胰腺星狀細胞對胰島β細胞胰島素釋放和合成的影響#

1、中國科技論文在線- PAGE 6 - FORMTEXT 胰腺星狀細胞對胰島細胞胰島素釋放和合成的影響基金項目：國家自然科學(xué)基金（30971399）高等學(xué)校博士學(xué)科點專項科研基金（200802860020） FORMTEXT 殷俊梅， FORMTEXT 孫子林， FORMTEXT 李鳳飛， FORMTEXT 陳碧君作者簡介：殷俊梅（1986-），女，碩士研究生，胰島纖維化相關(guān)機制通信聯(lián)系人：孫子林（1963-），男，主任醫(yī)師，胰島纖維化相關(guān)機制. E-mail: sunzilin1963SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGE

2、FORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET bkCompanyEN Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical Schoo

3、l, Southeast University,Nanjing,210009;Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009;Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009;Department of end

4、ocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009 * MERGEFORMATDepartment of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009;Department of endocrinology, Zhongda hospital, Institute of

5、Diabetes, Medical School, Southeast University,Nanjing,210009;Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009;Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,2

6、10009SET bkCompanyCHN 東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所;東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所;東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所;東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所 * MERGEFORMAT東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所;東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所;東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所;東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所SET bkPostcode 210009;210009; * MERGEFORMAT210009;210009;S

7、ET bkMobile13951749490; * MERGEFORMA13951749490;SET bkTelphone025-83275147; * MERGEFORMA025-83275147;SET bkAddress 江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科;江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科; * MERGEFORMAT江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科;江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科;SET bkEmail ;sunzilin1963;lifengf

8、ei2005;332335218 * MERGEFORMAT;sunzilin1963;lifengfei2005;332335218SET bkIntroduction 殷俊梅（1986-），女，碩士研究生，胰島纖維化相關(guān)機制;孫子林（1963-），男，主任醫(yī)師，胰島纖維化相關(guān)機制; * MERGEFORMAT殷俊梅（1986-），女，碩士研究生，胰島纖維化相關(guān)機制;孫子林（1963-），男，主任醫(yī)師，胰島纖維化相關(guān)機制;SET bkAuthorCHN 殷俊梅;孫子林;李鳳飛;陳碧君 * MERGEFORMAT殷俊梅;孫子林;李鳳飛;陳碧君SET bkAuthorEN YIN Junmei;

9、SUN Zilin;LI Fengfei;CHEN Bijun * MERGEFORMATYIN Junmei;SUN Zilin;LI Fengfei;CHEN BijunSET bkContact 孫子林 * MERGEFORMAT孫子林SET bkFund 國家自然科學(xué)基金（30971399）高等學(xué)校博士學(xué)科點專項科研基金（200802860020） * MERGEFORMAT國家自然科學(xué)基金（30971399）高等學(xué)校博士學(xué)科點專項科研基金（200802860020）SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMA

10、T1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET version 1.5 * MERGEFORMAT1.5SET bkReferencesInfo 1*|*期刊*|*Bachem MG,

11、 Schneider E, Gross H, et al. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology. 1998, 115(2):421-432.2*|*期刊*|*Lee H, Lim C, Lee J, Kim N, et al. TGF-beta signaling preserves RECK expression in activated pancreatic stellate cells. J Cell

12、Biochem. 2008, 104(3):1065-1074.3*|*期刊*|*Nomiyama Y, Tashiro M, Yamaguchi T, et al. High glucose activates rat pancreatic stellate cells through protein kinase C and p38 mitogen-activated protein kinase pathway. Pancreas.2007, 34(3):364-72.4*|*期刊*|*Hong OK, Lee SH, Rhee M, et al. Hyperglycemia and h

13、yperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: Possible explanation of islet-specific fibrosis in type 2 diabetes mellitus. J Cell Biochem. 2007, 101(3):665-675.5*|*期刊*|*Hong OK, Lee SH, Rhee M, et al. Hyperglycemia and hyperinsulinemia have addit

