促胰岛β细胞增殖中药的筛选和药效评价
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摘要
糖尿病(diabetes mellitus,DM)是由多种遗传和环境因素相互作用而引起的一组慢性代谢异常综合征。目前常用的口服降糖药有双胍类、磺脲类、噻磋烷二酮类等。这些化学降糖类药物尽管见效较快,但对肝、肾等脏器损害较大,甚至有发生低血糖的危险。一般这类抗高血糖药物仅仅只是降低异常升高的血糖,对于糖尿病患者起到治标不治本的作用。
为此,本课题着眼于其病理基础之一“胰岛损伤”,特别是晚期糖尿病,胰岛细胞功能严重衰退,β细胞大量凋亡。针对这一点本课题建立了一个细胞水平上的药物筛选模型,从我国传统中药库中筛选出具有促进β细胞增殖,抑制其凋亡的药物。从而达到治标又治本的目的。且纯天然提取物类药物具有作用温和、毒副作用小等优点,开发利用潜力很大。
首先,本论文以大鼠胰岛瘤细胞系RINm5f为基础材料,以GLP-1为阳性参照,用SRB法测定细胞生长曲线及增殖活性。确定了最佳细胞铺板浓度为104个/孔,阳性参照浓度50nmol/L,增殖率最高达30%。确定了中药样品的筛选浓度分别为100mg/L和10mg/L,最佳干预时间72小时,成功建立了体外药物筛选模型。并运用此模型对313味中药进行了初筛和复筛,筛选出蜈蚣、地龙两味中药具有促RINm5f细胞增殖的作用。
然后,我们对活性药物进行初步分离。动物类药材蜈蚣,地龙采用水提醇沉方法,然后分别对各分离组分进行细胞活性评价。结果显示只有蜈蚣的50%乙醇醇沉上清组分对RINm5f细胞的生长有显著的促进作用,其他各组分都无显著促增殖活性的作用。
最后,本论文选择促细胞增殖率相对较高的中药蜈蚣水提物进行了体内药效的评价。通过比较四氧嘧啶糖尿病小鼠服用蜈蚣水提物前后的生长情况,血糖值,胰岛β细胞修复情况等,初步考察中药蜈蚣水提物的降血糖功效。结果表明:蜈蚣水提物具有良好的体内降血糖功效。
Diabetes (diabetes mellitus, DM) is a group of chronic metabolic syndrome arising from the interaction of many genetic and environmental factors. Now commonly used oral hypoglycemic agents are glucophage, glibenclamide and so on. Although these chemical hypoglycemic drugs have fast effects, the liver, kidney and other organs were seriously injuried, or even there is risk of hypoglycemia. Generally such anti-high blood sugar drugs simply reduce the abnormal increased of blood sugar, but they can’t reduce islet damage in patients with diabetes.
To avoid such shortage mentioned above, this subject focus on one of the basic pathological event in "islet injury," especially the advanced diabetes, islet severe recession of cell function, large number ofβ-cells apoptosis. In order to figure out this problem, a drug screening model on cell level was established for screening drugs from traditional Chinese medicine with the function of promotingβcell proliferation, and inhibiting apoptosis .The aim is to gain profit from treating the symptoms and cause of disease. And pure extracts of natural drugs play modest role, have slight side effects, and have great potential on being developed and utilized.
First, in this experiment, RINm5f cells was basic materials, GLP-1 was positive reference, the activity of cell growth and proliferation determinated by SRB method. The best density of cells determined was 10,000 per hole, the GLP-1 concentration was 50 nmol / L, and the most proliferation rates was at 30%. The best secreen concentrations of Chinese medicine samples were 100 mg / L and 10 mg / L, the best interference time was 72 h. Successfully established a drug screening model in vitro at cell level. And 313 kinds of herbs were secreened by this model, and founed that two Chinese herbal medicines scolopendra and pheretima had the function of promoting proliferation in RINm5f cells.
Then, the two drugs which had the function of promoting proliferation in RINm5f cells were preliminary separated by water-alcohol-ways, and evaluated the activity in RINm5f cells. The results showed that only the components dissolved in 50 percents of ethanol of scolopendra had significant effect on promoting proliferation in RINm5f cell, the others had no significant function on promoting proliferation.
