RNA干扰Survivin基因沉默对卵巢癌SKOV3及SKOV3/ADM细胞凋亡的影响

背景与目的:RNA干扰技术被广泛应用于肿瘤基因治疗、抗病毒感染、基因药物筛选等许多领域。Survivin基因在卵巢癌细胞株SKOV3及其耐药株SKOV3/ADM中高表达,是进行卵巢癌基因治疗理想的靶点。本研究探讨Survivin基因沉默后对卵巢癌细胞SKOV3及其耐药株SKOV3/ADM凋亡的影响。方法:将重组质粒pshRNA鄄Survivin以脂质体转染至SKOV3、SKOV3/ADM细胞。分别用半定量RT鄄PCR、Westernblot检测细胞内SurvivinmRNA和蛋白表达的变化,吖啶橙/溴乙锭(AO/EB)染色法、TUNEL及流式细胞仪检测重组质粒诱导细胞凋亡的情况。结果:转染pshRNA鄄Survivin后,SKOV3、SKOV3/ADM细胞中SurvivinmRNA拷贝数明显减少,抑制率分别为83.79%和91.56%,蛋白质的表达水平显著降低;干扰组细胞凋亡率显著高于对照组(P<0.01)。流式细胞仪检测结果显示,Survivin沉默后48h,SKOV3、SKOV3/ADM的细胞凋亡率分别为14.05%和21.02%,而在对照组未检测到明显凋亡。结论:重组质粒pshRNA鄄Survivin可明显抑制SKOV3、SKOV3/ADM细胞内SurvivinmRNA和蛋白的表达,介导卵巢癌细胞凋亡。     

沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

目的 探讨沉默轴突导向蛋白4D(Sema4D)基因对卵巢癌SKOV3细胞增殖、侵袭、转移的影响,并验证Sema4D干扰RNA对人卵巢癌裸鼠移植瘤生长的抑制作用.方法 针对Sema4D基因构建短发夹RNA(shRNA)重组载体pshRNA Sema4D-A、pshRNASema4D-B和pshRNASema4D-C质粒,并行酶切鉴定和测序分析.采用逆转录聚合酶链反应(RT-PCR)法筛选RNA干扰效果最佳的重组载体,通过脂质体介导转染卵巢癌SKOV3细胞(重组载体组),并以空载体组(转染空载体pcDNA3.1质粒)和未转染组(未作任何处理)为对照.采用RT-PCR法和Westem blot法分别检测转染前后SKOV3细胞中Sema4D mRNA和蛋白表达,并榆测转染前后SKO3细胞的增殖、侵袭和转移能力.建立人卵巢癌裸鼠移植瘤模型,将满足成瘤标准的15只裸鼠随机分为pshRNASema4D组(重组载体组)、空载体组和未转染组,每组5只,分别向瘤块内注射pshRNASema4D-B(50μg/次)、空载体(50μg/次)和磷酸盐缓冲液(PBS,100μl/次),每3 d注射1次,共3周,观察移植瘤生长情况.结果 酶切鉴定和测序分析证实,靶向Sema4D的3个ShRNA重组载体pshRNASema4D-A、pshRNASema4D-B和pshRNASema4D-C质粒构建成功.RT-PCR法筛选显示,重组载体pshRNASema4D-B质粒干扰效果最佳.重组载体组转染24、48和72 h时,SKOV3细胞中Sema4DmRNA的相对表达水平分别为0.401.4±0.051、0.120±0.035和0.014±0.015,重组载体组转染72 h时的Sema4D mRNA相对表达水平明显低于空载体组(0.521±0.019)和未转染组(0.536±0.040,P<0.05).Western blot法检测显示,重组载体组转染24、48和72 h时,细胞中Sema4D蛋白的相对表达水平分别为0.196±0.023、0.074±0.015和0.040±0.014,重组载体组转染72 h时的Sema4D蛋门相对表达水平明显低于空载体组(0.275±0.009)和未转染组(0.282±0.015,P<0.05).与空载体组和未转染组比较,重组载体组细胞增殖、侵袭和迁移能力受到抑制(P<0.05).注射pehRNASema4D-B后,裸鼠移植瘤生长缓慢,瘤块重量较空载体组和未转染组明显降低(P<0.05).结论 RNA干扰技术能成功沉默SKOV3细胞中Sema4D基因的表达,通过下调Sema4D基因的表达呵抑制卵巢癌细胞增殖、侵袭及转移,Sema4D干扰RNA能抑制人卵巢癌裸鼠移植瘤的生长,Sema4D基因可能成为人卵巢癌基因靶向沉默治疗的重要候选基因之一.

