中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
50期
9898-9902
,共5页
舒肝颗粒%肝星状细胞%细胞因子%肝纤维化%TGF-β1%smad3%smad7
舒肝顆粒%肝星狀細胞%細胞因子%肝纖維化%TGF-β1%smad3%smad7
서간과립%간성상세포%세포인자%간섬유화%TGF-β1%smad3%smad7
背景:肝纤维化是一种可逆性的病变,及时地干预肝纤维化的病程能够减少肝硬化及其致命并发症的发生.肝星状细胞在肝纤维化发病机制中起主要作用.目的:采用舒肝颗粒作用于体外培养的大鼠肝星状细胞,观察其是否对转化生长因子β1促进肝星状细胞活化及跨膜信号转导有影响,以探讨其抗纤维化的作用机制.设计、时间及地点:对照观察细胞学实验,于2008-06／2009-02在泸州医学院分子中心实验室完成.材料:肝星状细胞株HSC-T6购自上海中医药大学肝病研究所,其表型为活化的肝星状细胞.舒肝颗粒由泸州医学院附属医院药物研究所提供,批号:20071120.方法:四甲基偶氮唑盐法测不同质量浓度(0.01,0.02,0.04,0.08 g/L)舒肝颗粒对HSC-T6细胞增殖情况的影响,选择对细胞增殖无影响的剂量(0.01 g／L).再将HSC-T6细胞种板培养后,分为4组,空白对照组培养液中不加其他处理因素.转化生长因子β1组培养液中加入5 μg/L转化生长因子β1液.舒肝颗粒组培养液中加入前面选择的0.01 g／L舒肝颗粒药液;联合用药组培养液中同时加入舒肝颗粒药液与转化生长因子β1液,剂量同上.主要观察指标:①肝星状细胞的形态.②免疫组织化学观察肝星状细胞表达Smad3和Smad7.③反转录-聚合酶链反应观察肝星状细胞活化及跨膜信号转导结果.结果:①转化生长因子β1组细胞形态类似于对照组,且伸展更明显,联合用药组,细胞形态更接近于转化生长因子β1组,各组均未见核固缩或凋亡现象.②免疫组织化学法测Smad3和Smad7在对照组分别表达较高;转化生长因子β1能轻度上调Smad3和Smad7的表达(P<0.05):而舒肝颗粒组和联合用药组与对照组相比,Smad7表达上调更为显著,为对照组的1.99倍(P<0.01).⑨反转录-聚合酶链反应结果显示与对照组相比,舒肝颗粒组上调Smad7mRNA表达(P<0.05),而Smad3mRNA的表达无明显变化.给予转化生长因子β1(5μg/L)作用后,Smad3和Smad7表达均上调(P < 0.05).联合组作用的影响是Smad7上升1.01倍(P < 0.05),但Smad3的转录水平无明显变化(P > 0.05).结论:① 转化生长因子β能刺激smad3和smad7的基因表达,使R-Smads/I-Smads之间的反馈调节机制重新达到平衡.②舒肝颗粒可能通过对肝星状细胞增殖的抑制而起到抗肝纤维化的作用,且抑制程度与药物剂量呈一定依赖关系.③舒肝颗粒可能通过上调smad7的表达,抑制TGF-β/smad信号转导途径.
