大豆多肽对前列腺癌PC-3细胞增殖及凋亡的影响

目的探讨大豆多肽对前列腺癌PC-3细胞增殖及凋亡的影响,为临床应用大豆多肽治疗前列腺癌提供实验依据。方法用不同浓度的大豆多肽(5、10、15、20μmol/L)处理前列腺癌PC-3细胞不同时间(24、48、72 h),采用MTT法检测细胞的增殖活力,倒置显微镜观察细胞的形态,流式细胞术检测细胞凋亡率及细胞周期。结果大豆多肽可抑制前列腺癌PC-3细胞的增殖,将细胞周期阻滞在G2/M期,诱导细胞凋亡,中晚期细胞凋亡趋势明显,且呈明显的剂量与时间依赖性;随着大豆多肽浓度的增加,前列腺癌PC-3细胞密度逐渐降低,边缘趋于圆滑,细胞间隙逐渐增大,局部可见部分已固缩的细胞及死亡的细胞碎片。结论大豆多肽可抑制前列腺癌PC-3细胞增殖,诱导细胞凋亡,在临床治疗前列腺癌方面具有广阔的应用前景。     

人前列腺癌PC-3细胞膜蛋白组学的分析

目的分析人前列腺癌PC-3细胞中的所有膜蛋白,全面展示PC-3细胞中的蛋白质表达谱。方法采用一维SDS-PAGE,结合高效液相色谱-串联质谱的方法,分离并鉴定PC-3细胞中的膜蛋白。结果在严格的过滤参数条件下,PC-3细胞中鉴定出2172种蛋白,其中1499种经过基因本体评注(GOA)显示为已知细胞组分,其余为未知细胞组分。在已知细胞组分中,564种(37.6%)蛋白为质膜蛋白,其中138种为跨膜蛋白。跨膜蛋白中,21.17%被预测至少有1个跨膜区,57.66%有1～3个跨膜区,30.66%有4～9个跨膜区,11.66%有不少于10个跨膜区。许多新的蛋白也被鉴定,其中包括假设蛋白和一些cD-NA序列。结论这些被鉴定蛋白的生物学功能和理化性质将有助于进一步理解前列腺癌侵袭和转移的分子机制,为寻找与前列腺癌转移相关的分子提供新的实验证据。     

Survivin基因干扰和过表达对人乳腺癌MCF-7细胞凋亡的影响

目的观察凋亡抑制蛋白Survivin基因干扰和过表达对人乳腺癌MCF-7细胞凋亡的影响。方法将Survivin基因shRNA干扰质粒和过表达质粒pcDNA3.1-GFP-Survivin在脂质体介导下转染MCF-7细胞,并设空白对照组和脂质体对照组,转染后48 h,荧光显微镜下观察转染效率;荧光定量RT-PCR法检测转染细胞中Survivin基因mRNA的转录水平;MTT法检测转染细胞的凋亡率。结果转染MCF-7细胞后48 h,Survivin基因RNAi质粒和过表达质粒的转染效率为50%～60%;shRNA质粒转染组MCF-7细胞中Survivin基因mRNA的转录水平明显低于空白对照组和脂质体对照组,而过表达质粒转染组明显高于空白对照组和脂质体对照组(P<0.05);shRNA质粒转染组MCF-7细胞的凋亡率明显高于空白对照组和脂质体对照组,而过表达质粒转染组明显低于空白对照组和脂质体对照组(P<0.05)。结论 Survivin基因对人乳腺癌MCF-7细胞的凋亡具有一定的抑制作用,可作为人乳腺癌生物治疗的候选基因。     

