Survivin基因RNAi对子宫颈癌裸鼠移植瘤生长与凋亡的影响

目的：观察survivin基因RNAi对宫颈癌裸鼠移植瘤生长与凋亡的影响。方法：随机将BALB/c裸鼠分4组,分别注射转染携survivin RNAi重组质粒的HeLas2细胞、转染阴性对照质粒细胞HeLaNC、转染空质粒细胞HeLaU6 neo及宫颈癌HeLa细胞,建立人宫颈癌裸鼠皮下移植瘤模型。观察survivin RNAi对人宫颈癌裸鼠皮下移植瘤生长的影响;免疫组化法检测移植瘤组织中survivin蛋白的表达和微血管密度（MVD）,HE染色及TUNEL染色观察survivin RNAi对人宫颈癌裸鼠皮下移植瘤凋亡的影响。结果：成功建立人宫颈癌裸鼠皮下移植瘤模型, HeLas2组裸鼠瘤重明显小于其他3组（P<0.05）;肿瘤生长抑制率为67.9%。免疫组化结果显示,HeLas2组裸鼠移植瘤组织中survivin蛋白表达显著下降; MVD值也显著降低（P<0.05）;HE染色、TUNEL染色结果显示,HeLas2组裸鼠移植瘤组织细胞凋亡明显增多（P<0.05）,AI值达(22.73±137)%。结论：Survivin基因RNAi可通过下调移植瘤组织survivin蛋白表达和降低其MVD抑制移植瘤生长并促进其凋亡。     

沉默H-ras基因对人涎腺腺样囊性癌裸鼠移植瘤的抑制作用

目的构建H-ras靶向短发夹RNA(short hairpin RNA,shRNA)质粒,研究其对人涎腺腺样囊性癌裸鼠成瘤的抑制作用及其对移植瘤细胞增殖活性及细胞凋亡率的影响.方法构建含H-ras靶向特定序列的真核表达质粒pHRAS,稳定表达pHRAS质粒的细胞为实验组,未处理的SACC-M细胞为对照组,建立裸鼠荷瘤模型,计算成瘤率;移植瘤原代培养检测质粒表达及瘤细胞增殖活性;免疫组织化学法检测瘤内H-ras蛋白表达;采用组织学观察、流式细胞术及TUNEL法检测肿瘤细胞凋亡情况.结果pHRAS质粒转染组成瘤率及肿瘤体积显著低于对照组,原代培养可见大量瘤细胞表达绿色荧光.实验组瘤内H-ras蛋白表达量明显减少,实验组肿瘤细胞凋亡率为(41.55±4.25 )%,明显高于对照组的(4.73±1.35) %(P＜0.05).结论shRNA沉默H-ras基因能抑制SACC-M细胞裸鼠皮下移植瘤的形成,在体内有效下调H-ras蛋白的表达,且对肿瘤细胞具有促凋亡作用.     

RNAi介导SALL4基因沉默对SKOV-3细胞裸鼠皮下移植瘤生长的影响机制

目的 探讨婆罗双树样基因4(SALL4)对人卵巢癌SKOV-3细胞裸鼠皮下移植瘤生长的影响及可能机制.方法 通过SALL4短发夹RNA(shRNA)慢病毒感染构建稳定低表达SALL4基因的SKOV-3细胞株,分为对照组(blank)、载体组(NC shRNA)和干扰组(SALL4 shRNA);采用qRT-PCR和蛋白...     

