Increased potency of 1,25-dihydroxyvitamin D3 on human osteoblast-like cells following structural side-chain modification

Structural modifications of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) appear to alter its biological activity. We have investigated whether the position of the C=C bond in the side chain of fluorinated analogues can alter the spectrum of activity of 1,25(OH)₂D₃. For this purpose we compared the actions of 26,27-hexafluoro-1,25-dihydroxy-Δ²²-vitamin D₃ (1,25(OH)₂26,27F₆Δ²²D₃), 26,27-hexafluoro-1,25-dihydroxy-Δ²³-vitamin D₃(1,25(OH)₂26,27F₆Δ²³D₃) and 1,25(OH)₂D₃ on human osteoblast-like cells. Both analogues and 1,25(OH)₂D₃ stimulated the production of osteocalcin and alkaline phosphatase activity in a dose-dependent manner. Both analogues were markedly more potent than 1,25(OH)₂D₃ in these respects. At high concentrations the vitamin D₃ analogues and metabolite inhibited DNA synthesis in a dose-dependent manner. A correlation between the inhibition of cell growth and expression of the two osteoblast markers was observed, and apart from a difference in potency, did not differ from 1,25(OH)₂D₃. These studies indicate that hexafluorination and the C=C bond increase the potency of 1,25(OH)₂D₃ on human bone-derived osteoblast-like cells in vitro, but without changing their relative activity on these various aspects of osteoblastic function tested.     

胎盘源性干细胞在肝脏疾病中的研究进展

人类胎盘源性干细胞(hPDSCs)是干细胞的混合群.再生医学已将其用于某些功能衰竭和损伤器官的细胞再生、抗细胞凋亡、抗炎,抗肿瘤和细胞功能恢复研究.目前已有许多实验研究证明:胎盘间充质干细胞(PDMSCs)可以在体外分化为肝细胞样细胞,并于体内外促进干细胞增生和抗肝细胞凋亡,在动物肝损伤模型抑制肝纤维化.本文就胎盘干细胞的来源、分类、生物学特性以及胎盘干细胞在肝脏疾病中的治疗研究做一综述,以便为进一步探讨胎盘源性干细胞在肝脏疾病治疗中的应用提供新的思路.     

转染BIG-3基因的人骨髓基质干细胞向软骨细胞诱导分化的实验研究

目的研究转染BIG-3基因对人骨髓基质干细胞(hBMSCs)向软骨纠胞诱导分化的影响。方法将构建好的pMSCVpuro-BIG-3转染至建系的hBMSCs,5μg/mL嘌罗霉素筛选12 d后,扩增并收集细胞,电泳证实特定条带无误。实验分三组：hBMSCs-BIG-3组、hBMSCs-EV组和hBMSCs组,每组重复6次,将三组细胞制成微粒模拟三维培养模式,分别加入浓度为400 ng/mL的重组人骨形态发生蛋白-2(rhBMP一2)作用14 d后,检测甲苯胺蓝及Ⅱ型胶原mRNA,利用MPS60病理图象分析系统采集图像,并进行灰度分析,半定量比较基质表达量。结果灰度值：hBMSCs-BIG-3组为684 481 822,hBMSCs-EV组为439 101 780,hBMSCs组为441 082 183,hBMSCs-BIG-3组灰度值明显高于后两组(P＜0．05),hBMSCs-EV组和hBMSCs组之间差异无显著性意义(P=0．862)。结论BIG-3基因有助于hBMSCs向软骨细胞诱导分化。     

Absence of Heat Intolerance (Panting) Syndrome in Foot‐and‐Mouth Disease‐Affected Indian Cattle (Bos indicus) is Associated with Intact Thyroid Gland Function

Foot‐and‐mouth disease (FMD) is a highly contagious and economically important viral disease with high morbidity and reduced productivity of affected animals. We studied the heat intolerance (HI) (panting) syndrome and the effect of FMD virus (FMDV) infection on thyroid gland function in Indian cattle (Bos indicus). Experimental infection with FMDV Asia 1 resulted in a mild form of disease with superficial lesions. Heat intolerance syndrome and its signs were not observed among the recovered animals. Subtle changes in the serum level of thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄) were observed. However, there were no distinct histological changes in the thyroid gland, and FMDV antigens were not detected in the thyroid tissues. Our results thus suggest that the absence of panting syndrome in FMD‐affected Bos indicus cattle may be associated with intact thyroid gland function.     