T-cadherin基因在前列腺癌组织中的表达及其对前列腺癌细胞DU145增殖的影响

目的探讨T-cadherin基因在前列腺癌组织中的表达及其对前列腺癌细胞DU145增殖的影响。方法采用RT-PCR法检测40份前列腺癌及相应癌旁组织中T-cadherin基因mRNA的表达。分别用10 ml滴度为1.6×10~(12)pdf/ml的GFP-T-cadherin腺病毒(Ad-GFP-T-cadherin)和GFP腺病毒(Ad-GFP)感染前列腺癌DU145细胞后,MTT法检测T-cadherin对前列腺癌DU145细胞增殖的影响,Western blot法检测T-cadherin对P21和cyclin D1蛋白表达水平的影响,同时设空白对照组(未感染病毒)。结果 T-cadherin在38/40(95%)的前列腺癌中表达下调(P0.01),其表达与前列腺癌的分期、格里森评分及分化有关。Ad-GFP-T-cadherin组DU145细胞中P21蛋白表达水平明显高于Ad-GFP组及空白对照组(P0.01),而cyclin D1蛋白表达水平及增殖活性明显低于Ad-GFP组及空白对照组(P0.01);空白对照组DU145细胞的增殖活性及细胞中P21、cyclin D1蛋白表达水平与Ad-GFP组比较,差异无统计学意义(P0.05)。结论 T-cadherin的表达与前列腺癌的发生密切相关,且可通过上调P21和下调cyclin D1蛋白的表达抑制前列腺癌DU145细胞的增殖。     

丹皮酚对人卵巢癌SKOV3细胞凋亡的影响及其可能的机制

目的探讨丹皮酚对人卵巢癌SKOV3细胞的促凋亡作用及其可能的机制。方法用25、50、100、200、400μg/ml丹皮酚处理SKOV3细胞,设未经丹皮酚处理的细胞为对照组,24 h后,采用MTT法检测细胞增殖情况;用50、100、200μg/ml丹皮酚处理SKOV3细胞,设未经丹皮酚处理的细胞为对照组,24 h后,采用流式细胞术(flow cytometry,FCM)、Hoechst染色法检测细胞凋亡情况,Western blot法检测caspase3及survivin蛋白的表达情况。结果与对照组相比,各浓度丹皮酚对SKOV3细胞的增殖均有明显的抑制作用,呈浓度依赖性(P<0.05),IC50值为200.06μg/ml;100、200μg/ml浓度组中SKOV3细胞凋亡率分别为(35.33±1.32)%、(39.56±1.27)%,与对照组[(9.01±1.21)%]相比明显增加(P<0.05);丹皮酚100、200μg/ml浓度组中发生凋亡的细胞数量较对照组多;丹皮酚处理后细胞凋亡相关蛋白survivin表达降低,而caspase3表达增加,高浓度组与低浓度组相比,差异均有统计学意义(P<0.05)。结论丹皮酚能显著抑制人卵巢癌SKOV3细胞增殖,促进细胞凋亡,其机制可能与其调控凋亡相关蛋白survivin、caspase3表达变化有关。     

热休克蛋白70过表达对过氧化氢诱导BRL细胞caspase 3表达的影响

目的探讨热休克蛋白(heat shock protein,Hsp)70过表达对过氧化氢(hydrogen peroxide,H_2O_2)诱导Buffalo大鼠肝细胞(BRL细胞)cleaved caspase 3表达的影响。方法将BRL细胞分为6组:空白对照组、Ad-CMV-Null组、Ad-CMV-Hsp70组、H_2O_2组、Ad-CMV-Null+H_2O_2组和Ad-CMV-Hsp70+H_2O_2组,Ad-CMV-Null、Ad-CMV-Hsp70、AdCMV-Null+H_2O_2、Ad-CMV-Hsp70+H_2O_2组分别加入1×10~7 PFU/ml的Ad-CMV-Null或Ad-CMV-Hsp70培养48 h后,H_2O_2、Ad-CMV-Null+H_2O_2和Ad-CMV-Hsp70+H_2O_2组分别加入150μmol/L H_2O_2处理2 h,空白对照、Ad-CMVNull和Ad-CMV-Hsp70组分别加入培养液培养。分别采用实时荧光定量PCR检测细胞中Hsp70基因mRNA的转录水平,Western blot法检测细胞中Hsp70及cleaved caspase 3蛋白的表达水平。结果与H_2O_2组相比,Ad-CMVHsp70+H_2O_2组细胞中Hsp70基因mRNA的转录水平和蛋白表达水平均显著升高(P0.01),cleaved caspase 3蛋白表达水平显著下降(P0.01)。结论Hsp70可抑制caspase 3的激活最终保护BRL细胞,减少凋亡损伤。     

