荧光素酶标的人肝癌细胞裸鼠异种移植瘤模型的生物发光成像和PET-CT成像比较

目的利用荧光素酶基因标记的人肝癌细胞株BEL-7402建立裸鼠肝原位移植模型,及小鼠肝原位移植模型的生物发光和小动物PET-CT成像的比较。方法构建表达荧光素酶基因的真核表达载体并将其转入人肝癌细胞BEL-7402,经梯度浓度G418筛选获得稳定表达荧光素酶基因的细胞克隆并扩大培养。BALB/cA-nu裸鼠肝门静脉接种5×105个发光细胞使其成瘤,活体荧光成像和小动物PET-CT成像系统观察肿瘤的生长情况。结果获得了稳定表达Luc的人肝癌细胞株,将其接种到裸鼠体内,活体荧光成像系统观察发现能够成瘤,小动物PET-CT影像观察发现小鼠肝脏边缘对18 F-FDG有高摄取区域。结论利用荧光素酶基因标记的人肝癌细胞BEL-7402成功建立了原位肝癌裸鼠模型,小动物活体成像结合小动物PET-CT技术为原位肿瘤模型的建立提供了一种新的可靠的技术,为进一步研究肝癌生长转移机制和药物开发提供了新的有用工具。     

活体荧光成像对裸鼠肝癌细胞系MHCC97-H原位移植模型的动态量化分析

目的:利用生物自发光的裸鼠肝癌原位移植模型,以活体荧光成像技术对肝癌的生长和转移情况进行动态、量化分析.方法:将稳定转染了荧光素酶(luciferase)基因的人肝癌细胞株MHCC97-H-LUC细胞,移植至裸鼠肝脏包膜下,每周利用活体荧光成像系统对裸鼠体内移植瘤的生长部位和范围进行成像,测量肿瘤细胞生物发光量,动态观察肝癌细胞在裸鼠体内的肿瘤数量、生长速度和转移情况.结果:建立可稳定表达荧光素酶的人肝癌细胞株MHCC97-H-LUC并用于进行生物自发光的裸鼠原位移植模型;利用活体荧光成像系统对裸鼠体内的移植瘤成像,见发光部位由肝脏向腹腔扩散,发光量随时间呈指数级增长;病理学观察证实肿瘤细胞长.结论:利用活体荧光成像技术的动态量化分析可灵敏、准确地监测裸鼠肝癌原位移植模型中肿瘤细胞的生长及转移情况,为肿瘤发生、生长、转移机制及对抗肿瘤生长和转移的体内研究提供了科学的量化手段.     

基于活体成像的表达人PSCA抗原的小鼠肿瘤模型的建立

目的:探讨并建立可供药物评价或生物学功能研究的表达人PSCA抗原的荧光素酶小鼠肿瘤模型。方法:克隆人PSCA基因及荧光素酶基因luc,构建真核表达质粒pcDNA-PSCA及pcDNA-luc,共转染RM-1细胞株;照度计检测转染细胞的荧光素酶活性,RT-PCR及流式细胞术检测PSCA的表达,筛选出共表达荧光素酶及人PSCA的RM-PSCA/luc细胞株;将RM-PSCA/luc细胞接种C57BL/6小鼠,观察所致肿瘤的生长情况及小鼠存活状况,并对小鼠进行活体成像检测Luc的表达;采用免疫组织化学染色方法检测人PSCA在小鼠肿瘤组织中的表达。结果:筛选到了稳定共表达Luc及人PSCA抗原的RM-PSCA/luc细胞,接种实验小鼠全部成瘤,肿瘤生长迅速,小鼠平均存活38天;转染荧光素酶基因的肿瘤细胞生物特性稳定,活体成像仪检测到转染荧光素酶细胞在小鼠体内活体成像,荧光素酶表达活性的强弱能够反映肿瘤大小;免疫组织化学染色方法检测到小鼠肿瘤组织PSCA的表达。结论:成功构建了可用于活体成像表达人PSCA抗原的小鼠肿瘤模型,为相关肿瘤药物及疫苗的研发奠定基础。     