14、ive effects on activation and proliferation of pancreatic stellate cells: possible explanation of islet-specific fibrosis in type 2 diabetes mellitus. J Cell Biochem. 2007, 101(3):665-675.6*|*期刊*|*潘琳，李宏亮，楊文英等，胰腺星狀細胞活化在高脂飼養(yǎng)大鼠胰島纖維化形成中的作用。中華內(nèi)分泌代謝雜志。2009, 25(1):25-27.7*|*在線文獻*|*李鳳飛，孫子林，殷俊梅等. 胰腺星狀細胞在2型糖尿

15、病胰島纖維化進程中的影響OL. 中國科技論文在線2011-06-07/index.php/default/releasepaper/content/201106-70.8*|*期刊*|*Mizukami H, Wada R, Yonezawa A, et al. Suppression of post-prandial hyperglycaemia by pioglitazone improved islet fibrosis and macrophage migration in the Goto-Kakizaki rat. Diabetes Obes Metab. 2008, 10(9):

16、791-794.9*|*期刊*|*Tikellis C,Wookey PJ, Candido R, et al. Improved islet morphology after blockade of the renin-angiotensin system in the ZDF rat. Diabetes. 2004, 53(4):989-997.10*|*期刊*|*Masuyama T, Komeda K, Hara A, et al. Chronological characterization of diabetes development in male Spontaneously

17、Diabetic Torii rats. Biochem Biophys Res Commun. 2004, 314(2):870-877.11*|*期刊*|*Shinozaki S, Ohnishi H, Hama K, et al. Indian hedgehog promotes the migration of rat activated pancreatic stellate cells by increasing membrane type-1 matrix metalloproteinase on the plasma membrane. J Cell Physiol.2008,

18、 216(1):38-46.12*|*期刊*|*Yoshikawa H, Kihara Y, Taguchi M, et al. Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long-Evans okushima Fatty rats. Am J Physiol Gastrointest Liver Physiol. 2002, 282(3):549 -558.13*|*期刊*|*Olson LE, Soriano P. Increased PDGFRalpha activation disrupt

19、s connective tissue development and drives systemic fibrosis. Dev Cell. 2009, 16(2):303-313.14*|*期刊*|*Jacobsen ML, Rnn SG, Bruun C, et al. IL-1beta-induced chemokine and Fas expression are inhibited by suppressor of cytokine signalling-3 in insulin-producing cells. Diabetologia. 2009, 52(2):281-288.

20、 * MERGEFORMAT1*|*期刊*|*Bachem MG, Schneider E, Gross H, et al. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology. 1998, 115(2):421-432.2*|*期刊*|*Lee H, Lim C, Lee J, Kim N, et al. TGF-beta signaling preserves RECK expression in activated p

21、ancreatic stellate cells. J Cell Biochem. 2008, 104(3):1065-1074.3*|*期刊*|*Nomiyama Y, Tashiro M, Yamaguchi T, et al. High glucose activates rat pancreatic stellate cells through protein kinase C and p38 mitogen-activated protein kinase pathway. Pancreas.2007, 34(3):364-72.4*|*期刊*|*Hong OK, Lee SH, R

22、hee M, et al. Hyperglycemia and hyperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: Possible explanation of islet-specific fibrosis in type 2 diabetes mellitus. J Cell Biochem. 2007, 101(3):665-675.5*|*期刊*|*Hong OK, Lee SH, Rhee M, et al. Hyperglycemi

23、a and hyperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: possible explanation of islet-specific fibrosis in type 2 diabetes mellitus. J Cell Biochem. 2007, 101(3):665-675.6*|*期刊*|*潘琳，李宏亮，楊文英等，胰腺星狀細胞活化在高脂飼養(yǎng)大鼠胰島纖維化形成中的作用。中華內(nèi)分泌代謝雜志。2009, 25(1):25-27.7*|

24、*在線文獻*|*李鳳飛，孫子林，殷俊梅等. 胰腺星狀細胞在2型糖尿病胰島纖維化進程中的影響OL. 中國科技論文在線2011-06-07/index.php/default/releasepaper/content/201106-70.8*|*期刊*|*Mizukami H, Wada R, Yonezawa A, et al. Suppression of post-prandial hyperglycaemia by pioglitazone improved islet fibrosis and macrophage migration in the Goto-Kakizaki rat.

