四硫化四砷对人宫颈癌细胞Hela环氧合酶表达和PGE2产生的影响

目的研究中药雄黄主要成分四硫化四砷(As4S4)对子宫颈癌细胞Hela增殖和凋亡作用的影响及其作用机制。方法以不同浓度(7．5、15、30、60 mg/L)的As4S4对Hela细胞分别处理不同的时间(12、24、36、48、60 h),用四甲基偶氮唑蓝(MTT)法测定细胞增殖反应;流式细胞仪检测细胞凋亡率;Western blot检测环氧合酶-2(COX-2)蛋白的表达;用放射免疫法榆测细胞PGE2释放水平。结果As4S4对Hela细胞增殖有明显的抑制作用(P<0．01),并呈明显的时效和量效关系,作用24 h时IC50的As4S4作用浓度为30 mg／L。As4S4可诱导Hela细胞凋亡,与对照组相比,差异有极显著意义(P< 0．01),并呈浓度依赖性。As4S4可明显抑制Hela细胞COX-2的表达(P<0．05),随着As4S4浓度的增加,其COX-2蛋白表达水平逐渐降低。不同浓度As4S4作用24 h,可明显抑制Hela细胞PGE2的分泌水平,与对照组比较差异有极显著意义(P<0．01)。结论As4S4对Hela细胞增殖具有抑制作用,可促进细胞凋亡,其作用机制可能与抑制细胞COX-2表达和PGE2分泌水平有关。     

四硫化四砷抑制人胃癌SGC7901细胞增殖及其作用机制

目的：探讨四硫化四砷（As4S4）对人胃癌细胞系SGC7901增殖抑制作用及其作用机制.方法：MTT法检测四硫化四砷对体外培养SGC7901细胞的抑制作用,透射电镜观察细胞形态学改变,流式细胞仪检测细胞周期的变化,RT-PCR法检测bcl-2和bax mRNA的表达,Western blot法检测bcl-2和bax蛋白的表达.结果：四硫化四砷具有体外抑制SGC7901细胞增殖的作用,能诱导细胞凋亡,细胞中bcl-2的表达降低,bax 的表达增强.结论：四硫化四砷具有抑制胃癌细胞系SGC7901细胞生长的作用,其机制可能与诱导细胞凋亡和调节bcl-2与bax表达有关.     

硫化砷对白血病细胞的生长抑制及作用机制

 目的 研究硫化砷（As4S4）通过体外培养对白血病细胞HL-60细胞的生长抑制作用及其分子机制。方法 以不同浓度（7.5 ~ 60 mmol／L）的As4S4作用于体外培养HL-60细胞12 ~ 48 h,四甲基偶氮唑蓝（MTT）法检测细胞生长抑制率,流式细胞仪观察细胞凋亡率,免疫组化法检测bcl-2、p53蛋白表达,应用RT-PCR法检测HL-60细胞中p53 mRNA表达。结果 不同浓度的As4S4作用12 ~ 48 h后,可显著抑制HL-60细胞的生长,呈时间浓度依赖性,并诱导细胞发生凋亡,凋亡率为30.18 % ~ 70.98 %,不同作用时间、不同浓度间凋亡率差异有统计学意义（P＜0.01）,RT-PCR结果显示As4S4作用24 h后,下调p53 mRNA的表达。免疫组化结果显示,bcl-2、p53蛋白表达逐渐降低,并呈浓度依赖性。结论 As4S4能够显著抑制HL-60细胞的生长。诱导其凋亡,并下调bcl-2、p53的表达,可能是其重要机制。     