三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移的比较

目的采用活体成像技术比较三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移情况,为研究肿瘤转移提供理想的动物模型以及活体分析方法。方法以荧光素酶（luciferase,Luc）作为报告基因导入小鼠乳腺癌细胞4T1、66c14和4TO7中,经G418筛选获得稳定表达荧光素酶的细胞克隆并扩大培养。标记细胞稀释成1×107cells/mL,取0.1 mL进行乳腺原位及尾静脉接种BALB/c小鼠,制作小鼠乳腺原位和尾静脉移植瘤模型,比较三株细胞在小鼠体内生长及转移情况。结果获得稳定表达荧光素酶基因的细胞克隆,将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠乳腺原位接种后7 d,均有肿瘤生长,接种后28 d,4T1细胞乳腺原位移植瘤最大,66c14细胞瘤体次之,4TO7细胞瘤体最小;接种后35 d,三株细胞乳腺原位移植瘤大小较一致,但4T1和66c14原位移植瘤均发生转移,其中4T1细胞较66c14细胞转移严重,而4TO7细胞未见转移;接种后42 d,三株细胞乳腺原位移植瘤大小无明显差别,而4T1和66c14细胞随天数的增加,移植瘤转移程度逐渐严重,4T1较66c14细胞转移更严重,呈广泛性转移,4TO7细胞仍未见转移。将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠尾静脉接种后7 d,小动物活体成像发现小鼠肺部均能检测到荧光,其中4T1细胞接种的小鼠肺部荧光信号最强,且小鼠陆续死亡;4TO7细胞接种小鼠肺部荧光信号次之;66c14细胞接种小鼠肺部荧光信号最弱。尾静脉接种后14 d,4TO7和66c14细胞随着观察天数的增加,转移程度逐渐严重,4TO7细胞接种小鼠肺部荧光信号较66c14细胞强且小鼠陆续死亡。结论乳腺原位自发转移模型较尾静脉转移模型更真实反应了肿瘤细胞在体的转移特性,且能完整地呈现肿瘤转移的全过程,可作为研究肿瘤转移的最理想模型。     

不同接种量荧光素酶标记小鼠乳腺癌细胞4T1在小鼠体内生长及肺转移的比较

目的 采用活体成像技术比较四种剂量荧光素酶标记肿瘤细胞在小鼠体内生长及肺转移情况,为光学标记肿瘤模型的药物筛选或机制研究提供参考资料.方法 以荧光素酶作为报告基因导人小鼠乳腺癌细胞4T1中,经G418筛选获得稳定表达荧光素酶的细胞克隆并扩大培养.标记细胞稀释成1×107细胞/mL,2×107细胞/mL,5×107细胞/mL和1×108细胞/mL四种剂量,取0.1 mL接种子BALB/c小鼠右侧第二对乳腺脂肪垫内,制作小鼠原位乳腺癌模型,比较肿瘤细胞在小鼠体内生长及肺转移情况.结果获得稳定表达荧光素酶基因的细胞克隆,在致瘤性方面和亲代细胞无明显差别,四种剂量细胞接种BALB/c小鼠后,均有肿瘤生长,接种第28天时,四种剂量接种的原位移植瘤大小没有明显差别,但接种两个高剂量肿瘤细胞的小鼠组各有2只小鼠死亡;接种后31 d,发现四种剂量接种的原位移植瘤均发生不同程度的转移,随着观察天数的增加,转移程度逐渐严重,接种后42 d,小鼠陆续发生死亡.结论 根据转移和死亡情况,确定接种1×106个细胞/只不仅肺转移明显,而且存活时间一般超过45 d,比高剂量接种存活时间长,为最佳肺转移剂量.     

采用活体成像技术监测肿瘤生长及转移模型的建立

目的采用活体成像技术监测稳定高表达荧光素酶报告基因的肿瘤细胞在小鼠体内生长及转移情况,为肿瘤治疗的药物研发提供新的有用工具。方法采用lipofectamine2000介导的基因转染方法,将pcDNA3．1 7Luc载体转染小鼠高转移乳腺癌细胞株4T1、EMT-6及结肠癌细胞株CT26,经G418抗性筛选及有限稀释法获得可稳定高表达荧光素酶的单克隆细胞;MTT法测定各转染细胞对不同化疗药物的抗性,并采用活体成像的方法检测各转染细胞在小鼠体内的成瘤和转移。结果获得了可稳定高表达荧光素酶基因的单克隆细胞株,该单克隆细胞株具有与亲本细胞系相同的对化疗药物的敏感性;将单克隆细胞株植入小鼠皮下,可采用活体成像技术准确监测肿瘤细胞体内生长及转移。结论采用活体成像技术构建的肿瘤动物模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

荧光素酶标记人胃癌细胞裸鼠原位移植模型的建立

目的建立荧光素酶标记人胃癌原位异种移植模型。方法将萤火虫荧光素酶作为标记基因导入人胃癌MGC803细胞,建立稳定表达荧光素酶的细胞,将其接种裸鼠胃壁浆膜下,建立胃癌裸鼠原位肿瘤模型。用活体荧光成像系统检测肿瘤的发生发展,并进行小动物超声影像和病理学分析。结果裸鼠原位成瘤率为100%,活体荧光成像观察发现在接种第7天,就可以观察到肿瘤发光。21 d后肿瘤进入对数生长期,28 d后肿瘤出现明显坏死,平均荧光光子数呈现下降趋势。超声成像发现小鼠胃部有直径为8.39 mm,面积为28.92 mm2瘤块。结论荧光素酶标记可以实时监测原位异种移植人胃癌生长状况。     

