【作者】 史涛坪；

【导师】 张旭；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 研究背景：膀胱移行细胞癌(Transitional Cell Carcinoma of Bladder，TCCB)是泌尿系统最常见的肿瘤之一，是一多因素多阶段的发展过程，涉及抑癌基因的失活、癌基因的活化、细胞周期的调控、基因启动子的转录活化和表遗传学等诸多方面，而这些方面都与信号传导通路密切相关。Notch信号通路对肿瘤的发生发展具有广泛的多样化的影响，也是许多重要细胞信号通路的交汇点，以Notch为靶点的肿瘤基因治疗药物已被用于T细胞急性淋巴细胞白血病(T-cell acute lymphoblastic leukemias，T-ALL)的前期临床研究。然而Notch信号通路在TCCB发生发展中的作用并不明确。Notch信号通路是一条进化中高度保守的与细胞间通讯相关的通路，它在许多器官发育过程中起重要的作用，包括果蝇外周神经系统的发生、线虫的外阴发育和哺乳动物的淋巴系统发育。Notch信号转导通路由Notch受体、Notch配体和CSL(转录因子)三部分组成。人Notch受体家族包括4个成员(Notchl-4)和5种配体：Jagged1、Jagged2、Dll1、Dll3和Dll4。越来越多证据表明Notch信号通路与肿瘤发生发展有关，多数研究结果显示Notch具有促癌作用，但在皮肤癌和宫颈癌中为抑癌作用。方法：1．标本来源：收集本院2000至2008年膀胱移行细胞癌组织新鲜标本70例。其中男41例，女29例。年龄28～75岁，平均59岁。所有标本均经病理学检查诊断为膀胱移行细胞癌(TCCB)，并进行病理学分级及分期。病理分级按WHO标准，Ⅰ级35例，Ⅱ-Ⅲ级35例。临床分期按UICC-TNM标准，非浸润性膀胱癌(Ta)35例，浸润性膀胱癌(T1～T3)35例。另取10例正常膀胱黏膜作为对照组，剔除吸烟嗜酒者。一份标本留取后立即置于液氮中保存。另一份经10％甲醛溶液固定，常规石蜡包埋、切片，用于免疫组织化学染色。2．采用免疫组化SP法定性检测Notch-1、Notch-2、Notch-3、Jagged-1和DLL-1蛋白在70例膀胱移行细胞癌和10例正常膀胱粘膜组织中的表达情况。以在细胞质或细胞膜出现棕黄色颗粒为阳性，显微镜下观察并计数阳性染色细胞数。其表达以细胞染色的比率和强度进行判断：一为＜10％的细胞胞质或胞膜上微弱染色，+为11％-20％的细胞胞质或胞膜上中度或强染色，++为21-60％的细胞胞质或胞膜上强染色，+++为＞60％的细胞胞质或胞膜上强染色。3．采用两步法RT-PCR检测Notch-1、Notch-2、Notch-3、Jagged-1和DLL-1基因在mRNA水平的表达。4．采用Western-Blot定量检测Notch-1、Notch-2、Notch-3、Jagged-1和DLL-1基因在蛋白质水平的表达。5．对70例患者进行随访分析，检测其肿瘤转移、复发时间及总生存时间，结合Notch-1、Notch-2、Notch-3、Jagged-1和DLL-1基因的表达分析其与术后生存时间的关系。结果1．在10例正常膀胱移行上皮中以上5种Notch信号通路基因均为深染(+++)。非浸润性膀胱癌组织中染色主要集中于细胞膜上，染色较淡，浸润性膀胱癌中染色部位分布于细胞质中，呈片状浓聚。Notch信号通路基因分别根据其临床分期和病理分级，非浸润性膀胱癌和浸润性膀胱癌以及低级别和高级别膀胱癌的染色率有显著性差异(p＜0．05)。2．通过RT-PCR检测正常膀胱移行上皮、非浸润性膀胱癌组织和浸润性膀胱癌组织中Notch信号通路各基因Notch-1、Notch-2、Notch-3、Jagged-1和DLL-1的mRNA表达差异有统计学意义。3．Western-Blot检测正常膀胱移行上皮、非浸润性膀胱癌组织和浸润性膀胱癌组织中Notch信号通路各基因Notch-1、Notch-2、Notch-3、Jagged-1和DLL-1的蛋白质表达差异有统计学意义。4．非浸润性膀胱癌患者的术后无瘤生存时间与Notch-1和Jagged-1基因的表达状态相关(p值分别为0．024和0．003)。结合不同Notch-1和Jagged-1基因的表达发现，两基因表达均较低者术后无瘤生存时间明显较短(28．09±8．84月)，而两基因表达均较高者术后无瘤生存时间明显较较长(88．78±6．81月)。四组患者术后无瘤生存时间差异有统计学意义(p=0．002)。结论：1．在正常膀胱粘膜上皮、非浸润性膀胱癌和浸润性膀胱癌中Notch信号通路基因的表达呈现3种模式。2．非浸润性膀胱癌中Notch-1和Jagged-1基因的低表达与患者预后不良相关。3．Notch信号通路在非浸润性膀胱癌中可能起抑癌基因的作用。研究目的：输尿管梗阻可加速肾结核的进展，而针对结核的抗痨治疗可促进输尿管的纤维化，使梗阻加重，最终导致肾功能严重损害。本课题拟明确早期留置输尿管双J管在肾结核治疗中的作用。方法：进行多中心随机临床病例研究，以比较单纯抗痨治疗和早期留置双J管对肾结核治疗的作用。随访时间至少为24个月，判断治疗失败的主要标准为患者患侧肾小球滤过率(GFR)降至10 ml／min。结果：共298名患者，被随机分配到单纯抗痨治疗和早期留置双J管组。随机分组24个月后，早期留置双J管组患者肾功能优于单纯抗痨组(p=0．012)，留置双J管组治疗失败的比例较低(p=0．001)。随机分组4周后，留置双J管组患者生活质量评分(Medical Outcomes Study 36-Item Short-Form General Health Survey questionnaire)也优于单纯抗痨治疗组(p=0．001)。结论：在肾结核患者中早期留置双J管，可有效保护肾功能，减少肾切除的可能性并提高生活质量。更多还原

【Abstract】 Introduction: Bladder cancer is the 5th most common malignancy and the 4th leadingcause of cancer death of men in the United States. Clinically, the most important distinctionis that between papillary and invasive TCCB. Papillary cancers typically exhibit activationof the MAPK pathway, as a consequence of oncogenic mutations in FGFR3 or H-ras, withincreased Cyclin D1 expression. In the other way, invasive TCCB are characterized bysevere disturbances in proximate cell cycle regulators, e.g. Rb1 and CDKN2A/p16^INK4A,which decrease dependency on mitogenic signaling. In addition, these disturbances permit,promote and are in turn exacerbated by chromosomal instability, which is further enhancedby loss of TP53 function. Intriguingly, neither canonical WNT/b-Cateninn nor hedgehogsignaling appear to play major roles in TCCB. This may reflect its origin from moredifferentiated urothelial cells possessing a high regenerative potential rather than a stem cellpopulation.Like urothelia, keratinocytes is not directly originated from stem cells and bothWNT and hedgehog signaling which have important crosstalk with Notch family are notactive. Notch