膀胱ICCs细胞SCF/c-kit信号通路研究现状

膀胱ICCs细胞是Smet和Jonavicius等于1996年首先在豚鼠和人逼尿肌发现的类似于胃肠道Cajal间质细胞（In-terstitial cells of Cajal,ICC）的一种特殊间质细胞。它是泌尿系平滑肌蠕动的起搏者,在膀胱逼尿肌兴奋收缩过程中起着重要作用。最新研究表明SCF/c-kit通路与ICCs细胞形态和功能的维持密不可分,信号通路的异常会导致ICCs表型和功能的转变,这一发现为之后的尿动力方面研究提供了新的平台。     

膀胱逼尿肌细胞的培养及鉴定

目的：建立膀胱逼尿肌细胞的分离、培养、扩增的常规方法。方法：采用胶原酶消化法分离大鼠膀胱逼尿肌细胞，在含15％胎牛血清DMEM中培养，观察细胞形态及扩增情况，用特异性抗α-SM-Actin抗体进行免疫组化鉴定细胞类型。结果：膀胱逼尿肌细胞生长照好，培养12h后细胞开始贴附瓶底，3～4d后可观察到细胞融合现象，7d可覆盖培养瓶底部75％～80％以上。α-Actin免疫组化染色鉴定，光镜观察梭形细胞胞浆内见纵行排列的棕黄色丝状物。结论：该培养方法简单、可靠，短期内可获得大量较纯的膀胱逼尿肌细胞。     

动脉平滑肌细胞和心肌细胞钾通道的膜片钳全细胞记录技术

目的：介绍一种动脉平滑肌、心室肌细胞急性酶分离方法，并在该分离技术的基础上采用膜片钳全细胞记录方式研究细胞钾离子通道的特性。方法：采用胶原酶Ⅰ(1mg／ml)、木瓜蛋白酶(5mg／ml)消化分离大鼠肺内动脉平滑肌细胞；用链霉蛋白酶E(1mg／ml)、胶原酶Ⅰ(1mg／ml)消化分离大鼠尾动脉平滑肌细胞；用链霉蛋白酶E(0．5mg／ml)灌流消化、分离得到豚鼠心室肌细胞。结果：以上分离的细胞数量多，形态正常，胞壁光滑完整，活性好。于4℃无钙液中保存，8h内可用于膜片钳全细胞记录。记录出的外向电流可被钾通道阻断剂CsCl及TEA所阻断。结论：用酶急性分离的动脉平滑肌和心室肌细胞应用于膜片钳研究中具有快速、经济、简便易行等优点。     

PBOO对豚鼠膀胱卡哈尔间质细胞形态结构的影响

目的探讨豚鼠膀胱流出道梗阻(PBOO)对膀胱卡哈尔间质细胞(ICC)形态结构的影响。方法建立雌性豚鼠PBOO模型,并根据尿动力学检测结果将模型分为PBOO后逼尿肌不稳定(DO)组和PBOO后逼尿肌稳定(DS)组,以假手术组作为对照,饲养4周后取膀胱组织,制成全层组织铺片和冰冻组织切片,c-kit免疫荧光染色分别观察各组膀胱肌层和黏膜下ICC的形态和分布情况。结果PBOO4周后,DO组豚鼠膀胱出现排尿频率、排尿压力的增加(n=12,P<0.05),并且观察到明显的储尿期膀胱不稳定收缩。对照组、DS组和DO组豚鼠膀胱均观察到c-kit染色阳性且呈典型长梭形有突起的ICC,主要分布于平滑肌肌束间和黏膜下层;在DO组豚鼠的膀胱中c-kit染色阳性的ICC分支突起增加且互相连接呈网络状,而在对照组和DS组中ICC呈散在分布,细胞之间联系较松散。结论在PBOO所致DO豚鼠膀胱中ICC发生了形态和结构的变化,相互之间连接呈网络状,ICC的形态和结构的变化可能是逼尿肌不稳定的重要发病基础。     

