New approaches to moderate CRISPR-Cas9 activity: Addressing issues of cellular uptake and endosomal escape

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The prognostic value of lymph node ratio in Medullary thyroid carcinoma: A multi-center study

IntroductionThe lymph node ratio (LNR), which represents the proportion of metastatic lymph nodes resected, has been found to be a prognostic variable in several cancers, but data for Medullary thyroid carcinoma (MTC) are sparse. The aim of this study was to determine the value of the LNR in predicting outcome in patients with MTC.Materials and methodsA retrospective multicenter study design of 107 patients with MTC who underwent total thyroidectomy with neck dissection between 1984 and 2016. The association of LNR with patient and tumor characteristics and prognostic factors was evaluated.ResultsStudy population consisted of 53.3% female, mean age at diagnosis was 50.3 ± 18.4 years; 16.8% had inherited MTC. LNR was positively correlated with tumor size (p = 0.018) and inversely correlated with age at diagnosis (p = 0.024). A higher LNR was associated with extrathyroidal extension (p < 0.001), multifocality (p = 0.001), bilateral tumor (p = 0.002), distant metastases (p < 0.001), and tumor recurrence (OR = 14.7, p < 0.001). LNR was also correlated to postoperative calcitonin levels (p < 0.001) and carcinoembryonic antigen (p = 0.011). LNR >0.1 was associated with shorter disease-specific survival in patients at risk: tumor larger than 20 mm at diagnosis (p = 0.013), sporadic MTC (p = 0.01), and age above 40 years at diagnosis (p = 0.004). Cox multivariate survival analysis revealed LNR as the only significant independent factor for disease free survival (p = 0.005).ConclusionsThis study showed that LNR correlates well with patient and tumor characteristics and prognostic variables. We suggest that LNR should be considered an important parameter for predicting outcome in MTC.     

NRAGE影响人食管癌细胞放射抗性的作用及其机制研究

背景与目的：食管癌是全球威胁人类生命和健康的常见恶性肿瘤之一，中国每年食管癌发病人数占全球发病总人数的一半以上，以食管鳞癌最为常见。放疗是食管癌三大治疗手段之一，而放射抗性是导致其治疗失败的主要原因。神经营养因子受体相互作用MAGE类药物（neurotrophin receptor-interacting MAGE homolog，NRAGE）在放射抗性细胞株TE13R120中表达量明显高于亲本TE13细胞，且NRAGE亚细胞定位变化可能参与食管癌细胞放射抗性的形成。通过基因转染构建NRAGE稳定表达的食管癌细胞系，以进一步明确NRAGE基因与食管鳞癌细胞放射抗性的关系，分析该基因影响放射抗性的具体机制。方法：通过基因转染构建NRAGE稳定表达的食管癌细胞系。采用细胞克隆形成实验检测细胞放射敏感性，采用流式细胞术检测细胞周期及凋亡；细胞划痕、Transwell侵袭实验检测细胞迁移、侵袭能力，采用实时荧光定量聚合酶链反应（real-time fluorescence quantitive polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）检测细胞中β-catenin表达情况。组间差异采用t检验或方差分析。结果：实验分为转染过表达组（Eca109/NRAGE组）和空白对照组（Eca109组）。Eca109/NRAGE细胞中NRAGE的表达量明显高于Eca109（t=29.65，P<0.05）。照射后Eca109/NRAGE细胞的放射抗性显著高于Eca109。流式细胞术检测结果显示，Eca109/NRAGE细胞中对射线抵抗性最强的S期细胞比例增加，对射线最敏感的G ₂ /M期减少。Eca109/NRAGE细胞凋亡率较Eca109细胞降低（t=3.268，P<0.05）。Eca109/NRAGE细胞迁移和侵袭能力均高于Eca109。RTFQ-PCR和Western blot检测结果显示，β-catenin的mRNA表达及蛋白水平在Eca109/NRAGE细胞中明显高于Eca109（t=15.87，P<0.05）。结论：NRAGE参与Eca109细胞放射抗性的形成，改变细胞的细胞周期分布和凋亡情况，影响细胞迁移及侵袭能力并可能影响食管癌细胞的放射敏感性，该作用可能与激活Wnt/β-catenin信号转导通路有关。     