半胱氨酸酶在NS-398诱导HepG2细胞凋亡中的作用

目的 探讨选择性环氧合酶(COX-2)抑制剂NS-398诱导肝癌HepG2细胞凋亡的分子机制。方法 采用流式细胞术测定细胞凋亡情况;Western blot法检测不同浓度NS-398处理后凋亡相关蛋白Bcl-2、Caspase3表达的变化; 并以流式细胞术检测半胱氨酸酶-3(Caspase-3) 酶活性的变化。结果 流式细胞术显示NS-398(0、100、200、300、400μmol/L)作用HepG2细胞24h后,对照处理组没有出现凋亡峰,其余各组(100、200、300、400μmol/L)出现明显的凋亡峰,其凋亡率分别为(10.51±1.04)%、(27.79±2.40)%、(45.72±3.32)%,(60.22±2.03)%(P<0.01),不同浓度NS-398处理后凋亡相关蛋白Bcl-2表达下降,Caspase-3蛋白表达增加,随着NS-398处理浓度的增加,表达活性Caspase3的细胞百分率分别为(2.67±0.22)%、(9.53±0.15)%、(21.28±0.43)%、(39.63±0.8)%、(63.40±0.69)%(P<0.01)。结论 选择性COX-2 抑制剂可能通过调节Bcl-2蛋白表达活化Caspase3,从而诱导肝癌细胞HepG2凋亡。     

雄黄抑制卵巢癌细胞株SKOV3增殖和诱导凋亡的体外研究

目的 研究中药雄黄主要成分As₄S₄对卵巢癌细胞株SKOV3细胞的增殖抑制和诱导凋亡的作用及其机制。方法 不同浓度As₄S₄(20 μmol/L、40 μmol/L、60 μmol/L、80 μmol/L),处理SKOV3细胞24 h,48 h,72 h后,透射电镜观察细胞形态变化;MTT法测定细胞增殖抑制率;流式细胞仪检测细胞凋亡率和细胞周期变化;RT-PCR法和Westernblot法检测As₄S₄对卵巢癌细胞株SKOV3细胞Bcl-2和Bax mRNA和蛋白的表达影响。结果 不同浓度As₄S₄处理的卵巢癌细胞株SKOV3,细胞增殖受到抑制,作用呈明显的浓度、时间依赖性,差异有统计学意义(P＜0.01);流式细胞仪进行细胞周期DNA成分分析结果显示,随着As₄S₄浓度的增加SKOV3细胞G₀/G₁期细胞百分比明显降低,S期细胞百分比上升;Bcl-2的表达随药物作用浓度的增加而递减,Bax的表达随药物作用浓度的增加而增强。结论 四硫化四砷具有抑制SKOV3细胞生长增殖的作用,其作用机制与诱发细胞凋亡和调节Bcl-2与Bax表达有关。     

Radiation induces upregulation of cyclooxygenase-2 (COX-2) protein in PC-3 cells

PURPOSE: To investigate the impact of gamma-irradiation on cyclooxygenase-2 (COX-2) expression and its enzymatic activity in PC-3 cells. Cell cycle redistribution, viability, and apoptosis were quantitated in control and irradiated cells with or without the COX-2 inhibitor NS-398. METHODS AND MATERIALS: Western blot analysis was used to assess COX-2 protein expression. Prostaglandin (PGE(2)) was measured after addition of arachidonic acid (AA) using a Monoclonal Immunoassay Kit. Cell cycle and apoptosis were assessed using flow cytometry. RESULTS: We observed a dose-dependent increase in COX-2 of 37.0%, 79.7%, and 97.5% following irradiation with 5, 10, and 15 Gy, respectively. The PGE(2) level of irradiated cells was higher than in controls (1512 +/- 157.5 vs. 973.7 +/- 54.2 rhog PGE(2)/mL; p < 0.005, n = 4) while cells irradiated in the presence of NS-398 had reduced PGE(2) levels (218.8 +/- 80.1 rhog PGE(2)/mL; p < 0.005; n = 4). We found no differences in cell cycle distribution or apoptosis between cells irradiated in the presence or absence of NS-398. CONCLUSIONS: COX-2 protein is upregulated and enzymatically active after irradiation, resulting in elevated levels of PGE(2). This effect can be suppressed by NS-398, which has clinical implications for therapies combining COX-2 inhibitors with radiation therapy.     