25、Diabetes Obes Metab. 2008, 10(9):791-794.9*|*期刊*|*Tikellis C,Wookey PJ, Candido R, et al. Improved islet morphology after blockade of the renin-angiotensin system in the ZDF rat. Diabetes. 2004, 53(4):989-997.10*|*期刊*|*Masuyama T, Komeda K, Hara A, et al. Chronological characterization of diabetes d

26、evelopment in male Spontaneously Diabetic Torii rats. Biochem Biophys Res Commun. 2004, 314(2):870-877.11*|*期刊*|*Shinozaki S, Ohnishi H, Hama K, et al. Indian hedgehog promotes the migration of rat activated pancreatic stellate cells by increasing membrane type-1 matrix metalloproteinase on the plas

27、ma membrane. J Cell Physiol.2008, 216(1):38-46.12*|*期刊*|*Yoshikawa H, Kihara Y, Taguchi M, et al. Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long-Evans okushima Fatty rats. Am J Physiol Gastrointest Liver Physiol. 2002, 282(3):549 -558.13*|*期刊*|*Olson LE, Soriano P. Increa

28、sed PDGFRalpha activation disrupts connective tissue development and drives systemic fibrosis. Dev Cell. 2009, 16(2):303-313.14*|*期刊*|*Jacobsen ML, Rnn SG, Bruun C, et al. IL-1beta-induced chemokine and Fas expression are inhibited by suppressor of cytokine signalling-3 in insulin-producing cells. D

29、iabetologia. 2009, 52(2):281-288.SET bkAuthorsInfo |1|殷俊梅|YIN Junmei|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|殷俊梅（1986-），女，碩士研究生，胰島纖維化相關(guān)機制|江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科|21000915105177678*|

30、2|孫子林|SUN Zilin|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|孫子林（1963-），男，主任醫(yī)師，胰島纖維化相關(guān)機制|江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科|210009|sunzilin196313951749490|3|李鳳飛|LI Fengfei|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所

31、|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|lifengfei2005|4|陳碧君|CHEN Bijun|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,21000

32、9|332335218| * MERGEFORMAT|1|殷俊梅|YIN Junmei|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|殷俊梅（1986-），女，碩士研究生，胰島纖維化相關(guān)機制|江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科|21000915105177678*|2|孫子林|SUN Zilin|東南大學(xué)附屬中大醫(yī)

33、院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|孫子林（1963-），男，主任醫(yī)師，胰島纖維化相關(guān)機制|江蘇省南京市東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科|210009|sunzilin196313951749490|3|李鳳飛|LI Fengfei|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinol

34、ogy, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|lifengfei2005|4|陳碧君|CHEN Bijun|東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所|Department of endocrinology, Zhongda hospital, Institute of Diabetes, Medical School, Southeast University,Nanjing,210009|332335218|SET bkTitleIn

35、fo 胰腺星狀細胞對胰島細胞胰島素釋放和合成的影響|Effect of pancreatic stellate cell on the secretion and expression of insulin of Beta-Cell|國家自然科學(xué)基金（30971399）高等學(xué)校博士學(xué)科點專項科研基金（200802860020） * MERGEFORMAT胰腺星狀細胞對胰島細胞胰島素釋放和合成的影響|Effect of pancreatic stellate cell on the secretion and expression of insulin of Beta-Cell|國家自然科學(xué)基金

