体外诱导大肠癌细胞分化及其碱性磷酸酶活性的变化

目的 观察单独和联合使用全反式维甲酸(ATRA)和1，25-二羟维生素D3对大肠癌细胞株LoVo碱性磷酸酶(ALP)活性的变化的影响。方法 用不同剂量的ATRA和1，25-二羟维生素D3单独或联合在体外作用于LoVo细胞2、4和6天，用脱氧胆酸钠溶解细胞，动力学方法测定ALP活性，紫外吸收法铡定蛋白质含量。结果 单独或联合使用ATAR和1，25-二羟维生素D3均可使LoVo细胞的肠型ALP活性逐渐升高，而以联合用药效果更好(P＜0.05)。结论 联合使用ATRA和1，25-二羟维生素D3诱导LoVo细胞比单独用药效果更好，对于大肠癌的诊疗具要的指导意义。     

全反式维甲酸诱导大肠癌细胞株LoVo凋亡的初步研究(摘要)

目的:细胞凋亡的调节紊乱与肿瘤的发生密切相关,已发现维甲类化合物全反式维甲酸ATRA在一些实体瘤细胞系中具有诱导凋亡作用。但在大肠癌中无报道,我们就此作一研究。材料和方法:结肠癌细胞株Lo-Vo在1640培养液中常规培养,取对数生长期细胞进行实验,ATPA溶解在无水乙醇中,实验和对照组乙醇终浓度均为0.05%。①MTT法反映活细胞数量,用MTT法测细胞生长抑制率;②流式细胞仪(FCM)分析细胞周期。FCM测定采用单染色单激光法。标本为10~(-5)molATRA作用后2、4、6d和传2、4、6代的LoVo细胞株;③IXl0~(-5)mol、IXl0~(-6)molATRA作用6d后在电镜下观察凋亡细胞;④用DNA断端标记(TDT)法测定凋亡细胞:凋亡细胞DNA断裂,末端被FITC标记,在荧光显微镜下计算凋亡细胞;⑤核酸电泳:按照Wessenlborg方法进行。结果:①ATRA浓度在10~(-5)~10~(-7)mol对LoVo细胞均有生长抑制作用(P<0．01),是有效药物浓度,生长抑制显示时间剂量依赖性;②流式细胞仪分析细胞周期,结果ATRA能改变LoVo细胞的细胞周期,使G_0/G_1期比例增高,S期下降,细胞被阻滞于G_1:期。10(-5)mol组第6天及传代2、4、6代后G_0/G_1期细胞比例下降,S期逐渐上升并出现亚“G_0/G_1”凋亡峰;③透射电镜下见特征性凋亡细胞,其特点是核膜完整染色质固缩,形成新月形,而对照组     

全反式维甲酸对人胰腺癌细胞PC-3诱导分化作用及其对Survivin表达的影响

维甲酸类药物是一类细胞诱导分化剂，能够抑制多种肿瘤细胞的增殖，调节着细胞的生长和分化，全反式维甲酸（A—TRA）作为维甲酸类药物的代表，具有显著的抗肿瘤作用。我们用ATRA诱导人胰腺癌细胞PC-3，观察细胞周期的改变和Survivin基因表达的变化，以及ATRA对人胰腺癌细胞增殖的抑制作用，为维甲酸类药物临床应用于胰腺癌的治疗提供实验依据。     

全反式维甲酸对人胃癌裸小鼠移植瘤的抗增殖及诱导分化作用

用裸小鼠胃癌细胞移植瘤作为体内诱导分化研究模型，采用移植瘤生长状态、移植瘤组织学、超微结构、细胞周期动力学变化及移植瘤ｐ５３、ｐ２１蛋白表达作为指标，研究了不同剂量的全反式维甲酸（ＡＴＲＡ）对裸小鼠胃癌细胞移植瘤的抗增殖及诱导分化作用。结果发现：在ＡＴＲＡ３００～１０００μｇ／只·ｄ的剂量下，对移植瘤具有明显生长抑制作用，并对移植瘤细胞有较强的诱导分化作用；同时可抑制移植瘤癌细胞ｐ５３、ｐ２１的表达。本实验结果提示：ＡＴＲＡ在体内对人胃癌细胞有较强的抗增殖和诱导分化作用，这种抗癌作用可能与ＡＴＲＡ抑制癌细胞ｐ５３及ｐ２１表达，促进癌细胞凋亡有密切关系。     