factors in keratinocytes act as tumor suppressors, which is quite differentfrom other tumors. It inspired us to investigate the function of Notch family in TCCB.Notch signaling participates in the development by maintaining the self-renewalpotential of some tissues and can positively or negatively influence proliferation,differentiation, and apoptosis depending on the cell types. To date, 4 Notch receptors havebeen identified （Notch 1-4） in humans, with corresponding ligands including Delta-like-1,Delta-like-3, Delta-like-4, Jagged-1 and Jagged-2. Involvement of Notch in cancer was firsthighlighted in human T-cell leukaemia, fuelling the notion that aberrant Notch signalingpromotes tumorigenesis. However, there is mounting evidence that Notch signaling is notexclusively oncogenic. It can instead function as a tumor suppressor in the skin. Theseapparently contradictory functions of Notch signaling strongly indicate that the outcome ofNotch activation is dependent on the cellular context. To date, the Notch pathway has been rarely associated with TCCB. In this study, theexpression of Notch ligands and receptors was detected in normal and tumorous humanbladder transitional epithelia, respectively. The relationship between the expression patternof them and prognosis of bladder cancer patient was also evaluated.MATERIALS AND METHODS: Patients and Tissue Samples: Fresh tumor tissuesnap-frozen in liquid nitrogen in the operating room was obtained from single hospitalbetween January 2000 and December 2006. All tumors were primary and diagnosedaccording to the WHO/ISUP classification of urothelial tumors and staged according to theTNM system as TaG1 in 35, T1G2 in 2, T2G2 in 16 and T3G3 in 17. These tissues werefrom 41 men and 29 women with the mean age at diagnosis being 59 years old （range 28 to75）. None of them had received any preoperative therapy. For comparison, 10 cases ofnormal urothelia were obtained from surgical specimens. The pathological characteristics ofthese urothelia were confirmed by hematoxylin and eosin staining.Immunohistochemical staining: Section preparation andimmunostaining were done aspreviously described. Two observers evaluated the staining pattern of the protein separatelyand scored each specimen for the percentage of positive cells identified. An average of 20fields was observed for each specimen. The immunoreactivity of a tissue sample wasconsidered negative （-） if less than 10% of the cells showed the same staining intensity asthe positive control in arterial endothelia, low expression level （+） if 11%-20% of the cellsshowed the staining above, medium expression level （++） if 21%-60% of the cells showedthe staining above and high expression level （+++） if more than 60% of the cells showedthe staining above.Total RNA was extracted with TRIzol （Invitrogen） reagent. Two-step RT-PCR wascarried out in 25μl volume on indicated amounts of RNA by use of a gene specific primer.Western Blotting: Protein levels were determined by the Bradford assay. Protein extractswere mixed with SDS sample buffer and electrophoresed in 10%. The signals werevisualized with the ECL system （DuPont NEN, Wilmington DE, USA）.Statistical Analyses: The association between the expression of Notch factors intumors and the various clinicopathological variables was examined using the chi-square testor Fisher’s exact test. Mann-Whitney U test was performed for continuous variables.Survival analyses were conducted according to the Kaplan-Meier method and survival characteristics were compared using log rank tests. Disease-flee survival was defined as theperiod between surgery and the detection of initial