脱细胞软骨基质来源的多孔支架复合山羊髓核细胞体内初步构建组织工程髓核的实验研究

[目的]探讨脱细胞软骨基质多孔支架复合PKH26标记的山羊髓核细胞体内异位构建组织工程髓核的可行性.[方法]制备脱细胞软骨基质来源的多孔支架,扫描电镜(scanning electron microscope,SEM)观察、天狼星红染色、HE染色观察、MTT毒性检测;分离山羊髓核细胞,通过倒置显微镜观察、番红O染色、Ⅱ型胶原免疫组化染色进行鉴定;将PKH26标记的山羊髓核细胞接种支架上,体外培养3d后进行LIVE/DEAD活性染色,将细胞支架复合物置入裸鼠皮下,培养6周,病理切片,荧光显微镜下观察,进行番红O、Ⅰ、Ⅱ型胶原免疫组化染色.[结果]扫描电镜观察支架孔隙相连通且分布均匀,天狼星红染色支架呈黄绿相间色,HE支架淡染,MTT检测细胞增殖曲线无统计学差异(P＞0.05);P1代髓核细胞呈软骨样细胞形态,番红O染色、Ⅱ型胶原免疫组化染色均阳性,PKH26标记后的细胞呈红色荧光;体外LIVE/DEAD染色细胞呈绿色荧光,体内培养6周后,带红色荧光的细胞填满支架孔隙,番红O、Ⅱ型胶原免疫组化染色阳性,Ⅰ型胶原免疫组化染色弱阳性.[结论]以脱细胞软骨基质多孔支架复合山羊髓核细胞在体内能够形成组织工程髓核样组织.     

人类肥大乳房乳腺上皮细胞的原代培养

目的 探讨人类肥大乳房乳腺上皮细胞的原代培养方法.方法 采用胶原酶消化培养法,在体外进行人类肥大乳房乳腺上皮细胞的分离培养,用倒置显微镜观察、细胞爬片、HE染色和细胞角蛋白免疫组织化学染色的方法对分离培养的细胞进行形态学观察和鉴定.结果 倒置显微镜下观察细胞形态呈鹅卵石型或多角型,部分形态不规则,增殖的过程中形成岛屿状闭合型的细胞群,细胞之间连接紧密.细胞HE染色可见细胞胞质呈粉红色或浅紫色,细胞核呈蓝紫色,圆形或椭圆形,核中可见深蓝色的染色体.免疫组化鉴定结果显示,培养的细胞胞浆内可见棕黄色的细胞角蛋白着色,表达上皮细胞特异的细胞角蛋白18.结论 应用酶消化法和条件培养液可以获得纯度较高的人类肥大乳房乳腺上皮细胞.     

组织工程骨-软骨复合组织种子细胞的获取与培养

目的:探索体外培养组织工程骨-软骨复合组织种子细胞的条件,并观察其部分生物学活性。方法:采用机械-酶消化法对3周龄新西兰大白兔耳软骨和关节软骨消化来获得软骨细胞,采用全骨髓贴壁法来获得骨髓间充质干细胞,对两种细胞进行原代和传代培养,通过倒置相差显微镜观察细胞形态、绘制生长曲线、免疫组化染色等对细胞进行生物学特性分析。结果:原代培养的软骨细胞以多角形或三角形为主,传代3次以后出现去分化。形态学和免疫组化显示细胞3代以内可以保持表型稳定,具有较强的增殖及分泌细胞外基质的能力,而且耳软骨及关节软骨细胞在实验中没有表现出明显的生物学差别。采用贴壁筛选法获得的BMSCs呈长梭形或多边形,生长曲线呈"S"形,Ⅱ型胶原免疫组化染色阳性,细胞生长增殖能力旺盛。结论:体外分离培养的软骨细胞和骨髓间充质干细胞符合组织工程要求,能够作为骨-软骨复合组织的种子细胞。     

