]  [DOI]

31.	Banan A, Zhang Y, Losurdo J, Keshavarzian A. Carbonylation and disassembly of the F-actin cytoskeleton in oxidant induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor alpha in a human colonic cell line. Gut. 2000;46:830-837.  [PubMed]  [DOI]

32.	Banan A, Fields JZ, Talmage DA, Zhang Y, Keshavarzian A. PKC-beta1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants. Am J Physiol Gastrointest Liver Physiol. 2001;281:G833-G847.  [PubMed]  [DOI]

33.	Hahm KB, Im YH, Parks TW, Park SH, Markowitz S, Jung HY, Green J, Kim SJ. Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut. 2001;49:190-198.  [PubMed]  [DOI]

34.	Beck PL, Podolsky DK. Growth factors in inflammatory bowel disease. Inflamm Bowel Dis. 1999;5:44-60.  [PubMed]  [DOI]

35.	Sandborn WJ, Targan SR. Biologic therapy of inflammatory bowel disease. Gastroenterology. 2002;122:1592-1608.  [PubMed]  [DOI]

36.	Wiercińska-Drapało A, Flisiak R, Prokopowicz D. [The role of transforming growth factors beta in pathogenesis of ulcerative colitis]. Pol Merkur Lekarski. 2001;10:177-179.  [PubMed]  [DOI]

37.	Whiting CV, Williams AM, Claesson MH, Bregenholt S, Reimann J, Bland PW. Transforming growth factor-beta messenger RNA and protein in murine colitis. J Histochem Cytochem. 2001;49:727-738.  [PubMed]  [DOI]