熊果酸抑制胃癌细胞SGC7901增殖和诱导细胞凋亡的机制

背景与目的:研究表明熊果酸(ursolicacid,UA)可抑制多种肿瘤细胞的增殖并诱导凋亡,但目前有关UA作用于胃癌细胞的报道较为少见。环氧合酶-2(cyclooxygenase-2,COX-2)在多种癌前病变及癌组织中高表达。本研究旨在探讨熊果酸抑制人胃癌细胞SGC7901增殖和诱导凋亡的机制。方法:MTT法检测0、10、20、30、40!mol/LUA作用不同时间对SGC7901细胞增殖的影响;荧光染料Hoechst33258染色观察不同浓度UA作用24h细胞凋亡情况;流式细胞仪检测细胞周期变化及凋亡率;Westernblot法检测COX-2蛋白以及凋亡相关蛋白Bcl-2、Bax表达。放射免疫分析法测定COX-2催化产物前列腺素E2(prostaglandinE2,PGE2)。结果:20～40!mol/LUA可抑制SGC7901细胞的增殖,并呈浓度和时间依赖性,作用12、24、36、48h的半数抑制浓度(IC50)分别为(57.50±1.18)!mol/L、(34.28±2.05)!mol/L、(27.54±1.11)!mol/L、(24.83±1.02)!mol/L;20～40!mol/LUA作用24h后,SGC7901细胞被阻滞于G0/G1期,细胞凋亡率分别为(9.10±2.39)%、(26.30±1.25)%、(35.20±2.26)%;同时COX-2蛋白表达及其催化生成产物PGE2浓度下降,凋亡相关蛋白Bcl-2表达减少,Bax无明显变化。结论:熊果酸对SGC7901细胞具有增殖抑制及诱导凋亡作用,其机制可能与阻滞细胞周期、抑制COX-2表达进而减少PGE2生成以及下调凋亡相关蛋白Bcl-2表达有关。     

p38MAPK 在结肠癌细胞凋亡中的作用及与COX-2 的关系

 目的 探讨结肠癌细胞p38MAPK介导celecoxib（COX-2选择性抑制剂）抗肿瘤的作用及与COX-2的关系。方法 用MTT法检测celecoxib对人结肠癌HT-29细胞生长的作用，用Western blot法测定各组细胞COX-2和Phosph—p38MAPK蛋白表达量，采用流式细胞术检测celecoxib和SB203580（p38MAPK特异性抑制剂）作用后HT-29细胞凋亡和细胞周期分布。结果 p38MAPK和COX-2蛋白表达量与对照组（0．23±0.12）（0.95±0．14）相比，celecoxib可使p38MAPK蛋白表达水平明显升高（0．62±0．11），而使COX-2蛋白表达水平降低（0、44±0．11）；SB203580使p38MAPK（0.12±0．05）及COX-2蛋白（0、23±0．13）表达水平均降低；SB203580和celecoxib共同作用后，p38MAPK表达量介于celecoxib和SB203580作用之间（0．43±0．12），COX-2表达量下降最为显著（0．15±0．10））。celecoxib和eeleeoxib＋SB203580均可显著诱导HT-29细胞凋亡（P〈0．01和P〈0．05），与对照纽（4．31％）相比，其凋亡率分别为40．95％、26．24％。结论 在HT29细胞中，celecoxib可通过活化p38MAPK而诱导结肠癌细胞凋亡，p38MAPK是COX-2的上游激酶，COX-2的表达水平受p38MAPK调控，并且COX-2可能对p38MAPK有负反馈调节作用。celecoxib是通过COX-2及其以外的p38MAPK通路诱导肿瘤细胞凋亡而发挥抗肿瘤作用的。     

Inotodiol Inhabits Proliferation and Induces Apoptosis through Modulating Expression of cyclinE,p27, bcl-2, and bax in Human Cervical Cancer HeLa Cells

Inonotus obliquus is a medicinal mushroom that has been used as an effective agent to treat various diseasessuch as diabetes, tuberculosis and cancer. Inotodiol, an included triterpenoid shows significant anti-tumor effect.However, the mechanisms have not been well documented. In this study, we aimed to explore the effect of inotodiolon proliferation and apoptosis in human cervical cancer HeLa cells and investigated the underlying molecularmechanisms. HeLa cells were treated with different concentrations of inotodiol. The MTT assay was used toevaluate cell proliferating ability, flow cytometry (FCM) was employed for cell cycle analysis and cell apoptosis,while expression of cyclinE, p27, bcl-2 and bax was detected by immunocytochemistry. Proliferation of HeLacells was inhibited by inotodiolin a dose-dependent manner at 24h (r=0.9999, p<0.01). A sub-G1 peak (apoptoticcells) of HeLa cells was detected after treatment and the apoptosis rate with the concentration and longerincubation time (r=1.0, p<0.01), while the percentage of cells in S phase and G2/M phase decreased significantly.Immunocytochemistry assay showed that the expression of cyclin E and bcl-2 in the treated cells significantlydecreased, while the expression of p27 and bax obviously increased, compared with the control group (p<0.05).The results of our research indicate that inotodiol isolated from Inonotus obliquus inhibited the proliferationof HeLa cells and induced apoptosis in vitro. The mechanisms may be related to promoting apoptosis throughincreasing the expression of bax and cutting bcl-2 and affecting the cell cycle by down-regulation the expressionof cyclin E and up-regulation of p27. The results further indicate the potential value of inotodiol for treatmentof human cervical cancer.     

Cyclooxygenase-2 (COX-2) inhibitors sensitize tumor cells specifically to death receptor-induced apoptosis independently of COX-2 inhibition

Cyclooxygenase-2 (COX-2) is involved in diverse processes such as inflammation, carcinogenesis and apoptosis. As COX-2 inhibitors interfere with these processes, inhibition of COX-2 has been suggested as a promising anticancer treatment. However, the role of COX-2 in modulation of apoptosis as well as the death pathways affected by COX-2 inhibitors are poorly characterized. Here we demonstrate that the selective COX-2 inhibitors NS-398 and nimesulide increased TNF sensitivity of TNF-resistant HeLa H21 and TNF-sensitive HeLa D98 cells, although this cytokine induced significant COX-2 activity, as judged by prostaglandin E(2) (PGE(2)) production, only in H21 cells. TNF did also not induce PGE(2) production in MCF-7/casp-3 cells stably expressing COX-2; however, nimesulide strongly enhanced TNF-induced apoptosis in these cells. Furthermore, COX-2 activity in HeLa H21 cells could be inhibited by NS-398 concentrations that were 10 000-fold lower compared to those required for the induction of cell death. Most intriguingly, sensibilization to apoptosis was specifically observed in response to activation of death receptors. Not only TNF-induced cell death but also apoptosis triggered by the CD95 and TRAIL receptors was enhanced by nimesulide. In contrast, apoptosis induced by the anticancer drugs doxorubicine and etoposide that target the mitochondrial death pathway remained unaffected. Together, our data suggest that COX-2 inhibitors overcome apoptosis resistance and selectively sensitize tumor cells to the extrinsic death receptor-induced apoptotic pathway independently of COX-2.     

