兔脑种植VX2肿瘤动物模型的建立

目的建立兔VX2肿瘤脑内种植动物模型,观察其生长特性。方法采用兔脑内VX2肿瘤组织块种植法将VX2肿瘤组织块种植入24只成年New Zealand大白兔右侧大脑皮质内,用B超检测肿瘤的生长情况,在实验兔在肿瘤种植后第13、171、9、21、232、5天取材,进行组织学观察。并观察实验兔在种植VX2肿瘤后的生存期及出现厌食、偏瘫等神经系统体征和死亡的时间。结果VX2肿瘤组织块种植入脑内后荷瘤兔的中位生存期为24.5 d,平均生存期为24.8 d,肿瘤体积随种植后的时间在对数坐标中接近一条直线。VX2肿瘤种植入兔脑17 d后血供较丰富、呈鱼肉状生长,与正常脑组织边缘界限不清楚,第17~19天肿瘤中心出现坏死并出现腹腔内转移。光镜下从VX2肿瘤种植后第17天开始肿瘤细胞向正常脑组织浸润,并形成瘤巢,瘤周脑组织形成水肿带。结论在缺乏兔源性脑肿瘤的情况下,采用兔VX2肿瘤组织块颅内种植能较好模拟颅内肿瘤生长,为对脑肿瘤的某些实验研究提供条件。     

Antitumor effects of MutT homolog 1 inhibitors in human bladder cancer cells

ABSTRACT

As standard second-line regimen has not been established for patients who are refractory to or relapse with cisplatin-based chemotherapy, an effective class of novel chemotherapeutic agents is needed for cisplatin-resistant bladder cancer. Recent publications reported that MutT homolog 1 (MTH1) inhibitors suppress tumor growth and induce impressive therapeutic responses in a variety of human cancer cells. Few studies investigated the cytotoxic effects of MTH1 inhibitors in human bladder cancer. Accordingly, we investigated the antitumor effects and the possible molecular mechanisms of MTH1 inhibitors in cisplatin-sensitive (T24) and – resistant (T24R2) human bladder cancer cell lines. These results suggest that TH588 or TH287 may induce cancer cell suppression by off-target effects such as alterations in the expression of apoptosis- and cell cycle-related proteins rather than MTH1 inhibition in cisplatin-sensitive and – resistant bladder cancer cells.

Abbreviations: MTH: MutT homolog; ROS: reactive oxygen species; CCK-8: cell counting kit-8; DCFH-DA: dichlorofluorescein diacetate; PARP: poly (ADP-ribose) polymerase     

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.     

Age-Related Alterations in the Biochemical and Functional Properties of the Bladder in Type 2 Diabetic GK Rats

Previous studies have demonstrated that experimental type 1 diabetes induced by streptozotocin causes alterations in the biochemical and functional properties of several receptor systems in the rat bladder. However, the exact mechanism involved in the pathophysiology of voiding dysfunction in type 2 diabetic patients is unknown. Because the GK rat is a widely accepted genetically determined rodent model for human type 2 diabetes, we investigated diabetes-induced changes in the bladder smooth muscle of the GK rats at several time points. Male GK rats and age-matched Wistar rats, as controls, were maintained for 4, 8, 16, and 32 weeks. Contractile responses to KCl, carbachol, ATP, and electrical field stimulation (EFS) were measured by using the isolated muscle bath techniques. Acetylcholine (ACh) release induced by EFS from bladder muscle strips was measured by using high-performance liquid chromatography coupled with a microdialysis procedure. Maximum contractile responses to carbachol and ATP, the release of ACh, and tissue sorbitol levels were similar in bladders from GK and control rats until 8 weeks of age. At 16 weeks of age, however, the contractile responses to carbachol and ATP, and tissue sorbitol levels were increased, and the EFS-induced ACh release was decreased in GK rats compared with controls. Although the maximum contractile responses to EFS were unchanged until 16 weeks of age, they were decreased in 32-week-old GK rats, compared with controls. Our data indicate the presence of age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats.     