食管癌TIL两种分离诱导培养方式效果的比较

目的　寻求有效地从肿瘤手术标本中分离出较多的TIL前体细胞并保持高度增殖能力和抗瘤生物活性的方法。方法　利用文献介绍的直接分离诱导培养法和酶消化分离诱导培养法对食管癌手术标本进行TIL细胞分离 ,将分别获得的TIL前体细胞数 ,诱导培养增殖速度及抗癌细胞生物活性的效果等进行比较。结果　直接分离诱导培养法获得的前体TIL细胞数少 ,但诱导培养增殖的速度快。而酶消化分离诱导培养法 ,获得的前体TIL细胞数多 ,但诱导培养增殖的速度慢。两法分离诱导培养增殖的TIL抗肿瘤活性是一致的 ,经统计学处理无显著性差异 (P <0 .0 5 )。结论　直接分离诱导培养法 ,操作简单 ,节省酶试剂 ,并能保持TIL的抗肿瘤生物活性 ,是一种较理想的TIL分离方法。     

改良法体外培养大鼠成肌细胞的实验研究

目的:探索一种新型体外分离培养新生大鼠骨骼肌成肌细胞的途径.方法:取新生Wistar大鼠的四肢骨骼肌适量,采用混合酶一步消化分离获取成肌细胞,将差速贴壁法和胰酶消化法相结合进行纯化,观察分离纯化成肌细胞的形态学特点、生长状况,绘制细胞生长曲线,利用扫描电镜观察成肌细胞表面结构并进行结蛋白免疫细胞化学特异性鉴定.结果:采用改良方法分离成肌细胞产量高、耗时短;在培养过程中双重纯化获取的细胞生长状态良好,接种后3～5 d增殖达高峰;在扫描电镜下可观察到成肌细胞膜表面有微绒毛突起;体外培养的成肌细胞中94%的细胞胞质呈结蛋白抗体染色强阳性.结论:改良方法分离纯化大鼠骨骼肌成肌细胞效果理想,是成肌细胞体外培养的有效途径之一.     

子宫内膜异位症异位内膜细胞原代培养方法的改良

目的：建立一种简易的人异位子宫内膜细胞体外原代培养方法。方法：通过胰蛋白酶消化、贴壁纯化等技术,分离及培养10例子宫内膜异位症患者的异位子宫内膜细胞。在光学显微镜下观察细胞形态,以免疫细胞化学法鉴定细胞类型。结果：8份标本获得成功,培养的异位内膜细胞均有腺上皮细胞和间质细胞两种形态细胞。膜上皮细胞和基质细胞分别经角蛋白单抗和波形蛋白单抗免疫组化染色为阳性,腺上皮细胞平均生长时间为5周,间质细胞平均生长时间为14周。结论：成功建立了子宫内膜异位症异位内膜细胞体外原代培养方法,该改良培养法经济、简单、高效。     

豚鼠前庭单个毛细胞的分离

从７只豚鼠前庭中取出壶腹嵴、位觉斑，放置在胶原酶溶液中孵育５０ｍｉｎ～６０ｍｉｎ后行机械分离。可获得均含有皮板和纤毛的两种类型活性毛细胞８３（±２３）个，其中Ⅰ型毛细胞呈烧瓶状，细胞中部有明显的颈；Ⅱ型毛细胞呈圆形或椭圆形，胞体比Ⅰ型毛细胞小；Ⅰ、Ⅱ型毛细胞的比例是６２１９；分离后５ｈ仍有４０％的毛细胞具有活性。另外，笔者还观察到失活的早期变化是胞内出现颗粒。实验结果表明，酶消化后辅以机械分离，可获得大量单个、离体的活性毛细胞，并且能够进行哺乳动物前庭的生理学研究。     

豚鼠壶腹Cajal样细胞的分布及Oddi括约肌肌电活动观察

目的 研究成年豚鼠壶腹处Cajal样细胞(ICLC)的分布及Oddi括约肌(SO)的肌电活动.方法 取成年豚鼠壶腹全层铺片,c-Kit免疫细胞化学染色观察ICLC的分布情况;采用黏膜接触电极记录豚鼠SO的肌电活动.结果 壶腹的外侧壁和内侧壁均可观察到c-Kit阳性ICLC,位于环形和纵行平滑肌层内以及两层平滑肌之间.肌内的ICLC细胞呈梭形.肌间的ICLC胞体呈椭圆形或三角形,发出3个以上的有分支的突起,这些细胞相互连接排列成网络状,与十二指肠的肌间丛Cajal间质细胞(IOCs)十分相似.在壶腹外侧壁的内面ICLC与十二指肠的深肌丛ICCs相似.壶腹的内侧壁肌层有大量的ICLC,与平滑肌细胞平行走行.在壶腹开口处,ICLC围绕开口形成一个ICLC环.在SO记录到自发节律性的肌电活动波.结论 壶腹内的ICLC可能参与SO自主节律性运动的调控并与SO运动障碍的发生有关.     