丁酸钠对CHO细胞生长及rhEPO表达量的影响

目的:通过对比实验的方式,在培养基中添加不同浓度的丁酸钠(NaBu),以此获取CHO细胞生长与产物rhEPO表达量的影响.方法:在24孔板上采用不同浓度的丁酸钠(0mM、0.25mM、0.5mM、0.75mM)培养CHO细胞生长,设定对照组与研究组,并借助血球计数板测定细胞密度,以此保持各组相同的细胞接种数据,测定实验...     

雷公藤红素对卵巢癌SKOV3/DDP细胞的抑制作用及其机制

目的探讨雷公藤红素(celastrol)对卵巢癌顺铂(cisplatin,DDP)耐药细胞SKOV3/DDP的抑制作用及其机制。方法 MTT法检测不同浓度雷公藤红素对SKOV3/DDP细胞增殖的影响;显微镜下观察不同浓度雷公藤红素对SKOV3/DDP细胞形态的影响;流式细胞术检测不同浓度雷公藤红素对SKOV3/DDP细胞周期及凋亡的影响;细胞划痕试验检测不同浓度雷公藤红素对SKOV3/DDP细胞迁移能力的影响;Transwell体外侵袭试验检测不同雷公藤红素对SKOV3/DDP细胞侵袭能力的影响;Western blot法检测SKOV3/DDP细胞中凋亡及侵袭相关蛋白的表达水平。结果经雷公藤红素作用后,SKOV3/DDP细胞的增殖抑制率显著上升(P0.05);镜下观察SKOV3/DDP细胞数目显著减少(P0.05);G1期细胞比例明显升高(P0.05);细胞凋亡率显著增加(P0.05);细胞迁移距离明显减小(P0.05);穿膜细胞数目明显减少(P0.05);SKOV3/DDP细胞中Cyclin A、p-AKT、NF-KB、MMP-2、MMP-9和P-GP蛋白的表达水平均明显下降(P0.05)。结论雷公藤红素对SKOV3/DDP细胞抑制作用显著,为临床晚期失去手术机会卵巢癌患者的治疗提供了参考。     

白藜芦醇基于PI3K/Akt通路对人结肠癌细胞HCT-116凋亡的影响

目的：探究白藜芦醇基于PI3K/Akt通路对人结肠癌细胞HCT-116凋亡的影响及其相关分子机制。方法：通过MTT实验检测白藜芦醇对HCT-116细胞的增殖抑制效应;流式细胞仪检测白藜芦醇对HCT-116的促凋亡效应;蛋白免疫印迹实验检测HCT-116细胞中Caspase-3、Bcl-2、Bax、PI3K、Akt表达情况的影响。结果：白藜芦醇对HCT-116有明显的抑制增殖作用,并有效促进了其凋亡,且引起Caspase-3与Bax表达上调,Bcl-2表达下调并抑制了PI3K和Akt的磷酸化。结论：白藜芦醇可诱导HCT-116细胞发生凋亡,且与抑制PI3K/Akt信号通路的激活相关。     

乳酸对重组CHO细胞生长代谢及EPO表达的影响

利用脉冲实验方法研究了乳酸和渗透压对重组CHO细胞生长代谢和EPO表达的影响 ,证明乳酸分子本身对重组CHO细胞的生长代谢和产物表达没有明显的影响 .乳酸浓度从 0增加到 6 9mmol·L^-1对应于渗透压从 30 0增加到 4 2 0mOsm ,细胞的比生长速率下降了 33% ,葡萄糖和谷氨酰胺的比消耗速率均增加了 2 3% ,细胞对葡萄糖和谷氨酰胺的得率系数分别下降了 4 5 %和 4 7% ,乳酸的比生成速率则保持恒定 ,表明在高渗透压下营养物的利用更倾向于能量代谢过程 ,好氧能量代谢过程所占的比例随着渗透压的增加而增加 ;另一方面 ,氨对谷氨酰胺的得率系数增加了 4 4 % ,丙氨酸对谷氨酰胺的得率系数下降了 2 4 % ,表明谷氨酰胺经谷氨酸脱氢酶途径的代谢流量随着渗透压的增加而增加 .EPO的比生成速率随着渗透压的增加而下降     