毛囊单位促进组织工程皮肤形成的体外实验研究

目的 通过插入分离的毛囊单位,构建带有皮肤附属器的组织工程皮肤,探讨毛囊对组织工程皮肤形成的作用.方法 将头皮分离成毛囊单位,插入由Ⅰ型鼠尾胶原、成纤维细胞和角质形成细胞构建的组织工程皮肤模型中,浸没培养1周,气-液界面培养3周.通过镜下观察、HE染色和免疫组化检测组织工程皮肤和毛囊的生长状态.结果 相对于悬浮培养的毛囊,实验组毛囊体外生长期延长,结构更完整.HE染色显示,实验组组织工程皮肤可见毛囊和皮脂腺存在.免疫组化染色显示,带有毛囊的组织工程皮肤中可见反映基底膜完整性的Laminin和Ⅳ型胶原呈线状连续分布于表皮、真皮连接处;而反应表皮成熟度的CK4和CK10/13则呈阳性分布于基底上层非角化细胞.结论 复合毛囊单位可以促进组织工程皮肤表皮组织的分化和成熟.本方法为组织工程皮肤的构建和毛囊的体外培养提供了新的思路和方法.     

食管小细胞癌临床病理分析

目的探讨食管小细胞癌的临床病理特征及组织起源。方法收集食管小细胞癌9例，分析临床病理资料，进行光镜观察及免疫组化ABC法标记7种抗体。结果患者以中老年多见，肿瘤位于中下段，镜下形态分为燕麦细胞型、非燕麦细胞样型和混合细胞型小细胞癌，免疫组化染色CK、EMA均阳性，Syn、CgA 5例阳性。结论食管小细胞癌是一种高度恶性肿瘤，组织发生可能来自全能干细胞。     

子宫颈小细胞癌病理特点及诊断分析

目的:探讨宫颈小细胞癌(SCCC)的临床病理特点及诊断.方法:对10例SCCC进行组织形态学、免疫组化观察和文献复习.结果:组织学显示为3种不同分化的细胞区域,其中大部分为形态较一致的小细胞区,细胞散在、片状或梁索状,浸润性生长;少数区域呈鳞状细胞癌和腺癌分化.免疫组化:小细胞成分CK5/6、LCA、desmin阴性,CK8、Cg-A、synapsin,、NSN阳性.结论:SCCC是一种少见的高度恶性肿瘤,具有特征性的组织学改变,应与宫颈的小细胞鳞状细胞癌、类癌等进行鉴别,伴有鳞状细胞癌及腺癌分化的SCCC预后要比单纯小细胞癌差.     

睾丸大细胞钙化型支持细胞瘤临床病理观察

目的:探讨睾丸大细胞钙化型支持细胞瘤的临床及病理组织学特征。方法:运用组织学、免疫组化及组织化学技术对1例睾丸大细胞钙化型支持细胞瘤进行光镜观察及免疫标记,并结合相关文献对其临床表现、组织形态、免疫组化特点及治疗和预后等进行分析。结果:患者为青年男性,病理组织学表现为肿瘤细胞排列成巢状、梁状,细胞呈多角形,胞质嗜酸,核大空泡状,伴间质广泛钙化。免疫组化染色显示肿瘤细胞inh ib in(+)、S-100(+)和vim entin(+),PLAP(-)、SMA(-)、CK(-)、AFP(-)。组织化学染色阿辛蓝(+)、过碘酸雪夫氏反应(PAS)阴性。结论:大细胞钙化型支持细胞瘤是一种罕见的睾丸性索间质肿瘤;免疫组化有助于睾丸大细胞钙化型支持细胞瘤的诊断,鉴别诊断包括精原细胞瘤的管状型、睾丸间质细胞瘤(Leyd ig细胞瘤)、普通型及硬化型支持细胞瘤、雄激素不敏感综合征以及隐睾中的支持细胞结节等,手术切除预后良好。     