Abstract：

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

通过小干扰RNA沉默mdr-1基因逆转卵巢癌耐药细胞株SKOV3／TAXOL多药耐药的研究

 目的　构建靶向于卵巢癌耐药细胞株中高表达的多药耐药-1（mdr-1）基因的短发夹状RNA重组质粒,探讨RNAi沉默技术逆转卵巢上皮性癌（卵巢癌）细胞多药耐药的可行性。方法　运用基因克隆技术构建靶向于mdr-1基因的重组质粒PGPU6／GFP／Neo-mdr-1并转染至卵巢癌耐药细胞株SKOV3／TAXOL,反转录聚合酶链反应检测到mdr-1基因表达。CCK-8检测对SKOV3／TAXOL细胞增殖的抑制作用。结果　构建的重组质粒PGPU6／GFP／Neo-mdr-1能明显抑制mdr-1基因的表达。蛋白质印迹结果表明,转染重组质粒PGPU6／GFP／Neo-mdr-1的SKOV3／TAXOL靶细胞的抑制率显著提高。RT-PCR检测mdr-1基因mRNA转录水平下降;pPGPU6／GFP／Neo-mdr-1明显抑制SKOV3／TAXOL细胞的增殖。结论　重组质粒PGPU6／GFP／Neo-mdr-1可有效地抑制SKOV3／TAXOL细胞内源mdr-1基因的表达和mRNA的转录,并抑制细胞的增殖和促进凋亡的发生,应用RNAi技术,能够逆转卵巢癌细胞对化疗药物的多药耐药。为化疗耐受的肿瘤细胞提供了新的基因治疗手段。     

小干扰RNA介导的间皮素沉默促进顺铂诱导的卵巢癌耐药细胞SKOV3/cDDP的凋亡

目的:探讨小干扰RNA(small interference RNA,siRNA)介导的间皮素(mesothelin,MSLN)沉默对顺铂(cisplatin,cDDP)诱导的卵巢癌耐药细胞SKOV3/cDDP凋亡的影响。方法:应用蛋白质印迹法检测SKOV3/cDDP细胞MSLN蛋白的表达。通过RNA干扰(RNA interference,RNAi)技术干扰耐药细胞SKOV3/cDDP MSLN表达,建立稳定感染的细胞株SKOV3/cDDP-LV-MSLN-RNAi。应用MTT法和集落形成实验检测SKOV3/cDDP-LV-MSLN-RNAi细胞对cDDP的敏感度,FCM检测cDDP诱导的细胞凋亡。结果:SKOV3/cDDP细胞MSLN蛋白的表达水平明显高于SKOV3细胞(P=0.000);cDDP对干扰组SKOV3/cDDP-LV-MSLN-RNAi细胞的半数抑制浓度(half inhibition concentration,IC50)值明显低于阴性组SKOV3/cDDP-LV-MSLN-neg和空白组SKOV3/cDDP细胞(P=0.000);干扰组细胞的集落形成抑制率和凋亡率明显高于阴性组和空白组,差异有统计学意义(P<0.05)。结论:MSLN沉默可促进cDDP诱导的卵巢癌耐药细胞凋亡,在一定程度上逆转cDDP耐药。MSLN可能在卵巢癌cDDP耐药中发挥重要作用。     

小干扰RNA沉默survivin基因对结肠癌Caco-2细胞增殖和凋亡的影响

目的:观察survivin在人结肠癌细胞Caco-2中的表达及小干扰RNA(small interfering RNA,siRNA)沉默survivin基因对survivin蛋白表达、细胞增殖及凋亡的影响.方法:以脂质体LipofectAMINE 2000为载体,将针对特异靶点survivin基因的siRNA转染入人结肠癌细胞Caco-2.应用免疫细胞化学SP法和Western印迹法检测survivin蛋白表达的变化,MTT法检测细胞增殖,FCM法检测细胞凋亡.结果:免疫细胞化学检测结果显示,survivin蛋白在Caco-2细胞空白对照组、脂质体对照组及阴性错配对照组的细胞质中均呈强阳性表达,而在survivin-siRNA转染组细胞质中呈弱阳性表达;Western印迹法检测结果显示,survivin-siRNA转染组细胞的蛋白条带亮度明显低于空白对照组、脂质体对照组和阴性错配对照组;MTT检测结果显示,与阴性错配对照组相比, survivin-siRNA转染组细胞生长出现明显的抑制(P＜0.05),且不同时段(24、48和72 h)的肿瘤细胞增殖抑制率之间差异有统计学意义(P＜0.05);FCM检测结果显示,survivin-siRNA转染组细胞出现明显的亚二倍体峰,细胞凋亡率(9.72%)明显高于空白对照组(P＜0.01).结论:siRNA沉默survivin基因可抑制结肠癌细胞增殖,促进细胞凋亡.推测survivin基因可能成为结肠癌基因治疗的一个新靶点.     