人脂肪组织来源内皮祖细胞的分离、培养及鉴定方法的实验研究

目的探讨人脂肪组织来源的内皮祖细胞的分离、培养及鉴定的方法。方法脂肪抽吸术获取人体脂肪组织,消化法得到的细胞接种在Fibronectin包被的培养皿内,用含2%胎牛血清的DMEM培养,传至第2代,分诱导组(EGM-2,2?S,VEGF,bFGF等)和非诱导组(DMEM,2?S)两组进行培养。免疫细胞荧光分别检测两组的CD34、vWF和PECAM-1表达;流式细胞仪分别检测两组的CD34、CD45、CD133和PECAM-1表达率;荧光显微镜观察细胞摄取DiI-ac-LDL的功能;诱导组细胞接种于甲基纤维素半固体培养基进行三维培养,观察血管样结构形成情况。结果诱导组细胞12d后呈现内皮细胞典型的铺路石样形态,未诱导组细胞未观察到这种变化;免疫细胞荧光显示诱导组vWF、PECAM-1表达阳性,未诱导组细胞CD34表达阳性;流式细胞仪检测显示诱导组PECAM-1阳性率为(67.41±13.35)%,明显高于非诱导组的(6.73±2.21)%(P<0.01),非诱导组CD34阳性率为(72.39±13.45)%,明显高于诱导组的(16.06±3.86)%(P<0.01);荧光显微镜观察显示诱导组细胞具有摄取DiI-ac-LDL的功能;诱导组细胞三维培养形成"树枝样"分叉结构。结论建立了一种从人脂肪组织分离、培养EPCs的方法,并对其表型进行鉴定,有望为血管组织工程提供新的种子细胞来源。     

ADAMTS-1在人卵泡颗粒细胞和卵泡液中的表达研究

目的探讨Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS-1)mRNA和蛋白在人卵泡壁颗粒细胞(GCs)和卵丘颗粒细胞(CCs)中的表达水平及变化。方法收集2014年3月至2016年6月期间在华中科技大学同济医学院生殖医学中心接受ICSI治疗的患者卵泡液,其中大卵泡(直径≥18 mm)和小卵泡(≤10 mm)各86份,离心后上清用于Western Blot和ELISA实验,沉淀用密度梯度离心法分离纯化得到GCs;收集脱颗粒细胞的培养液直接离心法获得CCs。GCs和CCs均分别提取mRNA和蛋白,荧光定量PCR检测GCs和CCs中ADAMTS-1 mRNA水平,Western Blot检测ADAMTS-1蛋白水平,比较ADAMTS-1 mRNA和蛋白在不同颗粒细胞中的表达差异。结果 ADAMTS-1 mRNA在大卵泡GCs、小卵泡GCs、CCs中均有表达,且其在小卵泡GCs中表达量最高,显著高于CCs(P<0.05)。ADAMTS-1蛋白在大卵泡GCs、小卵泡GCs、CCs及卵泡液中均有表达,且存在成熟ADAMTS-1蛋白(85 KDa)及前体(110 KDa)两种形式;...     

Shiga toxin-1 affects nitric oxide production by human glomerular endothelial and mesangial cells

Acute renal failure hallmarks the pathogenesis of the epidemic form of hemolytic uremic syndrome (D+HUS), which is caused by E. coli strains that produce Shiga-like toxin (Stx). In this study, we investigated the influence of Stx-1 on nitric oxide (NO) production by human glomerular microvascular endothelial cells (GMVEC) and human mesangial cells. NO synthesis by human mesangial cells is in the micromolar range and that of GMVEC in the picomolar range. Stx-1 reduced NO production in non-stimulated GMVEC (5 nmol/l Stx-1 required) without inhibition of protein synthesis. In non-stimulated and TNFα-pretreated mesangial cells, NO production was reduced with a maximal reduction at 10 fmol/l shiga toxin. The cellular iNOS antigen content in mesangial cells was reduced in a concentration-dependent way (10 fmol/l-100 pmol/l), while partial inhibition of protein synthesis required 10 nmol/l Stx-1 in these cells. Our in vitro data suggest that Stx may reduce NO synthesis during the course of HUS development, contributing to the aggravation of the thrombotic microangiopathy and renal failure as observed in HUS.     

Modification of gene expression induced in human osteogenic and osteosarcoma cells by culture on a biphasic calcium phosphate bone substitute

Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-β1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.     