揹景:肝纖維化是一種可逆性的病變,及時地榦預肝纖維化的病程能夠減少肝硬化及其緻命併髮癥的髮生.肝星狀細胞在肝纖維化髮病機製中起主要作用.目的:採用舒肝顆粒作用于體外培養的大鼠肝星狀細胞,觀察其是否對轉化生長因子β1促進肝星狀細胞活化及跨膜信號轉導有影響,以探討其抗纖維化的作用機製.設計、時間及地點:對照觀察細胞學實驗,于2008-06／2009-02在瀘州醫學院分子中心實驗室完成.材料:肝星狀細胞株HSC-T6購自上海中醫藥大學肝病研究所,其錶型為活化的肝星狀細胞.舒肝顆粒由瀘州醫學院附屬醫院藥物研究所提供,批號:20071120.方法:四甲基偶氮唑鹽法測不同質量濃度(0.01,0.02,0.04,0.08 g/L)舒肝顆粒對HSC-T6細胞增殖情況的影響,選擇對細胞增殖無影響的劑量(0.01 g／L).再將HSC-T6細胞種闆培養後,分為4組,空白對照組培養液中不加其他處理因素.轉化生長因子β1組培養液中加入5 μg/L轉化生長因子β1液.舒肝顆粒組培養液中加入前麵選擇的0.01 g／L舒肝顆粒藥液;聯閤用藥組培養液中同時加入舒肝顆粒藥液與轉化生長因子β1液,劑量同上.主要觀察指標:①肝星狀細胞的形態.②免疫組織化學觀察肝星狀細胞錶達Smad3和Smad7.③反轉錄-聚閤酶鏈反應觀察肝星狀細胞活化及跨膜信號轉導結果.結果:①轉化生長因子β1組細胞形態類似于對照組,且伸展更明顯,聯閤用藥組,細胞形態更接近于轉化生長因子β1組,各組均未見覈固縮或凋亡現象.②免疫組織化學法測Smad3和Smad7在對照組分彆錶達較高;轉化生長因子β1能輕度上調Smad3和Smad7的錶達(P<0.05):而舒肝顆粒組和聯閤用藥組與對照組相比,Smad7錶達上調更為顯著,為對照組的1.99倍(P<0.01).⑨反轉錄-聚閤酶鏈反應結果顯示與對照組相比,舒肝顆粒組上調Smad7mRNA錶達(P<0.05),而Smad3mRNA的錶達無明顯變化.給予轉化生長因子β1(5μg/L)作用後,Smad3和Smad7錶達均上調(P < 0.05).聯閤組作用的影響是Smad7上升1.01倍(P < 0.05),但Smad3的轉錄水平無明顯變化(P > 0.05).結論:① 轉化生長因子β能刺激smad3和smad7的基因錶達,使R-Smads/I-Smads之間的反饋調節機製重新達到平衡.②舒肝顆粒可能通過對肝星狀細胞增殖的抑製而起到抗肝纖維化的作用,且抑製程度與藥物劑量呈一定依賴關繫.③舒肝顆粒可能通過上調smad7的錶達,抑製TGF-β/smad信號轉導途徑.
배경:간섬유화시일충가역성적병변,급시지간예간섬유화적병정능구감소간경화급기치명병발증적발생.간성상세포재간섬유화발병궤제중기주요작용.목적:채용서간과립작용우체외배양적대서간성상세포,관찰기시부대전화생장인자β1촉진간성상세포활화급과막신호전도유영향,이탐토기항섬유화적작용궤제.설계、시간급지점:대조관찰세포학실험,우2008-06／2009-02재로주의학원분자중심실험실완성.재료:간성상세포주HSC-T6구자상해중의약대학간병연구소,기표형위활화적간성상세포.서간과립유로주의학원부속의원약물연구소제공,비호:20071120.방법:사갑기우담서염법측불동질량농도(0.01,0.02,0.04,0.08 g/L)서간과립대HSC-T6세포증식정황적영향,선택대세포증식무영향적제량(0.01 g／L).재장HSC-T6세포충판배양후,분위4조,공백대조조배양액중불가기타처리인소.전화생장인자β1조배양액중가입5 μg/L전화생장인자β1액.서간과립조배양액중가입전면선택적0.01 g／L서간과립약액;연합용약조배양액중동시가입서간과립약액여전화생장인자β1액,제량동상.주요관찰지표:①간성상세포적형태.②면역조직화학관찰간성상세포표체Smad3화Smad7.③반전록-취합매련반응관찰간성상세포활화급과막신호전도결과.결과:①전화생장인자β1조세포형태유사우대조조,차신전경명현,연합용약조,세포형태경접근우전화생장인자β1조,각조균미견핵고축혹조망현상.②면역조직화학법측Smad3화Smad7재대조조분별표체교고;전화생장인자β1능경도상조Smad3화Smad7적표체(P<0.05):이서간과립조화연합용약조여대조조상비,Smad7표체상조경위현저,위대조조적1.99배(P<0.01).⑨반전록-취합매련반응결과현시여대조조상비,서간과립조상조Smad7mRNA표체(P<0.05),이Smad3mRNA적표체무명현변화.급여전화생장인자β1(5μg/L)작용후,Smad3화Smad7표체균상조(P < 0.05).연합조작용적영향시Smad7상승1.01배(P < 0.05),단Smad3적전록수평무명현변화(P > 0.05).결론:① 전화생장인자β능자격smad3화smad7적기인표체,사R-Smads/I-Smads지간적반궤조절궤제중신체도평형.②서간과립가능통과대간성상세포증식적억제이기도항간섬유화적작용,차억제정도여약물제량정일정의뢰관계.③서간과립가능통과상조smad7적표체,억제TGF-β/smad신호전도도경.