36、（30971399）高等學(xué)校博士學(xué)科點專項科研基金（200802860020）（東南大學(xué)附屬中大醫(yī)院內(nèi)分泌科，東南大學(xué)糖尿病研究所）SET bkTitleInfo * MERGEFORMAT SET bkAuthorsInfo * MERGEFORMAT 摘要： FORMTEXT 目的 觀察胰腺星狀細胞（pancreatic stellate cell, PSC）對胰島細胞株INS-1細胞釋放及合成胰島素的影響，探索PSC對胰島細胞功能的可能作用。方法 構(gòu)建INS-1和PSC細胞共培養(yǎng)系統(tǒng)，細胞分為INS-1正常糖組（5.5mM葡萄糖），INS-1高糖組（16.7mM葡萄糖）；共培養(yǎng)正常糖組（

37、5.5mM葡萄糖），共培養(yǎng)高糖組（16.7mM葡萄糖）。分別孵育不同時間，于1、2、6小時提取培養(yǎng)上清行放射免疫法檢測胰島素分泌水平，于6、12、24小時提取INS-1細胞RNA行RT-PCR檢測胰島素基因表達水平。結(jié)果 在不同孵育時間，正常糖濃度和高糖濃度下，共培養(yǎng)組胰島素分泌水平均低于INS-1單獨培養(yǎng)組1h（2.150.13 vs 1.310.16, 5.590.05 vs 2.620.04，P0.05），2h(5.550.11 vs 3.8020.12, 12.910.39 vs 9.910.19, P0.05), 6h(10.430.25 vs 4.980.18, 25.840.52

38、 vs 16.190.41, P0.05)。不同孵育時間，正常糖濃度下，共培養(yǎng)組胰島素表達水平與INS-1單獨培養(yǎng)組無明顯差異，但高糖濃度下，共培養(yǎng)組胰島素基因表達水平明顯低于INS-1單獨培養(yǎng)組：12h(1.730.09 vs 0.470.11, P0.05), 24h（1.940.12 vs 0.580.08，P0.05）。結(jié)論 PSC可抑制INS-1細胞在正常條件和高糖刺激下胰島素分泌，正常糖濃度下不影響胰島素的合成，但在高糖條件下可明顯抑制胰島素的合成。關(guān)鍵詞： FORMTEXT 胰腺星狀細胞；胰島細胞；胰島素中圖分類號： FORMTEXT 請查閱中國圖書館分類法SET bkAutho

39、rsInfo * MERGEFORMAT SET bkTitleInfo * MERGEFORMAT FORMTEXT Effect of pancreatic stellate cell on the secretion and expression of insulin of Beta-Cell FORMTEXT YIN Junmei, FORMTEXT SUN Zilin, FORMTEXT LI Fengfei, FORMTEXT CHEN Bijun(Department of endocrinology, Zhongda hospital, Institute of Diabete

40、s, Medical School, Southeast University,Nanjing,210009)Abstract: FORMTEXT Object To explore the effect of pancreatic stellate cells (PSCs) on the secretion and expression of insulin of Beta-Cell line Ins-1 under normal and high-concentration glucose. Methods We established a co-culture system using

41、activated PSC and INS-1 cell under 5.5mM glucose and 16.7mM glucose, separately. The supernatant was collected after 1h, 2h and 6h. The total RNA of INS-1 cell was extracted after 6h,12h and 24h. The secretion of insulin was detected by RIA. The gene expression of insulin was detected by RT-PCR. Res

42、ults The secretion of insulin was much lower in the co-culture group, both under 5.5mM glucose and 16.7mM glucose . 1h（2.150.13 vs 1.310.16, 5.590.05 vs 2.620.04，P0.05），2h(5.550.11 vs 3.8020.12, 12.910.39 vs 9.910.19, P0.05), 6h(10.430.25 vs 4.980.18, 25.840.52 vs 16.190.41, P0.05)。 The gene express

43、ion of insulin was lower in the co-culture group when cultured in 16.7mM glucose. 12h(1.730.09 vs 0.470.11, P0.05), 24h（1.940.12 vs 0.580.08，P0.05）Conclusion Activated PSC could inhibit the secretion of insulin of INS-1 cell both under normal or high glucose culture, and also inhibit the gene expres