Activation of NFKB-JMJD3 signaling promotes bladder fibrosis via boosting bladder smooth muscle cell proliferation and collagen accumulation

Chronic cystitis is characterized by the hyperplasia and fibrosis of the bladder wall as well as attenuated compliance of the bladder. To further unravel its underlying molecular mechanism, the role of NFκB-JMJD3 signaling pathway in cystitis induced bladder fibrosis was investigated. Jmjd3 and Col1/3 expression was detected in a cystitis mouse model that was developed by intraperitoneal injection of cyclophosphamide (CYP). Human bladder smooth muscle cells (hBSMCs) were stimulated in vitro with lipopolysaccharide (LPS), and the cell proliferation and collagen accumulation were detected using EdU, CCK8, flow cytometry, qPCR, western blotting and immunofluorescence assays. Furthermore, the effects of NFκB and JMJD3 on cell proliferation and collagen accumulation were investigated using its selective antagonists, JSH23 and GSK-J4, respectively. CYP induced cystitis significantly increased Jmjd3, Col1 and Col3 expression in the bladder muscle cells. Furthermore, LPS stimulation markedly activated NFκB signaling and elevated JMJD3 expression in hBSMCs, and the activation of NFκB-JMJD3 signaling significantly promoted cell proliferation and collagen accumulation by upregulating CCND1 and COL1/3 expression, respectively. Our study reveals the critical role of NFκB-JMJD3 signaling in cystitis induced bladder reconstruction by regulating hBSMC proliferation and extracellular matrix (ECM) deposition, and these findings provide an avenue for effective treatment of patients with cystitis.     

Circular RNA circMTO1 suppresses bladder cancer metastasis by sponging miR-221 and inhibiting epithelial-to-mesenchymal transition

Bladder cancer remains a leading cause of cancer-related death because of its distant metastasis and high recurrence rates. Deregulation of circular RNAs (circRNAs) can act either as tumor suppressors or oncogenes to control cell proliferation, migration, and metastasis. The role of circMTO1 in bladder cancer remain unknown. In this study, we investigated whether circMTO1 could use as a biomarker and therapeutic target for bladder cancer treatment. We first demonstrated that circMTO1 was an important circRNA frequently downregulated in bladder cancer tissue, and lower circMTO1 levels were positively correlated with bladder cancer patients' metastasis and poorer survival. Ectopic expression of circMTO1 in bladder cancer cells inhibited cell's epithelial-to-mesenchymal transition (EMT) and metastasis. In addition, we also revealed that circMTO1 was able to sponge miR-221 and overexpression of circMTO1 negatively regulated the E-cadherin/N-cadherin pathway to inhibit bladder cancer cells' EMT by competing for miR-221. In conclusion, our findings provide comprehensive evidences that circMTO1 is a prognostic biomarker in bladder cancer and suggest that circMTO1 may function as a novel therapeutic target in human bladder cancer.     

Serum immunoglobulin levels and the risk of bladder cancer in the AMORIS Cohort

BackgroundThe anti-tumour T-cell response in bladder cancer has been shown to correlate with response to treatment and prognosis. However, little is known about the role of humoral immunity in this highly immunogenic human cancer, which is characterised by a high mutation-associated neoantigen load and a strong response to immunotherapy. In the present study, we interrogated the Swedish Apolipoprotein Mortality Risk Study (AMORIS) to explore the relationship between pre-diagnostic serum immunoglobulin levels and the risk of developing bladder cancer.MethodsOur analysis included all AMORIS participants aged 20 years or older, who had all three major serum immunoglobulins (IgA, IgM, IgG) recorded at the same baseline measurement (n = 29,876). All participants were free from bladder cancer at the time of measurement. Samples were obtained between 1985–1996, with follow-up information until 2011. Multivariate Cox proportional hazards regression was used to investigate the association between bladder cancer risk and different levels of pre-diagnostic serum immunoglobulins.ResultsDuring a mean follow-up period of 15.31 years, 163 (0.5%) individuals were diagnosed with bladder cancer. Multivariate Cox regression showed an inverse association between pre-diagnostic serum IgM levels ≥ 1.4 g/L and bladder cancer risk compared to serum IgM levels < 1.4 g/L [HR: 0.68 (95% CI 0.45–1.03)]. Corresponding associations could not be established for serum IgA or IgG.ConclusionOur findings implicate serum IgM in the pathogenesis of bladder cancer and suggest that the concept of humoral immune surveillance against cancer warrants further mechanistic investigation.     