As2S2对人卵巢癌耐药株C13K/DDP细胞增殖和凋亡的作用

 目的：研究As₂S₂对卵巢癌耐药株C13K/DDP细胞增殖抑制和诱导凋亡的作用。方法：以 不同浓度(4、6、8、10μmol/L)的As2S2,分三个时间点(24、48、72h)干预C13K/DDP细胞,采用 四甲基偶氮唑蓝(MTT)法检测As2S2对C13K/DDP细胞的增殖抑制率;流式细胞仪(FCM)检测细胞凋 亡率;Western blot检测BCL-2、BAX、AKT的表达。结果：MTT结果显示不同浓度(4、6、8、10 μmol/L)的As₂S₂作用C13K/DDP细胞后,与DDP组相比其增殖受到抑制,作用呈明显的时效和量效 关系,差异有统计学意义(P<0.01);流式细胞仪的结果显示6、8μmol/L As₂S₂诱导细胞的24h凋 亡率分别为(16.05 ±2）%、( 22.30±3）%,DDP组为(9.45 ±2)%,对照组为( 7.82±1.2)% ;6、8μmol/L As₂S₂诱导细胞48h凋亡率分别为(28.94 ±1.8）%、(37.85 ±3）%,DDP组为 （14.74±3.2）%,对照组为( 9.80±2.6）%,各组比较,差异有统计学意义(P<0.05);Western blot结果显示BCL-2、AKT表达下调,BAX表达明显上调。结论：As₂S₂对人卵巢癌耐药株 C13K/DDP细胞具有增殖抑制和诱导凋亡的作用,可能与BCL-2下调、BAX上调或AKT下调有关。     

Synthetic and naturally occurring COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest

Epidemiological studies suggest that the use of NSAIDs and/or a high intake of fruit and vegetables reduce the risk of oesophageal adenocarcinoma. Since COX-2 is up-regulated in Barrett's oesophageal carcinogenesis, the protective effect of NSAIDs and natural food components might reflect COX-2 inhibition. We explored the effects of quercetin, a natural flavonoid with a potent COX-2 inhibitory activity, and two commercially available selective COX-2 inhibitors (NS-398 and nimesulide) on cell proliferation, apoptosis, PGE2 production and COX-2 mRNA expression in a human oesophageal adenocarcinoma cell line (OE33). Changes in the relative numbers of adherent and floating cells were quantified and apoptotic cells were identified using ethidium bromide and acridine orange staining under fluorescence microscopy. Flow cytometric analysis of adherent and floating cells was used to quantify apoptosis and to examine the effects of the agents on the cell cycle. After 48 h exposure at concentrations of > or =1 microM both COX-2 inhibitors and quercetin suppressed cell proliferation (P < 0.01) and increased the fraction of floating apoptotic cells. At higher concentrations (50 microM) and longer exposure (48 h) the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors (P < 0.01). Cell cycle analyses showed that quercetin blocked cells in S phase, while the selective COX-2 inhibitors blocked cells in G1/S interphase. COX-2 mRNA expression was suppressed by quercetin and the synthetic COX-2 inhibitors in a time- and dose-dependent manner. Quercetin and the synthetic COX-2 inhibitors (10 microM) suppressed PGE2 production by approximately 70% after 24 h exposure (P < 0.001). We conclude that OE33 is a useful model for the study of COX-2 expression and associated phenomena in human adenocarcinoma cells. Synthetic COX-2 inhibitors and the food-borne flavonoid quercetin suppress proliferation, induce apoptosis and cell cycle block in human oesophageal adenocarcinoma cells in vitro, and future studies should assess their effects in vivo.     

Mechanism of growth inhibitory effects of cyclooxygenase-2 inhibitor-NS398 on cancer cells

