本文選題：過氧乙酸 + 小腸粘膜下基質�。� 參考：《華中科技大學》2016年博士論文

【摘要】：尿道缺損是泌尿外科的最常見的疾病之一,可發(fā)生于先天性疾病如尿道下裂、尿道上裂等疾病,也可以發(fā)生于尿道損傷、感染,常伴有損傷后的尿道纖維化瘢痕狹窄。短段的尿道狹窄也可以通過尿道擴張、尿道狹窄切開及尿道狹窄段切除加端端吻合術予以治療。然而,長段的尿道狹窄或者尿道缺損,需要進行移植物進行修補。目前,臨床上常用的移植物包括舌粘膜、頰粘膜及陰莖皮膚。然而,口腔粘膜的取材會帶來一定的并發(fā)癥,如張口困難、感染、潰瘍、麻木、疼痛等。另外,長段的尿道狹窄術后易復發(fā),對于該類患者,往往面臨取材來源不夠。因此,需要尋求更廣泛來源的尿道替代移植物。組織工程技術的迅速發(fā)展,為尿道重建提供了新的治療途徑。然而,Chapple等最近認為在組織工程技術大規(guī)模的應用于臨床之前應該繼續(xù)研究以尋找出更加合適的組織工程材料。目前,報道較多組織工程材料的主要包括化學合成材料、天然生物基質類材料、天然生物材料的提取物,以及不同類材料的聚合物。其中,小腸粘膜下基質(SIS)屬天然生物基質類材料,具有天然的細胞與組織兼容性,且含括膠原、纖維連接蛋白、GAG及生長因子和信號蛋白等,能促進細胞生長與遷移。但其結構致密,不利于營養(yǎng)液的滲透與代謝物質的交換。另外,該類基質常規(guī)脫細胞后仍具有少量的異體細胞核成分的殘余,會引起宿主的慢性炎癥反應。理想的組織工程支架材料應該具有三維(3D)多孔結構及最少的異體細胞核成份的殘余。本實驗通過過氧乙酸氧化SIS制作出具有3D多孔結構的改性SIS,同時也最大限度地去除了異體的細胞核成分殘余。我們進一步評價膀胱移行細胞種植這種經過氧乙酸(Peracetic acid, PAA)改性的小腸粘膜下基質(SIS)在家兔尿道重建中的應用效果。本研究共分以下三部分：第一部分：PAA改性脫細胞SIS基質的制備目的：制作出3D多孔的SIS支架材料,并評估其表征、力學性能方法：取新鮮的豬小腸組織,機械方法游離出粘膜下層及漿肌層,去除上皮層,分離出小腸粘膜下基質,置入蒸餾水預脫細胞處理,PAA改性SIS組加5%過氧乙酸(PAA),而無PAA改性組加PBS液,然后兩組均置入1%Triton X-100。最后75%乙醇消毒,無菌純凈水保存?zhèn)溆�。取材行HE、MASSON染色、掃描電鏡觀察。應用密度瓶法測定兩組基質材料的空隙率。取材大小5mmx60mm,使用萬能試驗機測定其力學性能,包括最大拉長、最大強度及楊模量大小。統(tǒng)計分析兩組SIS材料在形態(tài)學和力學性能間的差異。結果：未經PAA處理的SIS結構致密,且有少量細胞成分殘余,而經PAA處理的SIS呈3D多孔狀,幾乎沒有細胞核成分殘余。SIS的上皮面電鏡結果顯示經5%PAA處理SIS表面孔徑明顯增加,經過測量分析后,脫細胞新鮮SIS的孔徑值為1.75±0.32μm,而經過PAA氧化處理后的SIS孔徑值為5.16±1.831μm,無PAA處理的SIS空隙率為18.5±2.6%,而經過PAA處理后的SIS空隙率66.8±3.9%,兩者差異均具有顯著統(tǒng)計學意義(P0.01)。經萬能拉伸儀測定,脫細胞無PAA處理SIS及經PAA處理的SIS的最大拉伸率分別為16.3±1.6%和15.0±1.2%,最大拉伸強度分別為35.1±7.2 Mpa和24.3±5.6 Mpa,楊氏模量分別為150.4±39.2Mp和118.5±28.4Mpa.結論：經PAA改性的后的SIS具有3D多孔狀結構,幾乎無異體細胞核成分殘余,且仍能維持一定的機械性能,可以作為理想的組織工程材料。第二部分：組織工程膀胱移行上皮細胞-SIS復合物的體外構建目的：體外構建膀胱移行上皮細胞-改性SIS復合物,觀察其形態(tài)、評估改性SIS的細胞兼容性。方法：取成年新西蘭大白兔膀胱壁0.6x0.6 cm,置入PBS液漂洗。將膀胱組織塊置入含有DMEM培養(yǎng)基的pronase E蛋白酶中,消化過夜,小心刮取膀胱粘膜層移行上皮細胞。加含有5%胎牛血清、1%青霉素-鏈霉素的ECM培養(yǎng)基培養(yǎng),0.05%胰酶傳代。制作細胞爬片,應用抗AE1/AE3抗體進行細胞免疫組織化學鑒定。制作PAA改性SIS支架材料和非PAA改性SIS支架材料浸提液,行MTT實驗記錄兩組細胞生長的OD值,了解兩組支架材料的細胞兼容性。收集細胞并種植于預先制好的PAA改性SIS支架材料和非PAA改性的SIS支架材料上,繼續(xù)培養(yǎng)至2周,取材行HE染色、電鏡觀察。利用RT-PCR技術和Western-blot技術檢測Uroplakin基因在UC-經PAA改性SIS復合物的表達變化。結果：膀胱移行上皮細胞在無PAA處理SIS及5%PAA處理后SIS浸提液中生長狀態(tài)良好,且均優(yōu)于單純ECM培養(yǎng)基的趨勢,但是差異均無統(tǒng)計學意義(p=0.35)。無PAA處理SIS組細胞生長以單層結構為常見,而在5%PAA處理后SIS組中,細胞生長連接成片,形成2-3層的復層結構。電鏡下,無PAA處理SIS上細胞呈多角形,扁平狀,伸出偽足,緊密貼附于支架材料上。而在5%PAA處理SIS支架上,細胞分布更加緊密,相互融合呈多層面三維立體結構,表層細胞多呈圓形、橢圓形及立方形。經PAA改性SIS-UC復合物較無PAA改性-UC復合物高表達Uroplakin基因。結論：采用酶消化法聯(lián)合機械的刮擦法能分離培養(yǎng)具有活性的膀胱移行上皮細胞,陽性表達AE1/AE3。未經PAA處理的脫細胞SIS與經5%PAA改性處理SIS支架材料與膀胱移行上皮細胞均具有良好的細胞兼容性。經5%PAA改性處理SIS能更好的促進細胞在支架材料上生長并形成復層結構,且能促進膀胱移行上皮向終末分化。第三部分：組織工程膀胱移行上皮-SIS復合物修補家兔尿道缺損模型的建立與重建效果觀察目的：觀察組織工程膀胱移行上皮-SIS復合物修補家兔尿道缺損的效果方法：健康成年新西蘭家兔18只,分成三組,膀胱移行上皮細胞種植PAA改性的SIS支架組、膀胱移行細胞種植無PAA改性的SIS材料組,無膀胱上皮細胞種植的經PAA改性SIS組,每組各6只。按照第二部分的方法,構建組織工程膀胱上皮細胞-SIS復合物。再次麻醉家兔,游離腹側尿道海綿體至粘膜層,建立家兔尿道粘膜缺損模型1.5×0.8cm2,修剪支架材料大小為1.7×1cm2,以補片的方式進行修補。術后抗感染、尿管沖洗,手術后半年行尿道造影檢查,大體病理觀察及HE、MASSON、免疫組化檢測。結果：上皮細胞-5% PAA改性SIS復合物組均存活,尿道通暢,再生尿道粘膜完全再生。病理見復層移行上皮、上皮下平滑肌增生,血管增生。而上皮細胞-無PAA改性SIS復合物組,有尿瘺發(fā)生,再生尿道粘膜不完整。病理見上皮不規(guī)則,及慢性炎癥細胞浸潤。單純PAA改性SIS組有尿道狹窄,尿道粘膜蒼白、僵硬、攣縮,病理提示大量纖維結締組織增生,平滑肌和血管少見。上皮細胞-PAA改性SIS組的上皮及平滑肌表達定量顯著優(yōu)于上皮細胞-無PAA改性組(P0.05)。平滑肌及血管均優(yōu)于單純PAA改性SIS組(P0.05)結論：膀胱移行上皮細胞種植PAA改性SIS是進行長段尿道缺損修補的理想替代物。
