【作者】 林有智；

【导师】 陈孝平；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 目的：建立胆管癌Smad4KD稳定细胞系并对其进行鉴定。方法：采用用胆管癌细胞系HuCCTl和RBE作为研究对象,采用western blotting检测Smad蛋白的表达,并用外源性TGF-β刺激胆管癌细胞后检测Smad蛋白的磷酸化水平。从国外购买已被文献验证的有效pRetroSuper-puro Smad4质粒及其空载体。将质粒转染PT67细胞以包被逆转录病毒颗粒。收集病毒上清,分别感染野生型胆管癌细胞系,建立Smad4干扰组以及相应的空载体对照组。使用嘌呤霉素筛选获得表达耐药基因阳性的细胞系。以western blotting和Co-immunoprecipitation检测上述细胞系中Smad4蛋白的表达情况及Smad2/3/4复合物的形成情况。结果：HuCCT1和RBE细胞均表达TGF-βI型受体、Smad2、Smad3、Smad4和Smad7蛋白,而且外源性TGF-p时间依赖性地诱导Smad2和Smad3磷酸化。野生型和空载体对照组胆管癌细胞均表达Smad4,干扰组胆管癌细胞的Smad4表达量显著下降(P<0.01)。经外源性TGF-β刺激后,干扰组胆管癌细胞无法形成正常量的Smad2/3/4复合物。结论：已成功建立稳定干扰Smad4表达的胆管癌细胞系。目的：探讨Smad4对胆管癌细胞生长的作用。方法：将TGF-p和或TGF-p受体I抑制剂SB431542处理野生型HuCCT1细胞,分别于处理后的0d、1d、2d、3d、4d、5d和6d通过CCK-8试剂盒检测胆管癌细胞的增值情况。将TGF-p处理野生型RBE细胞,分别于处理后的Oh、6h、12h、24h、36h、48h、60h和72h通过CCK-8试剂盒检测胆管癌细胞的增值情况。在进行Smad4干扰后,将外源性TGF-p处理野生型、空载体对照组和干扰组细胞5d(HuCCT1)或48h (RBE),再通过CCK-8试剂盒检测各组胆管癌细胞的增值情况。采用western blotting检测各组HuCCT1细胞的cyclinA2和p21蛋白的表达水平,以及RBE细胞的phospho-Rb和p21蛋白的表达水平。分别将野生型、空载体对照组和干扰组HuCCT1细胞注射入裸鼠皮下,观察各组裸鼠的皮下成瘤情况。结果：TGF-β均能抑制HuCCTl和RBE细胞增殖。与野生型和空载体对照组细胞相反,干扰组RBE细胞对TGF-β抑制增殖的效应不敏感；TGF-β对干扰组HuCCT1细胞的增殖仅有轻度抑制作用,而且干扰组HuCCT1细胞在没有TGF-β作用下的生长速度快于野生型和空载体对照组细胞。无论是有或无TGF-β作用,干扰组HuCCT1细胞的cyclin A2蛋白的表达水平均高于野生型和空载体对照组细胞,TGF-β诱导干扰组HuCCT1细胞表达p21蛋白的水平明显低于空载体对照组；在TGF-β作用下,干扰组RBE细胞的Rb蛋白磷酸化水平高于野生型和空载体对照组,而p21表达量明显低于野生型和空载体对照组。与野生型和空载体对照组HuCCT1细胞所形成皮下瘤相比,干扰Smad4的HuCCT1细胞在裸鼠皮下形成的肿瘤体积更大。结论：Smad4介导胆管癌细胞的生长抑制,其机制可能是通过降低cyclin A2的表达和Rb蛋白的磷酸化水平,以及介导p21表达。目的：探讨Smad4对胆管癌细胞凋亡的作用。方法：将不同浓度的TGF-β处理野生型RBE细胞36h,以及将1ng/ml的TGF-β分别处理野生型RBE细胞0、12、24、36和48h,细胞经PI/Annexin-Ⅴ染色后进行流式细胞分析,检测细胞凋亡,并且通过western blotting检测不同时间点Bim和Bcl-2蛋白的表达以及caspase和PARP蛋白的激活情况；再将泛caspase抑制剂与TGF-β联合作用于野生型RBE细胞36h后,通过流式细胞术检测细胞凋亡。将1ng/ml的TGF-β分别处理空载体对照组和干扰Smad4组RBE细胞36h,采用同样的方法检测细胞凋亡。将TGF-β和/或JNK抑制剂SP600125作用于野生型RBE细胞36h后,通过流式细胞术分析比较各处理组细胞的凋亡,通过western blotting检测Bim和Bcl-2蛋白的表达以及caspase和PARP蛋白的激活情况；将泛caspase抑制剂与TGF-β和/或SP600125联合作用于野生型RBE细胞36h后,通过流式细胞术检测细胞凋亡。将TGF-β和/或SP600125分别作用于空载体对照组和干扰Smad4组RBE细胞36h,然后通过流式细胞术分析比较此两组细胞的凋亡,通过western blotting检测Bim和Bcl-2蛋白的表达以及caspase和PARP蛋白的激活情况。将TGF-β和/或SP600125作用于野生型RBE细胞6h后,通过western blotting检测Smad2和Smad3的磷酸化水平。将带有荧光素酶报告基因的质粒转入野生型RBE细胞,然后将TGF-β和/或SP600125处理细胞24h,最后分析TGF-β诱导报告基因的转录活性。将带有荧光素酶报告基因的质粒转入空载体对照组和干扰Smad4组RBE细胞,然后将TGF-β和/或SP600125分别作用于两组细胞24h,最后分析TGF-β诱导报告基因的转录活性。结果：TGF-β剂量依赖性地和时间依赖性地诱导野生型RBE细胞发生凋亡。与空载体对照组相比,shSmad4RBE细胞对TGF-β的促凋亡效应不敏感。SP600125增强TGF-β诱导野生型RBE细胞凋亡的效应。与空载体对照组相比,shSmad4RBE细胞对SP600125的促凋亡效应不敏感。接受泛caspase抑制剂处理的野生型RBE细胞对TGF-β和SP600125的促凋亡效应不敏感。TGF-β时间依赖性地诱导Bim表达上调、Bcl-2表达下调、以及caspase和PARP蛋白激活。SP600125增强TGF-β诱导的Bim表达上调、Bcl-2表达下调、以及caspase和PARP蛋白激活,该效应在干扰Smad4表达后被阻断。SP600125增强TGF-β诱导的Smad2和Smad3的磷酸化水平以及荧光素酶报告基因的转录水平。干扰Smad4表达能阻断SP600125对TGF-β诱导荧光素酶报告基因转录的增强效应。结论：Smad4介导TGF-p对RBE细胞的促凋亡效应,SP600125依赖Smad4介导增强TGF-β对RBE细胞的促凋亡效应,线粒体相关的caspase蛋白活化参与Smad4对凋亡的调控。目的：探讨Smad4在胆管癌细胞上皮-间质转化(epithelial-mesenchymal transition, EMT)、迁移和侵袭中的作用。方法：将TGF-β1分别作用于野生型RBE细胞0、6、12、24和48h,通过western blotting检测不同时间点N-cadherin和E-cadherin蛋白的表达。并于TGF-β1处理24h后在显微镜下观察野生型RBE细胞的形态。将TGF-β1分别作用于空载体对照组和干扰Smad4组RBE细胞24h,在显微镜下观察两组细胞的形态,通过western blotting检测两组细胞N-cadherin和E-cadherin蛋白的表达。