Mus81基因沉默对MDA-MB-231乳腺癌细胞增殖、凋亡及裸鼠成瘤能力的影响北大核心

目的:分析甲磺酸盐及紫外线敏感性81号基因(Mus81)在乳腺癌组织中的表达水平,观察Mus81基因敲减对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和裸鼠移植瘤形成能力的影响。方法:从TCGA数据库下载乳腺癌样本基因表达数据,应用perl及R软件整理数据筛选出每个样本Mus81基因表达量。通过慢病毒介导的小干扰RNA(short interfering RNA,siRNA)技术构建Mus81基因敲减的MDA-MB-231乳腺癌细胞系(即Mus81敲减组)和阴性对照组MDA-MB-231乳腺癌细胞系,以实时定量PCR法检测Mus81基因的敲减效率,再以MTT检测实验、克隆形成实验、细胞流式检测及实时定量PCR法检测两组MDA-MB-231细胞的生长、增殖、细胞周期分布、凋亡水平及Mus81下游基因表达水平。最后,向BALB/c裸鼠右侧腋下注射Mus81基因敲减组和阴性对照组MDA-MB-231乳腺癌细胞,观察Mus81基因沉默对MDA-MB-231细胞在裸鼠中成瘤能力的影响。结果:Mus81基因在乳腺癌组织中的平均表达量明显高于癌旁组织(P<0.05)。Mus81基因敲减组MDA-MB-231细胞中Mus81基因的表达水平明显低于阴性对照组(P<0.05),Mus81基因的敲减效率达70.7%。较之阴性对照组,Mus81基因敲减组MDA-MB-231细胞的生长速度明显减缓(P<0.05);形成的细胞克隆数也明显下降(P<0.05);细胞凋亡水平、G2/M期细胞比例则明显升高(P<0.05)。STC2等Mus81下游基因的表达水平在两组MDA-MB-231细胞间也有明显差异(P<0.05)。裸鼠成瘤实验显示,Mus81基因敲减组形成的裸鼠瘤体体积和重量均明显低于阴性对照组(P<0.05)。结论:Mus81基因在乳腺癌组织中的表达明显升高,且可能通过调控STC2等下游基因的表达促进三阴性乳腺癌细胞的生长增殖及裸鼠体内成瘤能力并抑制其凋亡,提示其可能是一个潜在的乳腺癌治疗靶点。     

沉默STAT3基因对人食管癌EC1细胞裸鼠移植瘤的抑制作用

目的构建STAT3靶向短发夹RNA（shRNA）质粒,研究其对人食管癌EC1细胞裸鼠皮下移植瘤的作用,探讨其在食管癌基因治疗中的可行性和特异性。方法构建STAT3的siRNA真核表达载体pSUPER-EGFP-STAT3,稳定表达该质粒的细胞为实验组,转染空质粒细胞及未处理细胞为对照组,建立裸鼠荷瘤模型;监测肿瘤生长变化,HE染色观察肿瘤病理学变化,RT-PCR、免疫组化方法检测STAT3mRNA和蛋白变化。结果裸鼠体内实验显示,实验组肿瘤增长受到明显抑制;HE染色显示对照组肿瘤体积大、异形明显;RT-PCR、免疫组化结果表明实验组STAT3mRNA和蛋白表达下调。结论shRNA沉默STAT3基因能抑制人食管癌EC1细胞裸鼠皮下移植瘤的形成,且能在体内有效下调STAT3mRNA和蛋白的表达,为食管癌的基因治疗提供了新的靶点,开辟了新的思路。     

RNAi沉默CXCR6基因对肺癌A549细胞增殖、体外侵袭力和裸鼠成瘤能力的影响

目的：探讨RNAi沉默CXC型趋化因子受体6（CXCR6）基因对A549细胞增殖、体外侵袭力和裸鼠成瘤能力的影响。方法 MTT法和Transwell小室模型检测稳定沉默CXCR6基因的A549细胞增殖和细胞侵袭迁移能力。将稳定沉默CXCR6的A549细胞与正常A549细胞分别皮下接种裸鼠,观察接种后肿瘤生长情况。结果稳定沉默CXCR6基因的A549细胞与正常A549细胞相比,前者增殖速率下降（P﹤0.05）,平均增殖抑制率为30.70%;前者的体外侵袭力也降低（P﹤0.05）,平均侵袭抑制率为74.93%,平均迁移抑制率为70.19%。裸鼠体内实验显示,稳定沉默CXCR6基因的A549细胞与正常A549细胞相比,其裸鼠体内成瘤能力降低（P﹤0.05）,瘤体重量下降了62.5%。结论 RNAi沉默CXCR6基因,可以抑制A549细胞的增殖、体外侵袭力和成瘤能力。     