Polyphenon E Effects on Gene Expression in PC-3 Prostate Cancer Cells

Polyphenon E (Poly E) is a standardized, caffeine-free green tea extract with defined polyphenol content. Poly E is reported to confer chemoprotective activity against prostate cancer (PCa) progression in the TRAMP model of human PCa, and has shown limited activity against human PCa in human trials. The molecular mechanisms of the observed Poly E chemopreventive activity against PCa are not fully understood. We hypothesized that Poly E treatment of PCa cells induces gene expression changes, which could underpin the molecular mechanisms of the limited Poly E chemoprevention activity against PCa. PC-3 cells were cultured in complete growth media supplemented with varied Poly E concentrations for 24 h, then RNA was isolated for comparative DNA microarray (0 vs. 200 mg/L Poly E) and subsequent TaqMan qRT-PCR analyses. Microarray data for 54,613 genes were filtered for >2-fold expression level changes, with 8319 genes increased and 6176 genes decreased. Eight genes involved in key signaling or regulatory pathways were selected for qRT-PCR. Two genes increased expression significantly, MXD1 (13.98-fold; p = 0.0003) and RGS4 (21.98-fold; p = 0.0011), by qRT-PCR. MXD1 and RGS4 significantly increased gene expression in Poly E-treated PC-3 cells, and the MXD1 gene expression increases were Poly E dose-dependent.     

Survivin启动子调控的EGFP基因真核表达质粒的构建及其在肺癌细胞中的特异性表达

目的构建由Survivin启动子调控的EGFP基因真核表达质粒,并检测其在人肺腺癌A549细胞中的特异性表达。方法以A549细胞基因组DNA为模板,PCR扩增Survivin启动子,替换pEGFP-C1载体中的CMV启动子,构建携带Survivin启动子的pEGFP-C1真核表达质粒pEGFP-C1/Surp,转染A549细胞及人胚肺成纤维细胞MRC-5,在荧光显微镜下观察EGFP的表达。结果重组表达质粒pEGFP-C1/Surp经双酶切和测序鉴定,证明构建正确。转染A549细胞和MRC-5细胞后,在荧光显微镜下可见A549细胞有较强的绿色荧光,而MRC-5细胞则未见绿色荧光。结论已成功构建以Survivin启动子为调控序列、EGFP为标示蛋白的真核表达质粒,且具有较强的肿瘤特异性启动活性,为进一步开发肿瘤靶向性基因治疗载体奠定了基础。     

Survivin在结直肠癌组织中的表达及其临床意义

目的探讨凋亡抑制因子Survivin在结直肠癌组织中的表达及其与各临床病理因素之间的相关性。方法应用RT-PCR技术检测58份结直肠癌组织及其癌旁正常组织标本中Survivin基因的表达,回顾性分析其与诸多临床病理因素间的相关性。结果在58份结直肠癌组织中,Survivin基因的阳性表达率为84.5%,而在癌旁正常组织中均不表达,二者差异有统计学意义。在癌组织中,Survivin的表达与患者的年龄、性别、肿瘤大小、部位及组织分化程度无明显相关性,但与Dukes分期及有无淋巴结转移密切相关。结论Survivin基因在结直肠癌组织中高表达,并与病理分期、有无淋巴结转移等恶性临床病理特征有密切相关性,提示Survivin在结直肠癌的发生、发展及预后中发挥重要作用。     

