CAJ | 학술논문

背景:传统方法多采用平滑肌细胞和移行上皮细胞构建组织工程膀胱,并进行支架材料的双面种植,但由于平滑肌细胞取材培养困难,且体外传代有限,双面种植较为困难.目的:实验拟验证以骨髓间充质干细胞及膀胱脱细胞基质构建组织工程膀胱的可行性.设计:基础实验研究.单位:中山大学附属第二医院林百欣医学研究中心.材料:实验于2006-03/2007-05在中山大学附属第二医院林百欣医学研究中心完成.实验室级别为卫生部部属医院开放实验室.1月龄SD大鼠,雌雄不限,体质量80～100g,由中山大学实验动物中心提供.新鲜猪膀胱取自南方医科大学动物实验中心.方法:采用全骨髓培养连续贴壁法体外培养大鼠骨髓间充质干细胞,并应用流式细胞仪检测其表面抗原.采用去污剂洗涤法制备猪膀胱脱细胞基质,并测定其纯度及特性.将第3代骨髓间充质干细胞接种到膀胱脱细胞基质上,以添加25 ng/L血管内皮生长因子(VEGF165)的培养液进行体外、体内复合培养,检测其相容性.体内实验以细胞单独培养为对照,体外实验以未植入细胞的材料为对照,并模拟合适的微环境诱导骨髓间充质干细胞生长分化,分别在4,8周后取出动物体内的复合材料行组织切片检查,并进行免疫组织化学角蛋白染色,检测上皮细胞再生情况.主要观察指标:骨髓间充质干细胞与膀胱脱细胞基质的生物相容性.结果:①采用全骨髓法成功培养出骨髓间充质干细胞,行流式细胞仪检测细胞表面抗原显示,传代第3代细胞CD29阳性细胞为99.43%.②制备的膀胱脱细胞基质具有良好的生物特性,镜下见均质状态的基质和细丝状的胶原纤维.体内外相容性实验表明骨髓间充质干细胞与膀胱脱细胞基质具有良好的相容性,细胞生长状态良好.③4周后组织切片检查可见组织中较多炎症细胞浸润,胶原及弹性纤维排列紧密,免疫组织化学角蛋白染色见有薄层、不连续的单层上皮生长,8周后可见组织中已无明显炎症细胞浸润反应,胶原及弹性纤维排列紧密,免疫组织化学角蛋白染色见有薄层、连续的多层上皮生长.结论:骨髓间充质干细胞与膀胱脱细胞基质具有良好的生物相容性,并且在体内与周围组织有良好的生物相容性,可以作为构建组织工程膀胱的材料. 揹景:傳統方法多採用平滑肌細胞和移行上皮細胞構建組織工程膀胱,併進行支架材料的雙麵種植,但由于平滑肌細胞取材培養睏難,且體外傳代有限,雙麵種植較為睏難.目的:實驗擬驗證以骨髓間充質榦細胞及膀胱脫細胞基質構建組織工程膀胱的可行性.設計:基礎實驗研究.單位:中山大學附屬第二醫院林百訢醫學研究中心.材料:實驗于2006-03/2007-05在中山大學附屬第二醫院林百訢醫學研究中心完成.實驗室級彆為衛生部部屬醫院開放實驗室.1月齡SD大鼠,雌雄不限,體質量80～100g,由中山大學實驗動物中心提供.新鮮豬膀胱取自南方醫科大學動物實驗中心.方法:採用全骨髓培養連續貼壁法體外培養大鼠骨髓間充質榦細胞,併應用流式細胞儀檢測其錶麵抗原.採用去汙劑洗滌法製備豬膀胱脫細胞基質,併測定其純度及特性.將第3代骨髓間充質榦細胞接種到膀胱脫細胞基質上,以添加25 ng/L血管內皮生長因子(VEGF165)的培養液進行體外、體內複閤培養,檢測其相容性.體內實驗以細胞單獨培養為對照,體外實驗以未植入細胞的材料為對照,併模擬閤適的微環境誘導骨髓間充質榦細胞生長分化,分彆在4,8週後取齣動物體內的複閤材料行組織切片檢查,併進行免疫組織化學角蛋白染色,檢測上皮細胞再生情況.主要觀察指標:骨髓間充質榦細胞與膀胱脫細胞基質的生物相容性.結果:①採用全骨髓法成功培養齣骨髓間充質榦細胞,行流式細胞儀檢測細胞錶麵抗原顯示,傳代第3代細胞CD29暘性細胞為99.43%.②製備的膀胱脫細胞基質具有良好的生物特性,鏡下見均質狀態的基質和細絲狀的膠原纖維.體內外相容性實驗錶明骨髓間充質榦細胞與膀胱脫細胞基質具有良好的相容性,細胞生長狀態良好.③4週後組織切片檢查可見組織中較多炎癥細胞浸潤,膠原及彈性纖維排列緊密,免疫組織化學角蛋白染色見有薄層、不連續的單層上皮生長,8週後可見組織中已無明顯炎癥細胞浸潤反應,膠原及彈性纖維排列緊密,免疫組織化學角蛋白染色見有薄層、連續的多層上皮生長.結論:骨髓間充質榦細胞與膀胱脫細胞基質具有良好的生物相容性,併且在體內與週圍組織有良好的生物相容性,可以作為構建組織工程膀胱的材料.
배경:전통방법다채용평활기세포화이행상피세포구건조직공정방광,병진행지가재료적쌍면충식,단유우평활기세포취재배양곤난,차체외전대유한,쌍면충식교위곤난.목적:실험의험증이골수간충질간세포급방광탈세포기질구건조직공정방광적가행성.설계:기출실험연구.단위:중산대학부속제이의원림백흔의학연구중심.재료:실험우2006-03/2007-05재중산대학부속제이의원림백흔의학연구중심완성.실험실급별위위생부부속의원개방실험실.1월령SD대서,자웅불한,체질량80～100g,유중산대학실험동물중심제공.신선저방광취자남방의과대학동물실험중심.방법:채용전골수배양련속첩벽법체외배양대서골수간충질간세포,병응용류식세포의검측기표면항원.채용거오제세조법제비저방광탈세포기질,병측정기순도급특성.장제3대골수간충질간세포접충도방광탈세포기질상,이첨가25 ng/L혈관내피생장인자(VEGF165)적배양액진행체외、체내복합배양,검측기상용성.체내실험이세포단독배양위대조,체외실험이미식입세포적재료위대조,병모의합괄적미배경유도골수간충질간세포생장분화,분별재4,8주후취출동물체내적복합재료행조직절편검사,병진행면역조직화학각단백염색,검측상피세포재생정황.주요관찰지표:골수간충질간세포여방광탈세포기질적생물상용성.결과:①채용전골수법성공배양출골수간충질간세포,행류식세포의검측세포표면항원현시,전대제3대세포CD29양성세포위99.43%.②제비적방광탈세포기질구유량호적생물특성,경하견균질상태적기질화세사상적효원섬유.체내외상용성실험표명골수간충질간세포여방광탈세포기질구유량호적상용성,세포생장상태량호.③4주후조직절편검사가견조직중교다염증세포침윤,효원급탄성섬유배렬긴밀,면역조직화학각단백염색견유박층、불련속적단층상피생장,8주후가견조직중이무명현염증세포침윤반응,효원급탄성섬유배렬긴밀,면역조직화학각단백염색견유박층、련속적다층상피생장.결론:골수간충질간세포여방광탈세포기질구유량호적생물상용성,병차재체내여주위조직유량호적생물상용성,가이작위구건조직공정방광적재료.
BACKGROUND:Smooth muscle cells and transitional epithelial cells were traditionally used to construct tissue-engineered bladder and to perform double-sided implantation of scaffold.However,double-sided implantation is difficult to perform,because smooth muscle cells are difficult to isolate or culture in vitro and passage is limited.OBJECTIVE:To verify the feasibility of tissue-engineered bladder reconstruction with bone marrow mesenchymal stem cells(BMSCs)and bladder acellular matrix(BAM).DESIGN:A basic empirical study.SETTING:Linbaixin Medical Research Center,Second Affiliated Hospital,Sun Yat-sen University.MATERIALS:Experiments were performed at the Linbaixin Medical Research Center,Second Affiliated Hospital,Sun Yat-sen University from March 2006 to Mav 2007.The laboratory was the Opening Laboratory of Hospital Affiliated to Health Department of China.One-month old SD rats of either sex,weighting 80-100 g were provided by Animal Experimental Center of Sun Yat-sen University.Fresh porcine bladders were offered by Animal Experimental Center of Southern Medical University.METHODS:Whole bone marrow culture and successive adherence method was used to culture rat BMSCs in vitro.Flow cytometry was employed to detect surface antigen.Eradicator washing method was applied to prepare porcine BAM and measure its purity and characteristies.Third passage of BMSCs were inoculated in BAM and cultured in a medium containing vascular endothelial growth factor(VEGF165)(25 ng/L)in vive and in vitro to test compatibility.Cells cultured alone were considered to be controls for the in vivo trial,and materials non-implanted with cells were considered to be controls for in vitro trial.Suitable microenvironment was simulated to induce the differentiation of BMSCs.Four weeks and eight weeks later,compound materials were respectively removed to perform tissue section test.Simultaneously,immunohistochemistry keratin staining was conducted to examine regeneration of epithelial cells.MAIN OUTCOME MEASURE:Biocompatibility of BMSCs and BAM.RESULTS:①BMSCs were cultured by whole bone marrow method.Flow cytometry demonstrated that third passage of cells were positive for CD29(99.43%).②BAM had good biological characteristics.Homogen matrix and byssoid collagen appeared under a microscope.Compatibility trials showed good compatibility of BMSCs and BAM and well-growth cells.③Four weeks later,histological section test confirmed inflammatory cell infiltration,closely-arranged collagen and elastic fiber.Immunohistochemistry keratin staining showed lamellar and discontinuous simple epithelium.Eight weeks later,no inflammatory cell infiltration was found,and closely-arranged collagen and elastic fiber were detected.Immunohistochemistry keratin staining showed lamellar and continuous multiple epitheliums.CONCLUSIoN:With good compatibility,BMSCs and BAM appear to be an ideal material for bladder tissue engineering.