良性胆管瘢痕狭窄的临床预防与差异蛋白组学研究
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摘要
目的
1.评价磁共振胰胆管成像(MRCP)在预防腹腔镜胆囊切除(LC)手术并发症中的价值。
2.探讨三种腹腔镜胆囊切除手术入路是否增加胆管损伤的发生率。
3.探讨人良性胆管瘢痕成纤维细胞的培养和鉴定方法.
4.研究人良性胆管瘢痕成纤维细胞与人胚肺成纤维细胞的蛋白表达差异并鉴定差异蛋白质。
5.分析TAGLN及FKBP1A在良性胆管瘢痕成纤维细胞中的功能。
方法
1.回顾性分析2006年1月至2010年6月我院收治的1079例拟行LC治疗的患者临床资料。根据不同时期行MRCP与否,分为非MRCP组(-=523)和MRCP组(n=556),对2组患者胆管损伤及胆总管结石残余情况进行比较。
2.回顾性分析我院2009年1月至12月经腹腔镜胆囊切除术治疗150例胆囊良性疾病的临床资料。
3.取临床人良性胆管瘢痕组织,组织块培养法培养；绘制生长曲线、形态学观察、免疫荧光及RT-PCR检测。
4.体外培养人良性胆管成纤维细胞和人胚肺成纤维细胞株,提取两种细胞的总蛋白,进行蛋白定量,通过双向凝胶电泳,图像分析,获得差异表达蛋白点,用质谱仪绘制肽质量指纹谱,通过计算机软件及网络信息鉴定差异表达的蛋白。通过western blot及免疫细胞荧光验证人良性胆管瘢痕成纤维细胞中FKBPIA、NME1以及TAGLN、Moesin的表达情况。
5.构建pcDNA3.1-TAGLN真核表达质粒、筛选FKBP1A-siRNA干扰片段,转染人良性胆管瘢痕成纤维细胞,通过MTT法及流式细胞仪观察细胞的增殖及凋亡情况,通过实时定量PCR及免疫蛋白印迹检测该细胞中FKBPIA、TAGLN、 smadl、smad5在基因水平及蛋白水平的表达变化。
结果
1.非MRCP组患者术中、术后发现胆管损伤5例和胆总管结石残余27例,MRCP组患者术中、术后均未发现胆管损伤和胆总管结石残余,2组患者在胆管损伤和胆总管结石残余方面的差异具有统计学意义(P<0.05)。MRCP组患者术前MRCP发现双胆囊1例,Mirizzi综合征8例,胆囊管变异34例,副肝管28例,胆总管结石27例,与术中结果相比,其诊断的准确性分别为87.5%、94.1%、89.3%和88.9%。
2.150例腹腔镜胆囊切除术中三孔法46例,二孔法54例,经脐入路50例,三组病例的手术成功率、手术时间、术中出血、术后肠功能恢复情况无差异,术后住院天数以经脐入路组(1.3±0.4d)为短(P<0.05)、术后疼痛以经脐入路组为轻(P<0.05)。三组病例出院后随访均无腹痛、黄疸、胆漏、出血、腹壁切口疝等并发症。
3.原代培养5天后可见梭形细胞移出、贴壁,14天细胞连接成片,传代后生长良好,2-3天可传代,细胞生长良好；电镜可见微丝,免疫荧光和RT-PCR检测提示该细胞有波形蛋白的表达和波形蛋白mRNA。
4.发现差异蛋白28种,其中人良性胆管瘢痕成纤维细胞表达上调9种,下调19种。其功能与细胞代谢、凋亡等相关；western blot及免疫细胞荧光提不FKBP1A、NME1以及TAGLN、Moesin的表达情况与差异蛋白组学分析结果一致。
5.转染pcDNA3.1-TAGLN和FKBP1A-siRNA后人良性胆管瘢痕成纤维细胞的增殖均受到抑制并出现凋亡,该细胞中smadl.smad5在基因水平(P<0.05)及蛋白水平的表达均有增强,而且该细胞在经过FKBP1A-siRNA作用后TAGLN在基因水平(P<0.05)及蛋白水平的表达均有增强。
结论
1.LC患者术前行MRCP有利于预防胆管损伤及胆总管结石残余的发生。
2.在选择病例中,三种手术入路均能安全完成腹腔镜胆囊切除术,并不增加胆管损伤的发生率。
3.原代培养人良性胆管瘢痕成纤维细胞成功。
4.人良性胆管瘢痕成纤维细胞与人胚肺成纤维细胞KMB17的蛋白表达存在差异,其中FKBP1A、NME1以及TAGLN、Moesin可能成为临床预测胆管瘢痕狭窄形成的分子标记和治疗胆管瘢痕狭窄的分子靶点。
5. TAGLN及FKBPIA在良性胆管瘢痕成纤维细胞中发挥重要作用,FKBP1A通过TGF-β/smads信号通路调控TAGLN的表达。
Objective1. To evaluate value of magnetic resonance cholangiopancreatography on
prevention of complications in laparoscopic cholecystectomy.2. To disscuss the application of three approach of laparoscopic cholecystectomy.3. To explore the method of cultivation and identification for human benign biliary
duct cicatrix fibroblasts.4. To investigate the people benign bile duct scar stricture fibroblasts and embryonic
lung fibroblasts protein expression differences and to identify the differential
protein.5. To analyze the function of TAGLN and FKBP1A in the fibroblasts of benign bile
duct scar.
Method
1. The clinical data of1079patients who would be performed by LC from January2006to June2010in our hospital were retrospective analysed. According to performing MRCP or not in the different period, the patients were divided into non-MRCP group and MRCP group. The data of bile duct injuries and bile duct stone residue were contrasted between two groups.