组织工程软骨构建的临床初步研究

目的:探讨利用组织工程技术在人体内构建软骨组织的可行性。方法:将体外培养扩增的第二代人肋软骨细胞,以细胞浓度50×106/ml与30%Pluronic-F127混匀,注入人耳后皮下或耳屏部位,3～6个月后在二期手术时部分取材,通过大体观察、组织学、电镜以及免疫组化等方法对新生软骨进行初步评价。结果:在3～6个月的随访中,观察到注射于耳后皮下的患者皮下有明显隆起,组织学检测显示有软骨陷窝和同源软骨细胞现象为成熟软骨组织,免疫组化染色有特异性的Ⅱ型胶原分泌。结论:采用体外培养的第二代人肋软骨细胞和Pluronic在人体内可构建组织工程软骨,证实了组织工程技术在人体内构建软骨组织的可行性。     

脱矿脱细胞骨基质环支架体外构建组织工程化椎间盘纤维环

目的:探计以脱矿脱细胞骨基质环为支架、纤维环细胞为种子细胞体外培养构建组织工程化椎间盘纤维环的可行性.方法:取兔椎间盘纤维环细胞培养,应用甲苯胺蓝染色和Ⅰ型、Ⅱ型胶原免疫组织化学染色进行鉴定.用纤维蛋白凝胶接种技术将兔椎间盘纤维环细胞接种到经脱矿脱细胞制备的骨基质环支架材料上,体外培养3个月.每月取培养的细胞支架复合体进行大体形态、HE染色光镜检查和扫描电镜观察,并用生化方法榆测羟脯氨酸、氨基葡聚糖(GAG)、脱氧核糖核酸(DNA)含量;逆转录聚合酶链反应(RT-PCR)方法检测Ⅰ、Ⅱ型胶原信使核糖核酸(mRNA)表达;免疫组化和蛋白质印迹方法检测Ⅰ、Ⅱ犁胶原蛋白表达.结果:培养的第1代细胞甲苯胺蓝染色呈异染性,Ⅰ型、Ⅱ型胶原免疫组化染色均可见阳性表达,表明培养的第1代细胞具有椎间盘纤维环细胞的表型特点.构建的复合体体外培养1、2、3个月时大体呈白色半透明样环状,有光泽,质韧,具有一定弹性,可扭曲;HE染色光镜下见支架孔洞被红染的组织填充,且空洞内的细胞密度逐渐增加;扫描电镜观察材料表面逐渐被组织填充;Ⅰ型、Ⅱ型胶原免疫组化染色均为阳性;培养2个月时复合体羟脯氨酸、GAG、DNA含量明显高于1个月时(P<0.01),3个月时与2个月时比较无显著性差异(P>0.05).各时间点复合体羟脯氨酸、GAG、DNA含量均低于正常纤维环(P<0.05或<0.01);培养1个月时复合体可检测到Ⅰ、Ⅱ型胶原mRNA和蛋白的表达.2个月时与1个月时比较有显著性差异(P<0.01),3个月时与2个月时比较无显著性差异(P>0.05).结论:以脱矿脱细胞骨基质环为支架、纤维环细胞为种子细胞构建的复合体在体外培养时,细胞能够保持表型特点、逐渐增殖和行使功能,此复合体可被鉴定为类纤维环组织,用其构建组织工程化椎间盘纤维环可行.     

骨髓间充质干细胞与骨组织共培养模型的建立

目的:建立骨髓间充质干细胞(BMSCs)与骨组织共培养模型,模拟体内成骨环境,以全面研究骨髓间充质干细胞及细胞支架复合物的生物性状.方法:通过插入式培养皿和培养板来构建细胞一组织共培养的模型,将新鲜兔骨碎粒及骨块与骨髓间充质干细胞共培养,观察共培养条件下细胞的形态学变化、ALP活性及矿化能力,免疫组化检测Ⅰ型胶原、骨钙素.结果:构建出骨髓间充质干细胞与骨组织共培养的模型.共培养的间充质干细胞同期ALP活性高于普通培养对照组,其Ⅰ型胶原、骨钙素免疫组化阳性,对照组Ⅰ型胶原免疫组化弱阳性、骨钙素免疫组化阴性.结论:体外构建的共培养模型部分模拟了体内成骨环境;经过共培养的细胞呈现成骨细胞的表型.     

