本文選題：堿性成纖維細(xì)胞生長因子 + 膠原　； 參考：《南京大學(xué)》2013年博士論文

【摘要】：膽管損傷后膽管的再生能力有限,修復(fù)術(shù)后往往伴隨嚴(yán)重的膽道并發(fā)癥。隨著組織工程學(xué)的發(fā)展,以可降解材料為基礎(chǔ)的半合成人工膽管的出現(xiàn),為解決這一難題帶來了新的希望。本研究運(yùn)用半合成人工膽管補(bǔ)片和管型對(duì)膽管側(cè)壁缺損和節(jié)段性缺損進(jìn)行修復(fù)重建。通過觀察比較空白膠原支架補(bǔ)片和管型及結(jié)合CBD-bFGF的膠原支架補(bǔ)片和管型對(duì)膽管上皮、腺體、微血管、平滑肌再生和瘢痕形成的影響,評(píng)價(jià)人工膽管材料對(duì)膽管缺損的修復(fù)效果和術(shù)后并發(fā)癥的預(yù)防作用。第一部分：人工膽管補(bǔ)片修復(fù)豬膽管側(cè)壁缺損的實(shí)驗(yàn)研究目的：探討結(jié)合CBD-bFGF的膠原支架補(bǔ)片對(duì)豬膽管側(cè)壁缺損的治療作用。方法：體外實(shí)驗(yàn)：掃描電鏡法檢測材料孔徑,液體置換法檢測材料孔隙率,膽汁浸泡實(shí)驗(yàn)檢測材料在膽汁中的體外降解速度。體內(nèi)實(shí)驗(yàn)：中華實(shí)驗(yàn)用豬38只,分為三組：膠原支架/PBS組12只,膠原支架/CBD-bFGF組14只,正常對(duì)照組12只。膠原支架/PBS組和膠原支架/CBD-bFGF組制備膽管側(cè)壁缺損后分別予空白膠原支架補(bǔ)片和結(jié)合CBD-bFGF的膠原支架補(bǔ)片進(jìn)行修復(fù),正常對(duì)照組不予任何處理,隨前兩組一同飼養(yǎng),按期處死。術(shù)后4周、8周和12周,對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行肝功能和血常規(guī)檢查。術(shù)后12周,對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行經(jīng)膽囊膽管造影檢查。術(shù)后4周、8周和12周處死的實(shí)驗(yàn)動(dòng)物,先進(jìn)行大體觀察,再獲取膽管進(jìn)行爆破壓測定。膽管修復(fù)區(qū)域經(jīng)中性甲醛溶液固定后,石蠟切片行HE染色、Masson染色及免疫組化CK19、vWF、Desmin和a-SMA染色檢查,對(duì)比評(píng)價(jià)修復(fù)效果。膠原支架/CBD-bFGF組的2只實(shí)驗(yàn)動(dòng)物飼養(yǎng)至24周進(jìn)行肝功能和血常規(guī)檢查,并進(jìn)行經(jīng)膽囊膽道造影檢查后,處死,再進(jìn)行大體觀察,最后獲取膽管進(jìn)行HE染色評(píng)價(jià)遠(yuǎn)期修復(fù)效果。結(jié)果：體外實(shí)驗(yàn)測得材料孔徑為186.65±45.37μm,孔隙率為84.31士6.5%,材料在膽汁中3w左右降解。體內(nèi)實(shí)驗(yàn)中膠原支架/PBS組和膠原支架/CBD-bFGF組的實(shí)驗(yàn)動(dòng)物均健康存活至實(shí)驗(yàn)完成。雖然這兩組實(shí)驗(yàn)動(dòng)物的肝功能和血常規(guī)術(shù)后均在正常范圍內(nèi),但是在術(shù)后4周、8周和12周,膠原支架/CBD-bFGF組的GGT和TBA指標(biāo)均顯著低于膠原支架/PBS組。