对野生型HuCCT1细胞划痕,经TGF-β1和／或TGF-β受体I抑制剂SB431542处理24h后在显微镜下拍照。将TGF-β1和／或SB431542分别处理野生型HuCCT148h和72h后,通过western blotting检测N-cadherin、MMP-9和Vimentin的表达。对空载体对照组和干扰Smad4组HuCCT1细胞划痕,经TGF-β1和／或SB431542处理24h后在显微镜下拍照。将空载体对照组和干扰Smad4组HuCCT1细胞加入transwell小室内,用TGF-β1和／或SB431542处理细胞24h,经过固定和结晶紫染色后在显微镜下计数并拍照。将空载体对照组和干扰Smad4组HuCCT1细胞加入事先包被Matrigel的transwell小室内,用TGF-β1和/或SB431542处理细胞48h,经过固定和结晶紫染色后在显微镜下计数并拍照。用TGF-β1/或SB431542处理空载体对照组和干扰Smad4组HuCCT1细胞48h和72h,通过western blotting检测N-cadherin、MMP-9和Vimentin的表达。将野生型、空载体对照组和shSmad4组HuCCT1细胞分别注射入裸鼠腹腔,于8周后观察肿瘤细胞播散至膈肌并形成癌灶的情况。结果：TGF-β1诱导RBE细胞的形态由上皮样向间质样转变,促进N-cadherin表达增加和E-cadherin表达降低。在无TGF-β1处理时,Smad4干扰组细胞比空载体组细胞的形态更趋于上皮样,E-cadherin表达水平也高于空载体组。在TGF-β1处理24h后,Smad4干扰组细胞形态的间质样变化程度、N-cadherin表达量明显低于空载体组;空载体组细胞E-cadherin的表达缺失,而Smad4干扰组细胞仍表达E-cadherin。在划痕实验中,TGF-β1促进野生型HuCCT1细胞迁移；在有／或无TGF-β1刺激下,shSmad4HuCCT1细胞的迁移能力均弱于空载体对照组细胞。在transwell实验中,TGF-β1诱导shSmad4HuCCT1细胞直接穿出transwell小室底膜的细胞数明显少于空载体对照组细胞；无论有无TGF-β1作用,shSmad4HuCCTl细胞通过Matrigel穿出transwell小室底膜的细胞数均明显少于空载体对照组细胞。TGF-β1诱导野生型HuCCT1表达N-cadherin、MMP-9蛋白并增强Vimentin蛋白的表达；无论有无TGF-β1作用,shSmad4HuCCT1细胞N-cadherin、MMP-9和Vimentin的表达水平均明显低于空载体对照组细胞。与野生型和空载体对照组细胞相比,shSmad4HuCCTl细胞从裸鼠腹腔播散至膈肌并形成癌灶的能力明显减弱。结论：Smad4介导RBE细胞发生上皮-间质转化；Smad4可能通过促进N-cadherin、MMP-9和Vimentin表达上调以介导HuCCT1细胞发生迁移和侵袭。更多还原

【Abstract】 Objective:To establish stable Smad4knockdown cholangiocellular carcinoma cell lines. Methods:We choosed human cholangiocellular cell lines HuCCTl and RBE as objects, detected the expression of TGBR I, Smad2, Smad3, Smad4and Smad7in these cells, and the phosphorylation of Smad2and Smad3after exogenous TGF-β by wstern blotting. To generate recombinant retroviruses, a pRetroSuper-puro Smad4(Addgene, United States) was transfected into PT67cells. A pRetroSuper-puro vector was transfected as control. Retroviral supernatants were collected for infection of HuCCTl and RBE cells, which were then selected with puromycin (0.2μg/ml and1μg/ml respectively) for2weeks. The expression of Smad4and Smad2/3/4complex were evaluated by western blotting and co-immunoprecipitation. Results:Both of wild type HuCCTl and RBE cells expressed TGBR Ⅰ, Smad2, Smad3, Smad4and Smad7, and exogenous TGF-β induced the phosphorylation of Smad2and Smad3of these cells in a time-dependent manner. Both of shSmad4HuCCTl and RBE cells habored a signifficantly decreased expression level of Smad4compared to wild type and vector control cells. In contrast to vector control cells, exogenous TGF-P could not cause observable Smad2/3/4complexes in shSmad4cells. Conclusion:Stable Smad4knockdown cholangiocellular carcinoma cell lines were successfully established. Objective:To investigate the role of Smad4in the proliferation of cholangiocellular carcinoma cells. Methods:HuCCTl cells were treated with TGF-β1and/or TGBR Ⅰ inhibitor SB431542for0,1,2,3,4,5and6d respectively, and the proliferation of these cells was detected by using the Cell Counting Kit-8. After treatment with TGF-β1and/or SB431542for0,6,12,24,36,48,60and72h respectively, the proliferation of RBE cells was