Behavior of epithelial differentiation antigens (carcinoembryonic antigen, epithelial membrane antigen, keratin and cytokeratin) in transitional cell carcinomas of the bladder.

Results of an immunohistochemical study in normal urothelium and transitional cell carcinomas of the bladder are presented. Paraffin-embedded material was confronted with immunoantisera against carcinoembryonic antigen (CEA), keratin (K), cytokeratin (CK) and epithelial membrane antigen (EMA). Immunohistochemical findings confirm the changes in reactivity of dysplastic urothelium and carcinoma in situ for CEA, CK and EMA, in comparison with normal urothelium. Statistically significant differences were also found, depending upon tumor stage, in staining of transitional cell carcinomas for K and CK. Expression of CK correlated with the tumor differentiation grade: normal urothelium and well-differentiated carcinomas showed a specific pattern of immunostaining for the basal cells, this pattern being lost in poorly differentiated carcinomas.     

膀胱肉瘤样癌的诊断与治疗（附2例报告）

目的 提高对膀胱肉瘤样癌的认识和诊治水平。方法 分析2例膀胱肉瘤样癌患者的临床资料并结合文献进行讨论。2例患者均有肉眼血尿或伴有膀胱刺激症，膀胱镜检查为实体瘤，表面有坏死组织；病理表现，肿瘤主要由移行上皮癌细胞和恶性间叶细胞（梭形或多形性细胞）组成，之间可见移行过渡；免疫组化CK（＋）、CEA（＋）、SMA（＋）。结果 2例Ⅰ期治疗均行经尿道膀胱肿瘤电切，术后常规膀胱灌注，1例术后5个月死于肿瘤复发及多处转移；1例半年后复发改行部分膀胱切除，至今健在。结论 膀胱肉瘤样癌是一种高度恶性、预后差的肿瘤。确诊需依赖病理和免疫组织化学检查，早期诊断和采用膀胱部分切除或根治性切除，是改善预后的关键。     

Epidermal growth factor--interactions with normal and malignant urothelium: in vivo and in situ studies

Epidermal growth factor (EGF) is excreted in urine in high concentrations and thus incubates with bladder epithelial cells continuously. However, it is not known whether any urothelial cells can bind urinary EGF or respond to it. Using a monoclonal antibody (528) to the binding portion of the human EGF receptor, immunoperoxidase staining demonstrated that the basal cell layer of normal urothelium is richly endowed with cell surface EGF receptors while the superficial cell layer is not. Alternatively, superficial cells of premalignant and malignant urothelium have many surface EGF receptors. Intravesical EGF induces in vivo activity of ornithine decarboxylase and DNA synthesis in rat bladders, with nuclear thymidine incorporation being limited to the basal epithelial cell layer. These studies indicate that urothelium can respond to urinary EGF and that this response parallels the distribution of EGF receptors. These findings combined with the difference in EGF-receptor expression between malignant and normal cells indicate that urinary EGF may play a role in bladder tumor development and/or growth.     

膀胱癌尿脱落细胞CK20表达的临床意义

目的　探讨尿脱落细胞CK2 0表达在膀胱癌诊断中的意义。　方法　应用免疫组化技术检测 4 0例膀胱癌患者和 2 0例非肿瘤患者尿脱落细胞CK2 0的表达。　结果　非肿瘤组尿脱落细胞无CK2 0表达 ,肿瘤组尿脱落细胞CK2 0表达率为 5 3% ,CK2 0与HE染色结合检测 ,阳性率达90 %。复发组尿脱落细胞CK2 0表达率 80 % ,明显高于初发组的 4 3% (P <0 .0 5 )。CK2 0表达率与肿瘤分级、分期无相关性。　结论　尿脱落细胞CK2 0表达检测特异性高 ,可用于膀胱癌的早期诊断和术后随访     

Histogenesis of nonurothelial carcinomas of the urinary bladder from pre-existent transitional cell carcinomas. A histopathological and immunohistochemical study

