【作者】 王振显；

【导师】 蔡文清；

【作者基本信息】 河北医科大学 ， 外科学， 2009， 博士

【摘要】 第一部分大鼠骨髓间充质干细胞的分离、培养以及诱导、分化成平滑肌样细胞的实验研究目的:采用全骨髓培养法结合贴壁法从大鼠骨髓中分离骨髓间充质干细胞,在体外培养、纯化、扩增、传代,应用1%二甲基亚砜(DMSO)和大鼠膀胱组织匀浆上清液诱导分化成平滑肌样细胞,探讨为组织工程膀胱的构建提供种子细胞的可能性。方法:无菌条件下取大鼠双侧股骨、胫骨和胸骨,用吸取D-Hank’s液的注射器冲出骨髓细胞,置于含10%FBS、100u/ml双抗的L-DMEM培养基中,37℃、5%CO2环境下进行培养,应用全骨髓培养法结合贴壁分离的方法进行纯化。待细胞达95%融合时,用0.125%的胰酶-0.02%的EDTA消化2-3min后进行传代。倒置相差显微镜观察原代及传代后的细胞生长特点。HE染色光镜观察细胞形态特征。扫描电镜(SEM)观察细胞表面的超微结构特征。利用MTT法测定大鼠骨髓间充质干细胞生长曲线,了解细胞生长特性。取第4代大鼠骨髓间充质干细胞用1%二甲亚砜(DMSO)预诱导8小时,然后应用大鼠膀胱组织匀浆上清液诱导7天。倒置相差显微镜观察诱导分化后的大鼠骨髓间充质干细胞形态学的变化并运用免疫细胞化学方法检测特异性蛋白标志物α-平滑肌肌动蛋白的表达。选用未进行诱导的MSCs作为阴性对照组。结果:原代培养的骨髓细胞成分复杂, 1-2天开始有少量细胞贴壁,形状不规则,有长梭形、多角形等。半量换液后,克隆生长的细胞团块开始迅速增殖,以集落生长为主。7-9天左右细胞可长满培养瓶底,达95%以上融合,主要呈长梭形,其中散在少量折光性强的小圆形细胞。细胞整体排列更趋于规律性,呈漩涡状,辐射状或鱼群样排列。传代后的细胞贴壁及生长更加迅速,2h后部分细胞即开始贴壁,24h内即可完全贴壁,6～7天可铺满培养瓶底。细胞形态更均一,长梭形,整体呈漩涡状,辐射状或鱼群样排列。HE染色示胞体以纺锤形居多,有突起,细胞核大、深染,核仁明显。扫描电镜观察主要为三种形态的细胞:宽大扁平形的细胞为主,长梭形和圆球形细胞占少数。表面可见大量微绒毛样突起,类似树突。P2、P4、P6细胞生长曲线基本相同,分为潜伏期(第1-2天)、对数生长期(第3-5天)和平台期(第6-7天),且几代细胞间生长状况稳定。第4代大鼠骨髓间质干细胞经过1%二甲基亚砜(DMSO)和大鼠膀胱组织匀浆上清液诱导可分化成为梭形平滑肌样细胞,融合后形成峰谷状排列,部分细胞表达平滑肌特异性蛋白标志物α-肌动蛋白。其诱导分化率为(45.6±3.5)%,阴性对照组的分化率为(3.8±0.77)%,二者相比差异有统计学意义(p<0.05)。结论:1.应用全骨髓培养法结合贴壁分离法可以快速的分离、纯化大鼠骨髓间充质干细胞,并使其在体外迅速得到了增殖,而且性状稳定。2. P2、P4、P6大鼠骨髓间充质干细胞生长曲线基本相同,细胞增殖快,且几代细胞间生长状况稳定。3.大鼠骨髓间充质干细胞在体外经膀胱组织匀浆上清液诱导可分化为平滑肌样细胞,可作为膀胱修复的种子细胞进一步研究,从而为后续实验研究打下基础。第二部分生物支架材料—人脱细胞羊膜的制备目的:探讨人脱细胞羊膜的制备方法及其作为生物支架材料应用于组织工程膀胱构建的可行性。方法:取新鲜人羊膜,钝性分离羊膜与绒毛膜组织,去除羊膜基底面残存的绒毛膜和血管组织,采用去污剂洗涤法和酶消化进行脱细胞处理。处理后的材料进行HE染色及电镜检查有无细胞成分残留。以诱导分化的大鼠骨髓间充质干细胞为鉴定细胞接种于96孔板中,分别采用人脱细胞羊膜浸提液及完全培养基进行培养,应用MTT法进行细胞毒性测定。将6-8片处理好的单层脱细胞羊膜按彼此纤维方向垂直的位置重叠,以200W的可见光照射3h,使其交联增厚,增加张力。结果:制备好的单层人脱细胞羊膜为白色半透明的薄膜,有一定弹性及韧性,厚约0.1mm。光镜观察无细胞成分残留,扫描电镜观察为有胶原蛋白形成的多微孔结构,无细胞残留。细胞毒性测定为Ⅰ级,细胞相容性好。交联后的人脱细胞羊膜厚度0.6-0.8mm,张力大大增加,能耐受缝合和牵拉。结论:采用去污剂洗涤和酶消化可以对新鲜人羊膜进行脱细胞处理获得脱细胞羊膜。所获支架材料具有一定的弹性、韧性及多微孔结构,为种子细胞的贴附和代谢提供了较好的物质基础。同时该支架材料细胞毒性低、具有良好的组织相容性。交联后的脱细胞羊膜厚度及张力大大增加,能耐受缝合和牵拉,是理想的组织工程用生物支架材料。第三部分组织工程膀胱的体外构建及大鼠膀胱替代实验目的:通过采用诱导分化的大鼠骨髓间充质干细胞(MSCs)和人脱细胞羊膜(HAAM)构建组织工程膀胱的实验研究,探讨应用组织工程膀胱进行大鼠膀胱替代修复的可行性。方法:以诱导分化成功的大鼠骨髓间充质干细胞(MSCs)作为种子细胞,以制备好的人脱细胞羊膜(HAAM)作为生物支架材料,将MSCs静态接种于HAAM表面,进行体外培养5天,获得组织工程膀胱。倒置相差显微镜观察细胞贴附情况。HE染色及扫描电镜(SEM)观察组织工程膀胱情况。将雄性SD大鼠30只随机分为A、B、C 3组,A、B两组分别12只,C组6只,均行半膀胱切除术,然后分别采用负载MSCs的HAAM(A组)、单纯HAAM(B组)以及直接缝合(C组)的方法进行膀胱修补。于术后2、4、8周分别测定膀胱最大容积及解剖取材观察膀胱组织再生情况,8周时行膀胱造影检查。结果:HAAM与MSCs复合培养第1～2天细胞增殖不明显,第3天时开始有细胞直接贴附于HAAM。随时间延长,贴附的细胞逐渐增多。复合培养第5天,HE染色可见MSCs贴附于羊膜,胞体较大,呈长梭形,细胞形态一致。扫描电镜观察可见细胞散在贴附在材料表面,伸有伪足,并生长入支架材料的孔隙中。大鼠膀胱重建术后2周时A组膀胱的Volmax为1.21±0.60ml,B组为1.12±0.53ml,两组比较无统计学意义(p>0.05),C组为0.75±0.41ml,与A、B两组比较差别有统计学意义(p<0.01)。随时间延长,4周、8周时大鼠的膀胱最大容积略有增加,但A、B两组仍无差别。术后8周时膀胱造影检查可见A、B两组膀胱形态大致正常,而C组膀胱形态明显缩小。大体标本检查: A、B组术后2周膀胱修补吻合区愈合良好,无漏尿;4周时膀胱修补吻合区与正常组织无界限。8周时已经近似正常膀胱,基本为正常组织所替代。膀胱腔内均无结石形成。修复区病理组织学观察:2周时A、B两组支架材料即已降解吸收,修补区大量炎性细胞浸润,内侧可见膀胱移行上皮细胞生长。A组可见平滑肌细胞形成,B组无明显平滑肌细胞。4周时浸润的炎性细胞明显减少,大量毛细血管长入。A组平滑肌细胞增多,B组稀疏可见平滑肌细胞。8周时膀胱的三层结构明显,炎性反应基本消失,A组吻合修补区较厚,平滑肌细胞规则且丰富。接近正常膀胱。B组修补区较薄,平滑肌细胞不规则。而C组仅2周时有轻微炎性反应,4、8周炎性反应消失。膀胱三层结构均正常。结论:利用MSCs和HAAM采用静态接种培养的方法在体外成功地构建了组织工程膀胱。所构建的组织工程膀胱进行大鼠膀胱替代实验,支架材料降解吸收迅速,平滑肌细胞再生良好,是理想的膀胱替代修补材料。更多还原