Inhibition of SULT2B1b expression alters effects of 3beta-hydroxysteroids on cell proliferation and steroid hormone receptor expression in human LNCaP prostate cancer cells

BACKGROUND: Sulfation is an important steroid inactivation in human tissues. Sulfotransferase (SULT) 2B1b selectively conjugates 3beta-hydroxysteroids and is expressed in epithelial cells of normal and cancerous prostate tissues. Dehydroepiandrosterone (DHEA) and Delta(5)-androstenediol (Delta(5)-Adiol) sulfation prevents their conversion to more potent androgens and estrogens in tissues although both compounds may also be biologically active. METHODS: SULT2B1b expression and activity were inhibited >85% in human LNCaP prostate adenocarcinoma cells using short interference RNA (siRNA). The effects of treating control and SULT2B1b-deficient LNCaP cells with DHEA, Delta(5)-Adiol, and 5alpha-androstane-3beta-17beta-diol (Anstane-diol) on cellular proliferation, estrogen receptors (ERs), androgen receptor (AR), and prostate specific antigen protein levels were examined. RESULTS: Physiological concentrations of DHEA and Delta(5)-Adiol increased proliferation of control cells and the proliferative effects were significantly increased in SULT2B1b-siRNA cells. DHEA, but not Delta(5)-Adiol increased AR levels at concentrations >/=1,000 nM in SULT2B1b-siRNA cells but not in control LNCaP cells. ER-alpha levels were not affected with any of the compounds tested. Physiological concentrations of DHEA and Delta(5)-A-diol decreased ER-beta levels in control cells and had significantly greater effects in SULT2B1b-siRNA cells. In contrast, Anstane-diol had no effect on AR or ER-alpha levels but induced more elevation of ER-beta levels in SULT2B1b-siRNA cells at concentrations >/=1,000 nM. CONCLUSIONS: SULT2B1b is involved in regulating prostate cell responsiveness to DHEA and Delta(5)-Adiol. Inhibition of SULT2B1b increased cell proliferation and ER-beta repression after treatment with physiological levels of DHEA and Delta(5)-Adiol indicating that SULT2B1b has an inhibitory effect on DHEA and Delta(5)-Adiol activity.     

长期体外培养对人脂肪源性间充质干细胞生物学特性的影响

目的通过观察长期体外培养对人脂肪源性间充质干细胞(ADSCs)生物学特性的影响,鉴定ADSCs作为组织工程种子细胞的优越性。方法通过流式细胞技术观察长期体外培养对人ADSCs表面抗原表达和凋亡的影响。通过碱性磷酸酶染色、Von Kossa染色及RT-PCR检测长期体外培养对人ADSCs成骨分化潜能的影响。结果原代ADSCs表面高表达间充质干细胞表面标记物,而不表达血源性细胞表面标记物,标记物不随传代次数变化。早期ADSCs凋亡率为1%～2%,随传代次数增多凋亡率逐渐增加．但幅度不大。碱性磷酸酶染色、Von Kossa染色和RT-PCR检测显示ADSCs传至第8代时仍能保持成骨分化潜能。结论ADSCs生物学特性稳定,是较为理想的组织工程及再生医学研究的种子细胞。     

人骨形态发生蛋白-2基因转染兔骨髓基质干细胞及其表达

目的探讨含有人骨形态发生蛋白-2(hBMP-2)cDNA的真核表达载体在兔骨髓基质干细胞(MSCs)中的转录及表达情况,为进一步进行多基因转染MSCs复合纳米仿生骨治疗骨缺损实验提供材料。方法用电穿孔方法将真核表达载体pIRES2-EGFP-hBMP2转染至兔MSCs中,荧光显微镜及流式细胞学检查观察转染效果、检测转染效率,定量RT-PCR方法观察hBMP-2cDNA在兔MSCs中的转录情况,免疫组织化学及westernblot方法检测hBMP-2蛋白表达情况,ALP活性定量测定检测hBMP-2蛋白功能。结果电穿孔方法将重组质粒转染至兔MSCs中,荧光显微镜下观察到绿色荧光蛋白表达,流式细胞仪检测转染效率为(42.7±2.1)%,转染24h后定量RT-PCR方法检测到hBMP-2cDNA在兔MSCs中的转录片断,免疫组织化学观察到hBMP-2蛋白呈强阳性表达,westernblot方法可见18KDhBMP-2蛋白条带,转染组细胞ALP活性明显提高(P>0.05)。结论pIRES2-EGFP-hBMP2通过电穿孔方法导入兔MSCs,并在其中有效表达,发挥生物学作用。     

Influence of vitamin D and retinoids on the gammacarboxylation of osteocalcin in human osteosarcoma MG63 cells