体外转染Cyclin A2基因对原代大鼠心肌细胞增殖的影响

目的探讨体外转染细胞周期素A2(Cyclin A2)基因对原代大鼠心肌细胞增殖的影响,为心脏再生提供细胞学依据。方法 SD乳鼠12只,分离、培养、鉴定原代乳鼠心肌细胞并分为3组,实验组:转染带有强化绿色荧光蛋白(GFP)和Cyclin A2基因的重组腺病毒(Ad-Cyclin A2-GFP);空病毒组:转染不含目的基因带有GFP的重组腺病毒(Ad-Null-GFP);阴性对照组:未做转染处理,仅加入等量的培养基。利用GFP示踪技术,评估原代心肌细胞转染效率;转染后的细胞继续体外培养3~5d,利用免疫荧光技术分别检测Cyclin A2、磷酸化组蛋白H3(H3P)、心肌肌钙蛋白-T(c Tn T)。结果 1.荧光GFP示踪表明原代心肌细胞的转染效率高达(97±0.74)%;2.免疫荧光标记显示,空病毒组和对照组结果相似;心肌细胞特异性标记蛋白c Tn T分布于细胞质,原代心肌细胞纯度高达(95±0.62)%;心肌细胞Cyclin A2主要在胞核内聚集,少数分布于细胞质。H3P为核蛋白在细胞核内分布。3.转染Cyclin A2后,实验组H3P阳性率明显高于空病毒组和对照组,差异具有统计学意义(P0.05);实验组可见大量多核细胞,以双核为主,伴少量3核细胞。结论腺病毒作为转染载体对原代心肌细胞有很好的侵染效率;Cyclin A2超表达促进原代心肌细胞形成双核。     

Expression of exogenetic enhanced green fluorescent protein in rat endocranium through lentivirus infection

The study aims to investigate whether exogenetic green fluorescent protein is able to express in the endocranium of rats, and to establish a method for further study in exogenetic gene knock-in or gene overexpression. Forty female Sprague Dawley (SD) rats were randomly divided into 4 groups with 10 in each: low and high dose groups, treated with 10% and 100% EGFP-lentivirus, respectively; negative control group, treated with virus enhancer; sham group, treated with normal saline. Seven days later, half rats’ brain tissues were perfusion fixed and fresh brain tissues were obtained from the rest after euthanasia in each group. Immunohistochemical analysis, Western blotting and RT-PCR were respectively performed to detect the site where EGFP expressed and its levels. Immunohistochemical analysis demonstrated that EGFP was successfully expressed in brain tissue of those rats infected with EGFP-lentivirus. Both Western blotting and RT-PCR showed that EGFP was expressed after treatment with EGFP-lentivirus, and the expression level increased with the dosage of the vector. Exogenetic EGFP gene can express in brain tissue of the rat, which laid a solid foundation for future studies in exogenetic gene knock in or gene overexpression.     

真核表达载体pIRES2-EGFP-hBMP2的构建和鉴定

目的:构建并鉴定人hBMP2基因的真核表达载体pIRES2-EGFP-hBMP2。方法:采用RT-PCR方法从人成骨肉瘤细胞中克隆hBMP2基因,连接T载体并测序正确后与真核表达载体pIRES2-EGFP连接,并进行酶切鉴定,最后将pIRES2-EGFP-hBMP2转染入HEK293细胞中,利用荧光显微镜和Western blot进行表达鉴定。结果:成功克隆了人BMP2基因,酶切鉴定和测序均正确,构建的真核表达载体pIRES2-EGFP-hBMP2能够正确表达hBMP2蛋白。结论:构建了hBMP2基因的真核表达载体pIRES2-EGFP-hBMP2,为研究该基因的功能奠定了基础。     