shRNA沉默Survivin基因对人骨肉瘤MG-63细胞成瘤能力的影响

目的:研究Survivin-shRNA对骨肉瘤MG-63细胞成瘤能力的影响,并探讨其可能的机制.方法:采用皮下异种移植法构建裸鼠骨肉瘤模型,骨肉瘤MG-63细胞培养成功后,重组腺病毒Survivin-shRNA转染.细胞以1:5比例进行传代,挑选稳定转染的MG-63细胞并扩增,Survivin-shRNA组为Survivin-shRNA转染的细胞,GFP组和CON组为阴性对照组.每3天用卡尺测定肿瘤体积,绘制肿瘤生长曲线.颈椎脱臼法处死裸鼠,取骨肉瘤标本称重.Western blot法检测SUV、VEGF、PCNA及CAS-3蛋白的表达.结果:腺病毒介导的RNA干扰构建Survivin-shRNA载体,Survivin-shRNA组转染细胞的胞质及胞核可见绿色荧光,并伴有少量小颗粒物质.骨肉瘤生长曲线显示,与CON和GFP组相比,Survivin-shRNA组的肿瘤增长缓慢,表明Survivin-shRNA在体内能抑制骨肉瘤的生长.肿瘤的重量结果显示,Survivin-shRNA在体内能抑制骨肉瘤重量的增加.Western blot结果显示,SUV、VEGF的表达在Survivin-shRNA组中明显低于对照组,而CAS-3表达相反.结论:Survivin-shRNA可抑制骨肉瘤细胞增殖及成瘤能力,其机制可能是通过调节SUV、VEGF、CAS-3的表达及抑制肿瘤新生血管形成等来实现的.     

RNAi沉默MCM7基因对人肝癌细胞SMMC-7721裸鼠移植瘤影响研究

目的探讨微小染色体维持蛋白7(miniehromosome maintenanceprotein7,MCM7)基因RNAi(RNA interference)的重组慢病毒载体,对人肝癌细胞SMMC-7721 MCM7基因表达和裸鼠移植瘤生长的影响。方法构建重组逆转录慢病毒载体MCM7-shRNA,以MCM7基因沉默重组慢病毒颗粒(LV-shRNA-MCM7)感染SMMC-7721细胞,作为实验组;以对照慢病毒颗粒(LV-shRNA-NC)感染SMMC-7721细胞,作为阴性对照组;空白对照组常规培养,不做任何处理。通过嘌呤霉素筛选出稳定转染细胞株。3组细胞分别接种至裸鼠皮下,建立人肝癌裸鼠移植瘤模型。观察裸鼠成瘤情况、移植瘤生长情况并绘制肿瘤生长曲线;4周后测定肿瘤体积和质量,并用RT-PCR、实时荧光定量PCR、蛋白质印迹法及免疫组织化学法检测移植瘤中MCM7的表达情况。结果 MCM7-shRNA慢病毒载体构建成功。裸鼠接种癌细胞后第6天均有肿瘤形成,与空白对照组和阴性对照组相比,实验组的肿瘤生长速度明显减慢,实验组、阴性对照组和空白对照组的瘤体平均体积分别为(27.72±7.80)、(81.86±10.91)和(79.75±16.61)mm3,差异有统计学意义,F=61.949,P<0.05;实验组、阴性对照组和空白对照组的瘤体平均质量分别为(0.19±0.06)、(0.501±0.14)和(0.509±0.18)g,差异有统计学意义,F=18.41,P<0.05。实验组MCM7mRNA的相对表达量为0.253±0.198,阴性对照组1.213±0.548,空白对照组1.201±0.744,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=4.091,P<0.05;实验组MCM7蛋白相对表达量为0.207±0.015,阴性对照组1.116±0.062,空白对照组1.088±0.040,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=292.778,P<0.05。MCM7蛋白在阴性对照组和空白对照组中阳性表达率均为100%(10/10),在实验组中为30%(3/10),实验组明显低于阴性对照组和空白对照组,差异有统计学意义,χ2=18.261,P<0.001。结论慢病毒沉默SMMC-7721细胞MCM7基因表达能有效抑制人肝癌裸鼠移植瘤的生长,MCM7基因可能成为肝癌基因治疗的有效靶点。     

IL-27基因对Eca109细胞在裸鼠体内的成瘤抑制作用及其机制

背景与目的:细胞因子为主的肿瘤生物治疗已成为肿瘤研究领域热点之一。本研究观察白细胞介素(interleukin,IL)-27基因转染人食管癌Eca109细胞株后在裸鼠体内的成瘤抑制作用及其机制。方法:以逆转录病毒为载体,采用基因转染的方法用G418梯度筛选法建立转染IL-27基因的Eca109细胞,RT-PCR检测其基因导入情况,ELISA法检测IL-27的分泌和其诱导外周血单个核细胞(peripheral blood mononuclear cells,PBMC)产生γ-干扰素(interferon,IFN)的能力,MTT法观察Eca109/IL-27细胞生长情况。将Eca109/IL-27、Eca109/LXSN和Eca109细胞接种于裸鼠皮下,观察其成瘤性、移植瘤的生长情况并计算抑瘤率。流式细胞技术检测移植瘤组织肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)中CD16、FasL的表达和肿瘤细胞上Fas的表达。结果:RT-PCR示Eca109/IL-27细胞中有IL-27p28和IL-27EBI3亚基基因表达,Eca109/LXSN和Eca109细胞中未见表达(P<0.01),从而成功建立稳定转染的Eca109/IL-27细胞株,ELISA检测Eca109/IL-27中IL-27的分泌量高于Eca109/LXSN和Eca109细胞(P<0.01)。IL-27基因转染不影响人食管癌细胞株的体外生长(P>0.05),Eca109/IL-27诱导PBMC产生IFN-γ含量高于Eca109/LXSN和Eca109[(56.28±1.61)pg/mL vs.(12.70±0.82)pg/mL]和(11.06±0.64)pg/mL,P<0.01),Eca109/IL-27移植瘤体积较Eca109和Eca109/LXSN减小,瘤重减轻,有抑瘤作用(P<0.05);Eca109/IL-27细胞接种的肿瘤组织TIL中CD16阳性率升高,FasL的表达增高(P<0.05),肿瘤细胞表面Fas表达增加(P<0.05)。结论:IL-27基因修饰Eca109细胞在裸鼠体内产生了抑制肿瘤生长的作用,其机制可能是通过活化自然杀伤细胞,以Fas/FasL的途径产生的。     

YH-16对宫颈癌Hela细胞及其裸鼠皮下移植瘤作用研究

目的:初步探讨YH-16(重组内皮抑素)对宫颈癌Hela细胞及其荷瘤生长抑制作用及作用机制.方法: 用MTT法检测YH-16对Hela细胞生长抑制作用;用透射电镜观察YH-16处理Hela细胞后的凋亡情况、流式细胞仪检测细胞凋亡率及细胞周期;将Hela细胞移植至裸鼠皮下成瘤,观察不同浓度YH-16 (0mg/kg、0.4mg/kg、0.75mg/kg和1.5mg/kg)对荷瘤鼠皮下移植瘤生长的影响;TUNEL法观察肿瘤细胞的凋亡;免疫组化方法检测裸鼠移植瘤组织中肿瘤微血管密度(MVD).结果: YH-16具有体外抑制Hela细胞增殖的作用(P<0.01);能诱导Hela细胞凋亡;YH-16能有效抑制裸鼠皮下移植瘤的生长(P<0.05);YH-16 15mg/kg组的肿瘤MVD计数较对照组和其它治疗组明显降低(P<0.05).结论:YH-16具有抑制宫颈癌Hela细胞及其皮下移植瘤生长的作用.     

