pEGFP-IL-24对B16F0细胞抑制效应的实验研究

目的:构建IL-24真核表达质粒,研究其体内外表达对肿瘤细胞生长的抑制作用。方法:用重组DNA技术构建IL-24真核表达质粒pEGFP-IL-24。用脂质体法将重组质粒及空载体体外转染B16F0细胞后,经激光扫描共聚焦显微镜观察其表达,用MTT法检测B16F0细胞的体外增殖能力。用小鼠实体瘤模型研究基因转染对肿瘤生长的抑制。结果:pEGFP-IL-24转染B16F0细胞后,LSM可观察到其表达。IL-24基因转染的B16F0细胞体外增殖能力明显受到抑制。转染IL-24的B16F0细胞体内致瘤率降低。结论:IL-24基因转染的肿瘤细胞体外生长受抑。IL-24基因转染的肿瘤细胞体内生长能力显著降低。     

人HSC70基因真核表达载体的构建和表达

目的构建HSC70（HSC）真核表达质粒，并在HepG2细胞中表达。方法采用RT—RCR方法从正常人肝细胞系QZE中得到HSC70的cDNA序列，然后克隆入pcDNA^TM3．1／V5-His^A载体，构建重组质粒pcDNA-HSC70；经酶切和测序鉴定后，将pcDNA-HSC70体外转染HepG2细胞系，瞬时表达带有His标签的HSC70融合蛋白；并通过免疫细胞化学方法检测蛋白表达情况。结果RT—PCR扩增到1960bp的HSC70 cDNA片断，经测序分析与GenBank中已知序列一致；重组质粒pcDNA—HSC70转染后的HepG2细胞中，可检测到瞬时表达的HSC70-His融合蛋白。结论成功构建重组质粒pcDNA—HSC70，并可以在真核细胞HepG2中瞬时表达。     

人CD154基因真核表达载体的构建及在Eca109细胞中的表达

目的：构建人CD154基因真核表达质粒pcDNA3．1-CD154,初步观察转染该质粒后Eca109细胞的细胞生物学特性。方法：人外周血单个核细胞体外经PHA激活后提取总RNA,用RT—PCR技术扩增人CD154cDNA全长并构建真核表达质粒pcDNA3.1—CD154,用Lipofectamine^TM 2000介导pcDNA3．1-cDl54质粒转染食管癌Eca109细胞株,RT—PCR检测转染细胞中CD154 mRNA的表达,观察细胞生长特征。结果：用T7／SP6通用引物测序证实重组质粒中插入片段为正向插入的人CD154基因。pcDNA3．1-CD154转染Eca109细胞后细胞异型性明显改善,生长速率减慢。结论：pcDNA3．1-CD154构建成功;转染该质粒的Eca109细胞生物学特性发生改变。     

重组凋亡素真核表达载体诱导人卵巢癌细胞凋亡

目的 观察凋亡素基因（VP3）在卵巢癌细胞株CoCl中诱导凋亡的情况。方法 用Kpn Ⅰ、Xba Ⅰ分别双酶切pMD18-CAVP3和pcDNA3．1（＋），分别回收365bp和5．0kb大小的片断，然后常规行连接反应，构建重组凋亡素真核表达栽体pcDNA3．1-VP3。采用Lipofectamine^TM 2000介导的基因转染法，在体外将pcDNA3．1-VP3转染入卵巢癌细胞株CoCl中，RT—PCR检测凋亡素在细胞中的表达，用流式细胞仪检测细胞凋亡。结果 酶切鉴定及测序结果表明重组凋亡素真核表达载体pcDNA3．1-VP3构建成功。RT—PCR证实VP3基因在COCl中存在并在转录水平表达。转染pcDNA3．1-VP3细胞凋亡率明显高于转染空质粒组（P〈0．01）。结论 凋亡素可有效诱导卵巢癌细胞COCl凋亡。     

恢复野生型PTEN基因表达对炎性乳腺癌MDA-MB-468体外生物学行为的影响

目的研究野生型PTEN基因转染对人炎性乳腺癌MDA—MB-468细胞体外生物学行为的影响及机制。方法应用基因重组技术构建重组质粒pcDNA3．1-PTEN，用脂质体转染乳腺癌细胞株MDA—MB-468，用RT—PCR、免疫组化及免疫印迹方法分析目的基因及其蛋白表达，并用免疫印迹方法检测转染后，在表皮生长因子的诱导下，磷酸化蛋白激酶B（PKB／Akt）的表达，四甲基唑蓝（MTT）法分析细胞增殖。Annexin V—FITC和PI双染流式细胞术检测细胞凋亡。结果RT—PCR、免疫组化及免疫印迹显示pcDNA3．1-PTEN转染组有PTEN mRNA及其蛋白表达，且显示转染组在表皮生长因子的诱导下，p-Akt蛋白表达下调。MTT检测表明pcDNA3．1-PTEN转染组活细胞数较其它各组均低（P〈0．01），流式细胞分析其凋亡率达21．68％。结论野生型PTEN基因依赖其磷酸酶活性可诱导乳腺癌MDA—MB-468细胞凋亡。     