人宫颈癌细胞Hela移植模型肿瘤生长的荧光分析和卡尺测量的比较

目的建立稳定表达绿色荧光蛋白的人宫颈癌细胞系,建立移植瘤模型并比较移植模型肿瘤生长的荧光分析和卡尺测量的优缺点。方法以Lipofectamine 2000介导chickenβ-actin-GFP-NEO转染人宫颈癌细胞Hela,经梯度浓度G418筛选获得稳定表达绿色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×10^6个发光细胞使其成瘤,利用活体荧光成像系统和游标卡尺观察肿瘤的生长情况。结果获得了稳定表达GFP的人宫颈癌细胞株,将其接种到裸鼠体内可成瘤。活体荧光成像观察发现,1至3周随着肿瘤体积逐渐增大,平均荧光光子数逐渐增加;4周时随着肿瘤出现明显坏死,平均荧光光子数呈现下降趋势,而游标卡尺测量结果显示肿瘤在4至5周仍然不断的增大。结论绿色荧光蛋白能够在人宫颈癌细胞Hela中长期稳定表达,用绿色荧光蛋白标记的人宫颈癌细胞Hela建立的裸鼠肿瘤模型可以为人宫颈癌研究提供理想的实验材料,应用小动物活体成像系统能够客观定量评价活的肿瘤细胞在动物体内的生长情况,而不是肿瘤体积的变化。     

SW1990和Capan-2红色荧光标记胰腺癌移植肿瘤模型的建立和比较

目的建立稳定表达红色荧光蛋白基因的人胰腺癌细胞系,为体内监测肿瘤的早期生长及抗肿瘤药物的药效评价建立一种新的肿瘤动物模型。方法以Lipofectamine 2000介导chickenβ-actin-RFP-NEO转染人胰腺癌细胞SW1990和Capan-2,经梯度浓度G418筛选获得稳定表达红色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×106个发光细胞使其成瘤,活体荧光成像系统观察肿瘤的生长情况。结果获得了稳定表达RFP的两种不同的人胰腺癌细胞株,将其接种到裸鼠体内可成瘤,利用活体成像系统观察了肿瘤的生长动态过程,并且SW1990肿瘤细胞的生长速度较Capan-2细胞快。结论用红色荧光蛋白标记的人胰腺癌细胞建立的裸鼠肿瘤模型为胰腺癌的研究和相关药物筛选提供了可进行荧光影像活体、动态分析的动物模型。     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。     

miR-424-5p通过下调BCL-2的表达抑制人肺癌A549细胞增殖

microRNAs(miRNAs)是一类在真核生物中广泛存在的长度约为20～22个核苷酸的单链非编码小RNA,通过与其靶基因mRNA的3′非翻译区（3′UTR）结合发挥转录后抑制作用,参与调节细胞生长增殖、细胞代谢、细胞凋亡以及肿瘤的发生发展等过程。为研究microRNA-424-5p（miR-424-5p）在肺癌细胞中的作用及机理,利用lipo2000转染试剂将miR-424-5p mimics转染入人的非小细胞型肺癌细胞(NSCLC)A549中,流式细胞术检测A549细胞的周期变化及凋亡情况,发现细胞生长阻滞于G1/G0期且凋亡率显著上升。利用克隆形成实验和CCK-8法分别检测,发现miR-424-5p导致A549细胞增殖能力及活力降低。用在线数据库预测出抗凋亡基因BCL-2可能是miR-424-5p的靶基因,随后扩增BCL-2 mRNA 的3′UTR,采用双荧光素酶报告实验及Western印迹检测证明BCL-2确为miR-424-5p的靶基因。构建BCL-2的真核表达载体pCMV-HA-BCL-2,与空载分别转染A549细胞后发现过表达BCL-2可抵消miR-424-5p引起的细胞周期阻滞及细胞凋亡。以上结果提示,miR-424-5p可以通过下调BCL-2的表达来抑制肺癌细胞增殖。     

Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay

DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.     

MicroRNA-574-5p Was Pivotal for TLR9 Signaling Enhanced Tumor Progression via Down-Regulating Checkpoint Suppressor 1 in Human Lung Cancer

Accumulating data suggested that functional expression of Toll-like receptors (TLRs) in tumor cells was involved in tumor progression. Our previous study demonstrated that TLR9 signaling could enhance the tumor progression of human lung cancer cells in vitro and in vivo. We further showed that miR-574-5p was the mostly up-regulated miRNA in human lung cancer cells under TLR9 signaling by miRNA array analysis. Here we characterized the potential role of miRNA-574-5p in enhanced tumor progression induced by TLR9 signaling in human lung cancer. We confirmed that TLR9 signaling effectively elevated the expression of miR-574-5p in human lung cancer cells. Notably, we found that down-regulation of miRNA-574-5p using miR-574-5p inhibitor in vitro or miR-574-5p sponge in vivo significantly abrogated the enhanced tumor progression induced by TLR9 signaling. Further studies showed that miR-574-5p was an important player associated with enhanced tumor progression of human lung cancer cells. Notably, we identified checkpoint suppressor 1 (Ches1) as the dominant direct target for miRNA-574-5p to confer the TLR9 signaling enhanced tumor progression. We revealed that over-expression of Ches1 significantly inhibited the cell cycle entry of human lung cancer cells. Finally, we revealed that the expression of miR-574-5p was positively correlated with TLR9 and reversely correlated with Ches1 in lung cancer patients. Our findings not only facilitated the further understanding of the crosstalk between miRNAs and TLRs in tumor biology, but also provided novel potential candidates for treatment of cancer.     

MicroRNA-7–regulated TLR9 signaling–enhanced growth and metastatic potential of human lung cancer cells by altering the phosphoinositide-3-kinase,regulatory subunit 3/Akt pathway

Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology.     

Targeting PRMT5/Akt signalling axis prevents human lung cancer cell growth

The emerging evidence reveals that protein arginine methyltransferase 5 (PRMT5) is involved in regulation of tumour cell proliferation and cancer development. Nevertheless, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remains largely obscure. Here, we showed that PRMT5 was highly expressed in human lung cancer cells and lung cancer tissues. Furthermore, we generated PRMT5 stable knockdown cell lines (A549 and H1299 cells) and explored the functions of PRMT5 in lung cancer cell proliferation. We found that the down‐regulation of PRMT5 by shRNA or the inhibition of PRMT5 by specific inhibitor GSK591 dramatically suppressed cyclin E1 and cyclin D1 expression and cell proliferation. Moreover, we uncovered that PRMT5 promoted lung cancer cell proliferation via regulation of Akt activation. PRMT5 was directly co‐localized and interacted with Akt, but not PTEN and mTOR. Down‐regulation or inhibition of PRMT5 markedly reduced Akt phosphorylation at Thr308 and Ser473, whereas the expression of PTEN and mTOR phosphorylation was unchanged, indicating that PRMT5 was an important upstream regulator of Akt and induced lung cancer cell proliferation. Altogether, our results indicate that PRMT5 promotes human lung cancer cell proliferation through direct interaction with Akt and regulation of Akt activity. Our findings also suggest that targeting PRMT5 may have therapeutic potential for treatment of human lung cancer.     

阳离子脂质体介导RNA干扰的效果评价

考察自制的肽型阳离子脂质体CDO14作为RNA转染载体的细胞毒性及其运载si RNA进行RNA干扰的效果。通过MTT法检测脂质体对稳定表达荧光素酶的肺癌A549(Luc-A549)细胞的毒性。以脂质体为载体将荧光素酶si RNA(Luc-si RNA)转染至Luc-A549细胞内,用发光仪检测转染细胞内荧光素酶含量,BCA法检测细胞内总蛋白含量。在裸鼠腋下接种Luc-A549细胞,成瘤后尾静脉注射Luc-si RNA和脂质体的复合物,利用活体成像系统检测模型小鼠体内荧光素酶的表达量。细胞毒性实验表明,自制脂质体的毒性与商品脂质体DOTAP相近,低于商品脂质体Lipo2000;细胞转染实验表明自制脂质体作为基因转染载体的转染效率高于DOTAP;体内转染实验表明CDO14作为载体转染效果优于DOTAP。结果表明,肽型阳离子脂质体CDO14具有毒性小、转染效率高等优点,有望作为转染载体用于基因治疗。     

MicroRNA-7 Compromises p53 Protein-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1

We previously demonstrated that the epidermal growth factor receptor (EGFR) up-regulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3′UTR of SMARCD1 and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3′UTR of SMARCD1 and down-regulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 down-regulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering with the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that although SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was down-regulated in patients with non-small cell lung cancer and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated microRNA pathway in the SWI/SNF chromatin remodeling complex suggests that EGFR-mediated miR-7 suppresses the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells.