人工胆管构建与应用的实验研究

发布时间：2018-05-27 11:04

本文选题：碱性成纤维细胞生长因子 + 胶原　；参考：《南京大学》2013年博士论文

【摘要】：胆管损伤后胆管的再生能力有限,修复术后往往伴随严重的胆道并发症。随着组织工程学的发展,以可降解材料为基础的半合成人工胆管的出现,为解决这一难题带来了新的希望。本研究运用半合成人工胆管补片和管型对胆管侧壁缺损和节段性缺损进行修复重建。通过观察比较空白胶原支架补片和管型及结合CBD-bFGF的胶原支架补片和管型对胆管上皮、腺体、微血管、平滑肌再生和瘢痕形成的影响,评价人工胆管材料对胆管缺损的修复效果和术后并发症的预防作用。第一部分：人工胆管补片修复猪胆管侧壁缺损的实验研究目的：探讨结合CBD-bFGF的胶原支架补片对猪胆管侧壁缺损的治疗作用。方法：体外实验：扫描电镜法检测材料孔径,液体置换法检测材料孔隙率,胆汁浸泡实验检测材料在胆汁中的体外降解速度。体内实验：中华实验用猪38只,分为三组：胶原支架/PBS组12只,胶原支架/CBD-bFGF组14只,正常对照组12只。胶原支架/PBS组和胶原支架/CBD-bFGF组制备胆管侧壁缺损后分别予空白胶原支架补片和结合CBD-bFGF的胶原支架补片进行修复,正常对照组不予任何处理,随前两组一同饲养,按期处死。术后4周、8周和12周,对实验动物进行肝功能和血常规检查。术后12周,对实验动物进行经胆囊胆管造影检查。术后4周、8周和12周处死的实验动物,先进行大体观察,再获取胆管进行爆破压测定。胆管修复区域经中性甲醛溶液固定后,石蜡切片行HE染色、Masson染色及免疫组化CK19、vWF、Desmin和a-SMA染色检查,对比评价修复效果。胶原支架/CBD-bFGF组的2只实验动物饲养至24周进行肝功能和血常规检查,并进行经胆囊胆道造影检查后,处死,再进行大体观察,最后获取胆管进行HE染色评价远期修复效果。结果：体外实验测得材料孔径为186.65±45.37μm,孔隙率为84.31士6.5%,材料在胆汁中3w左右降解。体内实验中胶原支架/PBS组和胶原支架/CBD-bFGF组的实验动物均健康存活至实验完成。虽然这两组实验动物的肝功能和血常规术后均在正常范围内,但是在术后4周、8周和12周,胶原支架/CBD-bFGF组的GGT和TBA指标均显著低于胶原支架/PBS组。术后8周和12周,胶原支架/CBD-bFGF组和胶原支架/PBS组的实验动物的新生胆管均能承受6KPa的爆破压。术后12周,这两组实验动物经胆囊胆道造影均未见狭窄和结石。Masson染色提示胶原支架/CBD-bFGF组的胶原结构比胶原支架/PBS组更规则,更接近于正常,并且胶原沉积量随时间呈下降趋势。免疫组化染色提示,胶原支架/CBD-bFGF组胆管上皮覆盖、腺体再生、微血管再生和平滑肌再生速度均显著快于胶原支架/PBS组,肌成纤维母细胞含量显著低于胶原支架/PBS组。术后4周,胶原支架/CBD-bFGF组的黏膜下腺体计数显著高于胶原支架/PBS组。相关性分析表明,胶原支架/PBS组、胶原支架/CBD-bFGF组和正常对照组在术后4周、8周和12周,胆管黏膜下腺体计数和MVD值之间均呈正相关。术后24w长期观察结果发现,胶原支架/CBD-bFGF组无狭窄及结石,胆管结构与正常胆管无差别。结论：1.采用胶原支架补片能完成长度为2cm,宽度为1/3-2/3胆管周径的胆管侧壁缺损的重建。2.结合CBD-bFGF的胶原支架补片能够显著地促进猪胆管侧壁缺损区域胆管上皮、腺体、微血管和平滑肌的再生,并降低胆管壁纤维性增厚引起胆管瘢痕性狭窄的风险。3.胆管黏膜下腺体计数和MVD值之间呈正相关,CBD-bFGF可能通过促进血管新生的方式促进胆管腺体的增生。第二部分：人工胆管管型修复猪胆管节段性缺损的实验研究目的：比较两种降解速度不同的胶原支架管型对猪胆管节段性缺损修复的影响；探讨结合CBD-bFGF的胶原支架管型对猪胆管节段性缺损的治疗作用。方法：体外实验：检测比较两种胶原支架的密度、孔径和孔隙率,以及在胆汁中的体外降解速度。其中胶原支架A为第一部分膜性修复研究采用的胶原支架,胶原支架B为在胶原支架A基础上改良的胶原支架。体内实验：中华实验用猪共35只,其中8只分为2组：胶原支架A组4只,胶原支架B组4只。胶原支架A组为采用胶原支架A构建的胶原支架管型替代胆管节段性缺损,胶原支架B组为采用胶原支架B构建的胶原支架管型替代胆管节段性缺损。观察4周内动物的一般情况、肝功能和血常规、HE染色,比较两种胶原支架管型对猪胆管节段性缺损修复的影响,并筛选出可供进一步研究的支架材料。其余27只分为3组：胶原支架/PBS组9只,胶原支架/CBD-bFGF组9只,正常对照组9只。胶原支架/PBS组和胶原支架/CBD-bFGF组分别采用筛选出来的空白胶原支架管型和结合CBD-bFGF的胶原支架管型替代胆管节段性缺损,正常对照组不予任何处理,随前两组一同饲养,按期处死。术后4周、8周和12周统计比较术后并发症的发生情况,并对实验动物进行肝功能和血常规检查。术后12周对实验动物进行经胆囊胆道造影检查。所有实验动物处死后行大体观察,获取胆管予中性甲醛固定后,石蜡切片行HE染色、Masson染色及免疫组化CK19、Ki67、vWF、 Desmin和a-SMA染色,对比评价修复效果。结果：通过体外实验测得胶原支架B材料的密度显著高于胶原支架A,但两种材料的孔径和孔隙率无差异。胆汁浸泡实验表明胶原支架B较胶原支架A在胆汁中的体外降解速度明显减缓。动物实验结果提示术后4w内,胶原支架A组的实验动物因胆漏、感染和营养不良等原因全部死亡,而胶原支架B组全部存活至观察的时间点,因此胶原支架B更适合用于进一步研究。结合CBD-bFGF的胶原支架B管型动物实验结果：术后并发症统计结果表明,胶原支架/CBD-bFGF组胆道梗阻的发生率显著低于胶原支架/PBS组,但两组死亡率、胆道感染发生率相比差异无统计学意义。肝功能和血常规提示：术后4周和8周,胶原支架/CBD-bFGF组部分实验动物出现了修复区域材料发生塌陷,继发了胆管狭窄和胆管感染导致肝功能受损,营养不良等情况；但是存活至术后12周的实验动物中,胶原支架/CBD-bFGF组的感染情况和肝功能受损情况较胶原支架/PBS组明显减轻,营养状况较胶原支架/PBS组明显改善。术后12周经胆囊胆道造影结果表明,胶原支架/CBD-bFGF组未出现明显胆道狭窄；胶原支架/PBS组则提示肝内外胆管显著扩张,胆管远端狭窄。HE染色提示,胶原支架/CBD-bFGF组瘢痕大小和胆管厚度均显著低于胶原支架/PBS组。Masson染色提示,胶原支架/CBD-bFGF组的胶原结构比胶原支架/PBS组更规则,更接近于正常；而胶原支架/PBS组则见大量的炎性细胞浸润。免疫组化染色结果提示,胶原支架/CBD-bFGF组胆管上皮覆盖、腺体再生、微血管再生和平滑肌再生速度均显著快于胶原支架/PBS组,肌成纤维母细胞含量显著低于胶原支架/PBS组。结论：1.采用改良过的空白胶原支架管型修复长度为2cm的胆管节段性缺损,仅能完成瘢痕性修复,无法预防胆管狭窄的发生。2.与改良过的空白胶原支架管型相比,结合CBD-bFGF的胶原支架管型能够显著地促进猪胆管节段性缺损胆管结构的再生,并降低了肌成纤维母细胞的含量,但存在材料降解速度和胆管再生速度不匹配,材料塌陷的风险。有进一步改进的空间。