evaluated by using the Cell Counting Kit-8. Cholangiocellular carcinoma cells in wild type, vector control and shSmad4groups were treated with or without TGF-β1for5d (HuCCT1) or48h (RBE), followed by analysis of their proliferation by using the Cell Counting Kit-8. The expressions of cyclin A2and p21in HuCCTl cells and phospho-Rb and p21in RBE cells from different groups were determined by western blotting. HuCCTl cells form wild type, vector control and shSmad4groups were injected subcutanously into nude mice, and subcutanous trasplanted tumors were monitored. Results:TGF-β1inhibited the proliferation of both HuCCTl and RBE cells. In contrast to wild type and vector control cells, shSmad4RBE cells were insensitive to cytostatic effect of TGF-β1. TGF-β1exerted only minimal inhibitory effect on the proliferation of shSmad4HuCCT1cells compared with wild type and vector control cells, and shSmad4HuCCT1cells grew faster than wild type and vector control cells in the absence of TGF-β1. Treated with or without TGF-β1, shSmad4HuCCT1cells expressed higher level of cyclin A2than wild type and vector control cells. The expression of p21induced by TGF-β1in shSmad4HuCCTl cells was significant lower than that in vector control cells. After TGF-β1stimulation, the expression level of p21in shSmad4RBE cells was lower than that in wild type and vector control cells, but the phosphorylation level of Rb was higher than that in wild type and vector control cells. Compared with wild type and vector control HuCCTl cells, shSmad4HuCCTl cells formed greater subcutanous trasplanted tumors in nude mice. Conclusion:Smad4is involved in the inhibition of proliferation of cholangiocellular carcinoma cells, possiblely by decreasing the expression of cyclin A2and the phosphorylation of Rb and by mediating the induction of p21by TGF-β1. Objective:To investigate the role of Smad4in TGF-P-induced apoptosis of cholangiocellular carcinoma cells. Methods:Wild type RBE cells were treated with different concentration of TGF-β1for36h, and with lng/ml TGF-β1for different time periods, followed by staining with PI/Annexin-Ⅴ and analysis of apoptosis by flow cytometer. The expression of Bim and Bcl-2and the activation of caspase cascade and PARP of wild type RBE cells treated with TGF-β1for different time periods were evaluated by western blotting. After stimulated with or without TGF-β1in the presence of pan-caspase inhibitor Z-VAD-fmk for36h, the apoptosis of wild type RBE cells was tested by flow cytometer. Vector control and shSmad4RBE cells were treated with TGF-β1for36h, followed by analysis of apoptosis by flow cytometer. Wild type RBE cells were treated with TGF-β1and/or JNK inhibitor SP600125for36h, followed by analysis of the apoptosis by flow cytometer and evaluation of the expression of Bim and Bcl-2and the activation of caspase cascade and PARP by western blotting. After stimulated with TGF-β1and/or SP600125in the presence of Z-VAD-fmk for36h, the apoptosis rates of wild type RBE cells were determined by flow cytometer. Vector control and