術(shù)后8周和12周,膠原支架/CBD-bFGF組和膠原支架/PBS組的實(shí)驗(yàn)動(dòng)物的新生膽管均能承受6KPa的爆破壓。術(shù)后12周,這兩組實(shí)驗(yàn)動(dòng)物經(jīng)膽囊膽道造影均未見狹窄和結(jié)石。Masson染色提示膠原支架/CBD-bFGF組的膠原結(jié)構(gòu)比膠原支架/PBS組更規(guī)則,更接近于正常,并且膠原沉積量隨時(shí)間呈下降趨勢。免疫組化染色提示,膠原支架/CBD-bFGF組膽管上皮覆蓋、腺體再生、微血管再生和平滑肌再生速度均顯著快于膠原支架/PBS組,肌成纖維母細(xì)胞含量顯著低于膠原支架/PBS組。術(shù)后4周,膠原支架/CBD-bFGF組的黏膜下腺體計(jì)數(shù)顯著高于膠原支架/PBS組。相關(guān)性分析表明,膠原支架/PBS組、膠原支架/CBD-bFGF組和正常對(duì)照組在術(shù)后4周、8周和12周,膽管黏膜下腺體計(jì)數(shù)和MVD值之間均呈正相關(guān)。術(shù)后24w長期觀察結(jié)果發(fā)現(xiàn),膠原支架/CBD-bFGF組無狹窄及結(jié)石,膽管結(jié)構(gòu)與正常膽管無差別。結(jié)論：1.采用膠原支架補(bǔ)片能完成長度為2cm,寬度為1/3-2/3膽管周徑的膽管側(cè)壁缺損的重建。2.結(jié)合CBD-bFGF的膠原支架補(bǔ)片能夠顯著地促進(jìn)豬膽管側(cè)壁缺損區(qū)域膽管上皮、腺體、微血管和平滑肌的再生,并降低膽管壁纖維性增厚引起膽管瘢痕性狹窄的風(fēng)險(xiǎn)。3.膽管黏膜下腺體計(jì)數(shù)和MVD值之間呈正相關(guān),CBD-bFGF可能通過促進(jìn)血管新生的方式促進(jìn)膽管腺體的增生。第二部分：人工膽管管型修復(fù)豬膽管節(jié)段性缺損的實(shí)驗(yàn)研究目的：比較兩種降解速度不同的膠原支架管型對(duì)豬膽管節(jié)段性缺損修復(fù)的影響；探討結(jié)合CBD-bFGF的膠原支架管型對(duì)豬膽管節(jié)段性缺損的治療作用。方法：體外實(shí)驗(yàn)：檢測比較兩種膠原支架的密度、孔徑和孔隙率,以及在膽汁中的體外降解速度。其中膠原支架A為第一部分膜性修復(fù)研究采用的膠原支架,膠原支架B為在膠原支架A基礎(chǔ)上改良的膠原支架。體內(nèi)實(shí)驗(yàn)：中華實(shí)驗(yàn)用豬共35只,其中8只分為2組：膠原支架A組4只,膠原支架B組4只。膠原支架A組為采用膠原支架A構(gòu)建的膠原支架管型替代膽管節(jié)段性缺損,膠原支架B組為采用膠原支架B構(gòu)建的膠原支架管型替代膽管節(jié)段性缺損。觀察4周內(nèi)動(dòng)物的一般情況、肝功能和血常規(guī)、HE染色,比較兩種膠原支架管型對(duì)豬膽管節(jié)段性缺損修復(fù)的影響,并篩選出可供進(jìn)一步研究的支架材料。其余27只分為3組：膠原支架/PBS組9只,膠原支架/CBD-bFGF組9只,正常對(duì)照組9只。膠原支架/PBS組和膠原支架/CBD-bFGF組分別采用篩選出來的空白膠原支架管型和結(jié)合CBD-bFGF的膠原支架管型替代膽管節(jié)段性缺損,正常對(duì)照組不予任何處理,隨前兩組一同飼養(yǎng),按期處死。術(shù)后4周、8周和12周統(tǒng)計(jì)比較術(shù)后并發(fā)癥的發(fā)生情況,并對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行肝功能和血常規(guī)檢查。