再造组织工程尿路移行上皮组织的实验研究

目的采用组织工程技术，将培养扩增的膀胱移行上皮细胞与可降解的聚乳酸／聚羟基乙酸共聚物（polylactical／glycolic acid copolymer，PLGA）支架材料复合并植入裸鼠体内进行培育，探讨构建尿路移行上皮组织的可行性。方法取幼兔膀胱，机械分离与酶消化法获取膀胱移行上皮细胞，并在体外行原代培养与传代扩增，将第4～5代扩增的上皮细胞接种至8个PLGA聚合材料的表面作为实验组，4个单纯支架材料作为对照组。实验组：细胞材料复合体培育4周和8周各4个，对照组：单纯支架培育4周和8周各2个，分别植入各组裸鼠体内进行培育，按各时间点取出标本后进行大体观察、组织学及免疫组织化学检测。结果实验组标本经HE和马森染色，4周显示在材料表面形成数层上皮细胞；8周移行上皮细胞进一步增殖形成上皮组织，抗角蛋白免疫组织化学染色，胞浆呈棕黄色阳性。对照组4周和8周经HE染色材料表面见少量成纤维细胞沉积，马森染色呈蓝色，经抗角蛋白免疫组织化学染色为阴性。结论采用组织工程技术可再造人尿路移行上皮组织，为尿路组织工程进一步实验研究奠定了基础。     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的：研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架。方法：应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物。采用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性;采用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态。结果：网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性。尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞,长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架贴附紧密,适度伸展并有基质分泌。结论：网状尿道支架适合尿道移行上皮细胞黏附生长．可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道。     

采用组织工程技术在裸鼠体内再造膀胱组织结构的研究

目的：探讨应用组织工程的方法在裸鼠体内再造膀胱的可行性。方法：体外分离、培养、扩增犬的膀胱移行上皮细胞和平滑肌细胞，分别接种于预制成囊样结构的聚乳酸-聚羟乙酸（PLGA)聚合物支架材料的内、外两侧表面，形成细胞-支架材料复合体，体外37％℃培育48h后植入到裸鼠背部皮下，20、40和60d时分别取材，进行组织学和免疫组织化学分析。结果：细胞-支架复合体经体内培育20-60d，在外观上形成了与支架材料形状基本一致的新生组织，组织学和免疫组织化学分析结果表明，其外壁由多层的平滑肌细胞构成，内壁由数层移行上皮细胞层构成，在组织结构上类似于正常膀胱壁。结论：在裸鼠体内构建了具有膀胱结构的基本特征的膀胱器官，提示采用组织工程技术构建膀胱器官是可行的。     

大鼠膀胱移行上皮细胞的原代培养研究

为建立膀胱移行上皮细胞的体外培养方法 ,我们对Wistar大鼠膀胱粘膜上皮细胞的原代培养方法进行研究。材料与方法　Wistar大鼠雌 5只、雄 15只 ,平均体重 30 0 g左右。采用乙醚吸入麻醉 ,无菌条件下游离大鼠全膀胱 ,标本放入含青霉素、链霉素的PBS液中漂洗。采用两种方法分别收集膀胱移行上皮细胞 :(1)改良的Roszell方法[1] 暴露膀胱粘膜 ,加入DispaseI酶 4℃消化 ,用器械刮取上皮 ,收集于混合消化液中 ,37℃振荡消化 ,用吸管吹打成单细胞悬液 ,PBS洗涤 2遍 ,收集上皮细胞 ,加入无血清培养液。细胞悬液接种于预涂胶原的培养板中 ,孵箱…     