44、sion of insulin of INS-1 cell when cultured in high glucose medium.Key words: FORMTEXT pancreatic stellate cell;pancreatic beta-cell;insulin引言胰腺內(nèi)外分泌腺不僅在解剖結(jié)構(gòu)上緊密相鄰，在生理功能上也緊密相關(guān)，哺乳動物胰島的內(nèi)分泌受多種復(fù)雜因素的控制。胰腺星狀細胞是參與胰島外胰腺纖維化的主要始動和效應(yīng)細胞，其活化后可分泌多種細胞因子，發(fā)揮致纖維化和致凋亡作用1-2。近年來有報道高糖、高胰島素可刺激PSC活化并分泌致病細胞因子3-4，這促使我們探討PSC對內(nèi)分

45、泌腺的影響。本實驗擬通過體外構(gòu)建PSC與胰島細胞株INS-1細胞共培養(yǎng)系統(tǒng)，觀察PSC對胰島細胞功能的影響。1 材料與方法材料RPMI 1640培養(yǎng)液（Gibco 公司）；DMEM/F12培養(yǎng)液（Gibco 公司）；胎牛血清（Hyclone 公司）； transwell 共培養(yǎng)小室（corning 公司）；Trizol試劑（invertrogen 公司）；大鼠胰島素放射免疫試劑盒購自Linco公司；RT試劑盒（formentas 公司）。方法PSC分離及鑒定取體質(zhì)量為150g左右的SD大鼠麻醉后無菌條件下打開腹腔充分暴露胰腺，用眼科剪及鑷子小心將其游離，迅速取出放入盛有4預(yù)冷GBSS緩沖液的培

46、養(yǎng)皿中。在培養(yǎng)皿中仔細剔除胰腺周圍的被膜結(jié)締組織，將其粗剪成小塊，GBSS緩沖液漂洗3次以去掉表面血污，滴管吸凈漂洗液，換用眼科剪再次反復(fù)剪切胰腺組織至0.5 mm0.5 mm0.5 mm大小為止。用彎頭吸管小心吸取組織塊，將其置于15cm2培養(yǎng)皿中，擺布在皿底部，小塊間相互間距為0.5cm左右。輕輕翻轉(zhuǎn)培養(yǎng)皿，令皿底朝上，靜置4h。4h后小心加入含有15%胎牛血清的DMEM/F12培養(yǎng)液10mL，讓培養(yǎng)液覆蓋附于皿底部的組織小塊，置入37、5%CO2 恒溫培養(yǎng)箱中培養(yǎng)。每天觀察組織塊、細胞及培養(yǎng)液情況，做好記錄。3天后細胞從組織塊長出，原皿換液，7天后原皿傳代，除去漂浮的組織塊。待細胞達80

47、%融合后傳代至培養(yǎng)瓶中培養(yǎng)，每瓶加4ml含15%胎牛血清的DMEM/F12培養(yǎng)液，每2-3天換液。經(jīng)特異性標記物-SMA、desmin、vimentin 免疫組化檢測鑒定，分離出細胞即為PSC。細胞培養(yǎng)INS-1細胞（由江蘇省人民醫(yī)院內(nèi)分泌科楊濤教授惠贈）培養(yǎng)于含15% FBS的RPMI1640培養(yǎng)基中，含有（11.1 mmol/L葡萄糖，10 mmol/L Hepes，1 mmol/L丙酮酸鈉，50mol/L -巰基乙醇，100 U/ml青霉素和100g/ml的鏈霉素）。PSC采用第3-7代細胞，換用RPMI1640培養(yǎng)基，添加15% FBS。置于37含5% CO2 的飽和濕度培養(yǎng)箱中培養(yǎng)，