2. From Jan to Dec2009,150cases laparoscopic cholecystectomy were performed in our hospital. The clinical data and surgical outcomes were retrospectively analysed.
3. The benign biliary duct cicatrix tissue which sampled from clinic was cultured for fibroblasts by primary explant culture. The performance of fibroblasts was observed with light microscope, TEM and SEM. The cells'growth was observed with making cell growth curve. The cultrued cells were identified by vimentin immunofluorescence. The cells'vimentin mRNA was dectedted by RT-PCR.
4. Human benign bile duct stricture fibroblast cell line and human embryonic lung fibroblasts were cultured in vitro and total protein of two fibroblasts was extracted and quantified. Then the protein was anlysised by two-dimensional gel electrophoresis, image analysis, obtained differentially expressed proteins. Peptide mass spectrometer is used to draw the quality fingerprint spectrum, through the computer software and network information to identify differentially expressed proteins. The expression of FKBP1A, NME1TAGLN and Moesin were analysis by western blot and immune cell fluorescence in two fibroblasts.
5. To build pcDNA3.1TAGLN eukaryotic expression plasmid, and screen FKBP1A siRNA interference fragment, then they were transfected to two fibroblasts. The cell proliferation and apoptosis were determined by MTT method and flow cytometry instrument. The expressions of FKBP1A, TAGLN, smadl, smad5were detected by real-time quantitative PCR and immune protein imprinting in two fibroblasts.
Results
1. The intraoperative and postoperative bile duct injury were found in5patients and the residual common bile duct stones were found in27patients in non-MRCP group. The intraoperative and postoperative bile duct injury and the residual common bile duct stones were not found in MRCP group. The data of bile duct injuries and bile duct stone residue were statistic difference between two groups (P<0.05). There were found double gallbladder in1patient by MRCP, Mirizzi syndrome in8patients, variant cystic duct in34patients, accessory hepatic duct in28patients, and complicating common bile duct stones in27patients in MRCP group. The diagnostic accuracy was87.5%,94.1%,89.3%and88.9%, respectively.
2. There were46patients which were performed laparoscopic cholecystectomy by tri-apporach,54patients by bi-apporach, and50patients by transumbilicar approach. All operations were successful. There were significant indifferences about operating time, blood loss, and recovery time of bowel function in the three groups. There were significant differences about postoperation hospital and pain in the three groups (P<0.05). They have not complications such as abdominal pain, jaundice, biliary leaks, bleeding and incisional hernia of abdominal wall when they are followed-up post-discharge.