【Abstract】 PartⅠThe studies on separation and cultivation of rat marrow mesenchymal stromal cells (MSCs) and their differentiation into smooth muscle cells by the supernatant of homogenized bladders in vitro.Objective: To investigate separation, cultivation and purification of murine marrow mesenchymal stromal cells(MSCs) in vitro and its differentiation into smooth muscle cells by the supernatant of homogenized bladder so as to contribute to the seed cells of bladder tissue engineering.Methods: MSCs were collected from degermed femurs, tibias and sternum of 4 to 6-week-old SD rat by flushing the shaft with buffer (D-Hank’s, PH 7.2) using a syringe with a No. 26 G needle. Cells were cultivated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 100u/mL penicillin-streptomycin). When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The morphologic characteristics of MSCs were studied by HE staining and scanning electron microscope (SEM). The growth curve was tested by MTT assay. The 4th MSCs were cultured in DMEM with 1% DMSO 8 hours before changing the supernatant of homogenized bladders to induce differentiation. Morphology of cells was observed and immunocytochemistry was performed to detect the expression of specificα-SMA.Results: The components of primarily cultured MSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clones began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. The HE staining result showed that the cell body was fusiform, and some of them had processes. The big and deep staining basophilia nucleus were in the middle of the cell body. Under SEM we could find three kinds of cell morphologies: the most were the large flattened cells and there were a few spindle-shaped and globe cells. On the cells surface we could see a large amount of short and thick microvillus. The growth curve of P2, P4, P6 were quite similar and the cells biological characters kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase. After induction, MSCs demonstrated smooth muscle cell-like morphology and a hill-and-valley growth pattern as well as expressed the specificα-SMA. Its differentiation rate was (45.6±3.5)% vs control group’s(3.8±0.77)% ,there was significant deviation between the two groups.Conclusion: 1.In this part, a simple new method which was used for the separation, purification and cultivation in vitro of MSC from SD rat bone marrow by total marrow culture associating with adhering to wall has been established. The MSCs could proliferate immediately and keep their biological character stable in vitro.2. The cells were noted to have a large expansive potential and a fusiform morphology. The growth curves of P2, P4, P6 were quite similar and the cell biological character kept stable.3. MSCs could be induced into smooth muscle cells by the supernatant of homogenized bladders in vitro, thus providing sufficient amount of seeds cells to bladder tissue engineering. Part II: Research the characteristic and preparation of human acellular amniotic membrane graft for tissue engineering applicationsObjective:To investigate the preparation method of human acellular amniotic membrane(HAAM) and evaluate the feasibility of using HAAM as biomaterial scaffold to construct tissue engineering bladder.Methods:Human amniotic membranes were decellularized by the method of enzyme digestion and eradicator washing. The human acellular amniotic membranes (HAAM) were then examined by HE staing and electron microscope to confirm no cell elements remained. Murine marrow mesenchymal stromal cells were implanted in 96-hole-plank and cultured by HAAM extract liquid or normal culture medium. The cytotoxcity of HAAM was tested through MTT assay.6-8 pieces of HAAM were overlapped with fiber trend and exposured under 200W light for 3 hours to increase its thickness and tension.Results:The HAAM was white and translucent, and the thickness was about 0.1mm. It was lubricous and had good elasticity and toughness. There were no cell elements remained under the examination of optical microscope and electron microscope. Many hole