兔原代胸主动脉平滑肌细胞培养方法及不同浓度血清培养基对其增殖的研究

目的探讨体外分离培养兔原代胸主动脉平滑肌细胞（vascular smooth muscle cells, VSMCs）技术及在不同浓度血清培养基下生长情况。方法采用组织贴块法进行原代培养,分别用不同浓度血清培养基（10％、15％、20％）对原代VSMCs进行培养．胰酶消化法传代,用倒置显微镜对原代培养兔平滑肌细胞进行形态学观察并鉴定,MTT法测定增殖能力。结果体积分数20％血清的DMEM培养液培养的原代细胞生长出现明显对数期。培养5代的兔平滑肌细胞纯度达97％以上：镜下可见平滑肌细胞生长,免疫组化提示平滑肌细胞肌动蛋白阳性表达,细胞生长第4～5d内光密度值变化较明显。结论本法能有效提高兔动脉平滑肌细胞原代培养的成功率,为研究血管平滑肌细胞生物学行为提供了有效模型。     

Tetrahydroporphyrin-tetratosylat (THPTS): A near-infrared photosensitizer for targeted and efficient photodynamic therapy (PDT) of human bladder carcinoma. An in vitro study

BackgroundEfficacy of PDT in muscle-invasive bladder cancer is hampered by low tissue penetration of most photosensitizers by short excitation wavelength. THPTS is excitable at near-infrared (760 nm) allowing tissue penetration up to 15 mm. We examined the cellular effects of THPTS-PDT in human bladder cancer cells.Material and methodsWe used four human transitional carcinoma cell lines, epithelial bladder progenitors (HBLAK) and bladder smooth muscle cells (HBSMC). We used flow cytometry to examine pharmacokinetics of THPTS, confocal laser scanning microscopy to analyze subcellular localization and production of reactive oxidative species (ROS), examined cytotoxicity and cell death pathways (qRT-PCR).ResultsTotal uptake varied between cell lines and was significantly high in HBLAK and HBSMC. Lysosomal localization was mainly seen in cancer cells and HBLAK, while THPTS was distributed throughout the cytoplasm in HBSMC. Significant ROS production was detected 30 min after THPTS-PDT. Growth arrest occurred within 4 h and resulted in apoptotic and necrotic cytotoxicity after 24 h. Cytotoxicity was dose-dependent and specifically high in cancer cells and HBLAK and significantly low in HBSMC.ConclusionTHPTS-PDT induces cellular mechanisms leading to cellular growth arrest, apoptosis and necrosis in human bladder cancer cells. These effects are only partly dependent on the total amount of THPTS uptake and rather dependent on its subcellular compartmentalization. HBSMC are hardly affected by THPTS-PDT confirming tumor specificity and safety. THPTS is a promising new photosensitizer with the unique advantage of deep tissue penetration allowing the treatment of solid tumors and warranting further animal studies.     

miRNA-218在膀胱癌中表达及意义

目的探讨miRNA-218在膀胱癌细胞中的表达及其对膀胱癌5637细胞活性与侵袭增殖能力的影响。方法采用RT-PCR法检测正常膀胱细胞与膀胱癌细胞中miRNA-218的表达情况;分别在膀胱癌5637细胞中加入miRNA-218序列模拟物(miRNA-218 mimics)与无关序列模拟物(scramble mimics)后,采用MTT法检测细胞活性、Transwell法检测细胞侵袭增殖能力。结果 miRNA-218在正常膀胱细胞中的表达明显高于低级别与高级别尿路上皮癌,差异有统计学意义(P<0.05)。与未处理的膀胱癌5637细胞比较,加入miRNA-218 mimics的膀胱癌5637细胞中,miRNA-218表达明显升高,差异有统计学意义(P<0.05);加入scramble mimics的膀胱癌5637细胞中,miRNA-218表达差异无统计学意义(P>0.05)。加入miRNA-218mimics的膀胱癌5637细胞的活性与侵袭增殖能力明显低于未处理的膀胱癌5637细胞与加入scramble mimics的膀胱癌5637细胞,差异有统计学意义(P<0.05)。结论 miRNA-218与膀胱癌的发生、侵袭转移有关,miRNA-218表达上调可抑制膀胱癌。     