雷公藤甲素对人前列腺癌PC-3细胞裸鼠移植瘤模型的作用

目的:探讨雷公藤甲素对前列腺癌PC-3细胞裸鼠移植瘤模型的作用。方法:构建前列腺癌PC-3细胞裸鼠皮下移植瘤模型,分为实验组和对照组,实验组给予雷公藤甲素治疗,0.4mg/(kg.d),皮下注射,连用15天,对照组仅给予含有等量DMSO的生理盐水,检测药物处理后肿瘤的变化,并通过免疫荧光染色的方法检测SUMO特异蛋白酶SENP1的表达。结果:雷公藤甲素能够显著抑制移植瘤的增长,并能抑制SENP1的表达水平。结论:雷公藤甲素可以显著抑制PC-3细胞裸鼠移植瘤的增长,并降低SENP1的表达水平。     

Metastases of human tumor xenografts in nude mice

In the present study, using systematic microscopic examination, we tried to determine the true incidence of metastases in nude mice bearing a wide variety of human tumors. A total of 63 malignant tumors were successfully transplanted subcutaneously and 831 nude mice bearing tumors were examined. It appeared that 17 of the 63 tumors (26.9%) retained their metastatic ability in nude mice. Most of these tumors were adenocarcinomas (11/17 cases). Generally the metastatic deposits in the lungs and, to a lesser extent, in the lymph nodes were small and thus only detectable on microscopic examination. We also found a positive correlation between the presence of metastases and neoplastic infiltration of the lymphatic and/or blood vessels around the subcutaneous tumors. Metastatic human tumors, including neoplastic cells from effusion, exhibited higher metastatic ability than primary tumors (p less than 0.005). However, the expression of this metastatic potential depends on several factors including tumor volume, survival time after inoculation and murine hepatitis infection. Thus, animals with metastases bore larger tumors (9.56 cm3) than those without metastasis (6.35 cm3; p less than 0.0001). Moreover, survival time after inoculation was longer in mice with metastases (104 days) than in mice without metastases (81 days; p less than 0.0001). A negative influence of viral hepatitis on the incidence of metastases was observed. This may simply be related to the shortened life span of the animals. Death due to this infection may precede the expression of the metastatic potential.     

Survivin基因沉默抑制结肠癌生长的动物实验研究

目的 探究survivin基因沉默对人结肠癌Lovo细胞裸鼠移植瘤生长的抑制作用.方法 构建靶向survivin的shRNA载体SUR和阴性对照质粒Neg,并将其转染人结肠癌Lovo细胞,分别种植到裸鼠皮下建立人结肠癌裸鼠移植瘤模型.随后观察各组裸鼠移植瘤生长情况,免疫组化方法检测移植瘤survivin蛋白的表达,TUNEL法检测肿瘤细胞的凋亡情况.结果 移植转染细胞8周,与空白对照组比较,SUR组移植瘤的体积和质量均有显著缩小(P<0.05),体积和质量抑瘤率分别为48.9%和51.2%.与空白对照组比较,SUR组移植瘤survivin表达显著下调,表达指数为31.9%;SUR组肿瘤细胞凋亡显著增加,凋亡指数18.47%(P<0.05).阴性对照Neg组的上述指标与空白对照组比较,差异均无统计学意义.结论 Survivin基因沉默能够抑制人结肠癌Lovo细胞裸鼠移植瘤的生长.     

Variation in agar growth of transformed 3T3 cells after tumor formation in nude mice

Cells from a cloned line of spontaneously transformed 3T3 cells had a colony-forming efficiency in agar (CFEag) of about 10-15% and induced poorly differentiated sarcomas when injected into nude mice. These tumor cells were recultured in vitro and tested for their ability to grow in suspension. Initially, the tumor cells had a CFEag which was a hundredfold to a thousandfold lower than the cells that had been grown only in vitro. After 5-8 further weekly passages, however, the tumor lines recovered their original ability to grow in agar. For determination as to whether this increased CFEag was due to selection of cells with a higher CFEag from the tumor cell population or to adaptation of many of the tumor cells to agar growth, clones were isolated directly from a primary tumor, and each was tested weekly for agar colony formation. All of the tumor clones, as well as the uncloned tumor line, were able to recover their original ability to grow in agar. However, one tumor clone had a relatively high CFEag in the first assay, so the selection hypothesis could not be totally excluded. The initial low CFEag of the recultured cells was not due to the presence of normal nude mouse cells in the population. Before in vivo growth no clones could be isolated from the sublines that had as low a CFEag as the tumor cells isolated after in vivo growth. Tumor cells that had been repeatedly passaged in vivo still had a much lower CFEag than the input cells upon explantation into culture. The results suggest that phenotypic alterations observed during tumor growth and subsequent cultivation have an epigenetic basis.     