The histogenesis of nonurothelial carcinomas of the urinary bladder is difficult to understand, since the bladder is normally lined exclusively by transitional cell epithelium. To gain more insights into the pathogenesis of nonurothelial carcinomas, the morphology and immunohistochemistry of transitional cell carcinomas (TCC), mixed transitional cell and nonurothelial carcinomas, and pure nonurothelial carcinomas were comparatively studied. Of papillary and of nonpapillary (solid) TCC (overall incidence 6.8%), 4.8% and 15.4%, respectively, disclosed foci of altered celllular and architectural phenotypes, consisting of squamous epithelium, pseudoglandular formations, and true glands with or without mucus production. The diverse phenotypic variants develop obviously by a metaplastic process as a result of the well-known inherent potential of the urothelium to undergo several pathways of cellular differentiation. There is strong evidence that squamous cell carcinomas arise secondarily from a squamous metaplasia and adenocarcinomas from metaplastic glandular epithelium within pre-existing TCC following complete carcinogenic transformation of the initially bland-looking metaplastic tumor cells. The metaplastic origin of nonurothelial bladder carcinomas is supported by immunohistochemical findings. The high molecular weight cytokeratin 34betaE12 identifies tumor cells with squamous characteristics, helping to explain the development of squamous cell carcinomas. Secretion of MUC5AC apomucin is assumed to play a central role in the histogenesis of nonurachal mucus-producing adenocarcinomas, including signet ring cell carcinomas. Metaplastic phenotypic variants of TCC should be recognized as distinct tumor entities with the potential to transform into nonurothelial carcinomas and thus possibly implying a poorer clinical outcome than typical, uniform TCC.     

Molecular pathways of urothelial development and bladder tumorigenesis

Bladder cancer is the fifth most common human malignancy and the second most frequently diagnosed genitourinary tumor after prostate cancer. The majority of malignant tumors arising in the urinary bladder are urothelial carcinomas. Clinically, superficial bladder tumors (stages Ta and Tis) account for 75% to 85% of neoplasms, while the remaining 15% to 25% are invasive (T1, T2–T4) or metastatic lesions at the time of initial presentation. Several studies have revealed that distinct genotypic and phenotypic patterns are associated with early vs. late stages of bladder cancer. Early superficial disease appears to segregate into 2 main pathways: (1) superficial papillary bladder tumors, which are characterized by gain-of-function mutations affecting oncogenes such as H-RAS, FGFR3, and PI3K, and deletions of the long arm of chromosome 9 (9q); (2) Carcinoma in situ, a “flat” high grade lesion considered to be a precursor of invasive cancer, is characterized by loss-of-function mutations affecting tumor suppressor genes, such as p53, RB, and PTEN. Based on these data, a model for bladder tumor progression has been proposed in which 2 separate genetic pathways characterize the evolution of early bladder neoplasms. Several molecular markers have been correlated with tumor stage, but the rationale for these 2 well-defined genetic pathways still remains unclear.Normal urothelium is a pseudo-stratified epithelium that coats the bladder, composed of 3 cell types: basal, intermediate, and superficial (“umbrella”) cells. We have identified a series of markers that are differently expressed in these distinct cells types, and postulated a novel model for urothelium development and configuration. Briefly, it is our working hypothesis that 2 distinct progenitor cells are responsible for basal/intermediate cells and “umbrella” cells, respectively. Basal and intermediate cells are characterized by a p63 positive phenotype, as well as expression of high molecular weight cytokeratins (CKs), such as CK5, CK10, and CK14. On the contrary, “umbrella” cells display a p63 negative phenotype and are characterized by expression of 2 specific low molecular weight CKs: CK18 and CK20. Neither urothelial stem cells nor bladder cancer stem cells have been identified to date. In this review, we will further expand on the issues discussed above.     