全反式维甲酸诱导前列腺癌DU145细胞凋亡中caspase-3及其细胞骨架蛋白F-actin的变化

目的探讨全反式维甲酸(ATRA)诱导雄激素非依赖型前列腺癌细胞系DU145凋亡过程中半胱氨酰天冬氨酸特异性蛋白酶(caspase)3及其细胞骨架蛋白F-actin的变化。方法应用吖碇橙(AO)染色,荧光显微镜观察细胞凋亡形态,流式细胞术(FACScan)测定ATRA对DU145细胞凋亡峰的形成,应用Westernblot测定ATRA在DU145细胞凋亡过程中caspase-3表达的变化,免疫荧光染色观察F-actin纤丝的形态。结果荧光显微镜观察细胞凋亡数目增多,48h时凋亡细胞比例为85.0%,流式细胞术检测可观察到凋亡峰,Westernblot检测显示裂解的caspase-3表达增多,免疫荧光染色可见F-actin的正常结构被破坏。结论ATRA诱导DU145细胞凋亡可能通过caspase-3介导,并导致细胞骨架蛋白破坏。     

rhBMP-2HACo复合材料异位诱导成骨中碱性磷酸酶活性的检测

目的：确定rhBMP-2/HA/Co复合制剂的生物活性,为其作为盖髓剂和根尖诱导剂提供理论依据。方法：用全自动生化仪检测rhBMP-2/HA/Co复合材料异位诱导成骨区样本单位湿重的碱性磷酸酶（ALP）活性,并与HA/Co组对照。结果：实验组样本的单位湿重碱性磷酸酶（ALP）活性的平均值均明显高于对照组。结论：rhBMP-2/HA/Co复合材料可作为一种新型的、具有良好生物活性的材料进一步研究,为将来的临床应用打下基础。     

维生素D3及其代谢物对成骨细胞分化及生物矿化的实验研究

目的研究体外维生素D₃及其代谢物在不同浓度下对成骨细胞分化和生物矿化的影响。方法采用胶原酶消化法分离成骨细胞;设立对照组,不同浓度VD₃、25(OH)VD₃和1α,25(OH)2VD₃药物处理组。采用CCK-8法检测细胞增殖,采用PNPP法测定ALP活性,采用钙含量测定、ELISA法和实时定量聚合酶链反应来评估成骨细胞对VD₃及其代谢物的反应,并对成骨标志物进行分析,评价VD₃及其代谢物对成骨细胞分化和生物矿化能力的影响。结果VD₃及其代谢物对成骨细胞无细胞毒性,只有200 nmol/L的VD₃能显著促进成骨细胞增殖,而25(OH)VD₃和1α,25(OH)2VD₃对成骨细胞增殖的促进作用不明显。25(OH)VD₃和1α,25(OH)2VD₃可以上调成骨细胞CYP24A1基因的转录活性,但不能直接代谢VD₃和25(OH)VD₃。25(OH)VD₃和1α,25(OH)2VD₃以剂量依赖性增加的方式促进早期成骨标志物(Runx2,ALP等)的表达。只有25(OH)VD₃能促进成骨细胞OCN基因和蛋白的表达,提高成骨细胞的生物矿化水平。结论体外25(OH)VD₃可诱导成骨细胞的分化和生物矿化,为其在临床骨质疏松症和骨组织工程中的应用提供依据。     

地塞米松和1，25(OH)2D3对体外人骨髓基质细胞成骨及成脂分化的影响

目的 观察地塞米松（Dex）和1，25（OH）2D3（D3）对骨髓基质细胞（MSCs）成骨及成脂分化的影响。方法 以离心法分离培养人MSCs，以10^-7mol／LDex和，或10^-8mol／Ll，25（OH）2D3作为分化诱导剂对细胞进行干预，分别用细胞碱性磷酸酶（ALP）染液试剂盒及苏丹Ⅲ染液对成骨细胞和脂肪细胞进行组织化学染色，计数；使用RT-PCR技术在转录水平检测成骨细胞标记物骨桥蛋白（OPN）及脂肪细胞标记物过氧化酶体增殖激活受体72（PPARγ2）mRNA的表达。结果细胞染色结果表明各干预组ALP^＋细胞百分比均较对照组增加，与对照组相比有显著性差异（P〈0．05），苏丹Ⅲ^＋细胞百分比Dex组较对照组增多，耽组较对照组减少，差异有显著性（P〈0．05），Dex＋D3组较Dex组苏丹Ⅲ^＋细胞数明显减少，两者相比差异有显著性（P〈0．05）；OPNmRNA及PPARγ2mRNA表达未在对照组测得，Dex诱导了OPNmRNA及PPARγ2mRNA表达，1，25（OH）2D3诱导OPNmRNA表达，并抑制Dex诱导的PPARγ2mRNA的表达。结论 Dex促进MSCs的成骨分化及成脂分化，1，25（OH），D，促进MSCs的成骨分化的同时抑制其成脂分化，与Dex合用抑制Dex成脂分化作用，强化了其成骨分化作用，反映了成骨细胞与脂肪细胞间存在的反变关系，表明两者来源于同一前体细胞的可能性。     