RNA干扰沉默EGFR基因对卵巢癌细胞侵袭力的影响

[目的]通过小于扰RNA（siRNA）沉默EGFR基因的表达，探讨其对卵巢癌SKOV3细胞体外侵袭力的影响。[方法]体外构建靶向EGFR基因的siRNA质粒和阴性对照质粒，Lipofectamine2000介导转染到SKOV3细胞中。实验分3组：空白对照组、非特异性转染组、特异性转染组。RT-PCR和免疫细胞化学法检测EGFR基因和蛋白水平表达。平板克隆形成法检测细胞生长增殖能力，Transwell侵袭实验检测细胞体外侵袭力。[结果]特异性转染组细胞EGFR基因mRNA和蛋白表达的抑制率分别为52．3％和51．8％．克隆形成抑制率和侵袭抑制率分别为47．4％和40．1％。[结论]siRNA可以有效抑制SKOV3细胞EGFR基因的表达，抑制其增殖能力和体外侵袭能力。     

Rab25基因siRNA诱导人类卵巢癌细胞A2780凋亡的实验研究

目的 观察Rab25基因的小分子干扰RNA(siRNA)对肿瘤细胞的诱导凋亡作用.方法 成功构建Rab25基因的siRNA,稳定转染靶细胞,MTT法检测细胞增生率;流式细胞仪(FCM)检测细胞生长周期和细胞凋亡率;DNA琼脂糖凝胶电泳法(DNAladder)确定凋亡.结果 细胞生长明显受到抑制,增生能力下降;阻断细胞生长于G1期(转染Psuper/Rab25 siRNA组细胞G1期达到88.6%);细胞凋亡增加,转染Psuper/Rab25 siRNA组细胞凋亡率为12.3%,与A2780组细胞及转染空载体组细胞相比,差异有统计学意义(P＜0.01);DNA凝胶电泳可观察到细胞凋亡特异的DNA梯形带.结论 Rab25基因的siRNA可抑制对肿瘤细胞的生长,诱导其凋亡,可为卵巢癌基因治疗开辟新途径.     

RNAi沉默Her-2基因对卵巢癌细胞SKOV3的动物实验研究

目的观察细胞株SKOV3/si RNA Her-2基因的沉默作用及生物学效应。方法分别将SKOV3/si RNA及对照组SKOV3种植NOD-SCI D小鼠皮下,监测两组瘤体积,3个月后处死NOD-SCI D小鼠,称瘤质量,并采用RT-PCR方法鉴定抑制Her-2表达的效果。结果建立的抑制Her-2基因表达的SKOV3细胞株(SKOV3/si RNA)成瘤力下降。结论动物实验证实RNAi沉默Her-2基因可影响卵巢癌的成瘤力,RNAi技术有可能成为治疗卵巢癌的新途径。     

抑制Survivin基因对人卵巢癌SKOV3细胞增殖和凋亡的影响

目的:探讨应用RNAi技术沉默Survivin基因对人卵巢癌SKOV3细胞的影响。方法:构建Survivin基因shRNA真核表达载体,转染人卵巢癌SKOV3细胞。RT-PCR及Western blot检测Survivin基因的表达,MTT实验、流式细胞仪检测细胞增殖、凋亡的变化。结果:siRNA实验组细胞Survivin蛋白及mRNA表达水平明显下降,细胞增殖能力显著降低,细胞凋亡率显著升高。结论:应用RNAi技术沉默Survivin基因可以降低卵巢癌SKOV3细胞Survivin基因的表达,进而抑制肿瘤细胞的生长、增殖并诱导细胞凋亡。因此,Survivin基因可能成为抗肿瘤治疗的潜在靶点。     