Radioimmunoimaging of human bladder tumor xenografts in nude mice by using monoclonal antibodies

Monoclonal antibodies HBJ127 and HBJ8, raised against T24 human bladder cancer cells, predominantly react with the cells in proliferating stages and with a portion of epithelial tumor cells, respectively. To investigate the in vivo localization of these monoclonal antibodies, the antibodies were labeled with radioiodine and indium-111 (111In) and injected into nude mice transplanted with human bladder tumors. The BT-11 bladder tumor had the highest concentration of radioiodinated HBJ127 and HBJ8 monoclonal antibodies, with 11.6 and 14.3% of the injected dose per gram and with a tumor-to-blood ratio of 2.6 and 1.6, respectively, at 4 days after the administration. An irrelevant monoclonal antibody did not show any specific accumulation in the BT-11 tumor. The 111In-labeled HBJ127 antibody was also localized in the tumor with a higher tumor-to-blood ratio than the radioiodinated antibody. The xenografted BT-11 tumor was successfully visualized with the radiolabeled HBJ127 and HBJ8 antibodies by scintigraphy. These monoclonal antibodies and the human bladder tumor xenografts may provide a good model for radioimmunoimaging and possibly therapy.     

Growth inhibition of human melanoma tumor xenografts in athymic nude mice by swainsonine

Swainsonine, an inhibitor of alpha-mannosidases, has been shown to block experimental metastasis of B16F10 melanoma and MDAY-D2 lymphoid tumor cells in syngeneic mice. In this report we demonstrate that swainsonine also reduces the growth rate of human melanoma cells in vitro and in vivo. Graded doses of swainsonine were administered either orally or via implanted Alzet miniosmotic pumps to athymic nude mice bearing subcutaneously implanted human MeWo melanoma cells. Swainsonine at 10 micrograms/ml in the drinking water or 0.5 mg/kg/day administered by miniosmotic pump reduced the growth rate of the MeWo tumors by approximately 50% and inhibited the expression of complex-type oligosaccharides in tumors and host intestine by only 10-20%. Swainsonine doses of 4 mg/kg/day reduced expression of complex-type oligosaccharides by 85% in vivo but afforded no additional inhibitory effect. A glycosylation mutant of MeWo called 3S5 has a defect in the synthesis of complex-type asparagine-linked oligosaccharides resulting in incomplete processing similar to that observed in swainsonine-treated MeWo tumor cells. Swainsonine did not inhibit the proliferation of 3S5 cells in vitro nor the growth of 3S5 tumors in nude mice. The results suggest that expression of highly branched complex-type oligosaccharides commonly associated with the malignant phenotype may provide the tumor cells with a growth advantage.     

Implantation of tumor xenografts in nude mice: an improved methodology

Xenografting of neoplasms is a common technique in cancer research. Implantation of non-uniform tumor tissue fragments often yields tumor nodules of irregular size and shape, which is disadvantageous. We therefore developed a device to standardize size and shape of such implants. This instrument is effective, inexpensive to fabricate, and simple to use.     

Isoenzyme pattern of enolase in human lung tumor xenografts in nude mice

A simple method is described which allows easy determination of neuroendocrine (NE) differentiation in human broncho-pulmonary tumor models grown in heterotransplanted nude mice. Enolase (EC 4.2.1.11) isoenzyme composition is studied using the electrophoretic method in xenograft tumor homogenates. The relatively large amount of alpha gamma and gamma gamma isoenzymes (neuron-specific enolase (NSE] is indicative of the neuroendocrine differentiation level of these tumors. The gamma gamma isoenzyme is present at a high level (M +/- SE: 10 +/- 2%) in all NE tumor models and absent in non NE tumor models. The alpha gamma isoenzyme is found in a significantly higher proportion in NE tumor models (30 +/- 2%) than in non NE tumor models (9 +/- 2%) (p less than 0.001). Moreover it is possible to discriminate between human and mice isoenzymes to estimate the proportion of mouse tissue hat is present in the xenograft.