[Abstract]:Urethral defect is one of the most common diseases in the Department of urology. It can occur in congenital diseases such as hypospadias, hypospadias and other diseases. It can also occur in urethral injury, infection, and urethral fibrosis scar stricture after injury. Short segment urethral stricture can also be treated with urethral dilatation, urethral stricture incision and urethral stricture excision. However, long segment urethral strictures or urethral defects need to be repaired by graft. Currently, the commonly used graft includes the mucosa of the tongue, the buccal mucosa, and the penis skin. However, the selection of the oral mucosa will bring some complications, such as oral difficulty, infection, ulcers, numbness, pain and so on. Long segment urethral stricture is easy to relapse. For this type of patients, it is often faced with insufficient sources of material. Therefore, a more extensive source of urethral replacement graft is needed. The rapid development of tissue engineering technology provides a new way of treatment for urethral reconstruction. However, Chapple and so on are most recently considered to be applied in a large scale in tissue engineering. The bed should continue to be studied to find more appropriate tissue engineering materials. At present, most of the reports of tissue engineering materials include chemical synthetic materials, natural biomaterials, extracts of natural biomaterials, and polymers of different materials. Among them, the submucosal matrix of the small intestine (SIS) is a natural biomaterial. It has natural cell and tissue compatibility, including collagen, fibronectin, GAG and growth factors and signal proteins, which can promote cell growth and migration. But the structure is dense and is not conducive to the infiltration of nutrient solution and the exchange of metabolic substances. In addition, this type of matrix often has a small number of allogenic cell nuclear components. The residual, cause the chronic inflammatory response of the host. The ideal tissue engineering scaffold material should have a three-dimensional (3D) porous structure and the least residual cell nuclear components. In this experiment, the modified SIS with 3D porous structure was produced by oxidation of peroxy acetic acid, and the residual cell nuclear components were removed to the maximum extent. We further evaluate the effect of Peracetic acid (PAA) modified small intestinal submucosal matrix (SIS) in the reconstruction of urethra in rabbits. This study is divided into three parts: Part I: preparation of PAA modified decellular SIS matrix: the preparation of 3D porous SIS scaffold materials, and evaluation of SIS Its characterization and mechanical properties: taking fresh pig small intestine tissue, removing the submucosa and muscularis layer mechanically, removing the epithelial layer, separating the submucous matrix of the small intestine, placing the distilled water preconditioning, the PAA modified SIS group and 5% peracetic acid (PAA), without the PAA modified group and PBS solution, and then placing the two groups in the final 75% of the X-100. 