光动力学疗法诱导人膀胱癌细胞凋亡的实验研究

目的:联合应用流式细胞仪和激光共聚焦显微镜研究叶绿素衍生物光动力学疗法诱导人膀胱癌细胞(T-24 and SCaBER)凋亡的现象.方法:光动力疗法体外作用于人膀胱癌T-24and SCaBER细胞,细胞凋亡指数采用双免疫荧光染色(Annexin V/PI)和流式细胞仪技术测定,凋亡细胞的形态学研究采用AO/EB双染色和激光共聚焦显微镜技术.结果:经光动力学作用后的膀胱癌细胞通过诱导凋亡而被抑制了细胞的活性.细胞被阻滞于G0～G1期.结论:叶绿素衍生物光动力学疗法通过诱导凋亡而抑制膀胱肿瘤的生长,激光共聚焦显微镜能提供更可靠的细胞早期凋亡的影像学资料.     

茶多酚对人膀胱癌细胞光动力学杀伤效应的影响

目的 :探讨茶多酚对人膀胱癌细胞叶绿素衍生物光动力学杀伤效应的影响。 方法 :采用MTT比色分析法 ,判断茶多酚影响体外人膀胱癌T -2 4和SCaBER细胞光动力学的杀伤效应。 结果:与单纯光动力学作用组比较 ,茶多酚能显著减弱叶绿素衍生物光动力学对两种人膀胱癌细胞的损伤 (P <0 0 5,0 0 1) ,茶多酚的作用时间与光动力学对T -2 4细胞杀伤效应呈负相关 ,而与SCaBER细胞却无相关性。 结论 :茶多酚具有较强的缓解膀胱肿瘤细胞光动力学杀伤效应的作用     

NO与cGMP协同降低细胞内钙离子的释放从而使阴茎勃起

阴茎海绵体平滑肌细胞（SMCs）强直性收缩使阴茎处于疲软状态，NO可使SMCs舒张，从而使阴茎勃起。Williams等研究了SMCs内NO受肾上腺素能刺激后的作用。从小鼠和人类分离新鲜的SMCs，利用Fura-2荧光标记法，测量细胞内钙离子浓度（[Ca^2＋]（i））。结果显示，不论细胞外是否存在钙离子，苯肾上腺素（PE）均能短暂性提高细胞内钙离子浓度，表明细胞内钙离子库有释放活动。     

高钾溶液对慢性非细菌性前列腺炎SD大鼠前列腺平滑肌细胞内钙离子浓度的影响及意义

目的 探讨慢性非细菌性前列腺炎SD大鼠前列腺平滑肌细胞(PSMC)内游离钙离子浓度([Ca2+]i)在高钾溶液作用下的变化及临床意义. 方法 SD大鼠40只,随机分为实验组和对照组,采用去势+雌二醇注射的方法诱导大鼠非细菌性前列腺炎,体外培养纯化PSMC,细胞经钙离子荧光探针Quest Fluo-8TM孵育后在激光共聚焦显微镜下连续动态扫描,然后加入高钾溶液250μl(60 mmol/L),观测细胞内钙离子荧光强度变化情况. 结果 光镜下实验组标本显示典型慢性前列腺炎的病理特征,对照组未见炎症表现.免疫细胞化学方法证实两组细胞均为PSMC.两组PSMC内钙离子荧光强度增加幅度分别为27.86±9.88和7.61 ±4.31,组间差异有统计学意义(P＜0.01).结论 高钾溶液导致细胞内钙离子浓度升高,可能会影响细胞及前列腺的各项电生理功能,在慢性前列腺炎发病机制中可能具有-定作用.     

草氨酸盐对人胰腺癌细胞内钙含量影响的研究

目的:探讨草氨酸盐(oxamate)对人胰腺癌细胞内钙含量的影响。方法:体外培养人胰腺癌细胞panc-1，用oxamate干预癌细胞后48h，行钙离子荧光指示剂Fluo-3/AM染色，激光共聚焦下观察不同浓度oxamate作用下细胞内钙含量的变化。结果:oxamate干预48h后，细胞内钙含量随着oxamate浓度的升高而致荧光强度逐渐增强，呈剂量-效应关系。结论:oxamate能增加panc-1细胞内钙离子含量，影响细胞内钙离子平衡，进而干涉细胞病理生理过程。     