鱼藤素通过调控miR-520a-3p影响卵巢癌SKOV3细胞的增殖和凋亡

目的：探讨鱼藤素通过调控miR-520a-3p表达对卵巢癌SKOV3细胞增殖和凋亡的影响。方法：将SKOV3细胞分为对照组（鱼藤素0μmol/L）、鱼藤素低剂量（5μmol/L）、中剂量（10μmol/L）、高剂量（20μmol/L）组,miR-NC组、过表达miR-520a-3p组,鱼藤素+anti-miR-NC组、鱼藤素+anti-miR-520a-3p组。CCK-8法、细胞集落形成实验、FCM以及qPCR法分别检测SKOV3细胞的增殖抑制率、细胞克隆形成数、凋亡率以及miR-520a-3p表达水平。结果：与对照组比较,鱼藤素（低、中、高剂量）组SKOV3细胞增殖抑制率、凋亡率、miR-520a-3p表达水平均显著升高（均P<0.05）,细胞克隆形成数显著减少（P<0.05）。与miR-NC组比较,过表达miR-520a-3p组SKOV3细胞的增殖抑制率、凋亡率均显著升高（均P<0.05）,细胞克隆形成数显著减少（P<0.05）。与鱼藤素+anti-miR-NC组比较,鱼藤素+anti-miR-520a-3p组SKOV3细胞的增殖抑制率、凋亡率均显著降低（均...     

短发卡状RNA抑制AKT2基因表达增强卵巢癌细胞对紫杉醇敏感性的研究

目的：构建AKT2基因特异性短发卡状RNA（shRNA）真核表达载体,观察RNA干扰（RNAi）技术,对人卵巢癌A2780和SKOV3细胞中AKT2蛋白激酶表达的抑制作用,并探讨其促进细胞凋亡的机制。方法：运用基因工程技术,设计合成针对AKT2mRNA的特异性shRNA,构建真核表达载体pAKT2-shRNA。将pAKT2-shRNA经脂质体（Lipofectamine^TM 2000）包裹转染人卵巢癌A2780和SKOV3细胞,荧光显微镜观察细胞内DsRed红色荧光蛋白的表达,计算转染效率,半定量RT—PCR（Semi-quantitative RT-PCR）、蛋白质免疫印迹（Western blot）检测AKT2的表达及干扰效率,流式细胞学检测法（FACS）检测细胞凋亡率,比较抑制AKT2基因前后卵巢癌细胞抵抗凋亡能力。结果：成功构建了PAKT2-ShRNA载体。A2780和SKOV3细胞转染pAKT2-shRNA后,荧光显微镜下观察转染效率约40％;Semi-quantitative RT-PCR和Western blot检测结果示,转染后AKT2 mRNA和蛋白表达均显著降低。FACS检测结果显示,转染pAKT2-shRNA后,紫杉醇25nmol／L处理细胞12h,与空白对照组和阴性对照组相比,细胞凋亡率显著增加,P〈0．05。结论：构建的pAKT2-shRNA真核表达载体能有效抑制AKT2基因表达;运用RNAi技术,降低卵巢癌细胞中AKT2基因表达水平,能增强肿瘤细胞对化疗药物的敏感性,促进肿瘤细胞凋亡。     

小分子RNA干扰Notch1基因对肾癌Caki-1细胞增殖的影响

目的：探讨Notch1在肾透明细胞癌株中的表达,采用短片段RNA（small interferhtg RNA,siRNA）干扰技术沉默肾透明细胞癌中Notch1的表达,并观察其对Caki-1增殖的影响。方法：体外培养人正常肾小管上皮细胞HK-2及人肾癌细胞786—0和Caki-1。实时荧光定量PCR（RT—PCR）检测Notch1 mRNA的表达,蛋白印迹法检测Notch1蛋白的含量。特异性Notch1 siRNA干扰肾癌Caki-1细胞,用Lipofectamine2000转染48h后,实时荧光定量PCR和蛋白印迹法分别检测其中Notch1 mRNA、蛋白的表达变化,CCK-8与克隆形成实验检测其增殖情况。结果：Notch1在肾癌细胞株中的表达要明显高于正常肾小管上皮细胞,差异具有统计学意义（P〈0.05）。siRNA—Notch1干扰以后可以明显降低Caki-1细胞中Notch1 mRNA和蛋白的表达,差异具有统计学意义（P〈0.05）,同时检测Caki-1细胞增殖能力也随之下降（P〈0.05）。结论：Notch1在肾癌透明细胞癌中高表达,下调Notch1信号通路可以抑制肾细胞癌Caki—1的增殖,调节肾癌的生长。     