3. The cells could climb and adherence from the tissue after5days, and connect total after14days in primary culture. The isolated fibroblast could grow, proliferate and passage in vitro. Microfilaments were observed in the cell plasm under TEM which was identified as fibroblast. The cell was positive in vimentin by immunofluorescence. The mRNA of vimentin was positive by RT-PCR.
4.28differences proteins were found by differential proteomics analysis.9species up-regulated and19species in human benign bile duct scar stricture fibroblasts. Its function is associated with cell metabolism, apoptosis, etc. The differential proteomics, FKBP1A, NME1TAGLN and Moesin were analysised by Western blot and immune cell fluorescence.
5. The proliferation of benign bile duct scar fibroblasts is restrained and apoptosis occurred after transfecting pcDNA3.1-TAGLN and FKBP1A-siRNA. Smad1, smad5of the fibroblast at the genetic level (P<0.05) and the expression of protein levels were increased. TAGLN at the genetic level (P<0.05) and the expression of protein levels were increased in the cells after transfecting FKBP1A-siRNA.
Conclusion
1. It could prevent the bile duct injury and residual common bile duct stones that the patients with LC were perfomed preoperative MRCP.
2. Laparoscopic cholecystectomy can safe perform by the three approachs, however, different patients should have been performed by different approach according to practical situation. The citerion by which to choose different approach should be safe operation.
3. The fibroblast of human benign biliary duct cicatrix was conveniently succeeded.
4. The protein expression human benign bile duct scar stricture fibroblasts and embryonic lung fibroblasts KMB17is different. FKBP1A, NME1and TAGLN, Moesin maybe become clinical prediction molecular markers forbile duct scar stricture formation and molecular targets for the treatment of bile duct scar stricture.
5. TAGLN and FKBP1A in benign bile duct scar fibroblasts play an important role. FKBP1A regulated the expression of TAGLN by the TGF-β/smads signal pathway.
1.评价磁共振胰胆管成像(MRCP)在预防腹腔镜胆囊切除(LC)手术并发症中的价值。
2.探讨三种腹腔镜胆囊切除手术入路是否增加胆管损伤的发生率。
3.探讨人良性胆管瘢痕成纤维细胞的培养和鉴定方法.
4.研究人良性胆管瘢痕成纤维细胞与人胚肺成纤维细胞的蛋白表达差异并鉴定差异蛋白质。
5.分析TAGLN及FKBP1A在良性胆管瘢痕成纤维细胞中的功能。
方法
1.回顾性分析2006年1月至2010年6月我院收治的1079例拟行LC治疗的患者临床资料。根据不同时期行MRCP与否,分为非MRCP组(-=523)和MRCP组(n=556),对2组患者胆管损伤及胆总管结石残余情况进行比较。
2.回顾性分析我院2009年1月至12月经腹腔镜胆囊切除术治疗150例胆囊良性疾病的临床资料。
3.取临床人良性胆管瘢痕组织,组织块培养法培养；绘制生长曲线、形态学观察、免疫荧光及RT-PCR检测。
4.体外培养人良性胆管成纤维细胞和人胚肺成纤维细胞株,提取两种细胞的总蛋白,进行蛋白定量,通过双向凝胶电泳,图像分析,获得差异表达蛋白点,用质谱仪绘制肽质量指纹谱,通过计算机软件及网络信息鉴定差异表达的蛋白。通过western blot及免疫细胞荧光验证人良性胆管瘢痕成纤维细胞中FKBPIA、NME1以及TAGLN、Moesin的表达情况。