shSmad4RBE cells were treated with TGF-β1and/or SP600125for36h, followed by analysis of apoptosis by flow cytometer and evaluation of the expression of Bim and Bcl-2and the activation of caspase cascade and PARP by western blotting. After stimulated with TGF-β1and/or SP600125for6h, the phosphorylation of Smad2and Smad3of wild type RBE cells was tested by western blotting. Wild type RBE cells were co-transfected with p3TP-Lux, which encodes firefly luciferase, and pRL-TK-luc, which encodes Renilla luciferase, and then were treated with TGF-β1and/or SP600125for24h, followed by analysis of the transcriptional activities of reporter by using a dual-luciferase reporter assay system. Vector control and shSmad4RBE cells were co-transfected with p3TP-Lux, which encodes firefly luciferase, and pRL-TK-luc, which encodes Renilla luciferase, and then were treated with TGF-β1and/or SP600125for24h, followed by analysis of the transcriptional activities of reporter. Results:TGF-β1induced the apoptosis of wild type RBE cells in a dose-and time-dependent manner. Compared with vector control cells, shSmad4RBE cells were insensitive to the pro-apoptotic effect of TGF-β1. SP600125enhanced the pro-apoptotic effect of TGF-β1on wild type RBE cells. Unlike the effect on vector control cells, SP600125could not increase the apoptotic rate of shSmad4RBE cells treated with TGF-β1. Z-VAD-fmk blocked the apoptosis of wild type RBE cells treated with TGF-β1or both TGF-β1and SP600125. TGF-β1led to increased expression of Bim, decreased expression of Bcl-2and activation of caspase cascade and PARP in a time-dependent manner. SP600125enhanced the TGF-β1-induced up-regulation of Bim expression, down-regulation of Bcl-2expression and activation of caspase cascade and PARP, which was blocked by knockdown of Smad4expression. SP600125increased the TGF-β1-induced phosphorylation of Smad2and Smad3, and enhanced the TGF-β1-induced transcriptional response. The effect of SP600125on transcriptional response was reduced by knockdown of Smad4expression. Conclusion:Smad4mediated the pro-apoptotic effect of TGF-β1on RBE cells. SP600125enhanced the pro-apoptotic effect of TGF-β1on RBE cells that involves Smad4-dependent mitochondria-related activation of caspase cascade. Objective:To investigate the role of Smad4in epithelial-mesenchymal transition (EMT), migration and invasion of cholangiocellular carcinoma cells. Methods:Wild type RBE cells were treated with TGF-β1for0,6,12,24and48h, followed by analysis of the expression of N-cadherin and E-cadherin by western blotting. The morphology of wild type RBE cells was observed after stimulation of TGF-β1for24h. Vector control and shSmad4RBE cells were treated with TGF-β1for24h, followed by observation of cell morphology and evaluation of the expression of N-cadherin and E-cadherin by western blotting. A straight wound line was made in wild type HuCCTl cells, which were photographed after treatment with TGF-β1and/or TGBR I inhibitor SB431542for24h. Wild type HuCCTl cells were treated with TGF-β1and/or