local recurrence, distant metastasis orstudy end. The Cox proportional hazards regression model was used to compare the relativeinfluences of different prognostic factors. The statistical software package SPSS 11.0 （SPSS,Inc., Chicago, Illinois） was used for all statistical analyses, with p＜0.05 indicatingstatistical significance.RESULTS: In the present study tumors were divided into 2 groups of papillary （pTa）and invasive （pT1-pT3）, respectively. All of the 5 kinds of Notch factors were intensivelystained in normal bladder transitional epithelia, but their expression was significantlydecreased in tumor tissues. Moreover, the expression level of the 5 proteins in the papillarytumors was lower than that in the invasive tumors （P＜0.05）. In tumor cells, the immuneactivity of Notch factors was observed on the membranes of papillary tumor cells, while itbecame diffused and turned to cytoplasm in invasive ones. The expression of Notch factorswas not related with patients’ age at diagnosis, sex, vascular invasion or lymph nodeinvolvement. No differences in immune activity of all the antibodies were found betweenthe basal cell layer and papillary layer of urothelia.To further confirm the IHC results, the tissue samples were subjected to RT-PCR andWestern blot analysis. The papillary tumors showed weak immunoreactive bands of theexpected Notch factors molecular weight in western blot, whereas the normal and invasivetissues had intense expression. Among the three groups, each two of them had statisticaldifference.Kaplan-Meier analyses revealed that the decreased expression of Notch-1 and Jagged-1was associated with an increased disease-free survival in papillary TCCB （p = 0.024 and0.003 respectively）. Survival time of patients with low expression of both Notch-1 andJagged-1 was significantly shorter than that with other expression patterns in papillarytumors （p = 0.002）. The expression pattern of other Notch factors was not correlated withthe survival time of the patients with invasive or all types of TCCB.CONCLUSION: There are two expression patterns of Notch family in TCCB betweenpapillary and invasive tumors. The expression of Notch-1 and Jagged-1 is correlated withdisease-survival of papillary TCCB patients. BACKGROUND: Tuberculous ureteral obstruction is a major cause of progressiveuropathy commonly complicates renal tuberculosis. The effect of chemotherapy andconcomitant fibrosis compounds the problem and results in renal loss. The possible benefitof early ureteral stenting in patients with renal tuberculosis is uncertain.METHODS: We performed a multicenter, randomized clinical trial on patients withrenal tuberculosis comparing the efficacy of medication only with medication plus earlyureteral stenting. The lest follow-up period was 2 years. The primary outcome was the timeof glomerular filtration rate （GFR） reduced to 10 ml/min.RESULTS: A total of 298 patients were randomly assigned to undergo either themedication only management or the early ureteral stenting procedure. The renal functioncould be better preserved by early ureteral stenting （P= 0.012）. The rate of treatment failurewas lower in patients who underwent the early ureteral stenting procedure than in thosereceiving medication only （P= 0.001）. After the first 4 week’s of assignment, patientssubject to early ureteral stenting procedure had higher scores on the Medical OutcomesStudy 36-Item Short-Form General Health Survey questionnaire （P = 0.003）.CONCLUSIONS: Early ureteral stenting in patients with renal tuberculosis wasbeneficial to preservation of renal function, decreased the possibility of renal loss andimproved life quality.更多还原