腺病毒介导的eNOS基因转移时血管平滑肌细胞增殖的抑制作用

目的：探讨腺病毒载体介导的内皮型一氧化氮合酶（eNOS）基因转染血管平滑肌细胞（VSMC）的效率及其对冠状动脉旁路移植术后再狭窄基因治疗的可行性。方法：构建带有eNOS基因的复制缺陷型重组腺病毒转染体外培养的VSMC，应用聚合酶（PCR），逆转录PCR（RT－PCR）技术和免疫组化法检测外源基因转染及表达效率；应用MTT法和流式细胞仪检测eNOS基因对VSMC的抑制作用。结果：外源性eNOS基因成功导入了VSMC；VSMC中有eNOS基因mRNA的表达；外源性eNOS蛋白呈高效表达。eNOS基因重组腺病毒载体转染细胞后，可显著抑制VSMC的生长，DNA合成减少。结论：腺病毒介导的eNOS基因可高效地转染VSMC，并可有抑制细胞生长。     

肺腺癌细胞A549 hnRNP B1基因RNA干扰的体外研究

目的 体外研究特异的小干扰RNA(siRNA)对肺腺癌细胞A549 hnRNP B1 基因表达及生长的抑制作用. 方法设计和构建由H1启动子驱动产生的特异的hnRNP B1 siRNA 表达质粒,转染至A549细胞,荧光定量PCR法及Western blot检测hnRNP B1基因的表达,MTT法检测该表达质粒转染后对A549细胞生长的影响.结果 针对hnRNP B1 基因构建的重组质粒具有明显的干扰效果,受特异hnRNP B1 siRNA 干扰的A549细胞hnRNP B1 mRNA及蛋白表达下调,细胞生长受到抑制.结论 应用RNA干扰技术可阻止hnRNP B1基因的表达,有望成为肿瘤基因治疗新的研究方向.     

红细胞分化去核因子在诱导小鼠红白血病细胞分化以及恶性逆转中的作用

目的：研究小鼠红细胞分化去核分化因子（MEDDF）在红细胞终末分化中的功能。方法：构建在真核细胞中表达的重组质粒pcDNA-MEDDF,用其转染小鼠红白血病细胞系（MEL）,通过分析细胞的生长曲线,分裂指数及在半固体培养基中的集落形成率比较细胞的生长线性,采用RT－PCR检测c-myc及β-珠蛋白（β-globin)基因的表达。结果：转染pcDNA-MEDDF与转染空载体（pcDNA3.1)的细胞相比较,细胞的增殖率减慢,分裂指数降低,在半固体培养其中形成集落的数量减少,转染细胞的联苯胺阳性率达32．8％,用RT－PCR检测发现,转染细胞β-珠蛋白的表达量上升了3．43倍,o-myc表达量下降了66．3％,。结论：MEDDF能命名小鼠红白血病细胞分化并抑制其恶性变。     

重组复制缺陷型腺病毒AdIL-10构建及其在HSC中的表达

目的利用AdEasy系统构建重组复制缺陷型腺病毒AdIL-10，并观察其在肝星状细胞(HSC)中的表达。方法从SD大鼠脾脏单核细胞中抽提总RNA，通过RT—PCR获得鼠IL-10 cDNA片段，插人穿梭质粒pAdTrack巨细胞病毒(CMV)启动子下游，构建重组穿梭载体pAdTrack-CMV-IL-10，线性化后与AdEasy-1电穿孔共转化BJ5183，获得重组腺病毒质粒pAdIL-10。pAdIL-10用Lipofectamin包装转染HEK 293细胞，获得重组腺病毒AdIL-10。将AdIL-10体外感染HSC，3d后抽提HSC总RNA，以RT—PCR检测IL-10的表达。结果通过酶切和测序法筛选出pAdIL-10；经293细胞包装，3d后观察到绿色荧光蛋白(GFP)表达；AdIL-10感染HSC3d后观察到GFP表达，通过RT—PCR可检测到IL-10的表达。结论利用AdEasy系统可制备重组腺病毒AdIL-10；AdIL-10感染HSC后可表达IL-10。     