出血性休克治疗后甲状旁腺对低血钙的反应

人们早已认识出血性休克复苏后出现低血钙这一事实,近年来研究的重点放到了离子钙的血管外移动上。间质内钙离子浓度正常时超过细胞内的浓度,其比例达10,000∶1,当出现出血性休克,随之而发生的钙离子自血管内移出,导致细胞内钙离子浓度升高,其结果可能是有害的。它可以影响细胞内代谢、是脑死亡的特殊有关因素,它也影响心肌功能,故有介绍在出血性休克复苏时应用钙阻断剂,以防止钙离子进入细胞内。尽管在治疗低血容量性休克中,围绕着钙的作用     

兰尼碱受体拮抗剂——丹曲洛林的细胞保护作用

作为第二信使，钙离子在多种细胞功能活动中均有重要作用。如果钙离子的细胞内稳态受到破坏，则可导致细胞毒性反应甚至细胞死亡。钙通道兰尼碱受体激活，可致细胞内钙库释放过多从而}I起细胞损伤。而缺血、缺氧、癫痫、创伤、麻醉及神经退变性疾病的动物模型和组织培养实验中皆可观察到钙离子内稳态异常。丹曲洛林属兰尼碱受体拮抗剂，最早用于治疗恶性高热。丹曲洛林可抑制细胞内主要钙库——内质网过多释放钙离子。丹曲洛林可能的抗损伤细胞保护作用已在不同的组织培养模型或动物疾病模型中得到广泛的研究，此类模型均涉及细胞内钙离子稳态破坏所介导的细胞毒效应。本文中我们综述了钙离子内稳态破坏在细胞死亡中的作用，丹曲洛林的药理学和药代动力学特点，丹曲洛林的细胞保护作用以及在众多应激和疾病模型中抑制细胞损伤的潜在应用价值。     

P物质对体外培养的胃癌细胞内游离钙浓度的影响

目的研究P物质（substance P，SP）对体外培养的人低分化胃癌细胞MKN45内游离钙浓度的影响。方法在RPMI1640培养液中培养人胃癌细胞系MKN45，应用钙特异性荧光指示剂Furu-3／AM负载细胞，用ASN-1377642（NK-1受体拮抗剂）、尼卡地平和不同浓度的SP作用于人胃癌细胞MKN45，采用激光扫描共聚焦显微镜观测胃癌细胞内游离钙浓度的变化。结果在Hanks液（含钙离子）中加入10、50及100nmol／LSP可以显著升高胃癌细胞内钙离子浓度（P〈0．05），并且呈剂量依赖性（P〈0．05）；在无外钙时，SP引起细胞内钙离子浓度升高的幅度低于有外钙时（P〈0．05）；在Hanks液中加入NK-1受体拮抗剂ASN-1377642和钙通道阻滞剂尼卡地平孵育后，SP引起胃癌细胞内钙离子浓度升高的幅度显著降低，与Hanks液组比较差异有统计学意义（P〈0．05）。结论SP可以显著升高胃癌细胞内钙离子浓度，细胞内钙离子浓度的升高源于内钙释放和外钙内流。     

钙浓度变化对体外培养人表皮角质细胞增殖的影响

目的:比较不同钙浓度培养的人表皮角质细胞(HEKs)增殖能力的差异,确定体外培养表皮角质细胞的最适钙浓度.方法:根据培养液中CaCl2浓度不同,将HEKs分为无钙培养组和0.1、0.3、0.5、1.0 mmol/L CaCl2培养组.细胞培养3 h后,加钙离子荧光染料Fluo-3-AM,显微镜观察细胞内的荧光强度,以反映细胞内钙离子浓度的变化;细胞培养2 d后加Hochest33258核染液,荧光显微镜下观察细胞生长形态;培养3 d时镜下观察细胞的生长情况,并分别用流式细胞术和MTT法分析细胞的增殖情况.结果:钙培养可使细胞内钙离子浓度增加,表现为细胞内Fluo-3-AM荧光强度增强,且随着CaCl2浓度的增加,荧光强度亦随之增加;在钙浓度为0.3mmol/L时,细胞的增殖指数最高,为(51.98±14.31)%,增殖活性最强;0.1mmol/L组次之;随着钙浓度的增加,细胞的增殖指数下降,增殖活性受到抑制,0.5～1.0mmol/L钙培养组的增殖指数和增殖活性均明显低于无钙培养组(P均<0.01).结论:不同钙浓度培养影响表皮角质细胞的增殖能力,钙浓度为0.3 mmol/L时细胞的增殖能力最强.     