[Abstract]:The regeneration of bile ducts after bile duct injury is limited and often accompanied by severe biliary complications after repair. With the development of tissue engineering, the emergence of semi synthetic artificial bile duct based on degradable materials has brought new hope to solve this problem. To evaluate the effect of artificial bile duct materials on the repair of bile duct defects and the prevention of postoperative complications by observing and comparing the effects of the blank collagen scaffold patch, tube type and CBD-bFGF collagen scaffold patch and tube type on the bile duct epithelium, gland, microvascular, smooth muscle regeneration and scar formation. Part 1: an experimental study of artificial bile duct patch to repair the side wall defect of pig bile duct: To explore the therapeutic effect of CBD-bFGF collagen scaffold patch on the side wall defect of porcine bile duct. Method: in vitro experiment: the pore size of material was detected by scanning electron microscope, the pore rate was detected by liquid replacement method, and the bile immersion test was used to detect the material in bile. In vitro experiment: 38 Chinese experimental pigs were divided into three groups: collagen scaffold /PBS group 12, collagen scaffold /CBD-bFGF group 14 and normal control group 12. Group /PBS of collagen scaffold and collagen scaffold /CBD-bFGF group were prepared with blank collagen support patch and collagen scaffold combined with CBD-bFGF, respectively. In the normal control group, the normal control group was not treated with any treatment. The liver function and blood routine examination were carried out for the experimental animals at 4 weeks, 8 and 12 weeks after the operation. The experimental animals were examined by cholangio cholangiography for 12 weeks after operation. The experimental animals were killed at 4, 8 and 12 weeks after the operation. The bile duct was observed and the bile duct was obtained at 4 weeks, 8 and 12 weeks after operation. After the neutral Formaldehyde Solution fixation, the paraffin section was stained with HE, Masson staining and immunohistochemical CK19, vWF, Desmin and a-SMA staining to evaluate the repair effect. The 2 experimental animals of the group /CBD-bFGF in the collagen scaffold were fed to the liver function and blood routine examination for 24 weeks, and the bile duct through the gallbladder was carried out. The results were obtained by HE staining of the bile duct. Results: the pore size of the material was 186.65 + 45.37 mu m, the porosity was 84.31, 6.5%, and the material was degraded in the bile of 3W in vitro. In the experiment, the experimental animals of the collagen support group /PBS group and the collagen scaffold /CBD-bFGF group were all in the experiment in vivo. Although the liver function and blood routine of these two groups were in normal range, the GGT and TBA indexes of the collagen scaffold /CBD-bFGF group were significantly lower than those of the collagen scaffold /PBS group at 4 weeks, 8 and 12 weeks after the operation. The experimental animals of the collagen supported /CBD-bFGF group and the collagen scaffold /PBS group were in the 8 and 12 weeks postoperatively. All the new bile ducts were able to withstand the pressure of 6KPa. 12 weeks after the operation, the two groups of experimental animals showed no stricture and.Masson staining of gallstones. The collagen structure of the collagen scaffold /CBD-bFGF group was more regular and closer to the normal, and the amount of collagen deposition decreased with time. In the collagen scaffold /CBD-bFGF group, the bile duct epithelium was covered, the gland regeneration, the microvascular regeneration and the smooth muscle regeneration were significantly faster than the collagen scaffold /PBS group. The muscle fibroblast content was significantly lower than that of the collagen scaffold /PBS group. The submucosal gland count of the collagen scaffold /CBD-bFGF group was significantly higher than that of the collagen scaffold /PBS group 4 weeks after the operation. The results showed that the collagen