多配体蛋白聚糖-1细胞定位对人肝癌细胞侵袭的影响

为研究SDC-1的细胞定位对人肝癌细胞侵袭能力的影响,利用HPSE小干扰RNA和HPSE抑制剂低分子量肝素处理高转移人肝癌细胞MHCC97-H,通过实时定量PCR方法检测HPSE的mRNA表达,应用免疫细胞化学方法分析SDC-1的细胞定位,通过基质胶侵袭实验观察细胞侵袭性的改变.结果显示:HPSE小干扰RNA能够显著干...     

乙酰肝素酶/多配体蛋白聚糖-1轴对癌细胞增殖的调控作用

多配体蛋白聚糖-1(Syndecan-1)是一种硫酸肝素类蛋白聚糖。乙酰肝素酶(Heparanase)是生物体内的内切糖苷酶,能够特异性降解Syndecan-1的硫酸乙酰肝素侧链。Heparanase在许多恶性肿瘤组织中表达增加,且Heparanase通过上调生长因子活性及改变Syndecan-1结构正调控肿瘤的恶性行为。肿瘤组织中Heparanase/Syndecan-1轴的形成在促进肿瘤组织的生长、扩散、血管生成等过程中发挥重要作用。针对Heparanase/Syndecan-1轴的抗肿瘤治疗在抑制恶性肿瘤生长方面具有良好的应用前景。     

以干细胞特异性转录因子诱导小鼠肝癌细胞Hepa1-6重新编程的研究

利用逆转录病毒携带小鼠Oct4、Klf4、Sox2和c-Myc基因介导Hepa1-6小鼠肝癌细胞系重新编程,得到一团自律性跳动的细胞团,及一群形态学上明显类似于小鼠胚胎干细胞的克隆样细胞团。经长期传代,克隆样细胞能保持其形态。检测证实,这些克隆样细胞团表达Oct4、Sox2蛋白。证实,Hepa1-6小鼠肝癌细胞可以被携带Oct4、Klf4、Sox2和c-Myc基因的逆转录病毒感染的方法诱导重新编程。     

以干细胞特异性转录因子诱导小鼠肝癌细胞Hepa1—6重新编程的研究

利用逆转录病毒携带小鼠Oct4、Klf4、SOx2和c-Myc基因介导Hepa1-6小鼠肝癌细胞系重新编程,得到一团自律性跳动的细胞团,及一群形态学上明显类似于小鼠胚胎干细胞的克隆样细胞团。经长期传代,克隆样细胞能保持其形态。检测证实,这些克隆样细胞团表达Oct4、Sox2蛋白。证实,Hepa1-6小鼠肝癌细胞可以被携带Oct4、KK4、SOX2和cMyc基因的逆转录病毒感染的方法诱导重新编程。     

拟南芥肌醇- 1-磷酸合酶类蛋白基因的克隆表达

以野生型拟南芥总RNA为模板,逆转录PCR反应获得拟南芥肌醇-1-磷酸合酶类蛋白基因cDNA（AtMIPS1）,开放读码框为1533bp,编码510个氨基酸．与已报道物种MIPS基因氨基酸序列比较分析表明,AtMIPS1与植物MIPS基因的氨基酸同源性和相似性较高达86％-90％与95％-96％,并含有MIPS基因的保守区域“334SYNHLGNNDG”．将该cDNA序列不改变阅读框架克隆到pET28a（＋）原核表达载体上,SDS—PAGE结果表明：在0．12g／LIPTG诱导2h的条件下得到最佳的蛋白表达效果,AtMIPS1的成功表达为其功能研究打下基础．     

TGF-β1对肝癌细胞系Hep3B中干性相关基因表达的影响

有研究表明TGF-β1可以诱导诸多上皮来源的癌细胞和正常细胞发生EMT并使其功能发生改变。实验就TGF-β1对肝癌细胞系Hep3B的作用展开研究:通过CCK8检测TGF-β1对Hep3B细胞增殖的影响,RT-PCR实验检测TGF-β1处理后细胞中EMT及干性相关基因的表达变化。结果表明:TGF-β1对Hep3B细胞的增殖无抑制作用;TGF-β1处理Hep3B细胞6d后EMT相关基因的mRNA表达水平并无显著改变,但TGF-β1可上调Hep3B细胞干性基因Oct-4,Klf-4,Nanog,C-myc的表达,并下调分化基因albumin的表达。结果提示TGF-β1一定程度上影响肝癌细胞系的干性基因表达,但并不一定是以发生EMT为前提的。     