48、每2-3天換液，待細胞80%融合后傳代。構(gòu)建共培養(yǎng)系統(tǒng)將處于對數(shù)生長期的INS-1和PSC經(jīng)胰酶消化后，制備成終濃度為1106/ml 的細胞懸液，并分別接種于六孔板和相應(yīng)大小0.4m孔徑的共培養(yǎng)小室，換用葡萄糖濃度為5.5mmol/L的RPMI 1640培養(yǎng)液，24小時后分別棄原培養(yǎng)液，將共培養(yǎng)小室置于六孔板內(nèi)，用無血清的RPMI 1640培養(yǎng)液設(shè)置共培養(yǎng)正常糖組（NG組，5.5mmol/L葡萄糖），共培養(yǎng)高糖組（HG組，16.7mmol/L葡萄糖）。并設(shè)置對照的單獨INS-1正常糖組（NG組，5.5mmol/L葡萄糖）和INS-1高糖組（HG組，16.7mmol/L葡萄糖）；分別于孵育1、2

49、、6小時收集培養(yǎng)上清，并于孵育6、12、24小時，收集各組INS-1細胞，提取RNA。RIA法檢測上清中胰島素分泌情況大鼠胰島素放射免疫試劑盒購自Linco公司，按說明書操作，江蘇省人民醫(yī)院核醫(yī)學(xué)科檢驗。RT-PCR檢測胰島素mRNA表達將共培養(yǎng)各組的INS-1細胞用Trizol裂解，按說明書進行操作分離RNA。將分離到的RNA用紫外光分光光度計檢測含量和純度，選取OD值260nm/280nm為1.82.0的總RNA用于實驗。取1ugRNA，1ul oligodT, 1ul逆轉(zhuǎn)錄酶和1ul核酸酶抑制劑加入20ul反應(yīng)體系，按RT試劑盒(formentas)進行逆轉(zhuǎn)錄反應(yīng)，合成cDNA。取2ul

50、 RT反應(yīng)液按照RT-PCR試劑盒說明書配置25ulPCR反應(yīng)液：mix12.5ul， RT產(chǎn)物2ul，上下游引物各0.5ul，加去離子水至25ul。取10ul產(chǎn)物進行2%瓊脂糖凝膠電泳，溴化乙錠顯色，凝膠成像分析系統(tǒng)對目的DNA條帶進掃描,用Quanti2ty One分析軟件測得電泳圖譜上每條基因條帶的光密度值。計算各個樣本強度與內(nèi)參的比值,從而反映胰島素mRNA的相對表達量。統(tǒng)計學(xué)分析實驗中每組設(shè)立3 個復(fù)孔，實驗重復(fù)3 次以上。應(yīng)用SPSS 17.0 分析，數(shù)據(jù)用 meanSD表示。多組間差異顯著性檢驗使用單因素方差分析，組間兩兩比較使用LSD 法，P 0.05差異具有統(tǒng)計學(xué)意義。2.結(jié)

51、果2.1 分離鑒定PSC 圖1(a) 圖1(b) 圖1：(a): 組織塊培養(yǎng)法分離PSC, 第3天。(b): 分離培養(yǎng)的PSC用-SMA免疫組化染色鑒定。2.2 不同時間上清中胰島素的分泌情況：在不同孵育時間，正常糖濃度和高糖濃度下，共培養(yǎng)組胰島素分泌水平均低于INS-1單獨培養(yǎng)組:表1 不同時間上清中胰島素含量組別無PSC有PSC 5.5mM 1h2.150.131.310.16 16.7mM 1h5.590.05 2.620.04 5.5mM 2h5.550.113.8020.12 16.7mM 2h12.910.399.910.19 5.5mM 6h10.430.254.980.18 1

52、6.7mM 6h25.840.5216.190.41 圖2 不同時間上清中胰島素含量:5.5mM糖濃度下，共培養(yǎng)組胰島素分泌低于對照組(* p0.05),16.7mM糖濃度下，共培養(yǎng)組胰島素分泌顯著低于對照組( p0.01)2.3 不同時間INS-1細胞中胰島素RNA表達情況：圖3 不同孵育時間，正常糖濃度下，共培養(yǎng)組胰島素表達水平與INS-1單獨培養(yǎng)組無明顯差異，但高糖濃度下，共培養(yǎng)組胰島素基因表達水平明顯低于INS-1單獨培養(yǎng)組：12h(1.730.09 vs 0.470.11, P0.05), 24h（1.940.12 vs 0.580.08，P0.05）3.討論：胰腺星狀細胞（panc