scaffold /PBS group, the collagen scaffold /CBD-bFGF group and the normal control group had a positive correlation between the count of the submucosal glands of the bile duct and the MVD value at 4 weeks, 8 and 12 weeks after the operation. After the long-term observation of 24W, there was no stenosis and stone in the /CBD-bFGF group of collagen scaffold and the structure of the bile duct was not different from that of the normal bile duct. Conclusion: 1. the collagen scaffold was used in the collagen scaffold. The reconstruction of the defect of the lateral wall of the bile duct with a width of 1/3-2/3 of the bile duct with a length of 2cm and a collagen scaffold patch with.2. combined with CBD-bFGF can significantly promote the regeneration of the bile duct epithelium, glands, microvessels and smooth muscle, and reduce the risk of scar stenosis caused by the fibrous thickening of the bile duct, and the risk of.3. gallbladder. There is a positive correlation between the number of submucosal glands and the value of MVD, and CBD-bFGF may promote the hyperplasia of bile duct glands by promoting angiogenesis. Second part: Experimental Study on repairing segmental defect of pig bile duct by artificial bile duct type: comparison of two kinds of collagen scaffold with different degradation rate for repair of segmental defect of pig bile duct The effects of the collagen scaffold combined with CBD-bFGF on the treatment of porcine bile duct segmental defects. Methods: in vitro experiments: the density, pore size and porosity of two collagen scaffolds, and the rate of degradation in bile in vitro were compared. The collagen scaffold A was the collagen scaffold and collagen used in the first membrane repair. The scaffold B was a modified collagen scaffold based on the collagen scaffold A. In vivo, 35 pigs were used in the experiment. 8 of them were divided into 2 groups: collagen scaffold A group 4, collagen scaffold B group 4. Collagen scaffold A scaffold was used as collagen scaffold to replace bile duct segmental defect, and collagen scaffold B group was used collagen scaffold B structure in collagen scaffold B group. To observe the general condition of the animals in 4 weeks, liver function and blood routine, HE staining, and compare the effects of two collagen scaffolds on the repair of segmental defect of the pig bile duct, and screen out the scaffold materials for further study. The other 27 were divided into 3 groups: collagen scaffold, group /PBS, 9 and collagen branches 9 rats in group /CBD-bFGF and 9 in normal control group. The collagen scaffold /PBS group and collagen scaffold /CBD-bFGF group were selected by the selected blank collagen scaffold and collagen scaffold to replace the bile duct segmental defect respectively. The normal control group was not treated with any treatment. The two groups were kept together with the first two groups. 4 weeks, 8 weeks and 12 weeks after the operation. The occurrence of postoperative complications was compared and the liver function and blood routine examination were performed on the experimental animals. The experimental animals were examined by gallbladder cholangiography at 12 weeks after the operation. All the experimental animals were subjected to general observation after death. After the bile duct was fixed with neutral formaldehyde, the paraffin sections were stained with HE, Masson staining and immunohistochemical CK19, Ki67, vWF. Desmin and a-SMA staining were used to evaluate the effect of repair. Results: the density of the collagen scaffold B was significantly higher than that of the collagen