犬膀胱移行上皮细胞的体外连续培养及生物学特征观察

目的探讨犬膀胱移行上皮细胞体外连续培养和纯化方法,为进一步构建组织工程尿路上皮组织提供实验依据。方法无菌获取幼犬膀胱组织,0.125%胰蛋白酶消化获取单细胞悬液,于上皮细胞无血清培养基中培养。采用0.05%胰蛋白酶和0.02?TA消化、纯化细胞,动态观察细胞形态变化和增殖情况,MTT法绘制生长曲线;透射电镜观察细胞超微结构;SABC法对培养的细胞进行免疫组织化学鉴定;并采用流式细胞仪检测不同代次细胞的细胞周期和倍体水平。结果采用酶消化法能从4～6cm2的膀胱黏膜组织中分离获取(1～5)×106个细胞。原代培养接种24h后可见大量细胞贴壁,第7天细胞生长融合达80%～90%,细胞排列呈典型的铺路石样。MTT法测定第3代细胞在传代培养7d生长达高峰;传代并纯化的细胞纯度高,无成纤维细胞污染,至第4～6代细胞仍保持良好形态。透射电镜下,可见上皮细胞特征性张力微丝和细胞间桥粒连接。细胞角蛋白AE1/AE3免疫组织化学染色,胞浆呈棕黄色阳性反应。不同代次细胞均为二倍体细胞。结论酶消化法能从较少的膀胱组织中分离获取足量、较纯的膀胱移行上皮细胞,细胞在无血清培养基中能连续传代扩增,可以满足进一步构建组织工程尿路上皮组织的需要。     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的:研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架.方法:应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液,接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物.应用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性,并用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态.结果:网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性.尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞;长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架紧密贴附,适度伸展并有基质分泌.结论:网状尿道支架适合尿道移行上皮细胞黏附生长,可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道.     

阴道粘膜上皮细胞的分离培养和鉴定

目的 初步建立阴道粘膜上皮细胞体外培养方法,为阴道粘膜上皮研究提供实验模型.方法 取雌性新西兰大白兔阴道粘膜组织小块,胶原酶Ⅳ和胰蛋白酶联合消化分离法收集上皮细胞,接种于角朊细胞无血清培养液中静置培养、传代.动态观察细胞生长增殖情况,扫描和透射电镜观察超微结构,流式细胞仪测定细胞增殖周期,并进行免疫组织化学鉴定.结果 体外培养的阴道粘膜上皮细胞为二倍体细胞,增殖状态良好,细胞间可见桥粒连接,免疫组化角蛋白染色阳性.细胞的超微结构和免疫组化染色均具有上皮细胞特征.结论 在本实验条件下,体外培养的阴道粘膜上皮细胞具有较好的增殖能力,可作为阴道粘膜上皮研究的理想实验模型.     

人体尿道粘膜上皮细胞的连续培养

目的：探讨人尿道粘膜上皮细胞体外培养技术，为构建组织工程化尿道提供种子细胞。方法：取尿道下裂患者尿道粘膜，经中性蛋白酶和胰蛋白酶消化，获取粘膜上皮细胞接种于含8％胎牛血清培养基中连续培养，观察细胞形态变化及生、增殖过程。结果：体外培养细胞长满后呈上皮细胞特有的铺路石样外观，体外可连续传9-10代，生长期50-60d。细胞角质蛋白免疫组织化学染色阳性，透射电镜下可见上皮细胞间特有的桥粒结构。结论：尿道粘膜上皮细胞可在体外连续培养，4代以内的培养细胞可用于组织工程化尿道的构建。     

以L929细胞为滋养层的尿道黏膜上皮细胞体外培养

目的 探索尿道黏膜上皮细胞体外培养的技术和方法，为进一步采用组织工程技术构建尿道黏膜组织奠定基础，并为尿道黏膜的生理、药理、毒理学及微生态研究提供实验模型。方法 取刚离乳的雄性新西兰幼兔尿道黏膜组织，分别以DispaseⅠ消化液和混合消化液消化成单细胞悬液，以差速贴壁法排除成纤维细胞，接种后以L929细胞为滋养层细胞进行培养，定期换液，细胞生长、增殖至80％～90％融合时传代。细胞进行常规HE染色、流式细胞仪检测，以扫描电镜、透射电镜观察其超微结构。再分别设立实验组(n=20)、阳性对照组(正常尿道黏膜组织石蜡切片，n=20)及阴性对照组(成纤维细胞铺片，n=20)行免疫组织化学染色。结果 原代培养10天左右细胞逐渐生长融合成片，如铺路石状，细胞大小均一。上皮细胞为二倍体细胞，生长期内均为单一的上皮细胞，无成纤维细胞混杂生长，细胞可传11～13代，成活50～60天。结论 新西兰幼兔尿道黏膜上皮细胞可在体外进行培养，在一定时间内保持增殖活力，为构建组织工程化尿道奠定了基础，且为尿道黏膜的体外研究提供了实验模型。     