siRNA下调LSD1基因对卵巢癌OVCAR-3细胞增殖和周期的影响

目的应用小分子干扰RNA（small interfering RNA,siRNA）抑制赖氨酸特异性组蛋白去甲基化酶1（lysine specific demethylase 1,LSD1）基因的表达,探讨其对卵巢癌OVCAR-3细胞增殖和生长周期的影响。方法采用脂质体介导法将LSD1-siRNA转染卵巢癌OVCAR-3细胞;实时荧光定量PCR（RFQ-PCR）和Western blot法检测基因的表达;MTT法检测细胞增殖能力;FCM分析细胞周期。结果成功转染LSD1-siRNA后,OVCAR-3细胞中LSD1 mRNA和蛋白表达均明显下降;细胞增殖明显抑制;G₂/M期细胞比例增加。结论 LSD1-siRNA可显著下调LSD1基因在卵巢癌OVCAR-3细胞中的表达水平,在一定程度上抑制卵巢癌细胞的增殖,导致细胞周期阻滞。     

RNA干扰Hedgehog信号通路对胃癌细胞增殖和凋亡影响及其机制的探讨

目的:观察RNA干扰Hedgehog信号通路关键因子Glil对胃癌细胞的生物学行为的影响并分析相关机制.方法:人工合成靶向Hedgehog信号通路活化关键因子Glil的siRNA(Glil siRNA)和对照无关siRNA(Con siRNA),应用脂质体将其转入人胃癌细胞SGC-7901,48 h后收集细胞,分别应用MTT法和流式细胞仪检测对细胞增殖、周期和凋亡的影响,同时应用TaqMan探针实时定量PCR检测靶基因Glil、Hedgehog活化标志基因PTCH1、细胞周期负调控蛋白基因CDKN1A(p21)和抗凋亡基因Bel-2的表达变化.结果:以单纯转染剂对照组为参照,siRNA转染后48 h,Glil siRNA和Con siRNA的抑制率分别为(53.33±6.06)%和(13.33±6.11)%,两组差异有统计学意义,t=5.701,P=0.005,n=3;G0/G1期分别为(80.67±4.51)%和(66.00±3.10)%,两组差异有统计学意义,t=5.500,P=0.005,n=3;凋亡率分别为(15.97±1.76)%和(7.77±1.11)%,两组差异有统计学意义,t=4.671,P=0.01,n=3;GlilsiRNA转染细胞内Glil、PTCH1、Bcl-2和CDKN1A(p21)基因的相对(参照单纯转染剂)表达分别为对照的0.24±0.11、0.43±0.09、0.52±0.13和2.10±0.30.分别与Con siRNA相比4条基因表达均差异有统计学意义,t=9.759,P=0.001,n=3;t=8.645,P=0.001,n=3;t=4.940,P=0.008,n=3;t=5.962,P=0.004,n=3.结论:应用RNA干扰技术可以阻断Hedgehog信号通路关键因子Glil的表达,抑制Hedgehog信号通路活化,从而有效地抑制胃癌细胞的增长,诱导凋亡,可能成为治疗胃癌新的生物靶向治疗途径.     

miR－300对人卵巢癌细胞增殖、周期及凋亡的影响

目的：研究 miR－300对人卵巢癌细胞增殖、凋亡和周期的影响。方法：通过荧光实时定量 PCR （qRT－PCR）检测卵巢正常上皮细胞及卵巢癌细胞中miR－300的表达水平和miR－300 siRNA的沉默效果;CCK－8法检测沉默miR－300对卵巢癌细胞增殖的影响;流式细胞术检测沉默miR－300对卵巢癌细胞凋亡和周期的影响。结果：miR－300在卵巢癌细胞中的表达显著高于卵巢正常上皮细胞;miR－300 siRNA可有效沉默卵巢癌细胞中miR－300的表达;沉默miR－300的表达可以明显抑制卵巢癌细胞的增殖能力,增强卵巢癌细胞的凋亡能力,并使卵巢癌细胞的周期阻滞于G1期。结论：miR－300在卵巢癌细胞的增殖、凋亡和周期过程中起着重要的调控作用。     