雄激素可与骨髓巨噬细胞膜结合引起细胞外钙离子的内流

目的:探讨雄激素作用于骨髓巨噬细胞(BMMs)的途径,研究雄激素与BMMs结合后对其产生的功能性影响。方法:体外诱导培养BMMs,提取细胞总RNA和蛋白,用RT-PCR和Western印迹方法观察BMMs中雄激素经典受体的表达情况。激光共聚焦显微镜观察T-BSA-FITC结合BMMs的情况。雄激素作用下,用Fura-2方法观察BMMs内钙离子浓度水平及特异性钙离子通道拮抗剂NiCl2对这种作用的影响,并用膜片钳方法观察BMMs膜表面离子电流的变化。结果:RT-PCR和Western印迹法都未检测到巨噬细胞中的雄激素受体,而阳性对照组(睾丸组织)的条带显著。用激光共聚焦显微镜观察到BSA-FITC能结合于巨噬细胞的细胞膜上。雄激素作用于BMMs后能迅速引起胞内游离钙离子浓度增加,这种作用可被NiCl2阻断。用膜片钳方法观察到雄激素作用于BMMs后引起胞外的阳离子内流,与Fura-2方法得出的结果一致。结论:雄激素可能作用于BMMs膜表面的非经典受体,诱发细胞外钙离子快速内流,使细胞内钙离子浓度升高,从而对巨噬细胞产生功能性影响。     

Effects of bradykinin on cytoplasmic calcium and motility in murine bladder tumor cells

PURPOSE: Bradykinin is known to mobilize calcium from an intracellular or extracellular source in several tumor cells. We evaluated whether bradykinin increases cytoplasmic calcium concentration ([Ca2+]i) and evokes locomotory movement in bladder cancer cells. MATERIALS AND METHODS: We studied the bladder cancer cell lines MBT-2, MB-49 and HT-1376, and the prostate cancer cell line PC-3 was used as a control. [Ca2+]i was measured with the fluorescent calcium indicator fura-2/AM. Bradykinin induced cell contraction was studied on only MBT-2 cells by taking microscopic photographs. Matrigel coated transwell chambers were used to test the invasive behavior of cancer cells. RESULTS: Bradykinin induced a transient increase in [Ca2+]i in the 3 bladder cancer cell lines, which was suppressed by a specific blocker of B2 receptors, the B2 inhibitor. Bradykinin did not induce an increase in [Ca2+]i in PC-3 cells. MBT-2 cells showed a contractile response to bradykinin. ML-9, a myosin light chain kinase inhibitor, or W-7, a calmodulin antagonist, completely abolished this bradykinin induced contraction, although a bradykinin induced calcium transient was consistently observed. When bladder cancer cells were incubated with bradykinin, the number of cells which migrated through a matrigel coated filter was significantly greater than that of the control without bradykinin. This bradykinin induced chemo-invasion was completely blocked by the B2 inhibitor. Bradykinin did not evoke the chemotactic response in PC-3 cells. CONCLUSIONS: Bradykinin can increase [Ca2+]i transiently in bladder cancer cells, which is mediated by B2 receptors. The contractile response of MBT-2 cells to bradykinin appears to occur as a result of the actin-myosin interaction caused by increased calcium. In addition, bradykinin can induce locomotory movement of bladder cancer cells through B2 receptors. It is difficult to explain this chemotactic response only by the calcium mobilizing effect of bradykinin.     