75%. HE, MASSON dyeing and scanning electron microscopy were used to determine the void ratio of two groups of matrix materials by density bottle method. The mechanical properties of the material were measured by the universal test machine, including the maximum elongating, maximum strength and young modulus. The morphology and force of the two groups of SIS materials were statistically analyzed. The results showed that the structure of SIS without PAA treatment was compact, and there was a small amount of residual cell components, while SIS treated with PAA was 3D multi pore, and there was almost no residual.SIS in the upper surface of the cell nucleus with electron microscopy. The pore size of SIS was obviously increased by 5%PAA treatment, and the pore size of the fresh SIS was 1.7 after measurement and analysis. 5 + 0.32 mu m, and after PAA oxidation, the SIS aperture value is 5.16 + 1.831 mu m, the SIS void ratio without PAA treatment is 18.5 + 2.6%, and the SIS void ratio after PAA treatment is 66.8 + 3.9%, and the difference is statistically significant (P0.01). The maximum tensile strength is 16.3 + 1.6% and 15 + 1.2% respectively, the maximum tensile strength is 35.1 + 7.2 Mpa and 24.3 + 5.6 Mpa respectively. The young's modulus is 150.4 + 39.2Mp and 118.5 + 28.4Mpa., respectively. After PAA modification, the SIS has 3D porous structure, and there is almost no residual cell nuclear composition, and still can maintain a certain mechanical properties, which can be used as an ideal group. Fabric engineering material. Second part: Construction of tissue engineering bladder transitional epithelial cell -SIS complex in vitro purpose: construct bladder transitional epithelial cell - modified SIS complex in vitro, observe its morphology, evaluate the cell compatibility of modified SIS. Methods: take 0.6x0.6 cm of adult New Zealand white rabbit bladder wall, put into the PBS solution and rinse the bladder group. The fabric was inserted into the pronase E protease containing the DMEM medium, digested for the night and carefully scraped the bladder mucosa layer to move the epithelial cells. The ECM medium containing 5% fetal bovine serum, 1% penicillin - streptomycin, 0.05% trypsin passages, and the cell climbing tablets were made, and the anti AE1/AE3 antibody was applied to the cellular immuno histochemical identification. The PAA modified SIS branch was made. The scaffold material and the non PAA modified SIS scaffold were extracted, and the MTT experiment was used to record the OD values of two groups of cell growth. The cell compatibility of the two groups of scaffolds was understood. The cells were collected and cultivated on the pre made PAA modified SIS scaffold material and the non PAA modified SIS scaffold material, and continued to be cultured for 2 weeks. R and Western-blot techniques were used to detect the changes in the expression of Uroplakin gene in UC- via PAA modified SIS complex. Results: the growth state of the bladder transitional epithelial cells in the SIS extract without PAA treatment SIS and 5%PAA was better than that of the pure ECM medium, but the difference was not statistically significant (p=0.35). Cell growth is common in single layer structure, and in SIS group after 5%PAA treatment, cell growth is connected to pieces to form 2-3 layers of complex layer structure. Under electron microscope, the cells on SIS without PAA treatment are polygonal, flat, protruding and attached to the scaffold material. On the 5%PAA treatment SIS scaffold, the