5.构建pcDNA3.1-TAGLN真核表达质粒、筛选FKBP1A-siRNA干扰片段,转染人良性胆管瘢痕成纤维细胞,通过MTT法及流式细胞仪观察细胞的增殖及凋亡情况,通过实时定量PCR及免疫蛋白印迹检测该细胞中FKBPIA、TAGLN、 smadl、smad5在基因水平及蛋白水平的表达变化。
结果
1.非MRCP组患者术中、术后发现胆管损伤5例和胆总管结石残余27例,MRCP组患者术中、术后均未发现胆管损伤和胆总管结石残余,2组患者在胆管损伤和胆总管结石残余方面的差异具有统计学意义(P<0.05)。MRCP组患者术前MRCP发现双胆囊1例,Mirizzi综合征8例,胆囊管变异34例,副肝管28例,胆总管结石27例,与术中结果相比,其诊断的准确性分别为87.5%、94.1%、89.3%和88.9%。
2.150例腹腔镜胆囊切除术中三孔法46例,二孔法54例,经脐入路50例,三组病例的手术成功率、手术时间、术中出血、术后肠功能恢复情况无差异,术后住院天数以经脐入路组(1.3±0.4d)为短(P<0.05)、术后疼痛以经脐入路组为轻(P<0.05)。三组病例出院后随访均无腹痛、黄疸、胆漏、出血、腹壁切口疝等并发症。
3.原代培养5天后可见梭形细胞移出、贴壁,14天细胞连接成片,传代后生长良好,2-3天可传代,细胞生长良好；电镜可见微丝,免疫荧光和RT-PCR检测提示该细胞有波形蛋白的表达和波形蛋白mRNA。
4.发现差异蛋白28种,其中人良性胆管瘢痕成纤维细胞表达上调9种,下调19种。其功能与细胞代谢、凋亡等相关；western blot及免疫细胞荧光提不FKBP1A、NME1以及TAGLN、Moesin的表达情况与差异蛋白组学分析结果一致。
5.转染pcDNA3.1-TAGLN和FKBP1A-siRNA后人良性胆管瘢痕成纤维细胞的增殖均受到抑制并出现凋亡,该细胞中smadl.smad5在基因水平(P<0.05)及蛋白水平的表达均有增强,而且该细胞在经过FKBP1A-siRNA作用后TAGLN在基因水平(P<0.05)及蛋白水平的表达均有增强。
结论
1.LC患者术前行MRCP有利于预防胆管损伤及胆总管结石残余的发生。
2.在选择病例中,三种手术入路均能安全完成腹腔镜胆囊切除术,并不增加胆管损伤的发生率。
3.原代培养人良性胆管瘢痕成纤维细胞成功。
4.人良性胆管瘢痕成纤维细胞与人胚肺成纤维细胞KMB17的蛋白表达存在差异,其中FKBP1A、NME1以及TAGLN、Moesin可能成为临床预测胆管瘢痕狭窄形成的分子标记和治疗胆管瘢痕狭窄的分子靶点。
5. TAGLN及FKBPIA在良性胆管瘢痕成纤维细胞中发挥重要作用,FKBP1A通过TGF-β/smads信号通路调控TAGLN的表达。
Objective1. To evaluate value of magnetic resonance cholangiopancreatography on
prevention of complications in laparoscopic cholecystectomy.2. To disscuss the application of three approach of laparoscopic cholecystectomy.3. To explore the method of cultivation and identification for human benign biliary
duct cicatrix fibroblasts.4. To investigate the people benign bile duct scar stricture fibroblasts and embryonic
lung fibroblasts protein expression differences and to identify the differential
protein.5. To analyze the function of TAGLN and FKBP1A in the fibroblasts of benign bile
duct scar.
Method
1. The clinical data of1079patients who would be performed by LC from January2006to June2010in our hospital were retrospective analysed. According to performing MRCP or not in the different period, the patients were divided into non-MRCP group and MRCP group. The data of bile duct injuries and bile duct stone residue were contrasted between two groups.
2. From Jan to Dec2009,150cases laparoscopic cholecystectomy were performed in our hospital. The clinical data and surgical outcomes were retrospectively analysed.
3. The benign biliary duct cicatrix tissue which sampled from clinic was cultured for fibroblasts by primary explant culture. The performance of fibroblasts was observed with light microscope, TEM and SEM. The cells'growth was observed with making cell growth curve. The cultrued cells were identified by vimentin immunofluorescence. The cells'vimentin mRNA was dectedted by RT-PCR.