大鼠胰腺癌细胞系R-PC的建立及其生物学特性

目的 建立1株大鼠源胰腺癌细胞系R-PC。方法 利用胰腺包膜下埋二甲基苯并蒽（DMBA）致癌药物的方法建立大鼠胰腺癌模型，用组织块法和胶原酶消化法进行原代培养，观察细胞形态学特征，研究细胞生长状态，测定细胞周期，计数染色体数目。组织化学染色和异种移植。结果 经过11个月的体外培养，连续传代80余次；细胞生长迅速，倍增时间为34h；染色体数目在42-76之间。免疫组织化学染色显示细胞角蛋白、细胞角蛋白19和细胞角蛋白20阳性；异种移植成瘤率为100％。结论 R-PC是高度恶性的大鼠胰腺癌细胞系，为体内和体外研究恶性细胞的增值和发展提供了优秀工具。     

两种体外培养兔膀胱变移上皮细胞方法的比较

目的 比较组织贴块法和酶消化法体外培养兔膀胱变移上皮细胞的优缺点,探索简单、高效培养膀胱变移上皮细胞的方法,为膀胱组织工程种子细胞的培养提供实验基础。方法 采用组织贴块法和酶消化进行兔膀胱变移上皮细胞体外培养,相差显微镜下观察其生长特点,应用细胞免疫荧光染色对细胞进行鉴定,测定细胞生长曲线了解细胞生长的基本规律。结果 培养的细胞具有上皮细胞的典型形态,生长曲线呈“S”形,角蛋白（CKAE1/AE3）单克隆抗体免疫荧光染色阳性,波形蛋白染色阴性。结论 组织贴块法和酶消化法均能成功培养出兔膀胱变移上皮细胞。组织贴块法原代培养时间长,但是成功率高。酶消化法可以在较短时间内获取大量细胞,但是操作步骤多,细胞容易污染或损伤。     

一氧化氮合成酶在膀胱癌中的表达及意义

目的　了解三种类型一氧化氮合成酶 (NOS)在膀胱癌中的表达情况 ,探讨其与肿瘤发生发展的关系。　方法　对 2 5例开放手术切除的膀胱癌组织标本进行三种NOS免疫组织化学染色 ,自动图像分析仪对染色程度分级 ;6例肾移植供体正常膀胱组织标本作为对照。　结果　诱导型NOS(iNOS)在肿瘤上皮细胞呈阳性表达 ,在正常膀胱移行细胞不表达或仅微弱表达 (P <0 .0 5)。内皮细胞型NOS (eNOS)在肿瘤基质毛细血管内皮细胞中的表达阳性率 1 0 0 % ,高于对照组中的 67%。两组标本中神经型NOS(nNOS)表达部位及强度相似。统计学分析显示iNOS和eNOS表达与肿瘤分级、分期无明显相关性 ,P均 >0 .0 5。　结论　iNOS及eNOS在膀胱癌的高表达可能与肿瘤的发生发展有关。     

Monoclonal antibodies against transitional cell carcinoma for detection of malignant urothelial cells in bladder washing

The diagnosis of transitional cell carcinoma by cytological examination of exfoliated urinary cells is important in the early detection and followup of patients with this disease. Proper interpretation requires a skilled pathologist. Accuracy also is influenced by collection methods and nonmalignant pathological conditions of the bladder. An immunocytochemical technique using monoclonal antibodies G4 and E7 successfully identified tumor-associated antigens on the surface of transitional carcinoma cells obtained by bladder washings. The method, which uses immunoperoxidase staining, was compared to conventional Papanicolaou staining of bladder washings from 75 patients with and without transitional cell carcinoma. Patients were divided into 4 groups: group 1 (nontumor control)--15 patients with no pathological condition of the bladder or nonmalignant urological diseases, group 2 (nontransitional cell carcinoma)--19 patients with other urological malignancies, group 3-18 patients with active transitional cell carcinoma and group 4-23 patients with a history of transitional cell carcinoma but no evidence of tumor at the time of the washing. The incidence of positive staining in these groups was 0, 5, 78 and 0 per cent, respectively. The diagnostic value of immunoperoxidase staining was similar to that of Papanicolaou staining in the control group and in patients with high grade transitional cell carcinoma, and provided specific morphological criteria not possible by conventional cytology studies. Interpretation of immunoperoxidase staining was difficult in washings with a large number of inflammatory cells if endogenous peroxidase activity was not blocked properly. The application of the immunoperoxidase staining method for diagnosis of low grade tumor is under further investigation.     