cells are more closely distributed and multilayered with each other. The surface cells are mostly round, oval and cubic. The PAA modified SIS-UC complex is more expressed than the Uroplakin gene without the PAA modified -UC complex. Conclusion: the enzyme digestion method combined with mechanical scraping can separate and culture the active cell cell of the bladder transitional epithelium, and the positive expression of AE1/AE3. has not been refined by PAA treatment. Cell SIS and 5%PAA modified SIS scaffold materials have good cellular compatibility with bladder transitional epithelial cells. 5%PAA modified SIS can better promote cell growth on scaffold material and form complex layer structure, and can promote the differentiation of bladder transitional epithelium to terminal differentiation. Third parts: tissue engineered bladder transitional epithelium -SIS compound Objective: To observe the effect of reconstruction and reconstruction of rabbit urethral defect model for repairing the rabbit urethra defect: observe the effect of tissue engineering bladder transitional epithelium -SIS complex to repair rabbit urethral defect: 18 healthy adult New Zealand rabbits were divided into three groups, the bladder transitional epithelial cells were planted with PAA modified SIS stents, and the implantation of bladder transitional cells was not modified by PAA. In the sex SIS group, no bladder epithelial cells were implanted in the PAA modified SIS group, each group was 6. The -SIS complex of the tissue engineered bladder epithelial cells was constructed according to the second part method. The rabbits were again anesthetized, free ventral urethral cavernous body to mucous layer, and the rabbit urethral mucosa defect model was set up 1.5 x 0.8cm2, the size of the pruning scaffold material was 1.7 X. 1cm2, repair with patch. After operation, anti infection, urethral rinse, half a year after the operation, urethrography examination, gross pathological observation and HE, MASSON, immunohistochemical detection. Results: epithelial cells -5% PAA modified SIS complex group survived, urethral smooth, regenerated urethral mucous membrane completely regenerated. Pathological findings of the epithelium, epithelial smooth epithelium, smooth epithelial smooth Muscle hyperplasia, vascular hyperplasia, and epithelial cells - no PAA modified SIS complex group, the occurrence of urinary fistula, regenerative urethral mucosa incomplete. Pathological epithelial irregular, and chronic inflammatory cell infiltration. Simple PAA modified SIS group with urethral stricture, urethral mucosa pale, rigid, contracture, a large number of fibrous connective tissue hyperplasia, smooth muscle, and less blood vessels The expression of epithelial and smooth muscle in the -PAA modified SIS group was significantly better than that of the epithelial cell without the PAA modified group (P0.05). The smooth muscle and blood vessels were superior to those of the PAA modified SIS group (P0.05). The implantation of PAA modified SIS in the transitional cell of the bladder was an ideal substitute for the repair of long segment urethral defects.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R318.08;R699.6

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