Finally, the water extracts of scolopendra which had higher rate of promoting the cell proliferation was used to evaluate its activity in vivo. The model of diabetes mellitus was induced by alloxan in mouse. The growth, blood sugar level,β-cell repairing were observed. The results showed that scolopendra had significant effect in decreasing blood glucose in diabetes mellitus mice.
为此,本课题着眼于其病理基础之一“胰岛损伤”,特别是晚期糖尿病,胰岛细胞功能严重衰退,β细胞大量凋亡。针对这一点本课题建立了一个细胞水平上的药物筛选模型,从我国传统中药库中筛选出具有促进β细胞增殖,抑制其凋亡的药物。从而达到治标又治本的目的。且纯天然提取物类药物具有作用温和、毒副作用小等优点,开发利用潜力很大。
首先,本论文以大鼠胰岛瘤细胞系RINm5f为基础材料,以GLP-1为阳性参照,用SRB法测定细胞生长曲线及增殖活性。确定了最佳细胞铺板浓度为104个/孔,阳性参照浓度50nmol/L,增殖率最高达30%。确定了中药样品的筛选浓度分别为100mg/L和10mg/L,最佳干预时间72小时,成功建立了体外药物筛选模型。并运用此模型对313味中药进行了初筛和复筛,筛选出蜈蚣、地龙两味中药具有促RINm5f细胞增殖的作用。
然后,我们对活性药物进行初步分离。动物类药材蜈蚣,地龙采用水提醇沉方法,然后分别对各分离组分进行细胞活性评价。结果显示只有蜈蚣的50%乙醇醇沉上清组分对RINm5f细胞的生长有显著的促进作用,其他各组分都无显著促增殖活性的作用。
最后,本论文选择促细胞增殖率相对较高的中药蜈蚣水提物进行了体内药效的评价。通过比较四氧嘧啶糖尿病小鼠服用蜈蚣水提物前后的生长情况,血糖值,胰岛β细胞修复情况等,初步考察中药蜈蚣水提物的降血糖功效。结果表明:蜈蚣水提物具有良好的体内降血糖功效。
Diabetes (diabetes mellitus, DM) is a group of chronic metabolic syndrome arising from the interaction of many genetic and environmental factors. Now commonly used oral hypoglycemic agents are glucophage, glibenclamide and so on. Although these chemical hypoglycemic drugs have fast effects, the liver, kidney and other organs were seriously injuried, or even there is risk of hypoglycemia. Generally such anti-high blood sugar drugs simply reduce the abnormal increased of blood sugar, but they can’t reduce islet damage in patients with diabetes.
To avoid such shortage mentioned above, this subject focus on one of the basic pathological event in "islet injury," especially the advanced diabetes, islet severe recession of cell function, large number ofβ-cells apoptosis. In order to figure out this problem, a drug screening model on cell level was established for screening drugs from traditional Chinese medicine with the function of promotingβcell proliferation, and inhibiting apoptosis .The aim is to gain profit from treating the symptoms and cause of disease. And pure extracts of natural drugs play modest role, have slight side effects, and have great potential on being developed and utilized.
First, in this experiment, RINm5f cells was basic materials, GLP-1 was positive reference, the activity of cell growth and proliferation determinated by SRB method. The best density of cells determined was 10,000 per hole, the GLP-1 concentration was 50 nmol / L, and the most proliferation rates was at 30%. The best secreen concentrations of Chinese medicine samples were 100 mg / L and 10 mg / L, the best interference time was 72 h. Successfully established a drug screening model in vitro at cell level. And 313 kinds of herbs were secreened by this model, and founed that two Chinese herbal medicines scolopendra and pheretima had the function of promoting proliferation in RINm5f cells.
Then, the two drugs which had the function of promoting proliferation in RINm5f cells were preliminary separated by water-alcohol-ways, and evaluated the activity in RINm5f cells. The results showed that only the components dissolved in 50 percents of ethanol of scolopendra had significant effect on promoting proliferation in RINm5f cell, the others had no significant function on promoting proliferation.