SB431542for48h and72h, followed by analysis of the expression of MMP-9and Vimentin by western blotting. After wounding, vector control and shSmad4HuCCT1cells were treated with TGF-β1and/or SB431542for24h and then photographed. Vector control and shSmad4HuCCTl cells were seeded into the upper chamber of transwells and were treated with TGF-β1and/or SB431542for24h, after fixing and staining with crystal violet, the cells which passed through the membrane were counted and photographed. Vector control and shSmad4HuCCTl cells were seeded into the upper chamber of transwells coated with matrigel and were treated with TGF-β1and/or SB431542for48h, after fixing and staining with crystal violet, the cells which passed through the membrane were counted and photographed. Vector control and shSmad4HuCCT1cells were treated with TGF-β1and/or SB431542for48h and72h, followed by analysis of the expression of N-cadherin, MMP-9and Vimentin by western blotting. Wild type, vector control and shSmad4HuCCT1cells were injected into the abdominal cavity of nude mice respectively, and the capacity of different group cells to disseminate to the diaphragm and form metastatic colonization was evaluated8weeks later. Results:TGF-β1induced the switch from epithelioid morphology to mesenchymal morphology in wild type RBE cells, and led to increased expression of N-cadherin and decreased expression of E-cadherin. In the absence of TGF-β1, shSmad4RBE cells showed more like epithelioids and higher expression level of E-cadherin than vector control cells. When treated with TGF-β1, shSmad4RBE cells were less prone to mesenchymal morphology accompanied with a lower expression level of N-cadherin, compared with vector control cells. Whereas vector control cells lost the expression of E-cadherin, Smad4knockdown cells still showed a considerable expression level of E-cadherin after TGF-β1treatment. In the wound healing assay, TGF-β1promoted the migration of wild type HuCCTl cells, and shSmad4HuCCTl cells showed lower capacity of migration than vector control cells. In cell migration assay by transwell, after treatment with TGF-β1, the number of shSmad4HuCCTl cells that passed through the membrane was smaller than that of vector control cells. In cell invasion assay by transwell, after treatment with or without TGF-β1, the number of shSmad4HuCCTl cells that passed through the membrane was smaller than that of vector control cells. TGF-β1induced the expression of MMP-9and increased the expression of Vimentin of wild type HuCCTl cells. The expression level of N-cadherin, MMP-9and Vimentin of shSmad4HuCCTl cells was lower than that of vector control cells in the presence or absence of TGF-β1. Compared with wild type and vector control cells, shSmad4HuCCTl cells showed a lower capacity to disseminate to the diaphragm and form metastatic colonization. Conclusion:Smad4mediates the epithelial-mesenchymal transition of RBE cells. Smad4mediates the migration and invasion of HuCCT1cells that involves up-regulation of N-cadherin, MMP-9and Vimentin expression.更多还原