Osteocalcin (OC) is a bone matrix protein, synthesized by osteoblasts, which contains three residues of gammacarboxyglutamic acid (GLA). A fraction of circulating OC, which is not fully carboxylated and does not bind to hydroxyapatite, is called undercarboxylated OC (ucOC). In elderly institutionalized women, we have shown an increase of circulating ucOC level which may result not only from vitamin K deficiency but also from vitamin D deficiency (Szulc et al., J Clin Invest 91: 1769; 1993). This intriguing finding prompted us to study the effect of vitamin D on the secretion of ucOC by osteoblastic cells in vitro in the presence of warfarin, an inhibitor of gammacarboxylation of GLA-containing proteins. The potential influence of retinoic acid (RA) was also studied, because its mechanism of action involves pathways that are similar to vitamin D. In the presence of warfarin (0.05 μg/mL), 1,25(OH)₂D (10⁻⁸-10⁻⁶ mol/L) decreased dose dependently ucOC secretion by human osteosarcoma MG63 cells (from 3.87 ± 0.96 to 2.12 ± 0.13 ng/10⁶ cells). When expressed as a fraction of total OC, secretion ucOC decreased from 47.4 ± 1.4% to 24.8 ± 3.2% in the MG63 cells. The secretion of total OC was stimulated by RA and by Ro 13-7410, which is a specific ligand of retinoic acid receptor (RAR), but not by 9-cis retinoic acid (9-cisRA), which is a physiologic ligand of retinoid X receptor (RXR). RA and Ro 13-7410 decreased ucOC secretion and ucOC% in warfarin-treated MG63 cells (RA: from 50.4 ± 13.3% to 13.5 ± 2.8%; Ro 13-7410: from 28.4 ± 8.2% to 11.3 ± 8.4%). 9-cisRA had no effect on OC gammacarboxylation. These results show that vitamin D, RA, and Ro 13-7410, but not 9-cisRA, may modify the gammacarboxylation of OC in human MG63 cells.     

骨形成蛋白-2基因在人骨髓基质细胞中的表达及对其成骨分化的作用

目的利用构建的人骨形成蛋白-2(BMP2)真核表达载体pcDNA3／BMP2，检测其转染人骨髓基质细胞后的表达及对其成骨分化的影响。方法酶切鉴定构建的真核表达载体pcDNA3／BMP2，利用脂质体介导的转染技术，将所构建的载体导入骨髓基质细胞中，体外单层培养。分别于转染后48h和4周采用原位杂交、免疫组化和碱性磷酸酶、钙化学染色方法检测BMP2的基因蛋白表达以及对骨髓基质细胞成骨分化的影响。结果pcDNA3／BMP2酶切片段的大小与理论相符。转染后细胞能检测到BMP2基因和BMP2蛋白表达，并促进成骨转化。结论pcDNA3／BMP2转染骨髓基质干细胞中可获得短暂和长期表达，并加强骨髓基质细胞的成骨分化能力。     

Parathyroid Hormone-Related Protein (1–37) Induces cAMP Response in Human Osteoblast-Like Cells

During recent years parathyroid hormone-related protein (PTHrP) research has been focused on the physiological functions of different fragments of the PTHrP molecule. Here we demonstrate that PTHrP (1–37) induced cyclic adenosine monophosphate (cAMP) response in primary human osteoblast-like cells, which were well characterized by the presence of alkaline phosphatase activity and osteocalcin production after stimulation with 1,25-dihydroxyvitamin D₃. However, there was no cAMP response to PTHrP (58–77). Furthermore, the response to PTHrP (1–37) was dose dependent, with a significant increase at 1 nM. The presence of PTHrP (1–37)-induced cAMP response in human osteoblast-like cells implies that aminoterminal PTHrP fragments may exert important functions in the bone. Received: 27 January 1997 / Accepted: 26 June 1997     