scaffold A in vitro, but the pore size and porosity of the two materials were not different. The bile immersion test showed that the degradation rate of collagen scaffold B in vitro was significantly slower than that of collagen scaffold A in bile. It was suggested that in 4W after operation, all the experimental animals in group A of collagen scaffold died of bile leakage, infection and malnutrition, and all the collagen scaffold B group survived to the time point of observation. Therefore, the collagen scaffold B was more suitable for further study. The results of B tube animal experiment with collagen scaffold of CBD-bFGF: statistical results of postoperative complications showed glue. The incidence of biliary obstruction in the original /CBD-bFGF group was significantly lower than that of the collagen stent /PBS group, but there was no significant difference in the mortality of the two groups and the incidence of biliary tract infection. The liver function and the blood routine showed that 4 weeks and 8 weeks after the operation, some experimental animals in the collagen scaffold /CBD-bFGF group appeared to collapse in the repair area and secondary bile duct stricture. And the bile duct infection resulted in impaired liver function and malnutrition, but in the experimental animals survived to 12 weeks after the operation, the infection of the collagen scaffold /CBD-bFGF group and the damage of the liver function were obviously less than that of the collagen scaffold /PBS group, and the nutritional status was obviously better than that of the collagen scaffold /PBS group. The results of gallbladder cholangiography at 12 weeks after the operation showed that the gel was adhesive. There was no obvious biliary stricture in the /CBD-bFGF group of the original stents, and the collagen stent /PBS group showed significant expansion of the intrahepatic and extrahepatic bile ducts. The.HE staining of the distal bile duct stricture showed that the size of the scar and the thickness of the bile duct in the collagen scaffold /CBD-bFGF group were significantly lower than that of the collagen scaffold /PBS group.Masson staining. The collagen structure of the group /CBD-bFGF group of the collagen scaffold was more than that of the collagen branch. The /PBS group was more regular and closer to normal, while a large number of inflammatory cells were found in the collagen scaffold /PBS group. The immunohistochemical staining results showed that the collagen scaffold /CBD-bFGF group was covered with bile duct epithelium, the gland regeneration was regenerated, and the rate of microvascular regeneration and smooth muscle regeneration was significantly faster than that of the collagen support group /PBS group, and the content of myofibroblast was significantly lower than that of the collagen scaffold. Group /PBS of collagen scaffold. Conclusion: 1. a modified blank collagen stent is used to repair the segmental defect of the bile duct with a length of 2cm, which can only complete the scar repair. It is impossible to prevent the occurrence of biliary stricture by.2. and the modified blank collagen stent type. The collagen scaffold combined with CBD-bFGF can significantly promote the pig bile duct segment. The regeneration of the bile duct structure of sexual defect and the reduction of myofibroblast content, but the risk of material degradation rate and the rate of bile duct regeneration, and the risk of material collapse. There is further improvement.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.1;R657.4

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