Galectin-3表达抑制对BT-549细胞系增殖和凋亡影响观察

目的：观察抑制Galectin-3表达与BT-549细胞系增殖和凋亡的关系,探讨利用RNA干扰技术作为三阴性乳腺癌基因治疗方法的可行性。方法：设计并化学合成Galectin-3小干扰片段并顺势转染乳腺癌细胞系BT-549（为Galec—tin-3siRNA组）,以未转染细胞为空白对照,以转染阴性干扰为阴性对照,荧光倒置显微镜和实时定量PCR验证干扰结果,XTT方法观察干扰后24、48、72和96h各组细胞增殖吸光度（A值）,流式细胞仪检测各组细胞凋亡情况,划痕实验和Tral2swell实验比较不同组间细胞迁移和侵袭情况,以上实验均重复3次。结果：空白对照组、阴性对照组和Galectin一3siRNA组Galectin-3mRNA相对含量分别为（0．078±0．003）、（0．069±0．004）和（0．015±0．001）,差异有统计学意义,F=486．97,P〈O．001。XTT法检测结果显示,同一组不同时间点之间差异有统计学意义,P值均〈O．00l;同一时间点各组之间差异亦有统计学意义,P值均〈O．001。随着时间推移,空白组和阴性对照组细胞继续生长,Galectin-3siRNA组细胞生长抑制,数量逐渐减少,干扰时间和实验分组之间存在交互效应,F=26．43,P〈0．001。流式细胞仪检测结果显示,Galectin-3siRNA组、空白对照组和阴性对照组凋亡率分别为（26．83±2．15）％、（2．73土0．18）％和（5．86土0．51）％,差异有统计学意义,F=24537．92,P〈0．00l。空白对熙组、阴性对照组和Galectin-3siRNA组愈合率分别为（91．47±4．39）％、（82．36土3．68）％和（52．26士1．95）％,差异有统计学意义,F=213．71,P〈0。001。转染Galectin-3siRNA的乳腺癌细胞浸润穿过Matrigel膜的细胞数（169．36±23．71）要比空白对照组（480．80土30．80）和阴性对照组（320．70±28．12）的细胞数显著减少,差异有统计学意义,F=115．20,P〈0．001。结论：Galectin-3siRNA成功转染入BT-549细胞,并抑制细胞增殖,诱导细胞凋亡,降低其迁移侵袭能力,为三阴性乳腺癌基因治疗提供实验依据。     

Rab25基因siRNA诱导人卵巢癌细胞A2780……

 目的 观察Rab25基因的小分子干扰RNA（siRNA）对肿瘤细胞的诱导凋亡作用。方法 成功构建Rab25基因的siRNA，稳定转染靶细胞，MTT法检测细胞增生率；流式细胞仪（FCM）检测细胞生长周期和细胞凋亡率；DNA琼脂糖凝胶电泳法（DNA ladder）确定凋亡。结果 细胞生长明显受到抑制，增生能力下降；阻断细胞生长于G1期（转染pSUPER／Rab25 siRNA组细胞G1期达到88.6 %）；细胞凋亡增加，转染pSUPER／Rab25 siRNA组细胞凋亡率为12.3 %，与A2780组细胞及转染空载体组细胞相比，差异有统计学意义（P＜0.01）；DNA凝胶电泳可观察到细胞凋亡特异的DNA梯形带。结论 Rab25基因的siRNA可抑制对肿瘤细胞的生长，诱导其凋亡，可为卵巢癌基因治疗开辟新途径     

婆罗双树样基因2干扰对宫颈癌HeLa细胞增殖和凋亡的影响

目的：研究婆罗双树样基因2（sal-like gene 2,SALL2）干扰对宫颈癌HeLa细胞增殖和凋亡的影响。方法：设计针对SALL2基因的3条小干扰RNA（siRNA-1、2、3）,并设载体组及未转染对照组,使用脂质体转染HeLa细胞。采用逆转录RT-PCR和Western blot检测转染后HeLa细胞中SALL2 mRNA和蛋白的表达以筛选沉默效果最好的siRNA片段。将最适siRNA片段转染HeLa细胞后,采用CCK-8检测细胞增殖率;流式细胞术检测细胞周期和凋亡率。结果：对照组HeLa细胞高表达SALL2。载体组和转染siRNA-3组SALL2的mRNA和蛋白表达与对照组相比,差异均无统计学意义（P均>0.05）,而siRNA-1和siRNA-2转染48 h后,HeLa细胞SALL2的mRNA和蛋白表达均降低,差异均有统计学意义（P均<0.05）,其中siRNA-1的沉默效率更好,故后续选择siRNA-1作为干扰组。与对照组相比,转染siRNA-1 48和72 h后细胞的增殖率均升高,差异均有统计学意义（P<0.05）;转染48 h后siRNA-1组处于G0/G1期的细胞比例减少,凋亡率明显降低（P均<0.05）。结论：SALL2基因干扰可明显提高HeLa细胞的增殖率,并降低其凋亡率。提示SALL2基因对宫颈癌HeLa细胞的增殖有抑制作用。