Characterization of insulin-like growth factor I binding sites in human bladder cancer cell lines

Summary The role of insulin-like growth factor I (IGF-I) in the growth and development of bladder cancer cells was investigated using cultured human cell lines representing differentiated (RT-4, 5637) or undifferentiated (T-24, J-82, TCC-SUP) transitional cell carcinoma (TCC). In the presence of 2% serum, IGF-I significantly stimulated the growth of all cell lines. The proliferation of T-24, 5637, and RT-4 cells was more sensitive to IGF-I than that of J-82 and TCC-SUP cells. [¹²⁵I]IGF-I binding to 5637 and J-82 cells was significantly higher than that to T-24 and TCC-SUP cells (P<0.001). RT-4 cells possessed the lowest binding capacity among the cell lines tested. Scatchard analysis of [¹²⁵I]IGF-I binding to four of the five cell lines indicated a single binding site for IGF-I, with apparent dissociation constants (K _d) of 1.27, 1.18, 1.34, and 1.39 nmol/l for TCC-SUP, J-82, 5637, and T-24, respectively. Therefore, the difference observed in [¹²⁵I]IGF-I binding among the bladder cancer cell lines was attributed to the difference of IGF-I binding sites and not to a change in receptor binding affinity. Cross-linking studies supported the suggestion that [¹²⁵I]IGF-I was bound to a receptor on these cells. The results indicate that cultured human bladder cancer cells contain functional IGF-I receptors. A differentiated cell line, RT-4, possesses significantly fewer IGF-I receptors than other cell lines. This suggests that the overexpression of IGF-I receptor may reflect the malignant potential of bladder cancer cells.     

Effect of 1,25-dihydroxyvitamin D3 on cytosolic calcium in dispersed parathyroid cells

We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cytosolic calcium ([Ca]i) of dispersed bovine parathyroid cells, using the fluorescent dye indo-1. The addition of 10(-8) M 1,25-(OH)2D3 caused an increase in [Ca]i by 23.4 +/- 2.7% over a 10 minute period. There was a significant increase in [Ca]i within two minutes of the addition of 1,25-(OH)2D3. 1,25-(OH)2D3 increased [Ca]i in a dose-dependent manner and this occurred with as little as 10(-10) M. Neither 10(-7) M 25-(OH)D3 nor 10(-7) M 24, 25-(OH)2D3 caused a significant increase in [Ca]i. Chelation of extracellular calcium with EGTA blocked the 1,25-(OH)2D3-induced increase in [Ca]i, suggesting that the increase was mainly from extracellular calcium. Neither 10(-5) M verapamil nor 10(-4) M diltiazem blocked the 1,25-(OH)2D3-induced increase in [Ca]i. The present data suggest that 1,25-(OH)2D3 might modify membrane permeability to calcium independent of voltage-dependent calcium channels sensitive to verapamil or diltiazem. The rapid effect of 1,25-(OH)2D3 raises the possibility that its mechanism is independent of genome activation, perhaps attributable to direct interaction with components of the parathyroid cell plasma membrane.     

Subsequent activation of mitogen-activated protein kinase after adhesion of transitional cell cancer cells to fibronectin

INTRODUCTION: In the process of tumor invasion and metastasis, interactions between tumor cells and extracellular matrix play a crucial role. Recently, it was shown that fibronectin binding to fibronectin receptor promotes mitogen-activated protein kinase (MAPK) activation after tyrosine phosphorylation of focal adhesion kinase (FAK). We investigated these signal transduction events in transitional cell cancer (TCC) cells. MATERIALS AND METHODS: (1) The adhesion of T24 cells, a fibronectin-receptor-positive TCC cell line, to fibronectin was investigated; (2) the MAPK activation after fibronectin stimulation in bladder cancer cell lines was examined by Western blotting using an antiactive MAPK antibody, and (3) FAK, Sos, and Grb-2 were also examined by Western blot analysis. RESULTS AND CONCLUSIONS: T24 cells adhered to fibronectin-coated dishes more quickly than to the noncoated dishes. Fibronectin stimulation induced activation of MAPK in T24, SCaBER, and HT1376 cells. However, activated MAPK was not detected in RT4 cells which do not express alpha(5)beta(1) integrin (major fibronectin receptor) after fibronectin stimulation. T24, SCaBER, and HT1376 expressed FAK and Sos. RT4 showed little FAK and Sos expression. Grb-2 was expressed in all cell lines. Adhesion of fibronectin-receptor-positive TCC cells to fibronectin activates the MAPK cascade, possibly resulting in activation of tumor cells.     