Survivin启动子调控sea基因真核表达质粒的构建及其在肺癌细胞中的特异性表达

目的构建Survivin启动子调控的葡萄球菌肠毒素A(SEA)基因真核表达质粒,并检测其在人肺腺癌A549细胞中的特异性表达。方法以金黄葡萄球菌(ATCC25923)基因组为模板,PCR扩增sea基因。用sea基因替代以Survivin启动子为调控序列的pGL-S-RED质粒中的Red基因,构建真核表达质粒pGL-S-SEA。酶切及测序鉴定后,用脂质体转染A549细胞和MRC-5人胚肺成纤维细胞,RT-PCR检测两种细胞sea基因的转录情况。结果Survivin启动子调控的、以超抗原SEA编码序列CDS为目的基因的真核表达质粒pGL-S-SEA构建正确,并在A549细胞中启动了sea基因的转录,其强度为内参基因(GAPDH)的65.96%,而在MRC-5细胞对照中未检测到sea基因的转录。结论已成功构建Survivin启动子调控的sea基因真核表达质粒,Survivin启动子可在转录水平特异性地调控sea基因在A549细胞内表达,为下一步以其作为基因疫苗治疗肺癌奠定了基础。     

Apoptotic Effects of Dietary and Synthetic Sphingolipids in Androgen-Independent (PC-3) Prostate Cancer Cells

Stress-induced activation and metabolism of plasma membrane sphingolipids results in intracellular ceramide accumulation and has been shown to induce apoptosis in human prostate cancer cells. This effect has been observed using synthetic ceramide analogs, such as C6-ceramide; however, the effects of naturally-occurring sphingolipids, such as C18-ceramide and sphingomyelin (CerPCho), on apoptosis and prostate cancer cell proliferation have not been examined. The results of the present study demonstrate that natural (CerPCho, C18-ceramide) and synthetic (C6-ceramide) sphingolipids reduced PC-3 cell proliferation by 15 ± 1.8, 17 ± 2.5, and 46 ± 2.1%, respectively (P < 0.05). These reductions in proliferation were due, in part, to increased cellular apoptosis. Treatment of PC-3 cells with CerPCho and C18-ceramide significantly increased apoptosis by 3.0 ± 0.8 and 3.6 ± 0.6%, respectively, compared to the untreated control, while the synthetic C6-ceramide significantly increased apoptosis by 55.7 ± 0.4%. C6-ceramide-induced apoptosis was associated with cell cycle arrest in the G₂/M phase, decreased extracellular signal-regulated kinase (ERK1/2) signaling and activation of the cell cycle regulatory protein, retinoblastoma (pRb). Treatment of PC-3 cells with C18-ceramide and CerPCho did not alter cell cycle distribution, pRb or ERK1/2 activation. Taken together, these results suggest that natural and synthetic sphingolipids induce apoptosis in PC-3 cells via distinct signaling mechanisms and potencies.     

凋亡抑制因子survivin在喉鳞癌中表达及其临床意义

目的　探讨凋亡抑制因子survivin在喉鳞癌中的表达及临床意义。方法　应用免疫组化SABC方法检测 5 4例喉鳞癌手术切除标本石蜡切片组织中survivin的表达 ,并以 10例声带息肉组织为对照。结果　survivin在声带息肉中不表达 ;在 5 4例喉鳞癌中 ,38例 ( 70 .4 % )表达阳性 ;survivin阳性表达率与喉癌患者的年龄、性别、肿瘤大小、临床分型无相关性 (P >0 .0 5 ) ,与喉癌临床分期、组织学分级、淋巴结转移呈正相关 (P <0 .0 5 )。结论　凋亡抑制因子survivin的异常表达所引起的凋亡抑制与喉鳞癌的发生发展相关 ,可作为肿瘤诊断、预后和治疗的一个新指标。     