靶向人端粒酶反转录酶RNA干扰载体质粒的构建与筛选

目的:设计靶向人端粒酶反转录酶的RNAi序列,构建shRNA表达载体质粒和hTERT真核表达质粒,采用共转染工具细胞筛选出有效靶点.方法:首先以EASYMsiRNA软件针对hTERT设计5个靶点siRNA序列和1条通用阴性对照序列,合成shRNA oligo表达框架,将其分别连接至经酶切消化的载体质粒、pGCsi-U6/Neo/GFP、pGCL-GFP,重组质粒转化1)H5α,阳性克隆进行PCR与测序鉴定;然后从cDNA文库中利用PCR钓取hTERT基因片段,与表达载体pEGFP-C1分别进行双酶切、纯化及定向连接、重组、转化,阳性克隆行PCR与测序鉴定,荧光镜检GFP表达;最后上述2个质粒系统共转染293T细胞,Western印迹检测hTERT蛋白表达水平,筛选最有效序列.应用SPSS16.0软件包对所得数据进行单因素方差分析.结果:BLAST检索5条hTERT siRNA序列均能100%命中,且不与其他基因同源:PCR鉴定及阳性克隆测序验证shRNA oligo与载体质粒成功连接、重组.hTERT基因PCR钓取片段产物大小1.4 Kp,经酶切与质粒连接,形成pEGFP-C1-hTERT表达载体,阳性克隆PCR、测序鉴定确定成功重组;转染293T细胞48h时荧光镜F可观察到GFP标记的hTERT融合蛋白.hTERT表达质粒和shRNA载体质粒共转染293T细胞,48h时Western印迹榆测结果显示:实验各组hTERT蛋白量均有不同程度减少,而内参对照B-aetin和GAPDH尤明显变化.经β-actin校正,得到各组hTERT蛋向相对表达量:KD No.1-5实验组与NC组相比,hTERT基因表达均得到不同程度的敲减(54.61%～76.84%.P＜0.05),其中No.4敲减效能最高.结论:5条RNAi设计序列均可有效、特异性地沉默hTERT基因的转录和表达水平,其中以5'-GCAAGTTGCAAAGCArrGGAA-3'敲减效能最优;构建外源基因的过表达载体质粒转染工具细胞,可作为RNAi敲减效率筛选的验绩标靶.     

cbfa1对人牙乳头细胞增殖和矿化能力的影响

目的 :观察核心结合因子 (cbfa1)对人牙乳头细胞增殖和矿化能力的影响。方法 :建立稳定表达cbfa1的人牙乳头细胞模型 ,分别采用MTT、FCM、cAMP检测以及ALP和骨钙素放射免疫等方法检测转染前后相关指数的变化。结果 :稳定转染cbfa1后 ,牙乳头细胞的MTT值、PrI指数明显降低 ,而cAMP含量、ALP活性和OC含量明显增高。结论 :稳定转染cbfa1后牙乳头细胞的增殖能力明显降低 ,矿化指标明显增高。     

脂质体介导反义hTR基因转染对腺样囊性癌细胞系SAcc83的影响

目的 检测SAcc83细胞系端粒酶活性并观察人端粒酶模板hTR封闭后细胞生长行为的改变及与凋亡的关系。方法TRAP-PCR-ELISA法检测SAcc83细胞系端粒酶活性,脂质体介导真核表达载体PBBS212-ahTR转染SAcc83,PCNA免疫组化染色以证实其增殖活性改变,TUNEL法及琼脂糖凝胶电泳检测细胞凋亡。结果转染反义hTR后SAcc83细胞增殖能力明显下降,群体倍增时间延长,转染细胞约30天后死亡。各项凋亡检测均证明细胞凋亡的存在。结论hTR反义基因通过封闭端粒酶模板RNA抑制了端粒酶活性,并可能通过某种途径激活细胞的凋亡程序,这为以端粒酶为靶子治疗恶性肿瘤提供了方向。     

Fas转染及抗体处理对舌鳞癌细胞Tca8113移植瘤形成和增殖的影响

目的　研究Fas转染和抗Fas单克隆抗体对舌鳞癌细胞系Tca8113裸鼠体内移植瘤形成和增殖的影响,探讨相关机制。方法　脂质体法以真核表达重组质粒pBK-fas转染舌鳞癌细胞系Tca8113。部分转染细胞行抗Fas单克隆抗体处理。未转染细胞、Fas转染细胞、Fas转染抗体处理细胞分别接种裸鼠皮下。观测移植瘤生长,绘制生长曲线。RT-PCR检测肿瘤细胞Fas mRNA表达。流式细胞术(FCM)检测肿瘤细胞凋亡、增殖及Fas蛋白表达。结果　Fas转染和抗体处理延迟肿瘤形成,抑制肿瘤增殖。Fas转染上调移植瘤Fas mRNA表达,提高Fas蛋白表达强度和凋亡指数,但不影响Fas蛋白阳性表达率和增殖指数。抗体处理不影响Fas mRNA和蛋白表达,但可提高凋亡指数,降低增殖指数。结论　Fas转染抑瘤效应是上调Fas转录和表达、促进凋亡的结果。抗Fas单克隆抗体抑瘤效应则与激活凋亡和抑制增殖有关。