Carcinosarcoma of the bladder: a case report

Carcinosarcoma of the urinary bladder is a uncommon tumor with characteristic histopathologic and immunohistochemical findings; his histogenesis have still not been clear; the prognosis seems to be improved by radical cystectomy and adjuvants therapies. We report a case of 47 years old women suffering from suprapubic pains, dysuria and hematuria of five months duration and had a 10 cm suprapubic mass that was found on physical examination. Radiographically, the tumor invaded the dome of the urinary bladder and causes bilateral hydronephrosis. Microscopically it was an urinary bladder carcinosarcoma. Our objective is to discuss the histogenesis, the anatomoclinical and prognosis of these rare tumors.     

Fibronectin distribution in normal and malignant urothelium

Fibronectin is a glycoprotein that mediates the attachment of BCG to the murine bladder. To assess the potential role of fibronectin on bladder cancer cells as a specific substrate for BCG binding in man, a semi-quantitative method was employed to evaluate the presence of fibronectin on normal urothelium and bladder cancer. Monoclonal anti-fibronectin binding to normal and malignant urothelial tissues was evaluated by an immunoperoxidase assay. Human tumor cell lines were evaluated with mixed hemadsorption and immunoperoxidase assays. In both systems, immunoreactive fibronectin had low expression on unfixed normal and malignant urothelium. With fixation, immunoreactive fibronectin decreased on supporting stroma and increased in normal and malignant urothelium. Fibronectin distribution did not show tumor specificity either with fixed or unfixed specimens.     

The expression of vascular endothelial growth factor in transitional cell carcinoma of urinary bladder is correlated with cancer progression

Vascular epithelial growth factor (VEGF) regulates neovascualrization in malignant cells. VEGF as a mitogen is thought to alter cancer cell formation and tumor progression. We aimed to investigative the expression of the VEGF gene to evaluate their clinical significance in transitional cell carcinoma (TCC) of urinary bladder. Tissue samples from 161 patients with TCC were examined with an immunohistochemical stain for the expression of the VEGF gene. The expression rate was compared to 32 normal bladder mucosal samples obtained from transurehtral surgery from noncancer patients. The results revealed significant differences between normal urothelium (0%) and cancer tissue (54.7%) for the positive staining of VEGF protein (P < 0.001). With the progression of tumor grade and clinical staging, the positive rate of VEGF gene expression significantly increased. Expression of the VEGF gene in the invasive group was greater than that in the noninvasive group (P < 0.001). The results revealed that expression of the VEGF gene is proportional to the formation and progression of TCC. Therefore, abnormal expression of VEGF genes can be used as a prognostic marker in TCC of urinary bladder.     

膀胱非上皮性肿瘤的诊治

目的 总结膀胱非上皮性肿瘤的诊治经验。方法 对1953～2002年收治的28例膀胱非上皮性肿瘤患者的诊治情况进行总结、分析。结果 膀胱非上皮性肿瘤的主要临床表现为血尿、盆腔肿块、尿频、排尿困难等症状。主要辅助检查为B超、CT、膀胱镜检查及镜下活检。本组28例中,经术后病理检查,恶性肿瘤17例(占61．7％),有7种病理类型,分别为膀胱横纹肌肉瘤、膀胱小细胞癌、膀胱平滑肌肉瘤、膀胱恶性淋巴瘤、膀胱恶性纤维组织细胞瘤、膀胱脂肪肉瘤、膀胱黑色素瘤;良性11例(占39．3％),有4种病理类型,分别为膀胱海绵状血管瘤、膀胱壁纤维瘤、膀胱平滑肌瘤、膀胱嗜铬细胞瘤。11例良性肿瘤均完整切除或电灼、电切。17例恶性肿瘤中,膀胱部分切除术7例、膀胱全切除术9例、无法切除1例,有7例恶性肿瘤因复发多次行手术切除。17例恶性肿瘤患者均获随访,3年存活率47．0％(8／17)。结论 膀胱非上皮性肿瘤临床少见,病理类型复杂,恶性居多且预后较差,良性肿瘤预后较好。术前诊断率低,膀胱镜下深部活检可提高诊断率。手术是该病的主要治疗方法。良性肿瘤应完整切除,恶性肿瘤应争取广泛切除,结合其病理特点辅助放化疗可能提高疗效。