膀胱移行细胞癌中促凋亡基因bak mRNA的表达及意义

目的：研究促凋亡基因bak在膀胱移行细胞癌组织中的表达。探讨bak基因在膀胱移行细胞癌发生演变过程中的意义。方法：应用原位分子杂交方法检测52例膀光移行细胞癌组织的bak mRNA的分布与表达。结果：52例膀胱移行细胞癌组织中bak基因mRNA表达的阳性率为57．7％,其阳 率随膀胱癌病理分级,临床分期的上升而降低,结论：促凋亡基因bak在大多数膀胱移行细胞癌细胞中有不同程度的表达。bak基因可能参与膀胱移行细胞癌的凋亡调节,并与膀胱移行细胞癌组织的分化有关。     

U100倍频双波长脉冲激光对兔膀胱黏膜的损伤效应

目的 观察不同频率、不同脉冲的U100倍频双波长脉冲激光对家兔膀胱黏膜的损伤效应。 方法 新西兰兔6只，避开膀胱三角区随机取四点进行实验，予置激光输出频率分别为3、10Hz，脉冲输出500、1000次，垂直接触照射膀胱黏膜，通过肉眼、光镜观察靶组织损伤的形态学改变。 结果 激光照射后，兔膀胱黏膜肉眼未见明显穿孔，光镜下可见激光照射处膀胱黏膜上皮细胞不同程度的脱落，固有层出血等病理改变，无肌层平滑肌细胞断裂等严重损伤；显示脉冲次数为500的损伤主要限于膀胱黏膜上皮浅层，脉冲次数为1000的损伤累及上皮全层。 结论 频率为10Hz，脉冲1000次的U100倍频双波长脉冲激光照射膀胱黏膜不会引起穿孔，但可引起上皮细胞脱落，固有层出血等病理改变。激光损伤程度与照射脉冲次数相关。     

地塞米松对哮喘豚鼠模型肺匀浆CC10的影响

目的:观察腹腔内注射地塞米松对豚鼠支气管哮喘模型肺组织Clara细胞蛋白10(CC10)的影响。方法:以27只健康豚鼠为实验动物,随机分为3组:即正常组、哮喘组和地塞米松(DEX)组。用卵蛋白致敏、雾化建立豚鼠哮喘模型,用酶联免疫吸附试验检测肺匀浆CC10的含量并观察其在组间的含量变化。结果:哮喘组豚鼠肺匀浆中CC10含量明显低于正常对照组(P<0.05);经DEX预处理后,肺匀浆CC10水平较哮喘组明显升高(P<0.05)。结论:CC10参与了哮喘的发病,可能是其中一种重要的内源性抗炎保护因子;而糖皮质激素在哮喘中的抗炎作用可能是通过提高CC10在肺组织的含量而实现的。     

31P NMR study of insulin effects on the isolated perfused rabbit urinary bladder

Insulin stimulates hexose transport, intermediary metabolism, and cell growth and development. These effects are well-documented in skeletal but not smooth muscle. 31P NMR spectroscopy was performed on rabbit urinary bladders (n = 4) to characterize insulin's actions on smooth muscle. The bladder and its vasculature were surgically isolated from the animal and perfused with a PSS/red blood cell perfusate. After a control steady state was achieved (approx 1-2 h), insulin (0.100 mU/ml) was added to the perfusate. Relative levels of intracellular phosphorylated compounds, pH, and free Mg2+ were measured and compared to control values. Also, extracellular pH and fractional volume were assessed using phenylphosphonate, a 31P NMR extracellular pH and volume indicator. Insulin induced significant increases in PCr (16 +/- 9%) at the expense of Pi, intracellular pH (delta pH 0.24 +/- 0.07), and fractional extracellular volume (49 +/- 1%). Intracellular free Mg2+ and extracellular pH did not change. These results indicate that in situ smooth muscle is sensitive to physiological levels of insulin. In fact, insulin improves the energy state of smooth muscle cells and the overall tissue perfusion.     