Cyclooxygenase (COX)-2 appears to play an important role in gastrointestinal carcinogenesis, and COX-2 overexpression has been demonstrated both in esophageal adenocarcinomas and lymph nodes metastasis. The aim of our study was to investigate the mechanism of growth inhibitory effect of selective inhibition of COX-2 by NS-398 on human cancer cells. The esophageal cancer cell lines (EC9706) that express COX-2 permanently and hepatocellular carcinoma cell lines (SMMC7721) while no expression of COX-2 were studied. Two kinds of cell lines were treated with various concentrations of NS-398 (selective for COX-2 inhibition) at 0.01-0.1 mM for 24 h, 48 h and 72 h. Antiproliferation effect was measured by 3H-TdR incorporation. The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis. Survivin was detected by immunocytochemical technique. The growth inhibition could be induced by NS398 in a dose- and time-dependent manner in two kinds of cell lines. FCM analysis revealed a high sub-G1 cell peak in EC9706 group. Agarose electrophroesis showed marked apoptosis ladder pattern, but no apoptosis by NS-398 in SMMC7721. The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23 +/- 1.08)% and (3.05 +/- 0.15)% (p < 0.001). After 24 h incubation with NS-398 at concentration of 0.1 dmM, the expression of survivin was markedly reduced in EC9706, but not in SMMC7721. We conclude that the administration of a selective inhibitor of COX-2 significantly decreases cell growth in cancer cell lines by different mechanism. NS-398 could inhibit cell proliferation in cancer cells whether or no COX-2 expression. Nevertheless, apoptosis in the cancer cells expressing COX-2 protein increase more than those lacking COX-2.     

超声靶向微泡破裂法介导RNA干扰沉默survivin基因对宫颈癌HeLa细胞凋亡的影响

目的:通过超声靶向微泡破裂(ultrasound targeted microbubble destruction ,UTMD)法介导基因转染,观察RNA干扰对宫颈癌HeLa细胞survivin基因的沉默效应和诱导细胞凋亡的作用.方法:构建靶向survivin基因的短发夹状RNA(short hairpin RNA,shRNA)表达质粒,将其和微泡共同处理后加入HeLa细胞并予以超声辐照,以空白细胞、单纯质粒、单纯超声辐照、质粒+超声辐照和质粒+Lipofectamine转染作为对照;采用荧光显微镜和FCM法评估基因转染效率,同时对超声辐照参数进行优化;应用FCM法和Hoechst染色及DNA ladder分析法检测细胞凋亡,应用RT-PCR检测survivin mRNA水平的变化.结果:经酶切鉴定及测序分析证实重组质粒构建成功.HeLa细胞采用UTMD处理后,基因转染率显著增加(P＜0.01),并在超声强度为1.0 W/cm2、辐照3 min的条件下最为显著(P＜0.01).PT-PCR检测结果显示,质粒与微泡共同作用加超声辐照组的survivin mRNA抑制率为(83.33±2.73)%,均显著高于各对照组(P＜0.01);FCM法检测结果显示,该组细胞的凋亡率同样显著高于各对照组(P＜0.01);Hoechst染色及DNA ladder检测结果显示,只有经脂质体或UTMD转染处理后的细胞中可检测到明显的细胞凋亡形态及凋亡梯带,其余各组均未检测到明显凋亡.结论:UTMD可有效增强表达质粒的转染效率,是一种新的、有应用前景的非病毒基因转移体系.UTMD介导RNA干扰沉默survivin基因可诱导明显的细胞凋亡,为肿瘤基因治疗及研究提供了新的方法.     