structure and remain figure were seen and rich collagen were observed by scanning electron microscope in HAAM. The cytotoxcity score was I, which indicated that HAAM had a good biocompatibility. The thickness of cross-link HAAM was about 0.6-0.8 mm and its tension was increased.Conclusion:HAAM can be obtained by enzyme digestion and eradicator washing method. This scaffold has good elasticity and toughness, on which the seeds can adhibit and metabolize. The HAAM has good biocompatibility. The cross-link HAAM is thick and tough to endure the suturation and drag. HAAM can be used as ideal biomaterial scaffold of bladder tissue engineering.Part III: Construction the tissue engineering bladder and replacement experiment for ratObjective: To investigate the feasibility of bladder repair and replacement by tissue engineering bladder through the construction of murine tissue engineering bladder by marrow mesenchymal stromal cells(MSCs) and human acellular amniotic membrane graft(HAAMG).Methods: The MSCs of the rats were implanted in HAAM and cultured for about 5 days. Then the tissue engineering bladders were harvested. The cells were observed with phase contrast microscope and the tissue engineering bladders were observed by HE staining and scanning electron microscope.30 rats were divided into 3 groups randomly and marked with A, B and C. There were 12 animals in group A and B, but 6 animals in group C. Hemicystectomies were performed in all 3 groups. The 12 defects of group A were repaired with the tissue engineering bladders, group B with HAAM grafts and the last 6 defects of group C were sutured directly. The rats underwent postoperative assessment of bladder volume and cystography after 2,4,8 weeks. The animal bladders were obtained after that, through which the tissue regeneration was examined.Results: There was no evident cell proliferation on the first and second day of coculture of HAAM and MSCs. The cells began to stick to HAAM on the third day and the number of adherent cells increased with the time. On the fifth day of coculture, by HE stain, it could be seen that MSCs adhered to amnion. The cells bodies were bigger, fusiform and uniform. Under scanning electron microscope, there were scattered cells adhered to the surface of material. At 2,4,8 weeks after surgery, the bladder volumes of the group A and B were different significantly as compared with that of the group C(p<0.01), but there is no significant difference between the group A and B(p>0.05 ).The Cystography indicated the morphology of the bladder was normal in A and B group at 8 weeks, but became small in C group. The tissue of the anastomoses area grew well after 2 week in A and B group. Portion of HAAM was absorbed after 2 weeks. The tissue structure of the replacement was somewhat the same as that of the normal one after 4,8 weeks. Through the pathological examination, there were lots of inflammatory cells in the replacement tissue in A and B group after 2 weeks, and these inflammatory cells vanished 8 weeks later. The musculature could be observed in A group after 2 weeks and increased along with the time, but little in B group. The epithelial cells could be observed in both A and B group after 2 weeks and increased with the time.Conclusion: After transplantation with MSCs/HAAM, epithelial cells and smooth muscle cells can regenerated in the graft, and the graft can be absorbed quickly. The tissue engineering bladder constructed by MSCs and HAAM can be used as an ideal biomaterial to replace and repair the bladder。更多还原