術(shù)后12周對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行經(jīng)膽囊膽道造影檢查。所有實(shí)驗(yàn)動(dòng)物處死后行大體觀察,獲取膽管予中性甲醛固定后,石蠟切片行HE染色、Masson染色及免疫組化CK19、Ki67、vWF、 Desmin和a-SMA染色,對(duì)比評(píng)價(jià)修復(fù)效果。結(jié)果：通過體外實(shí)驗(yàn)測得膠原支架B材料的密度顯著高于膠原支架A,但兩種材料的孔徑和孔隙率無差異。膽汁浸泡實(shí)驗(yàn)表明膠原支架B較膠原支架A在膽汁中的體外降解速度明顯減緩。動(dòng)物實(shí)驗(yàn)結(jié)果提示術(shù)后4w內(nèi),膠原支架A組的實(shí)驗(yàn)動(dòng)物因膽漏、感染和營養(yǎng)不良等原因全部死亡,而膠原支架B組全部存活至觀察的時(shí)間點(diǎn),因此膠原支架B更適合用于進(jìn)一步研究。結(jié)合CBD-bFGF的膠原支架B管型動(dòng)物實(shí)驗(yàn)結(jié)果：術(shù)后并發(fā)癥統(tǒng)計(jì)結(jié)果表明,膠原支架/CBD-bFGF組膽道梗阻的發(fā)生率顯著低于膠原支架/PBS組,但兩組死亡率、膽道感染發(fā)生率相比差異無統(tǒng)計(jì)學(xué)意義。肝功能和血常規(guī)提示：術(shù)后4周和8周,膠原支架/CBD-bFGF組部分實(shí)驗(yàn)動(dòng)物出現(xiàn)了修復(fù)區(qū)域材料發(fā)生塌陷,繼發(fā)了膽管狹窄和膽管感染導(dǎo)致肝功能受損,營養(yǎng)不良等情況；但是存活至術(shù)后12周的實(shí)驗(yàn)動(dòng)物中,膠原支架/CBD-bFGF組的感染情況和肝功能受損情況較膠原支架/PBS組明顯減輕,營養(yǎng)狀況較膠原支架/PBS組明顯改善。術(shù)后12周經(jīng)膽囊膽道造影結(jié)果表明,膠原支架/CBD-bFGF組未出現(xiàn)明顯膽道狹窄；膠原支架/PBS組則提示肝內(nèi)外膽管顯著擴(kuò)張,膽管遠(yuǎn)端狹窄。HE染色提示,膠原支架/CBD-bFGF組瘢痕大小和膽管厚度均顯著低于膠原支架/PBS組。Masson染色提示,膠原支架/CBD-bFGF組的膠原結(jié)構(gòu)比膠原支架/PBS組更規(guī)則,更接近于正常；而膠原支架/PBS組則見大量的炎性細(xì)胞浸潤。免疫組化染色結(jié)果提示,膠原支架/CBD-bFGF組膽管上皮覆蓋、腺體再生、微血管再生和平滑肌再生速度均顯著快于膠原支架/PBS組,肌成纖維母細(xì)胞含量顯著低于膠原支架/PBS組。結(jié)論：1.采用改良過的空白膠原支架管型修復(fù)長度為2cm的膽管節(jié)段性缺損,僅能完成瘢痕性修復(fù),無法預(yù)防膽管狹窄的發(fā)生。2.與改良過的空白膠原支架管型相比,結(jié)合CBD-bFGF的膠原支架管型能夠顯著地促進(jìn)豬膽管節(jié)段性缺損膽管結(jié)構(gòu)的再生,并降低了肌成纖維母細(xì)胞的含量,但存在材料降解速度和膽管再生速度不匹配,材料塌陷的風(fēng)險(xiǎn)。有進(jìn)一步改進(jìn)的空間。
[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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.1;R657.4

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