Selective inhibition of cyclooxygenase-2 enhances mitomycin-C-induced apoptosis

Purpose: Cyclooxygenase-2 (COX-2) is involved in antiapoptosis signaling, and its induction may require activation of protein kinase C (PKC). Safingol (SAF), a PKC inhibitor, has been shown to enhance apoptosis induced by mitomycin-C (MMC) in human gastric cancer MKN-74 cells. The aim of this study was to identify the role of COX-2 in MMC-induced apoptosis in MKN-74 cells. Methods: Protein expression of COX-2 and Bcl-2 and activation of PKCα were examined by Western blot analysis. Apoptosis induction was examined by staining with bisbenzimide trihydrochloride (Hoechst-33258) of condensed chromatin, which characterizes the cells undergoing apoptosis. COX-2 mRNA levels were examined by Northern blot analysis. Results: After exposure for 1–2 h to 1 μg/ml MMC, upregulation of COX-2 and Bcl-2 protein expression was noted. The activation of PKCα occurred within 1 h of MMC exposure, and temporally preceded the induction of COX-2. Similar results were observed in cells exposed to the PKC activator, 3-phorbol 12-myristate 13-acetate. Cotreatment with SAF and MMC abolished the induction of COX-2 by MMC. Furthermore, NS-398, a selective COX-2 inhibitor, significantly enhanced MMC-induced apoptosis by fivefold from 4 ± 2% (MMC alone) to 20 ± 2% (MMC plus NS-398). There was no discernible change in COX-2 mRNA levels after a 2-h exposure to MMC but a twofold increase after a 24-h exposure. Conclusions: MMC upregulates COX-2 expression, which appears to be an antiapoptotic signal downstream of PKC. Selective inhibition of COX-2 can therefore provide a novel way to enhance MMC-induced apoptosis independent of inhibiting PKC. Received: 6 July 1999 / Accepted: 19 November 1999     

活化T细胞核因子5 对人胃癌MGC803 细胞增殖及凋亡能力的影响

目的:探讨活化T 细胞核因子5（nuclear factor 5 of activated T cells, NFAT5）对人胃癌MGC803 细胞增殖及凋亡能力的影响及其可能的机制。方法：设计并合成3 条靶向NFAT5 基因的siRNA（siRNA2567、siRNA2714 和siRNA4562）及1 条与NFAT5 基因无同源性的阴性对照siRNA（NC-siRNA）,脂质体介导转染人胃癌MGC803 细胞后,采用Real-time PCR检测分析细胞中NFAT5 mRNA 表达水平的变化,进而筛选出有效抑制NFAT5 基因表达的siRNA（NFAT5-siRNA）。NFAT5-siRNA 转染MGC803 细胞48 h 后,进一步采用Real-time PCR和Western blotting 验证并检测细胞中NFAT5 和S100A4 mRNA及蛋白表达水平的变化,流式细胞术和CCK-8 法分析抑制NFAT5 表达对细胞增殖及凋亡的影响。结果: 转染siRNA2567 对NFAT5 mRNA的表达抑制最为明显（P<0.01）,siRNA2567 被验证为NFAT5-siRNA。转染NFAT5-siRNA 48 h 后,NFAT5 和S100A4 mRNA及蛋白的表达水平均明显降低（P<0.05）;与NC-siRNA 组相比较,NFAT5-siRNA 组MGC803 细胞的增殖率在72 h 和96 h 均显著降低（P<0.01）;NFAT5 基因沉默48 h 后,MGC803 细胞的凋亡率由（2.7±0.2）%上升至（7.9±0.2）%（P<0.01）。结论: NFAT5-siRNA 能有效沉默人胃癌MGC803 细胞中NFAT5 基因表达,在抑制细胞增殖率的同时能够有效促进细胞凋亡,该作用可能通过调控S100A4 表达实现。     