Finally, the water extracts of scolopendra which had higher rate of promoting the cell proliferation was used to evaluate its activity in vivo. The model of diabetes mellitus was induced by alloxan in mouse. The growth, blood sugar level,β-cell repairing were observed. The results showed that scolopendra had significant effect in decreasing blood glucose in diabetes mellitus mice.
引文
1.李连瑞,王秀玲.糖尿病的治疗药物进展[J].天津药学,2003,15(2):56
2.朱芸,刘金荣,张伟.降血糖药用植物资源概述[J].中西医结合学报,2004,2(1): 67-68
3.朱禧星.现代糖尿病学[M].上海:上海医科大学出版社,2000,4
4.余缁源,刘茂才,罗云坚.中西医结合内科学[M].北京:科学出版社,2003,616—618
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6.肖新华.1型糖尿病的治疗进展[J].国外医学内分泌分册,2004,24(4):281-282
7.严小惠,姚苏.糖尿病社区治疗药物及经济学评价[J].中国全科医学,2004,7(14): 259-260
8.曹秀虹,赵民牛,董凤英.口服抗糖尿病药物的临床应用[J].中围全科医学,2002,5(5):394-395
9.邱明才,马中书.2型糖尿病胰岛素强化治疗的新进展[J].中围全科医学,1999,2(1):6-7
10.古月娟娟,杜冠华.药物筛选模型研究进展[J].基础医学与临床,2001,21:302-305.
11.张志琪,张延妮,田振军.药物筛选模型和技术及其在中药活性成分研究中的应用[J].中围中药杂志,2003,28:907-910.
12.范益军.中药提取库中胰升血糖素样肤-1(GLP—1)受体激动剂的高通量筛选[D][硕士学位论文],重庆,重庆大学,2005.
13.曹仁贤.胰岛B细胞凋亡与糖尿病[J]围外医学·内分泌学手册,1997,17(2):83.
14. Porte D, Kahn SE. B-cell dysfunction and failure in type 2 diabetes potential mechanisms [ J ]. Diabetes, 2001, 50( 1 1) : 160 - 163
15.刘铮,只德贤.胰岛细胞系模型的研究进展[J]沈阳药科大学学报,2007,24.(1):62-68
16. Suzuki R,Okada N,Miyamoto H I. Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreaticβCell lines [J]. Life Sciences ,2002 ,71 :1717 - 1729.
17. Ishihara H ,Asano T , Tsu K ,et al. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose - stimulated insulin secretionsimilar to those of normal islets [J ] . Diabetologia ,1993 ,36 :1139 - 1145.
18. Persson-sjogren S ,Lejon K, Holmberg D ,et al. Expression of the NK-1 receptor on islet cells and invading immune cells in the non-obese diabetic mouse[J ] . Journal of Autoimmunity , 2005 ( 24), 4 : 269 -279
19.黎瑶,张磊.胰高糖素样肽1的研究进展[J]四川医学,2006,27(5):466-467
20.王毅飞,汪吉仪,王国华.胰高糖素样肽-1(7～36)NH2引起胰岛β细胞内游离钙的变化[J].广东医学,2000,21(4):286～288
21.王毅飞,汪吉仪,王国华.胰高糖素样肽(7～36)NH2引起胰岛β细胞内CAMP的变化[J].广东医学,2000,21(11):916～918
22. Edvell A ,Lindstrom p. Initiation of increased pancreatic islet growth in young normoglycemic mice (Umea + / ?) [ J ] .Endocrinology , 1999 , 140 : 778 - 783
23. Farilla L,Hui H ,Bertolotto C , et al. Glucagon-like Peptide - 1 promotes islet cell growth and inhibits apoptosis in zucker diabetic rats [J ]. Endocrinology ,2002 ,143 :4397~4408
24. Stofiefs DA ,Desia BM,Deleon DD , et al.Neonatal Exenitade 4 prevents the development of diabetes in the diabetes in the intrauterine growth retarded rat [J ].Diabetes ,2003 ,52 (3) :734~740
25. Buleau J, Roduit R, Susini S, et al.Glucagon-like peptide-l promotes DNA synthesis,activates phosphalidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene(PDX-l)DNA binding activity in beta(INS-l)cells. Diabetologia,1999,42:856-864
26. De L, Diva D,Deng SP, et al. Role of endogenous glucagons-like peptide-l in islet regeneration after partial pancreatectomy. Diabetes, 2003, 52(2): 365-371
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2.朱芸,刘金荣,张伟.降血糖药用植物资源概述[J].中西医结合学报,2004,2(1): 67-68
3.朱禧星.现代糖尿病学[M].上海:上海医科大学出版社,2000,4
4.余缁源,刘茂才,罗云坚.中西医结合内科学[M].北京:科学出版社,2003,616—618
5.万中林,肖德发.1998年中医药治疗2型糖尿病的进展[J]现代诊断与治疗,1999,l0(6):324-326
6.肖新华.1型糖尿病的治疗进展[J].国外医学内分泌分册,2004,24(4):281-282
7.严小惠,姚苏.糖尿病社区治疗药物及经济学评价[J].中国全科医学,2004,7(14): 259-260
8.曹秀虹,赵民牛,董凤英.口服抗糖尿病药物的临床应用[J].中围全科医学,2002,5(5):394-395
9.邱明才,马中书.2型糖尿病胰岛素强化治疗的新进展[J].中围全科医学,1999,2(1):6-7
10.古月娟娟,杜冠华.药物筛选模型研究进展[J].基础医学与临床,2001,21:302-305.