BACKGROUND: Hepatic fibrosis is a reversible disease, interfering in course of disease promptly can decrease hepatic cirrhosis and fatal complication. Hepatic stellate cells (HSCs) is a key factor in pathogenesy of hepatic fibrosis.OBJECTIVE: To investigate the effects of Shugan granule on HSCs activation and trans-membrane signal transduction stimulated by transforming growth factor beta 1 (TGF-β1) in rats, and to explore the anti-fibrosis mechanism.DESIGN, TIME AND SETTING: Controlled observational trials based on cytology were performed in the Central Laboratory of Molecules, Luzhou Medical College between June 2008 and February 2009. MATERIALS: HSC-T6 cell line was purchased from Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, its phenotype was the activated hepatic stellate cells. Shugan granule was offered by Drug Institute, Affiliated Hospital of Luzhou Medical College at a Batch No. 20071120.METHODS: The influence of different concentrations (0.01, 0.02, 0.04, 0.08 g/L) of Shugan granule on HSCs proliferation was determined by MTT. 0.01 g/L was defined as the dose of Shugan granule contributing no influence on cell proliferation. HSCs were cultured in a culture plate and then divided into 4 groups: control group without management, TGF-β1 group with 5 μg/L TGF-β1 solution in culture medium, Shugan granule group with 0.01 g/L Shugan granule in a culture medium, and TGF-β1 + Shugan granule group with 5 μg/L TGF-β1 solution and 0.01 g/L Shugan granule in culture medium. MAIN OUTCOME MEASURES: Morphological features of HSCs were detected by microscopic. Immunohistochemical method was used to detect the expression of Smad3 and Smad7 in HSCs. RT-PCR was applied to observe the HSCs activation and trans-membrane signal transduction. RESULTS: ①The cell morphology of TGF-β1 group was similar with that in the control group, and the extension was more obvious. In the TGF-β1 + Shugan granule group, the cell morphology was close to that in TGF-β1 group. There was no karyopyknosis or apoptosis observed in each group. ②Immunohistochemical method showed the expression of Smad3 and Smad7 in control groups were increased. TGF-β1 could slightly increase the expression of Smad3 and Smad7 (P < 0.05), while Shugan granule group and TGF-β1 + Shugan granule group increased the expression of Smad7 significantly, accounting for 1.99 times compared with control group (P < 0.01). ③RT-PCR result showed that Shugan granule could increase the expression of Smad7 (P < 0.05), but the expression of Smad3 was not regulated. 5 μg/L TGF-β1 could up-regulate the expression of Smad3 and Smad7 (P < 0.05). In the TGF-β1 + Shugan granule group, Smad7 expression was increased by 101% (P < 0.05), but Smad3 transcriptional level was not changed(P > 0.05). CONCLUSION: ①TGF-β can stimulate the gene expression of Smad3 and Smad7, it also obtain a balance of feedback regulation mechanism between R-Smads and I-Smads. ②Shugan granule may prevent and cure hepatic fibrosis through decreasing the proliferation of HSCs in a dose-dependent manner. ③Shugan granule can inhibit the TGF-β-Smad signaling pathway through increasing the expression of Smad7.