4. Human benign bile duct stricture fibroblast cell line and human embryonic lung fibroblasts were cultured in vitro and total protein of two fibroblasts was extracted and quantified. Then the protein was anlysised by two-dimensional gel electrophoresis, image analysis, obtained differentially expressed proteins. Peptide mass spectrometer is used to draw the quality fingerprint spectrum, through the computer software and network information to identify differentially expressed proteins. The expression of FKBP1A, NME1TAGLN and Moesin were analysis by western blot and immune cell fluorescence in two fibroblasts.
5. To build pcDNA3.1TAGLN eukaryotic expression plasmid, and screen FKBP1A siRNA interference fragment, then they were transfected to two fibroblasts. The cell proliferation and apoptosis were determined by MTT method and flow cytometry instrument. The expressions of FKBP1A, TAGLN, smadl, smad5were detected by real-time quantitative PCR and immune protein imprinting in two fibroblasts.
Results
1. The intraoperative and postoperative bile duct injury were found in5patients and the residual common bile duct stones were found in27patients in non-MRCP group. The intraoperative and postoperative bile duct injury and the residual common bile duct stones were not found in MRCP group. The data of bile duct injuries and bile duct stone residue were statistic difference between two groups (P<0.05). There were found double gallbladder in1patient by MRCP, Mirizzi syndrome in8patients, variant cystic duct in34patients, accessory hepatic duct in28patients, and complicating common bile duct stones in27patients in MRCP group. The diagnostic accuracy was87.5%,94.1%,89.3%and88.9%, respectively.
2. There were46patients which were performed laparoscopic cholecystectomy by tri-apporach,54patients by bi-apporach, and50patients by transumbilicar approach. All operations were successful. There were significant indifferences about operating time, blood loss, and recovery time of bowel function in the three groups. There were significant differences about postoperation hospital and pain in the three groups (P<0.05). They have not complications such as abdominal pain, jaundice, biliary leaks, bleeding and incisional hernia of abdominal wall when they are followed-up post-discharge.
3. The cells could climb and adherence from the tissue after5days, and connect total after14days in primary culture. The isolated fibroblast could grow, proliferate and passage in vitro. Microfilaments were observed in the cell plasm under TEM which was identified as fibroblast. The cell was positive in vimentin by immunofluorescence. The mRNA of vimentin was positive by RT-PCR.
4.28differences proteins were found by differential proteomics analysis.9species up-regulated and19species in human benign bile duct scar stricture fibroblasts. Its function is associated with cell metabolism, apoptosis, etc. The differential proteomics, FKBP1A, NME1TAGLN and Moesin were analysised by Western blot and immune cell fluorescence.
5. The proliferation of benign bile duct scar fibroblasts is restrained and apoptosis occurred after transfecting pcDNA3.1-TAGLN and FKBP1A-siRNA. Smad1, smad5of the fibroblast at the genetic level (P<0.05) and the expression of protein levels were increased. TAGLN at the genetic level (P<0.05) and the expression of protein levels were increased in the cells after transfecting FKBP1A-siRNA.
Conclusion
1. It could prevent the bile duct injury and residual common bile duct stones that the patients with LC were perfomed preoperative MRCP.
2. Laparoscopic cholecystectomy can safe perform by the three approachs, however, different patients should have been performed by different approach according to practical situation. The citerion by which to choose different approach should be safe operation.
3. The fibroblast of human benign biliary duct cicatrix was conveniently succeeded.
4. The protein expression human benign bile duct scar stricture fibroblasts and embryonic lung fibroblasts KMB17is different. FKBP1A, NME1and TAGLN, Moesin maybe become clinical prediction molecular markers forbile duct scar stricture formation and molecular targets for the treatment of bile duct scar stricture.
5. TAGLN and FKBP1A in benign bile duct scar fibroblasts play an important role. FKBP1A regulated the expression of TAGLN by the TGF-β/smads signal pathway.
引文
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