53、reatic stellate cell, PSC）PSC 于1998 年首先由Bachem 從胰腺基質(zhì)中分離并命名，該細胞主要定植于胰腺小葉之間及胰腺腺泡周圍1 。其活化后特異表達-SMA，并具備增殖、遷移及合成細胞外基質(zhì)成分的能力，是參與胰腺纖維化過程的主要始動與效應(yīng)細胞。近年來，不斷有學(xué)者研究認為PSC同時在胰島纖維化中發(fā)揮重要作用。Kim和潘琳5-6等先后在2型糖尿病（type 2 diabetes, T2DM）病人和高脂飼養(yǎng)SD大鼠胰島中發(fā)現(xiàn)-SMA染色陽性細胞，同時伴有胰島細胞數(shù)量減少和胰島素抵抗。我實驗組的前期研究也發(fā)現(xiàn)，活化PSC定植于纖維化胰島中，并可能是介導(dǎo)T2DM 胰島纖

54、維化發(fā)生發(fā)展的重要靶細胞7 。胰島纖維化是促使胰島 細胞功能進行性減退直至衰竭的重要病理生理機制之一。在T2DM 胰島纖維化進程中，細胞體積顯著縮小，纖維化區(qū)域出現(xiàn)明顯的胰島素分泌缺失10，而減輕胰島纖維化，可恢復(fù)胰島素一相分泌，改善糖代謝異常8-9。另一方面，高糖、高胰島素等眾多因素可刺激PSC活化、增殖，同時表達大量的細胞外基質(zhì)3-4。活化的PSC可發(fā)生遷移和聚集11，分泌眾多產(chǎn)物，可發(fā)揮多種生物學(xué)效應(yīng)，包括促纖維化（TGF-1、PDGF 等）12-13和促凋亡（TNF-、IL-1 等）作用12,14，從而可能導(dǎo)致胰島纖維化形成和胰島 細胞量減少、功能減退。因此，我們推測：活化的PSC可參

55、與胰島纖維化進程，并加速胰島細胞功能減退。本實驗利用INS-1 細胞株作為胰島 細胞模型，建立PSC與INS-1共培養(yǎng)體系，設(shè)立正常糖濃度和高糖濃度組，旨在模擬體內(nèi)生理及高血糖病理狀態(tài)，在體外初步探討PSC在T2DM病程中的可能作用。實驗結(jié)果表明，PSC在正常及高糖條件下均可抑制INS-1細胞胰島素的分泌，在高糖條件下還抑制其胰島素基因的表達，提示PSC在高糖條件下可明顯抑制胰島 細胞的胰島素分泌及表達功能，促使其功能減退。此結(jié)果進一步驗證我們的假說：活化的PSC可通過分泌致病因子、加速胰島纖維化等方式促使胰島 細胞功能障礙，加重糖尿病條件下胰島素分泌不足，最終導(dǎo)致胰島功能喪失。但其通過何種細

56、胞因子發(fā)揮作用，尚需進一步闡明。參考文獻 (References) FORMTEXT 1 Bachem MG, Schneider E, Gross H, et al. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology. 1998, 115(2):421-432.2 Lee H, Lim C, Lee J, Kim N, et al. TGF-beta signaling preserves RECK expression

57、 in activated pancreatic stellate cells. J Cell Biochem. 2008, 104(3):1065-1074.3 Nomiyama Y, Tashiro M, Yamaguchi T, et al. High glucose activates rat pancreatic stellate cells through protein kinase C and p38 mitogen-activated protein kinase pathway. Pancreas.2007, 34(3):364-72.4 Hong OK, Lee SH, Rhee M, et al. Hyperglycemia and hyperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: Possible exp