11.张志琪,张延妮,田振军.药物筛选模型和技术及其在中药活性成分研究中的应用[J].中围中药杂志,2003,28:907-910.
12.范益军.中药提取库中胰升血糖素样肤-1(GLP—1)受体激动剂的高通量筛选[D][硕士学位论文],重庆,重庆大学,2005.
13.曹仁贤.胰岛B细胞凋亡与糖尿病[J]围外医学·内分泌学手册,1997,17(2):83.
14. Porte D, Kahn SE. B-cell dysfunction and failure in type 2 diabetes potential mechanisms [ J ]. Diabetes, 2001, 50( 1 1) : 160 - 163
15.刘铮,只德贤.胰岛细胞系模型的研究进展[J]沈阳药科大学学报,2007,24.(1):62-68
16. Suzuki R,Okada N,Miyamoto H I. Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreaticβCell lines [J]. Life Sciences ,2002 ,71 :1717 - 1729.
17. Ishihara H ,Asano T , Tsu K ,et al. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose - stimulated insulin secretionsimilar to those of normal islets [J ] . Diabetologia ,1993 ,36 :1139 - 1145.
18. Persson-sjogren S ,Lejon K, Holmberg D ,et al. Expression of the NK-1 receptor on islet cells and invading immune cells in the non-obese diabetic mouse[J ] . Journal of Autoimmunity , 2005 ( 24), 4 : 269 -279
19.黎瑶,张磊.胰高糖素样肽1的研究进展[J]四川医学,2006,27(5):466-467
20.王毅飞,汪吉仪,王国华.胰高糖素样肽-1(7～36)NH2引起胰岛β细胞内游离钙的变化[J].广东医学,2000,21(4):286～288
21.王毅飞,汪吉仪,王国华.胰高糖素样肽(7～36)NH2引起胰岛β细胞内CAMP的变化[J].广东医学,2000,21(11):916～918
22. Edvell A ,Lindstrom p. Initiation of increased pancreatic islet growth in young normoglycemic mice (Umea + / ?) [ J ] .Endocrinology , 1999 , 140 : 778 - 783
23. Farilla L,Hui H ,Bertolotto C , et al. Glucagon-like Peptide - 1 promotes islet cell growth and inhibits apoptosis in zucker diabetic rats [J ]. Endocrinology ,2002 ,143 :4397~4408
24. Stofiefs DA ,Desia BM,Deleon DD , et al.Neonatal Exenitade 4 prevents the development of diabetes in the diabetes in the intrauterine growth retarded rat [J ].Diabetes ,2003 ,52 (3) :734~740
25. Buleau J, Roduit R, Susini S, et al.Glucagon-like peptide-l promotes DNA synthesis,activates phosphalidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene(PDX-l)DNA binding activity in beta(INS-l)cells. Diabetologia,1999,42:856-864
26. De L, Diva D,Deng SP, et al. Role of endogenous glucagons-like peptide-l in islet regeneration after partial pancreatectomy. Diabetes, 2003, 52(2): 365-371
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