人成骨样细胞和前破骨样细胞ER亚型、OPG/RANKL/RANK系统、IL-6及其受体以及破骨细胞标志基因表达的研究

目的分析比较两种人成骨样细胞系(U2-OS、MG-63)和两种人前破骨样细胞系(U937、HL-60)ER亚型、OPG/RANKL/RANK系统、IL-6及其受体以及破骨细胞标志基因表达的差异,为今后研究雌激素与IL-6等细胞因子在骨组织中相互关系提供适宜的细胞模型。方法RT-PCR和免疫印迹法检测ERα、ERβ的表达,ELISA法测定IL-6的分泌,OPG、RANKL、RANK及IL-6受体的表达采用免疫印迹法进行检测,TRAP、MMP-9则采用RT-PCR技术进行分析。结果(1)转录水平及蛋白水平证实四种细胞均表达ERα和ERβ;(2)四种细胞不同程度地表达OPG和RANKL,而RANK则仅见于U937细胞表达;(3)四种细胞均表达IL-6受体(IL-6Rα、gp130),除U2-OS细胞外其余三种细胞均组成型分泌IL-6;(4)破骨细胞标志基因TRAP在两种人前破骨样细胞U937、HL-60中均表达,且表达水平相近;而MMP-9仅在U937细胞中弱表达;两种人成骨样细胞U2-OS、MG-63未见有这两种基因表达。结论筛选出用于研究雌激素与IL-6等细胞因子在骨组织中相互关系的细胞模型,同时也为今后深入阐明骨靶向和其他新型抗骨质疏松雌激素的分子机制研究奠定基础。     

Evaluation of P‐glycoprotein (Pgp) expression in human osteosarcoma by high‐throughput tissue microarray

Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well‐established cause of MDR involves overexpression of P‐glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high‐throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre‐operative chemotherapy in osteosarcoma patients. Eighteen of the fifty‐seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606–1612, 2016.     

聚羟基烷酸酯与绵羊骨髓基质干细胞相容性的研究

目的评价聚羟基烷酸酯(PHBV)作为组织工程支架与绵羊骨髓基质干细胞(BMSCs)的生物相容性。方法原代培养绵羊BMSCs,传至2～3代后,接种至PHBV膜和泡沫样三维支架上,光镜和扫描电镜观察细胞形态,计数1、2、6 h时的细胞黏附率;并以接种至培养板上细胞为对照组,每日细胞计数,绘制生长曲线;按培养液量与支架体积10 mL／cm3为标准浓度制备浸提液,并制备标准浓度1／16～16倍的浸提液,以MTT法检测细胞毒性;流式细胞仪分析接种到材料上的细胞周期,计算增殖指数;BMSCs接种于PHBV三维支架上4、8、12 d,以Hoechst33258荧光法定量测定细胞内DNA含量, BCA法测定蛋白质含量。结果第3代BMSCs接种至PHBV膜上2 h后即大部黏附,黏附率75．6％,与对照组相比差异无统计学意义,绘制生长曲线见细胞生长与对照组无差异;MTT法检测见9个浓度梯度的浸提液毒性均为0级;光镜和扫描电镜观察见细胞接种于PHBV膜上2 h后大部分黏附,3 d后伸展良好,呈纺锤形或梭形,在三维支架的孔隙内立体生长,1周开始细胞间连接,3周广泛连接,分泌大量基质;流式细胞分析见接种于材料上的细胞周期无变化;接种至PHBV三维支架上的细胞内DNA、蛋白质浓度与对照组比较无差异。结论PHBV作为BMSCs的组织工程支架材料,具有良好的生物相容性。     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

The protective role of G protein-coupled estrogen receptor 1 (GPER-1) on methotrexate-induced nephrotoxicity in human renal epithelium cells

Nephrotoxicity is an important problem during methotrexate (MTX) treatment, which has been widely used for the treatment of several cancer types. Females are less susceptible to kidney diseases; however, the reason for this condition has not to be fully clarified. But sex hormones such as estrogen may have a protective effect on the kidney. We aimed to evaluate the possible protective role of estrogen on the MTX-induced renal epithelial cell death. Primary renal proximal tubular epithelial cells (RPTEC) were incubated with MTX (1, 10 and 100?μM), either alone or in combination with the 17β-estradiol, G protein-coupled estrogen receptor 1 (GPER1) agonist G-1, estrogen receptor alpha agonist propyl pyrazole triol (PPT), estrogen receptor beta agonist diarylpropionitrile (DPN). Cell viability was determined by MTT assays. Interleukin (IL)-1β, IL-6, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were determined in RPTEC. Approximately half of the cell death was observed with 10?μM MTX incubation for 48?h. The cell death was prevented by co-incubating with17β-estradiol, PPT and G-1. MTX was significantly induced IL-1β and IL-6.17β-estradiol, PPT and G-1 significantly decreased effects of MTX. SOD activity was significantly decreased treatment with MTX compared to control group. SOD activity was increased with co-incubation with 17β-estradioland G-1 compared to treatment with MTX. MDA levels significantly increased in treatment with MTX compared with the control group. Increased MDA levels by MTX-induced was decreased significantly by the treatment with 17β-estradiol and G-1. These data indicate that especially 17β-estradiol and G-1 may be useful in preventing undesirable effects of MTX in renal failure.