血管生成素-2在膀胱移行细胞癌中的表达及意义

目的　初步探讨血管生成素 2 (Ang 2 )在膀胱移行细胞癌组织中的表达及其与临床分期、病理分级的关系。方法　应用免疫组织化学S P法检测 4 3例初治膀胱癌及 2 8例正常膀胱组织中的Ang 2表达水平 ,并与临床资料对照进行统计分析。 结果　正常膀胱组织中未见Ang 2阳性染色 ;4 3例膀胱移行细胞癌中Ang 2阳性染色者 2 1例 ,Ang 2在许多膀胱癌细胞和癌组织中微血管内皮上呈强阳性染色 ,且染色率随膀胱癌病理分级、临床分期的上升而升高 (P <0 .0 5 )。结论　①Ang 2促进肿瘤新生血管形成 ,参与膀胱癌的发生和发展。②Ang 2的表达与膀胱移行细胞癌的临床分期、病理分级正相关。     

Growth characteristics of cultured human normal bladder epithelial cells: a comparison with urothelial carcinoma cell cultures

We present the in vitro growth characteristics and morphology of the first established long-term culture of human normal bladder epithelium. Normal bladder cells were grown with a mean generation time of 44 h and a life span of 33 population doublings in 12 weeks. The cells exhibited contact inhibition of growth. The cultures were morphologically examined and compared with transitional cell carcinoma in culture.     

一氧化氮合酶在膀胱癌中的表达及临床意义

目的：探讨一氧化氮（NO）在膀胱肿瘤中的作用及临床意义。方法：采用免疫组织化学方法对58例膀胱移行细胞癌标本（实验组）及14例良性膀胱组织标本（对照组）进行一氧化氮合酶（NOS）抗体染色，观察表达结果与肿瘤生物学特性之间的相关性。结果：诱导型NOS（iNOS)在实验组中的阳性表达率明显高于对照组（P＜0．01），其表达程度与肿瘤分级、分期无关（P＞0．05）；内皮型NOS（eNOS）在实验组和对照组血管内皮细胞都有表达，但前者表达较强；而在对照组中多呈不连续的表达。结论：NO在膀胱移行细胞癌的发生、发展中起重要的作用。     

Primary culture of human bladder carcinomas and establishment of human bladder carcinoma cell line by serum-free culture

It was possible to cultivate cells from bladder carcinoma tissues in 4 cases out of 6 without the overgrowth of fibroblast. A new human transitional cell carcinoma cell line (HAMT-1) was established in longterm tissue culture by using serum-free medium (BEM-841) which had been developed by us. The tissue for culture was taken from a 61-year-old Japanese male with grade 3 transitional cell carcinoma of the bladder. The microscopic features of the cell in cultures and of the tumors developed in nude mice resembled closely that of the original tumor. Electron microscopy of the cultured cells and the tumors developed in nude mice revealed characteristics of the epithelial origin of these cells with microvilli, junctional complex and scarce filament formation. Blood TPA level of the nude mouse with the transplanted tumor was equally high as that of the patient from whom the original tumor had been taken. The cells were anchorage independent in the serum-free medium but anchorage dependent in the medium containing 5% FCS. Anchorage dependency could not be restored by the addition of collagen and fibronectin. The doubling time of the cells were 18-20 hours. The chromosome counts of the cell line ranged from 59-78 with a modal count of 74.     

膀胱尿路上皮细胞癌CD44v6和CD44v8蛋白的表达及临床意义

目的 探讨C044v6和CD44v8蛋白在膀胱尿路上皮细胞癌中的表达及其与临床病理的相关性.方法 应用免疫组织化学技术检测60例膀胱尿路上皮细胞癌和10例正常膀胱粘膜CD44v6和CD44v8蛋白的表达.结果 CD44v6在10例正常膀胱粘膜无表达,60例膀胱尿路上皮细胞癌中阳性表达23例(38.3%),阳性表达与肿瘤的病理分级,TNM分期相关.CD44v8在10例正常膀胱粘膜无表达,60例膀胱尿路上皮细胞癌中阳性表达是29例(48.3%),阳性表达与肿瘤的病理分级,TNM分期相关.结论 膀胱尿路上皮细胞癌组织中CD44v6和CD44v8有相关性,可作为判断膀胱尿路上皮细胞癌分级,分期的一个参考指标,联合检测可以可提高判断的敏感性.     

Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder

OBJECTIVE: The insertion of absent or underexpressed genes into cancer cells to alter their malignant phenotype is an important potential application of available gene therapy technology. One of the more common viral vector systems that has been extensively studied for this purpose are the replication-deficient adenoviruses (Ad). Adenoviral infection of cells is mediated through a complex pathway, initiated following viral-cell attachment. Adenoviral-cell attachment occurs following interactions with a 46-kDa transmembrane protein with high affinity for both the Coxsackie and adenovirus, designated the CAR (Coxsackie and adenoviral receptor). Additional important cell-viral interactions that occur involve the alpha(v)-based integrins, specifically alpha(v)beta3 and alpha(v)beta5. The purpose of the present study was to determine the extent of expression and localization of the known Ad receptor proteins (CAR, alpha(v)beta3, and alpha(v)beta5) in normal and cancerous human bladders. MATERIAL AND METHODS: Frozen tissue samples of normal bladder and invasive transitional cell cancers of the bladder were evaluated. Tissue blocks containing muscle-invasive transitional cell carcinoma (TCC) were obtained following radical cystectomy, which were performed at our institution. Thirty-two invasive transitional cell bladder tumors were evaluated, each with a matched sample of histologically normal-appearing bladder used as a control. Four additional samples of normal bladder were obtained from patients with no evidence of disease of the bladder and served as further controls. Three additional cases of invasive bladder cancer with no matching normal tissue were also evaluated. Identification of the CAR receptor was performed using the anti-CAR mouse monoclonal antibody designated RmBC. The integrins alpha(v)beta3 and alpha(v)beta5 were identified using the mouse monoclonal antibodies designated LM609 and P1F6 respectively. All slides were evaluated by two of the authors (M.B., B.B.) without knowledge of the clinical and pathological data. RESULTS: Normal bladder: Normal bladder mucosa demonstrated a marked positivity for CAR in 29/35 (82.8%) cases. In contrast, normal transitional epithelial cells were uniformly negative when tested for the integrins alpha(v)beta3 and alpha(v)beta5. Subepithelial tissues, specifically the connective tissue components of the lamina propria and deep muscle wall of the bladder, were positive for alpha(v)beta3 and for alpha(v)beta5 in 61 and 75% of samples, respectively. Endothelial cells associated with the various layers throughout the bladder uniformly expressed both integrins and served as a consistent internal control for both antibodies. An almost identical staining pattern of the endothelium was observed using LM609 and P1F6 in all samples tested. Bladder transitional cell carcinoma: CAR immunoreactivity against TCC cells was uniformly decreased compared to normal transitional cells. Nine tumors exhibited a weak positivity for CAR while the remaining samples were negative. In some cases, the absence of CAR positivity was associated with histological evidence of carcinoma in situ. In 6 cases, it led to the identification of small regions of carcinoma in situ that were not noted on primary pathological evaluation. Peritumoral connective tissue expressed both integrins in the majority of cases, similar to the pattern described above for normal bladder. Transitional cell cancers demonstrated a similar pattern of expression of alpha(v)beta5, in which all tumor cells exhibited minimal or no staining. CONCLUSIONS: The success of all viral-mediated gene therapy strategies relies on the ability of the vector to efficiently deliver its genetic material to a target cell population. In the current study, we demonstrate that the bladder epithelial layer consistently expresses high levels of CAR. Deeper layers of the epithelium also express CAR, including the basal layer cells. A decrease in the expression of CAR appears as an early event in bladder carcinogenesis. We observed that both alpha(v)beta3 and alpha(v)beta5 are strongly expressed in muscle cells surrounding the neoplastic cells, as well as within the peritumoral connective tissue. In cases of invasive bladder cancer that have lost CAR expression, an adenoviral vector may still be utilized through the less efficient interactions with the integrins. Bladder tumor tissue may be less susceptible to an adenoviral-mediated gene therapy approach in which a significant percentage of tumor cells require transduction. Adenoviral uptake by tumor or peritumoral cells with subsequent gene transfer could be predicted by the level of CAR and alpha(v)-based integrin expression. This would enhance our ability to identify those patients whose tumors would be more susceptible to Ad-mediated gene delivery as part of an antitumor treatment.