5alpha-dihydrotestosterone inhibits 1alpha,25-dihydroxyvitamin D3-induced expression of CYP24 in human prostate cancer cells

BACKGROUND: A cross-talk between 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and 5alpha-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown. METHODS: The expression of 25-hydroxyvitamin D(3) 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1alpha,25-(OH)(2)D(3) was measured using a radioreceptor assay. RESULTS: Real-time RT-PCR showed that DHT at 1-100 nM significantly inhibited 1alpha,25-(OH)(2)D(3)-induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1alpha,25-(OH)(2)D(3) was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not. CONCLUSIONS: We demonstrated, for the first time, that DHT reduces the ability of 1alpha,25-(OH)(2)D(3) to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact.     

Enhancement of hepatic 4‐hydroxylation of 25‐hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug‐induced osteomalacia

Long‐term therapy with certain drugs, especially cytochrome P450 (P450; CYP)‐inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25‐hydroxyvitamin D₃ (25OHD₃) were evaluated after exposure to P450‐inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8‐fold increase in formation of the major metabolite of 25OHD₃, 4β,25(OH)₂D₃. This inductive effect was blocked by the addition of 6′,7′‐dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK‐2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)₂D₃ was the predominant monohydroxy metabolite produced from 25OHD₃, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)₂D₃ was increased 60% (p < 0.01) after short‐term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)₂D₃ (?10%; p = 0.03), and a nonsignificant change in 24R,25(OH)₂D₃ (?8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)₂D₃ and decrease in 1α,25(OH)₂D₃ levels. Examination of the plasma monohydroxy metabolite/25OHD₃ ratios indicated selective induction of the CYP3A4‐dependent 4β‐hydroxylation pathway of 25OHD₃ elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug‐induced osteomalacia. © 2013 American Society for Bone and Mineral Research.     

Contragestazol （DL111-IT） inhibits proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo

Aim： To evaluate the antiproliferative activity of contragestazol （DL111-IT） on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms. Methods： The cell killing ability of DL111-IT was measured by the 3-（4,5-dimethylthia-zol,2-yl）-2,5-diphenyltetrazolium bromide （MTT） reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma （pRb）, cyclin-dependent kinase 4 （CDK4） and cyclin D 1, was detected by Western blotting. Results： DL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50 % cell inhibition （IC50 value） was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT （1.25 mg/kg-20.0 mg/kg） given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50 %. Flow cytometric analysis indicated that DL111-IT could cause GI arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D 1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL 111- IT. Conclusion： DL111-1T inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.     

Effects of vitamin D3 analogues on parathyroid hormone secretion and calcium metabolism in vitamin D-deficient rats

22-Oxa-1α, 25-dihydroxyvitamin D₃ (OCT) and 2β-(3-Hydroxypropoxy)-1α, 25-dihydroxyvitamin D₃ (ED-71) are novel synthetic vitamin D₃ analogues. In order to examine their calcemic actions on intestine and bone, we have investigated the effects of OCT and ED-71 on intestinal Ca transport, bone mobilization and plasma parathyroid hormone (PTH) level in vitamin D-deficient rats. The vitamin D-deficient rats were intravenously given either 6.25μg/kg or 0.2μg/kg of 1,25-D₃, OCT or ED-71 and theirplasma Ca levels and intestinal Ca transport were measured periodically. At a high dose, 1,25(OH)₂D₃ and ED-71 showed a strong biphasic stimulation of intestinal Ca transport and bone mobilization, and reduced the plasma PTH levels to the normal level completely. On the other hand, OCT failed to suppress the PTH secretion although it exerted first phase action on the both intestinal Ca transport and bone mobilization in vitamin D-deficient rats. The reason why OCT failed to suppress the PTH secretion even at a high dose, has not yet been clarified, but it may be at least in part due to its weak calcemic action and short half-life in plasma.     

Comparison of ex vivo and in vitro intestinal cystic fibrosis models to measure CFTR-dependent ion channel activity

Background

New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis.