骨髓间充质干细胞和丝素蛋白/壳聚糖支架构建组织工程尿液流出道的研究

Study on the tissue-engineered urinary conduit constructed by bone marrow mesenchymal stem cells and silk fibroin/chitosan

目的探讨骨髓间充质干细胞(BMSC)和丝素蛋白/壳聚糖支架构建组织工程管状移植物在兔体内进行尿流改道的可行性和有效性。方法本研究共选取雄性SPF级新西兰大白兔36只。(1)取新西兰大白兔6只,行耳缘静脉麻醉后,提取BMSC和并进行体外培养,选取第3代BMSC,进行爬片,流式细胞仪检测细胞表面标志物;(2)另取6只新西兰大白兔,耳缘静脉麻醉后,取膀胱组织块,对尿路上皮细胞进行分离、培养及鉴定;(3)采用Transwell法,对BMSC悬液进行培养,采用免疫荧光检测诱导分化后BMSC是否向尿路上皮表型分化,同时采用蛋白质印迹法及聚合酶链式反应检测UPIa、角蛋白(CK)-18的表达情况;(4)制备丝素蛋白/壳聚糖支架,并对制备好的支架的形态、孔隙率进行评估;(5)将诱导分化14 d后的BMSC浓度调至4×106/mL,逐滴加入盖满整个丝素蛋白/壳聚糖支架材料,设为实验组,对照组为同样浓度的细胞悬液置于细胞培养瓶培养,取培养1、3、5、7、9 d复合物,扫描电镜观察、评估丝素蛋白/壳聚糖支架上细胞生长情况,使用噻唑蓝比色法检测细胞在支架上的增殖能力,并进行活细胞/死细胞测试;(6)取剩下的新西兰大白兔24只,随机分为实验组和对照组,每组各12只。麻醉后,分别将接种BMSC和未种BMSC的丝素蛋白/壳聚糖支架埋入实验组和对照组新西兰大白兔大网膜,网膜包裹促血管化2周后行苏木精-伊红染色,检测血管生成情况,行免疫组织化学染色观察细胞在支架表面生长情况;(7)对2组新西兰大白兔行尿流改道术,于术后1、2、4、8周进行取材,并分别行苏木精-伊红染色和免疫组织化学染色,术后10周利用静脉尿路造影检测流出道通畅情况。结果(1)分离出的BMSC呈圆形,培养3 d后以长梭状细胞形态为主;培养10 d,细胞融合度可达到90%,呈典型的纺锤样形态;获取第3代BMSC,流式细胞仪检测结果显示:约99.9%的细胞表面表达CD44和CD90,约97.0%的细胞表面不表达CD34和CD45。(2)尿路上皮细胞培养3 d,贴壁生长的细胞数目明显增多,细胞形态由类圆形变为扁平梭状,可见少量细胞集落形成;培养12 d,细胞融合度达到90%,细胞呈典型的铺路石样;CK(AE1/AE3)抗体鉴定结果显示,在荧光显微镜下可见大量多边形阳性细胞。(3)将诱导分化后的BMSC爬片行免疫荧光检测、蛋白质印迹法检测及聚合酶链式反应检测均提示:未诱导分化的BMSC不表达或低表达特异性UPIa蛋白,而诱导分化后细胞UPIa和CK-18蛋白表达上调。(4)制备好的冻干丝素蛋白/壳聚糖支架大体观察呈白色,可切成2 mm厚的薄片,扫描电镜下呈鱼网状结构,孔径大小为100~120μm,平均孔隙率为(91.2±4.3)%。(5)培养3 d,扫描电镜可见细胞黏附在丝素蛋白/壳聚糖支架上生长,较多的细胞伪足伸出,可见细胞之间的桥接现象;噻唑蓝比色法测试结果显示:细胞与支架材料共培养1、3、5 d,对照组的细胞增殖速度明显比实验组快,培养7 d,实验组和对照组细胞的增殖速度达到平衡,培养9 d实验组的细胞增殖速度加快,超过对照组;进行活细胞/死细胞测试,可观察到支架材料上90%以上的细胞为活细胞。(6)网膜包裹管状移植物促血管化2周后取材,肉眼可见移植物表面有新生血管生成;苏木精-伊红染色提示管状移植物腔面可见薄层、稀疏尿路上皮细胞覆盖;CD31免疫组织化学染色结果表明实验组可见明显的新生血管形成。(7)实验组12只新西兰兔尿流改道后均成活,未见输尿管扩张、肾积水现象,无尿外渗,流出道未见明显瘢痕形成挛缩。对照组12只新西兰兔在尿流改道术后4周内全部死亡,尸检可见流出道腔面塌陷,流出道内表面不平滑,有较多的结晶物附着。在尿流改道术后1、2、4、8周,苏木精-伊红染色可见上皮细胞逐渐完全覆盖流出道腔面;尿流改道术后1、2、4、8周,流出道表面可见上皮CK(AE1/AE3)和尿路上皮特异性蛋白UPⅢa表达逐渐增强;进一步检测连接蛋白ZO-1的表达情况与UPⅢa类似。术后10周,免疫组织化学染色显示CD31表达阳性;静脉尿路造影提示双肾分泌功能正常,肾盂、输尿管显影好,未见明显扩张现象,造影剂能够顺利通过流出道,流出道显影较好,未见明显狭窄、向外弥散现象。结论诱导分化后BMSC与丝素/壳聚糖支架共同构建组织工程尿液流出道是可行的,具有防止尿外渗、引流尿液的作用,为术后尿流改道提供了一种新思路。

Objective To explore the feasibility and effectiveness of tissue engineering tubular grafts constructed of bone marrow mesenchymal stem cell(BMSC)and silk fibroin/chitosan scaffolds for urinary diversion in rabbits.Methods A total of 36 male SPF New Zealand white rabbits were selected for this study.(1)Six New Zealand white rabbits were taken,after the ear vein anesthesia,the BMSC was extract and cultured in vitro,the third generation BMSC was selected,performed slides,and detected cell surface markers by flow cytometry.(2)Another 6 New Zealand white rabbits were taken,after the ear-marginal vein anesthesia,the bladder tissue block was taken and the urothelial cells were isolated,cultured and identified.(3)The Transwell method was used to culture the BMSC suspension,the immunofluorescence method was used to detect whether the induced cells differentiated into the urothelial phenotype,and the Western blotting and polymerase chain reaction were used to detect UPIa and cytokeratin(CK)-18 expression.(4)The silk fibroin/chitosan scaffold was prepared and the morphology and porosity of the prepared scaffold was evaluated.(5)Adjusted the BMSC concentration to 4×106/mL after induction of differentiation for 14 days,added drop by drop to cover the whole silk fibroin/chitosan scaffold material,set as the experimental group,and placed the control group with the same concentration of cell suspension on the cells culture flasks,scanning electron microscopy was used to observe the complexes cultured for 1,3,5,7 and 9 days,the growth of the cells on the silk fibroin/chitosan scaffold was evaluated,and the thiazole blue colorimetric method was used to detect the proliferation ability of the cells on the scaffold,and performed live cell/dead cell test.(6)The remaining 24 New Zealand white rabbits were taken and randomly divided into experimental group and control group,with 12 rabbits in each group.After the anesthesia,BMSC and unseeded BMSC silk protein/shell polysaccharide stent were buried in the experimental group and control group of New Zealand white rabbits retina,retinal wrap to promote angiochemical 2 weeks later,the angiogenesics were detected by hematoxylin-esin staining,and the chemical staining of immune tissue was used to observe the growth of cells on the surface of the stent.(7)Urinary diversion was performed on the 2 groups of New Zealand white rabbits.The samples were taken at 1,2,4,and 8 weeks after the operation,and hematoxylin-esin staining and immunohistochemical staining were performed respectively,and 10 weeks after operation,intravenous urography was used to detect the patency of the outflow tract.Results The isolated BMSC was round,and after 3 days of culture,they were mainly long spindle cells;after 10 days of culture,the cell fusion degree could reach 90%,showing a typical spindle-like morphology;the third generation BMSC was obtained,Cytometer test results showed that about 99.9%of the cells expressed CD44 and CD90 on the surface,and about 97.0%of the cells did not express CD34 and CD45.(2)Urothelial cells were cultured for 3 days,the number of adherent cells increased significantly,the cell morphology changed from round to flat spindle,and a small number of cell colonies could be seen;after 12 days of culture,the cell confluence reached 90%,and the cells showed a typical paving stone-like;the CK(AE1/AE3)antibody identification results showed that a large number of polygonal positive cells were visible under the fluorescence microscope.(3)Immunofluorescence detection,Western blotting detection and polymerase chain reaction detection of induced rabbit BMSC slides all indicated that uninduced differentiation of BMSC did not express or low-expressed specific UPIa protein,but after induction,the expression of UPIa and CK-18 protein were up-regulated.(4)The prepared freeze-dried silk fibroin/chitosan scaffold was white in general observation,and could be cut into 2 mm thick slices.It was a fishnet-like structure under a scanning electron microscope.The pore size was between 100 and 120μm,and the average porosity was(91.2±4.3)%.(5)After cultured for 3 days,scanning electron microscopy showed that the cells adhered to the silk fibroin/chitosan scaffold,more pseudopodia protruded,and the bridging phenomenon between the cells was visible;the results of the thiazole blue colorimetry test showed:cells and scaffold materials were co-cultured for 1,3,and 5 days,the proliferation rate of the control group was significantly faster than that of the experimental group.After cultured for 7 days,the proliferation rate of the experimental group and the control group reached a balance.After 9 days of cultivation,the proliferation rate of the experimental group increased faster than that of the control group;the live cell/dead cell staining was performed,and it was observed that more than 90%of the cells on the scaffold material were live cells.(6)Two weeks after omentum wrapped around the tubular graft vascularization,the material was taken.The naked eye showed neovascularization on the surface of the graft;hematoxylin-eosin staining indicated that the lumen of the tubular graft was covered with a thin layer and sparse urothelial cells;the results of CD31 immunohistochemical staining showed that the experimental group had obvious neovascularization.(7)All 12 New Zealand white rabbits in the experimental group survived after urinary diversion.There was no ureteral dilation,hydronephrosis,no urine extravasation,and no obvious contracture or scar formation in the outflow tract.The 12 New Zealand white rabbits in the control group all died within 4 weeks after urinary diversion.Autopsy showed that the cavity surface of the outflow tract was collapsed,the inner surface of the outflow tract was not smooth,and there were more crystals attached.At 1,2,4,and 8 weeks after urinary diversion,hematoxylin-eosin staining showed that epithelial cells gradually covered the lumen surface of the outflow tract;1,2,4,and 8 weeks after urinary diversion,the surface of the outflow tract could be seen that the expression of epithelial CK(AE1/AE3)and urothelial specific protein UPⅢa gradually increase;further detection of the expression of connexin ZO-1 was similar to UPⅢa.At 10 weeks postoperatively,immunohistochemical staining showed positive expression of CD31;intravenous urography showed that the secretory function of both kidneys was normal,and the renal pelvis and ureter were well developed without obvious expansion.The contrast agent could pass through the outflow tract smoothly,and the outflow tract was well developed.There was no obvious narrow and outward dispersion phenomenon.Conclusion It is feasible to construct a tissue-engineered urine outflow tract together with silk fibroin/chitosan scaffold after BMSC induction,which can prevent urine extravasation and drain urine,providing a new idea for postoperative urinary diversion.