Increased intracellular calcium is required for spreading of rat islet beta-cells on extracellular matrix

Rat islet beta-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet beta-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca2+ concentration [Ca2+]i as an intracellular step linking glucose stimulation and beta-cell spreading (inside-out signaling) was investigated. Purified rat beta-cells were attached to this matrix and incubated under various conditions known to affect [Ca2+]i. The effect of glucose on beta-cell spreading was mimicked by 25 mmol/l KCl (which induces calcium influx) and inhibited by diazoxide (which impairs depolarization and calcium entry) and by the L-type Ca2+ channel blocker SR-7037. When a 24-h incubation at 16.7 glucose was followed by 24 h at 2.8 mmol/l, beta-cells that had first spread regained a round phenotype. In the presence of thapsigargin, spreading progressed throughout the experiment, suggesting that capture of calcium by the endoplasmic reticulum is involved in the reversibility of spreading previously induced by glucose. Spreading was still observed in degranulated beta-cells and in botulinum neurotoxin E-expressing beta-cells when exocytosis was prevented. In summary, the results indicate that increased [Ca2+]i is required for the glucose-induced spreading of beta-cells on 804G matrix and that it is not a consequence of exocytotic processes that follow elevation of [Ca2+]i.     

Lithium effects on dispersed bovine parathyroid cells grown in tissue culture.

It is not clear whether hypercalcemia and hyperparathyroidism associated with lithium therapy are the result of an unmasking of preexisting disease or a direct effect of lithium on the parathyroid glands. To investigate this phenomenon, parathyroid hormone (PTH) secretion and cytosolic calcium concentrations [( Ca]i) were measured in normal and lithium-treated dispersed bovine parathyroid cells grown in tissue culture and incubated with varying concentrations of extracellular calcium [( Ca]e) (0.5 to 2.5 mmol/L). Results indicate that lithium has two effects on parathyroid secretory response: (1) a decrease in low calcium-stimulated PTH release and (2) a potentiation of PTH release at physiologic concentrations of extracellular calcium. [Ca]i was assessed by use of fura-2, a calcium-sensitive fluorescent indicator. Resting [Ca]i levels were unaffected by lithium (103 +/- 13 nmol/L in controls vs 101 +/- 5 nmol/l in lithium-treated cells, mean +/- SE). Subsequent increases in [Ca]i in response to increases in [Ca]e were significantly less in lithium-treated cells, with no difference at maximal [Ca]e. Increases in [Ca]i in response to a submaximal concentration of extracellular magnesium were also blunted in cells pretreated with lithium. In conclusion, our data suggest that, at physiologic calcium concentrations, lithium decreases parathyroid cell sensitivity to changes in [Ca]e, reducing [Ca]i levels and increasing PTH secretory response.     

钙离子浓度对人乳腺癌MCF-7细胞Caspase-3,Bax及Bcl-2表达的影响

目的 研究细胞内Ca^2 浓度[Ca^2 ]i的升高对人乳腺癌MCF—7细胞株中Caspase-3,Bax及Bcl—2表达的影响。方法 在体外培养下，经不同浓度thapsigargin(TG)处理的MCF—7细胞株用荧光指示剂Fura-2/AM于单波长荧光分光光度仪上测定细胞内钙离子浓度。用免疫组织化学方法检测Caspase-3，Bax及Bcl—2的表达。结果 随着细胞内钙离子浓度的提高，Bax表达逐渐增强，Bcl-2表达逐渐减弱，而Caspase-3始终呈阴性表达。结论 细胞内钙离子浓度的升高对MCF—7细胞株可诱发不通过Caspase-3途径的细胞凋亡。     

人膀胱移行细胞癌T24细胞中CK20 mRNA表达检测方法的建立

目的 建立巢式RT-PCR方法检测人膀胱移行细胞癌124细胞中CK20 mRNA表达进行分析的实验方法.方法 培养人膀胱移行细胞癌124细胞,利用普通RT-PCR及巢式RT-PCR方法分别检测其CK20mRNA的表达.结果 1.5%琼脂糖凝胶电泳发现普通RT-PCR及巢式RT-PCR结果均出现与预期结果一致的阳性条带.结论 巢式RT-PCR方法检测人膀胱移行细胞癌T24细胞中有CK20 mR-NA的表达.为进一步利用巢式RT-PCR方法研究临床膀胱移行细胞癌患者尿脱落细胞及肿瘤组织中CK20 mRNA的表达、提高检测敏感性及可靠性打下基础.为膀胱癌早期诊断和术后监测提供一种新的有效手段.