TGF—β1对肝癌细胞系Hep3B中干性相关基因表达的影响

有研究表明TGF-β1可以诱导诸多上皮来源的癌细胞和正常细胞发生EMT并使其功能发生改变。实验就TGF-β1对肝癌细胞系Hep3B的作用展开研究：通过CCK8检测TGF-β1对Hep3B细胞增殖的影响,RT-PCR实验检测TGF-β1处理后细胞中EMT及干性相关基因的表达变化。结果表明：TGF-β1对Hep3B细胞的增殖无抑制作用;TGF-β1处理Hep3B细胞6d后EMT相关基因的mRNA表达水平并无显著改变,但TGF-β1可上调Hep3B细胞干性基因Oct-4,Klf-4,Nanog,C-myc的表达,并下调分化基因albumin的表达。结果提示TGF-β1一定程度上影响肝癌细胞系的干性基因表达,但并不一定是以发生EMT为前提的。     

三种启动子shRNA表达框的制备及其在筛选有效SiRNA中的应用

为能快速经济地获取小干扰核糖核酸 (small interfering RNA siRNA)，设计采用特异性延伸引
物和上游、下游两条通用引物，通过重叠延伸一步聚合酶链反应(PCR)法一次性快速、简捷地制备包含U6
+1、H1或tRNAVal在内的三种人小RNA多聚酶III启动子 小发夹状RNA（shRNA）表达框.用该方法制备的增
强型绿色荧光蛋白（EGFP）特异性三种启动子 shRNA表达框在转染HepG2细胞后有效地抑制了EGFP转基因
表达，其中以tRNAVal shRNA表达框抑制效果最显著，且未检测到干扰素应答基因2'5'OAS mRNA的表达，
表明该表达框可被有效转染并启动产生特异基因的RNA干扰效应，进而用于快速筛选最佳siRNA位点及其最
适搭配启动子.     

人α-防御素HNP-1基因克隆及其结构分析

为了获得一种具有生物学活性的小分子多肽基因人α-防御素HNP-1，提取人外周静脉血液总RNA，反转录为cDNA作为模板，采用PCR的方法扩增得到长度约为380bp的防御素(Defensin)基因片段，并将该扩增产物连接入pMD18-T载体，转化大肠杆菌JM109感受态细胞，对PCR鉴定含有目的片段的克隆进行核苷酸序列测定，获得α-防御素HNP-1基因克隆。用序列处理在线工具包分析HNP-1基因结构。     

药用中间体1-甲基-5-巯基-1H-四氮唑相转移合成工艺研究

在水相中,相转移催化剂催化条件下合成1-甲基-5-巯基-1H-四氮唑,并对其工艺条件进行研究.采用IR与1 HNMR对产物结构进行了表征.结果表明催化剂的最优制备条件为:催化剂为环糊精、蒸馏水加入量为30mL、催化剂加入量为1%(占叠氮钠的摩尔百分数).在此催化条件下优化工艺条件为:异硫氰酸甲酯与叠氮钠的摩尔比为1.4∶1.0;滴加完异硫氰酸甲酯后的反应时间为2h;反应温度为75℃.产物收率为76.93%.     

Role of VEGF-A/C and CCR-7 in the enhanced metastasis of A549 cells induced by 2 and 4 Gy X-rays in vitro and in vivo

Radiotherapy is a common strategy in treating lung cancer.Accumulating evidence suggested that radiotherapy has the potential to promote the metastasis and invasion of carcinoma cells.In this study,we aimed to testify the role of vascular endothelial growth factor(VEGF)and C-C chemokine receptor-7(CCR-7)in the metastasis of human adenocarcinoma A549 cells in vivo and in vitro.Nude mice were injected with A549 cells irradiated by 0,2 and 4 Gy X-rays,respectively.Quantitative detections of VEGF-A/C and CCR-7 mRNA from lung sample were performed by real-time RT-PCR.Small interfering RNA(siRNA)transfection technology was further used to testify the role of the genes in the metastasis of A549 cells.VEGF and CCR-7mRNA could only be detected 10 weeks post injection in vivo when visible tumor foci scattered in lung.In addition,VEGF-A/C mRNA expressed significantly higher in mice injected with A549 cells irradiated by 2 and 4 X-rays than those with 0 Gy X-rays irradiation.On the other hand,A549 cells with or without X-rays irradiation transfected with VEGF siRAN and CCR-7 siRNA showed a dramatic decrease of invasiveness compared to normal A549 cells with or without irradiation.The migration indexes were?0.7,?0.48,?0.34 and?0.32 for A549 cells with CCR-7 siRNA,VEGF siRNA,X-rays combined CCR-7 siRNA and X-rays combined VEGF-siRNA respectively.These results demonstrated that X-rays could promote the metastasis of A549 cells,and VEGF-A/C and CCR-7 mRNA were closely related to the metastasis of A549 cells in vivo and in vitro.     