庆大霉素损伤后豚鼠前庭上皮STAT3mRNA的表达变化及意义

目的：观察庆大霉素损伤后豚鼠前庭上皮修复期细胞增殖和STAT3 mRNA的表达变化情况，探讨其生物学特性和意义。方法：采用原位杂交组织化学方法，检测庆大霉素损伤后豚鼠前庭上皮STAT3 mRNA的表达变化；采用Brdu体内标记和抗Brdu抗体免疫组化染色，检测庆大霉素损伤后豚鼠前庭上皮修复期细胞增殖情况。结果：在正常成年豚鼠前庭上皮中，STAT3 mRNA有低水平表达。庆大霉素损伤后1d组，STAT3 mRNA杂交信号最强，其后逐渐减少，21d组与正常对照组无显著差异。在正常对照组前庭上皮中无Brdu阳性细胞。各实验组均观察到Brdu阳性细胞，7d组最多。结论：庆大霉素损伤后，豚鼠前庭上皮钉AT3 mRNA表达增强，其时程变化与细胞增殖活动密切相关。STAT3在豚鼠前庭上皮损伤后的自发修复中可能起重要信号转导作用。     

Anwendung der optischen Kohärenztomographie (OCT) bei der Darstellung von Urothelerkrankungen der Harnblase

PURPOSE: The technique of optical coherence tomography (OCT) has significantly improved over the last few years. This new diagnostic procedure allows imaging of tissue structure of the bladder wall during cystoscopy with high resolution. MATERIALS AND METHODS: The penetration depth of OCT is limited to 2.5 mm. The resolution is approximately 15 microm. Fifty patients with different clinical conditions of the bladder were examined. Altogether 488 OCT images were generated. RESULTS: OCT of normal bladder mucosa clearly shows a differentiation between urothelium, lamina propria, and smooth muscle. Cystitis and metaplasia are characterized by blurring of the laminated structure and thickening of the epithelial layer. In malignant areas there is complete loss of the regular layered tissue structure. CONCLUSION: OCT improves the diagnosis of flat lesions of the urothelium. It has the potential for facilitating intraoperative staging of malignant areas in the bladder.     

Skeletal muscle metastasis from transitional cell carcinoma of the urinary bladder: clinicoradiological features

AIM: To define the clinicoradiological characteristics of skeletal muscle metastasis from transitional cell carcinoma of the urinary bladder. MATERIALS AND METHODS: A retrospective review of all patients with skeletal muscle metastasis was undertaken between January 1999 to December 2001. Patients suspected of having a metastasis on radiological examinations, and subsequently proven to have metastatic disease on histological examination were included in study. The clinical presentation and radiological features of five patients with skeletal muscle metastasis from bladder tumours were reviewed from hospital records. RESULTS: Twenty-four patients had skeletal muscle metastasis from various primaries. Of these five patients had previous or concurrent primary tumours in the bladder. Patients were aged between 27-70 years (mean 52 years), and all had persistent, localized pain with or without accompanying swelling. The muscles involved were psoas in three patients, adductor muscles of thigh in one and rectus abdominis in one. Four patients had radical cystectomy with urinary diversion (two ileal conduit and two orthotopic sigmoid neobladder). One patient presented with bladder tumour and concomitant muscular metastasis. All patients underwent helical computed tomography (CT) before confirmation of diagnosis by fine-needle aspiration (FNA) or biopsy. The typical appearance of low-density enhancing lesions on CT was mistaken for abscess in two patients and failure to respond to conservative treatment led to suspicion of metastasis. Diagnosis was proven histologically in all patients (FNA in three and biopsy in two). All patients had palliative chemotherapy (Mitomycin, Vincristine, Adriamycin and Cyclophosphamide). Two patients had local palliative 3500 rad radiotherapy for persistent pain. Mean survival was 8 months (range 6-12 months). CONCLUSION: Muscular metastasis from urothelial tumours typically presents with persistent localized pain with or without swelling. The characteristic low-density, ring-enhancing lesions on CT in a patient with previous or concomitant urothelial tumours should raise the suspicion of metastasis until proven otherwise. Prognosis is dismal.