Cancer incidence among Canadian Veterans: A matched cohort study

IntroductionOccupational exposures related to military service may increase the risk of cancer for military Veterans, while high levels of fitness during service may decrease risk. However, few studies have compared this post-career cancer risk directly to the employed general population.MethodsThis retrospective cohort study used linked administrative data. Canadian Armed Forces and Royal Canadian Mounted Police Veterans in Ontario, Canada were matched 1:4 on age, sex, geography, and community-level income to a group of non-Veterans most likely to have been employed during a period similar to the Veterans’ military service. Cancer diagnoses were identified using the Ontario Cancer Registry.ResultsDuring the study period, 642 of 30 576 included Veterans (2.1%) and 3408 of the 122 293 matched general population cohort (2.8%) experienced at least one cancer diagnosis. The crude rate of cancer was 153.5 per 100 000 person-years among Veterans vs. 205.9 per 100 000 person-years for the general population cohort. After adjusting for rurality and matching variables, Veterans had an 27% lower risk of developing any cancer than their matched comparators [hazard ratio = 0.73 (95% CI: 0.67–0.80)]. Among specific cancer types, the risk of lung and colorectal cancer was significantly lower for Veterans relative to the general population cohort; the risk of breast and prostate cancer was similar.DiscussionThis study adds to the growing international evidence suggesting that risk of many cancers among Veterans is lower or similar to the general population. Further understanding of the complex relationships among occupational exposures, environmental factors, and lifestyle factors is needed.     

探寻肝再生磷酸酶3在膀胱癌细胞T24中的作用

目的：已经有研究证明肝再生磷酸酶3（PhosphataseofRegeneratingLiver-3,PRL-3）在消化道肿瘤的发生发展过程中起到重要作用,但其在膀胱癌中发挥何种作用尚不清楚,本次研究主要探寻PRL-3在膀胱癌细胞T24中作用,以求为膀胱癌的治疗提供新思路。方法：采用siRNA干涉的方法下调T24中PRL-3蛋白表达水平,通过westernblot方法检测干涉效果,绘制细胞生长曲线、构建裸鼠种植瘤模型,检测PRL-3下调对细胞体外及体内增殖的影响。结果：我们所设计的siRNA能够下调PRL-3在A498细胞中的表达（F=7．26,P〈0．05）,细胞生长曲线显示下调PRL-3能够抑制细胞的增殖,这种作用从干涉后的第60h开始,随时间增加作用愈加明显（F≥8．35,P〈0．05）;动物实验表明,siRNA下调PRL-3能够抑制T24细胞在活体内的增殖,从干涉后第21天开始这种作用开始体现出来（F≥10．46,P〈0．05）,并且干涉组的肿瘤肿瘤明显低于两个对照组（F=13．63,P〈0．05）。结论：我们构建的siRNA能够在T24细胞中实现对PRL．3蛋白的下调,siRNA介导的PRL-3蛋白下调能够明显抑制T24细胞在活体外及活体内的增殖,这初步证明PRL-3在膀胱癌中发挥重要的作用。随着我们对PRL-3分子进一步深入的了解,揭示其发挥作用的具体机制,PRL-3很可能成为膀胱癌基因治疗的新靶点。     

卡介苗作用大鼠膀胱肿瘤细胞／正常移行上皮细胞后TNF—α．、IL-10、IFN-γ的检测及意义

目的：观察卡介苗（BCG）单独作用膀胱肿瘤细胞、正常膀胱移行上皮细胞及其代谢产物作用上述细胞后细胞生长情况及各自细胞培养液上清液中细胞因子（TNF-α．、IL-10、IFN-γ）浓度的变化,探讨其在卡介苗治疗膀胱肿瘤中可能的作用机制。方法：构建大鼠膀胱肿瘤模型,并原代培养大鼠膀胱肿瘤细胞及正常膀胱移行上皮细胞。分别用BCG,普通培养液和细胞培养的代谢产物作用上述细胞。酶联接免疫吸附剂测定法（ELISA法）检测各组细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）、干扰素-γ（IFN-γ）的浓度。结果：ELISA法检测各组细胞上清液中TNF-α、IL-10的浓度改变有显著差异,而IFN-γ的浓度无显著差异。结论：BCG可以直接刺激肿瘤细胞自身分泌细胞因子（TNF-α、IL-10）参与调节抑制肿瘤细胞的生长。