过表达Akt1细胞株的构建及鉴定

Akt1是细胞信号传导通路中的关键信号分子,具有促进细胞增殖、生长、迁移、侵袭,以及抑制细胞凋亡,抵抗化疗和放疗等重要作用。文章通过构建pLJM1-Akt1重组质粒,利用慢病毒侵染的方法将重组质粒转染至K562、Bel-7404细胞,用嘌呤霉素筛选得到Akt1稳定过表达的Bel-7404/Akt1、K562/Akt1细胞株,通过Western bloting分析细胞株中Akt1的表达情况。结果显示：Bel-7404/Akt1与K562/Akt1细胞中Akt1表达量明显高于野生型Bel-7404细胞与K562细胞,成功构建过表达Akt1的K562/Akt1、Bel-7404/Akt1稳转细胞株。Akt1过表达细胞株的成功构建为寻找和筛选高效、低毒、强特异性的Akt1抑制剂以及逆转细胞多药耐药（multidrug resistance,MDR）的研究提供了实验模型。     

Effects of Antihepatocarcinoma with Apatite Nanoparticles in vivo

The inhibition effect of hydroxyapatite （ HAP ） nanoparticles on hepatocarcinoma was investigated in vivo. The human hepatocarcinoma cell line Bel- 7402 was transplanted subcutaneously into nude mice. Hydroxyapatite nanoparticles suspension at a dose of 0. 2 mL was injected into the transplanted tumors every day for 2 weeks, and saline was used us control. The efficacy of hydroxyapatite nanoparticles on this carcinoma was surveyed and morphological changes of tissue and cells were observed by light microscopy and transmission electron microscopy （TEM）. Experimental results show that hydroxyapatite nanoparticles have a visible destructive effect on the structures of hepatocarcinoma cells and tissue. The inhibition rates of tumor growth were 77.21% and 51. 32% after intra-tumor injection of hydroxyapatite nanoparticles for 1 week and 2 weeks, respectively. Compared with the control group, hydroxyapatite nanoparticles can also prolong the survival time of the nude mice bearing this cancer significantly. This indicates that hydroxyapatite nanoparticles have the therapeutic potential on hepatoma in vivo.     

T C-1-HPV16 L1细胞模型与动物模型的建立与评价

为了解决人乳头瘤病毒16型L1蛋白(HPV16L1)为主要靶点的疫苗缺乏肿瘤模型细胞来验证疫苗的免疫效果的问题,构建了一个细胞模型并可以利用此细胞在实验动物体内形成肿瘤,用于以HPV16L1为主要靶点疫苗的验证实验。利用这个模型细胞或肿瘤模型可以在体内外检测疫苗的免疫学性质和保护功效。首先,利用聚合酶链式反应(polymerase chain reaction,PCR)的方法获得目标基因HPV16L1的基因序列并连接到载体质粒中,将质粒转染到细胞TC-1后在杀稻瘟菌素抗性压力筛选下获得稳定表达HPV16L1的细胞株。对TC-1和TC-1-HPV16L1两种细胞的生长增殖和成瘤特性进行比较发现,外源基因的加入对细胞特性没有影响。通过体外杀伤实验证实细胞TC-1-HPV16L1能够被HPV16L1靶点疫苗免疫过的小鼠脾淋巴细胞杀伤。实验动物被疫苗免疫后,进行攻瘤实验发现疫苗只对TC-1-HPV16L1组具有保护作用。综上,在本研究中成功地构建了可以检测HPV16L1疫苗的抑瘤效果的模型细胞TC-1-HPV16L1,为HPV疫苗的研究提供了一个模型细胞。     

稳定下调SIRT1基因表达的肝癌细胞系的构建

SIRT1(silent information regulator 1)与多种肿瘤的发病过程关系密切,但是其在肝癌中的作用尚不明确。为了研究SIRT1与肝癌的关系,通过构建慢病毒载体,病毒转染和包装,嘌呤霉素的筛选等实验方法,成功构建能稳定下调SIRT1基因表达的肝癌细胞系Hep3B和PLC/PRF/5,为后期进一步研究SIRT1在肝癌发病过程中的功能奠定了基础。     

A cell-based model for analyzing growth and invasion of tumor spheroids

Both chemical and mechanical determinants adapt and react throughout the process of tumor invasion. In this study, a cell-based model is used to uncover the growth and invasion of a three-dimensional solid tumor confined within normal cells. Each cell is treated as a spheroid that can deform, migrate, and proliferate. Some fundamental aspects of tumor development are considered,including normal tissue constraints, active cellular motility, homotypic and heterotypic intercellular interactions, and pressureregulated cell division as well. It is found that differential motility between cancerous and normal cells tends to break the spheroidal symmetry, leading to a finger instability at the tumor rim, while stiff normal cells inhibit tumor branching and favor uniform tumor expansion. The heterotypic cell-cell adhesion is revealed to affect the branching geometry. Our results explain many experimental observations, such as fingering invasion during tumor growth, stiffness inhibition of tumor invasion, and facilitation of tumor invasion through cancerous-normal cell adhesion. This study helps understand how cellular events are coordinated in tumor morphogenesis at the tissue level.