【作者】 严泉剑；

【导师】 邵国兴；

【作者基本信息】 中国人民解放军第四军医大学 ， 外科学， 2003， 博士

【摘要】 膀胱组织工程替代是目前比较新的的治疗膀胱缺损或功能衰竭的方法，包括利用种植或未种植自体细胞的支架材料治疗的多种替代方法。本试验尝试应用膀胱无细胞基质(BAMG)复合自体细胞或肝细胞生长因子(HGFs)构建组织工程膀胱在兔膀胱部分切除术后进行膀胱替代的应用，应用核移植技术拓展膀胱种子细胞来源。 目的：观察HGFs对膀胱平滑肌损伤的影响，探讨BAMG和HGFs在兔构建组织工程膀胱构建中的应用，尝试应用核移植技术拓展膀胱种子细胞来源。 方法：(1)制备新西兰兔单纯膀胱前壁肌层组织缺损创面3个，面积1cm~2大小，分别涂敷pUDKH，溶媒以及空载体，观察创面愈合速度，比较肥大指数(Hypertrophic index，HI)；(2)简化BAMG制备步骤，Masson’s染色，醋酸铀酰和柠檬酸铅染色，光镜及电镜检查制备效果；(3)用BAMG复合自体骨髓基质细胞，脂肪基质细胞，膀胱平滑肌细胞和上皮细胞，pUDKH+FB制备组织工程膀胱，分别用于兔膀胱次全切除替代，观察替代手术后的膀胱测压参数变化，影象学变化，组织形 第四军医人学烟十学位论义一态变化。（4）分别利用猪骨髓基质和脂肪基质细胞作为核供体，采用卵细胞胞质内直接注射法构建移核胚胎，比较囊胚形成率，观察孵化囊胚细胞染色体变化。 结果：（）新西兰兔单纯膀眺前壁肌层组织缺损模型上，溶媒对照组A，空载体对照组B以及pUDKH治疗组C创面面积7天时分别为44L8mmZ，43t9mm2和 2816mm2，14天时分别为 23t7mm2，22x6mm‘和4功mm‘，将初始创面面积，7天和 14天时创面面积数据应用重复测量方差分析，3组间愈合速度差异有统计学意义，F叫二36，P刃刀0 C组和A，B组之间比较，面积减少差异有统计学意义，尸刃刀0；A，B两组之间差异无统计学意义，P＝0石47。术后30天对横贯正常膀陇，过渡区和愈合创面的膀脱标本进行HE组织学检查，pUDKH治疗组与自体膀腕具有相似的正常的组织学结构，溶媒以及空载体对照组粘膜下纤维组织增生加厚而肌层较薄；三组的 HI分别为 1．7610二 7，1．83t0二 8和 1．11＊0．18（P－0＊0，C vs Aand B；尸＝0＊54，A vs B），提示 C组疲痕较 A，B组少。（2）预实验所用BAMG制备方法周期至少为60小时，金宁公司采用我们改进的方法制备BAMG周期最长是26小时。利用我们改进的方法制备的BAMG呈半透明状，抗漏尿能力好。改进的方法制备BAMG周期缩短34 ’J＼时，Masson’s染色，醋酸铀酚和柠檬酸铅染色，光镜及电镜检查。粗细不一的胶原纤维在兔BAMG内松散地堆积在一起，看不到细胞膜，细胞器和染色质的残留。u）分离的膀脱上皮细胞在倒置相差显微镜下观察，形状为多角形，鹅卵石状，AE ／AE3抗体染色膀眺上皮细胞呈阳性：膀肌平滑肌细胞形态为梭形，Q．肌动蛋白抗体染色膀脱平滑肌细胞阳性；倒置相差显微镜下兔MSCs与ASCs形态相似，呈梭状或纺锤状，易于贴壁，融合ASCs细胞爬片HE染色，部分分化细胞显示脂肪空泡不 －3． 第凹军医大学博上学位论义一染色，胞浆呈红色，胞核呈蓝色，未分化ASCs细胞看不到脂肪空泡。用BAMG复合自体膀肤平滑肌细胞和上皮细胞，骨髓基质细胞或脂肪基质细胞，pUDKH＋FB制备组织工程膀脱的时间分别为4周，3周，30分钟。术后12周进行膀眯测压显示：原位缝合组A动物能够维持23％的自体膀眺原有容量。单用BAMG组B，膀眯容量恢复到术前的85％。应用BAMG复合自体膀肌平滑肌细胞和上皮细胞，ASCs，MSCs或HGFs构建的组织工程膀脱组C、D、E、F，容量分别恢复到97O，940，950和 920。5组膀脱）l匝应性分别恢复到原来的 ZI0，1080，1060，102O，！07％和 104％。术前术后数掘 T检验，差异有统计学意义的是：A组膀眺容量（P—0．0053）及顺应性（P＝o．mm）均下降；B组术后LPP减少 （尸－0046）及PP减少（＊。o．034）：其他组差异无统计学意义。术前术后测量值按重复测量分析，总体分析统显示，手术前后测量值差异有统计学意义；组间比较差异无统计学意义。分组分析显示，差异有统计学意义的只有A组术后膀耽容量减少（P＝0刀35L顺应性下降（P－0刀36人手术后 12周，对A组的自体膀耽以及从 B、C、D、E和 F组再生膀脱区的标本进行横切片，HE或三色染色。A组显示正常的组织学分布。B组平滑肌纤维己经呈现有机的空间排列，形成了各种各样大小的束状，但再生不完全。C、D、E和F组，与自体膀脱具有相似的正常的组织学结构。术后12周，膀脓X线造影，B超显示，A组动物的膀眯而仅仅能够再生很小的储水能力。而组织工程膀眯在体积和轮廓上基本保持正常。 （4）猪MSCs、ASCs作为核供体，构建的移核胚胎存活率分别为79刀％、77．5％，以移核胚存活率数值为基线值，24小时卵裂率分别为45．3％。49．2％，36小时分另为的石％、58．2％。囊胚发育率分另为16刀％、13．4％，囊胚孵化率为分别为4．3％、3．4％。2组间率的比较差异无统计学意义更多还原