Effect of AQP9 Expression in Androgen-Independent Prostate Cancer Cell PC3

It is known that aquaporin 9 (AQP9) in the prostate was strictly upregulated by androgen and may represent a novel therapeutic target for several cancers, but whether AQP9 plays a role in the regulation of androgen-independent prostate cancer still remains unclear. In the present study, AQP9 was determined in prostate cancer and adjacent cancer tissues; AQP9-siRNA was applied to silencing AQP9 in androgen-independent prostate cancer cell PC3 cell line. Western blot and flow cytometry analysis were employed to detect changes in related-function of control and AQP9-siRNA groups. The results showed that AQP9 is significantly induced in cancer tissues than that in adjacent cancer tissues. Moreover, knockdown of AQP9 in PC3 androgen-independent prostate cancer cell prostate cancer cells increased inhibition rates of proliferation. In addition, knockdown of AQP9 resulted in a significant decrease in the expression of the Bcl-2 and with a notable increase in the expression of Bax and cleaved caspase 3, indicated that AQP9 knockdown promoted apoptosis in prostate cancer cells. From wound healing assay and matrigel invasion, we suggested that AQP9 expression affects the motility and invasiveness of prostate cancer cells. Moreover, In order to explore the pathway may be involved in AQP9-mediated motility and invasion of prostate cancer cells, the phosphorylation of ERK1/2 was significant suppressed in AQP9 siRNA-transfected cells compared with that in control cells, suggesting that AQP9 is involved in the activation of the ERK pathway in androgen-independent prostate cancer cells.     

Survivin-siRNA真核表达载体的构建及其对胃癌SGC-7901细胞增殖和凋亡的影响

目的构建Survivin-siRNA真核表达载体,并探讨其对胃癌SGC-7901细胞增殖和凋亡的影响。方法化学合成4条能转录出siRNA的模板DNA,各75个碱基,退火形成2条双链DNA,双酶切后插入pSUPER.basic载体。将阳性重组质粒转染SGC-7901细胞,进行细胞计数,并用MTT法检测细胞的增殖活性;半定量RT-PCR检测细胞Survivin基因mRNA的转录水平;流式细胞术检测细胞周期的变化。结果PCR和酶切鉴定表明Survivin-siRNA真核表达载体构建正确,其能下调SGC-7901细胞Survivin基因mRNA的转录水平,抑制SGC-7901细胞生长和增殖,并促进细胞凋亡,使G0/G1和亚G1期细胞增多,S期细胞减少。结论已成功构建了Survivin-siRNA真核表达载体,其能下调SGC-7901细胞中Survivin基因mRNA的转录水平,使细胞增殖减弱,凋亡增加,为RNA干扰技术应用于胃癌的基因治疗提供了一定的实验依据。     

In Vitro Antimetastatic Effect of Phosphatidylinositol 3-Kinase Inhibitor ZSTK474 on Prostate Cancer PC3 Cells

Tumor metastasis is the main cause of lethality of prostate cancer, because conventional therapies like surgery and hormone treatment rarely work at this stage. Tumor cell migration, invasion and adhesion are necessary processes for metastasis. By providing nutrition and an escape route from the primary site, angiogenesis is also required for tumor metastasis. Phosphatidylinositol 3-kinases (PI3Ks) are well known to play important roles in tumorigenesis as well as metastasis. ZSTK474 is a specific PI3K inhibitor developed for solid tumor therapy. In the present report, antimetastatic activities of ZSTK474 were investigated in vitro by determining the effects on the main metastatic processes. ZSTK474 exhibited inhibitory effects on migration, invasion and adhesive ability of prostate cancer PC3 cells. Furthermore, ZSTK474 inhibited phosphorylation of Akt substrate-Girdin, and the secretion of matrix metalloproteinase (MMP), both of which were reported to be closely involved in migration and invasion. On the other hand, ZSTK474 inhibited the expression of HIF-1α and the secretion of vascular endothelial growth factor (VEGF), suggesting its potential antiangiogenic activity on PC3 cells. Moreover, we demonstrated the antiangiogenesis by determining the effect of ZSTK474-reduced VEGF on tube formation of human umbilical vein endothelial cells (HUVECs). In conclusion, ZSTK474 was demonstrated to have potential in vitro antimetastatic effects on PC3 cells via dual mechanisms: inhibition of metastatic processes including cell migration, invasion and adhesion, and antiangiogenesis via blockade of VEGF secretion.