Toll样受体8在人宫颈癌细胞株HeLa中的表达及其激动剂对HeLa细胞增殖和凋亡的影响

目的 观察Toll样受体8(TLR8)在人官颈癌细胞株HeLa中的表达,探讨TLR8激动剂CL075对HeLa细胞增殖和凋亡的影响。方法 采用实时荧光定量聚合酶链反应(PCR)法,检测13种肿瘤细胞系中TLR8 mRNA和经CL075作用后HeLa细胞中环氧化酶2(COX-2)、Bcl-2和血管内皮生长因子(VEGF) mRNA的表达水平;采用免疫荧光技术对HeLa细胞中TLR8蛋白的表达进行定位观察;采用流式细胞术检测不同浓度CL075作用下HeLa细胞的细胞周期和凋亡的变化;采用四甲基偶氮唑蓝(MTT)法检测HeLa细胞的增殖状态。结果 与其他肿瘤细胞株相比,HeLa细胞中TLR8mRNA的表达水平最高,达703.7±20.6。TLR8蛋白主要定位于HeLa细胞的胞浆中。0.1、0.5和1.0 μg/ml的CL075分别作用于HeLa细胞48 h后,G2/M+S期细胞所占的比例逐渐增高,其中1.0μg/ml CL075作用组G2/M+S期细胞所占的比例最高,达(57.67±1.73)％,明显高于空白对照组[(39.02±2.33)％,P＜0.01]。经不同浓度的CL075处理后,HeLa细胞的凋亡水平与空白对照组相比,无明显改变(P＞0.05);但经顺铂处理后,凋亡细胞明显增多(P＜0.01)。MTT法检测结果显示,与空白对照组比较,CL075作用于HeLa细胞48和72 h后,HeLa细胞的增殖能力明显增强(P＜0.01)。CI075作用于官颈癌HeLa细胞24和48 h后,COX-2、Bcl-2和VEGF mRNA的表达水平明显升高(P＜0.05)。结论宫颈癌HeLa细胞中TLR8的表达水平及其与配体相互作用的信号,可能是肿瘤发生和发展的重要因素之一。TLR8可能是宫颈癌治疗的一个潜在靶点。     

hTERT反义寡核苷酸对HL-60细胞凋亡及细胞周期的影响

目的：研究hTERT基因反义寡核苷酸（ASODN）对HL-60细胞诱导凋亡作用及细胞周期的影响.方法：应用hTERT ASODN封闭HL-60细胞hTERT基因,RT-PCR方法检测目的基因的表达;流式细胞仪检测细胞凋亡及细胞周期分析,TUNEL方法检测细胞凋亡,琼脂糖凝胶电泳检测凋亡细胞DNA梯带.结果：hTERT ASODN作用细胞72h后,hTERT基因表达明显受到抑制（P＜0.01）.流式细胞仪检测显示：ASODN组细胞阻滞于G0/G1期,S、G2/M期细胞减少（P＜0.05）;细胞增殖指数（PI）明显降低（P＜0.05）;检测出早期凋亡峰.Annexin V/PI检测及TUNEL检测均显示：ASODN组凋亡细胞阳性率明显高于SODN组（P＜0.01）.琼脂糖凝胶电泳显示：ASODN组出现凋亡DNA梯带.结论：hTERT ASODN能够封闭目的基因表达,阻滞HL-60细胞于G0/G1期,并诱导细胞凋亡,对治疗白血病具有潜在应用价值.     

沉默Toll样受体4表达对肺腺癌A549细胞凋亡及细胞周期的影响

目的 探讨Toll样受体4（Toll-like receptor 4,TLR4）对肺腺癌A549细胞凋亡及细胞周期的影响。方法 体外化学合成针对TLR4的小干扰RNA（TLR4-siRNA）,通过脂质体介导转染人肺腺癌A549细胞。采用Real-time PCR和流式细胞仪检测分析干扰后A549细胞中TLR4 mRNA及蛋白的表达情况。另外,采用流式细胞仪检测分析干扰72 h后细胞凋亡率和细胞周期的变化。结果 与对照组比较,转染TLR4-siRNA后,A549细胞中TLR4 mRNA及蛋白的表达显著下降（P<0.05）,细胞的凋亡率明显增加[（1.73 ± 0.32）% vs.（7.40 ± 0.75）%（P<0.01）],细胞周期出现G0/G1期停滞[（48.21 ± 1.15）%vs.（66.26 ± 2.45）%（P<0.05）],同时S期细胞比例明显减少[（34.25 ± 1.46）% vs.（22.63 ± 3.39）%（P<0.05）]。结论 TLR4-siRNA能有效沉默A549细胞中TLR4的表达,在促进细胞凋亡的同时能够影响细胞周期分布从而抑制肿瘤细胞的生长,特异性干预TLR4基因的表达有望成为治疗肺癌的一种新手段。