【Abstract】 Bladder tissue engineering is a broad term used to describe the development of alternative tissue sources for diseased or dysfunctional native bladder. The techniques involved synthetic and natural biodegradable matrices alone, known as "unseeded" scaffolds, and the latest data on "seeded" scaffolds, which are impregnated with cultured cells from urologic or other organs.In this study, we address the function parameters of tissue engineered bladders with bladder accellular organ specific matrix graft (BAMG) seeded with hepatocyte growth factors or cultured cells from autologous urologic or other organs to replace a subtotal cystectomy. We also try to find a new source of "seed"cells with nuclear transplantation techneques.Aims: To study the HGFs function in bladder smooth muscle wound healing, identify the BAMG application in bladder tissues engineering construction, expand the source of "seed" cell with nuclear transplantation techneques.Methods: (1) A cyst-muscle-ectomy only rabbit model treated withmenstruum only A, 15ug pUDK B or 15ug pUDKH C. Gross, histologic and hypertrophic index (HI) analyses of the wound healing were performed postoperative on day 7 and day 14; (2) The adapted preparation is simplified and used to make the BAMG. The BAMG stained with Masson’ or in uranyl acetate and lead citrate, was observed with scanning electron microscopy and light microscopy examination; (3) Autologous MSCs, ASCs, smooth muscle and epthlial were isolated from biopsy respectly. Preparation the tissue engineering bladder with BAMG seeded without or with pUDKH + FB, autologous MSCs, ASCs, smooth muscle and epthlial from bladder biopsy respectly. The cystmetre of rabbits were performed preoperatively and postoperatively at 12 weeks after subsequent replacement of a subtotal cystectomy of rabbits. Graphy and histologic analyses were also performed; (4) Swine’s ASCs or MSCs were isolated and cultured to passage 3-5 and used as donor cells in nuclear transfer (NT) to produce the swine-mice reconstruction oocyte with direct intracystplasm microinjection techniques. Sire differences were noted in the ability of both MSCs and ASCs to form blastocysts. The chromosome analysis of hatching cells was also performed.Results: (1) A cyst-muscle-ectomy only rabbit model treated with menstruum only A, 15ug pUDK B or 15ug pUDKH C, the wound aera postoperatively was 44 ±8 mm2, 43 ± 9 mm2 and 28 ± 6 mm2 respectively on day 7, and 23 ±7mm2 , 22 ± 6mm2 和 4 ± 4mm2 respectively on day 14, analysis the data with repeat measure method, the wound aera reduce in C group did significant differ (F=16.236, p =0.00, C vs A and B). The hypertrophic index (HI) postoperatively at day 30 in three groups was 1.76±0.17, 1.83±0.18 and 1.11±0.18 respectively. Analyses of the HI showed that there were significant difference betweean the groups (.P=0.00, C vs A and B; P=0.054, A vs B). C group have little wound aera and HI; (2) Thepreparation time of BAMG, which typically indicates essentially no cell nuclei when stained with Masson’ dyes or uranyl acetate and lead citrate, was 26 huors with the adapted simple method; (3) The isolation bladder epithelial were cobblestone shapes under phase contrast microscope and positive staining with pancytokeration AE1/AE3, smooth muscle were shuttles forms and positive staining with anti- a -smooth muscle actin. The isolation rabbit MSCs and ASCs shapes like shuttles under phase contrast microscope. Preparation time of BAMG seeded with pUDKH + FB, MSCs, ASCs, smooth muscle and epthlial from bladder biopsy were 30 minutes, 3 weeks and 4 weeks respectly. Analysis of the the cystmetre data of rabbit performed preoperatively and postoperatively with repeat measure method showed that all parameter did not significant differ in tissue engineering bladder groups. T-test showed that the reduction of bladder volume and capacity did significant different between preoperative and postoperative. Histologically, group A, C, D, E, F retrieved bladders showed a normal cellular organization consisti更多还原