重组腺病毒载体介导人抑瘤素M基因对胰腺癌细胞体外增殖的抑制作用

目的构建了含人抑瘤素M（HOSM）基因的复制缺陷型重组腺病毒载体AD—HOSM，观察其对人胰腺癌细胞株Capan－2在体外生长抑制情况，为胰腺癌的基因治疗提供参考。方法通过同源重组方法构建含HOSM基因的缺陷型腺病毒载体AD—HOSM，报告基因AD—GFP检测腺病毒的对胰腺癌细胞系的转染效率；人胰腺癌细胞株Capan－2转染含HOSM基因的缺陷型腺病毒载体AD—HOSM后，用RT—PCR法检测外源HOSM基因在其中的表达；苔盘兰染色、细胞计数法检测AD—HOSM对Capan－2的体外增殖抑制情况。结果成功构建了重组腺病毒载体AD—HOSM，AD—HOSM对胰腺癌细胞Capan－2有较高的转染效率，HOSM基因在转染细胞能有效的表达。休外试验示AD—HOSM明显抑制CAPAN－2的的生长。结论重组腺病毒介导HOSM表达能有效抑制Capan－2在体外的增殖，提示重组腺病毒介导的HOSM基因治疗可能成为胰腺癌治疗的侯选方案。     

Construction and Expression of Eukaryotic Expressing Vector pCH510 of Polypeptide CH50 and Its Chemotaxis and Antitumor Function by in vivo Transfection

Fibronectin ( FN) is a high- molecular glycopro-tein with multiple- domain.FN fragments containingCell domain are chemotactic to macrophages[1] .CH5 0 polypeptide,a recombinant polypeptide weproduced by the recombination of Cell and Hep domains of human fibronectin and expression in E.coli,can activate macrophages and inhibitthe metas-tasis of tumors[2 -4 ] .To investigate the function ofCH5 0 expressed in vivo by gene transfection,explorethe feasibility of applying the polypeptide to gene…     

ephrinB2真核表达载体的构建及其在Hela细胞的表达

目的构建ephrinB2重组真核表达载体,研究该质粒在细胞中的表达,为研究ephrinB2对肿瘤生长及血管生成的影响奠定基础。方法逆转录获得人ephrinB2全长cDNA序列,连接到pEGFPN1上,转化大肠杆菌,脂质体转染Hela细胞。RT-PCR检测转录情况,WB鉴定目的蛋白表达。结果酶切和测序证实正确构建重组质粒,并在Hela细胞中表达。结论成功构建了pEGFPN1-ephrinB2真核表达载体,并在Hela细胞中有效表达,为进一步研究ephrinB2的功能奠定基础。     

小鼠CD160胞外段真核表达载体的构建和表达

目的 构建小鼠CD160胞外段的真核表达载体,并稳定转染CHO细胞进行真核表达.方法 从C57BL/6小鼠脾脏中提取总RNA,经RT-PCR扩增CD160胞外段,然后将其克隆到真核表达载体pcDNA3.1和pEGFP-N1两种质粒中,构建重组质粒psCD160和pEGFP-sCD160.重组质粒经双酶切鉴定及测序正确后,用脂质体转染CHO细胞,并用RT-PCR和Western blot检测CD160胞外段的表达,流式细胞术检测重组psCD160与其配体的结合能力.结果 获得长度约为520 bp的小鼠CD160胞外段基因,经测序证实其序列正确.用脂质体将含有CD160胞外段基因的重组质粒载体转染CHO细胞后.RT-PCR和Western blot分析证实转染的CHO细胞可表达重组的psCD160基因,并且表达的可溶性CD160可与其配体结合.结论 成功构建小鼠CD160胞外段基因的真核表达载体,并转染CHO细胞后表达出相应蛋白,为进一步研究CD160胞外段的功能效应奠定实验基础.     

Construction and expression of eukaryotic expressing vector pCH510 of polypeptide CH50 and its chemotaxis and antitumor function byin vivo transfection

Summary  To construct an eukaryotic expressing vector that expresses CH50, a recombinant Cell I-Hep I bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmidin vivo, the plasmid was constructed by DNA recombination. Gene transfection was performedin vitro andin vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfectionin vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3. 1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells andin vivo in mouse. The expression of the plasmidin vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor. This project was supported by a grant from the National Natural Science Foundation of China (No. 39870763) and a Funding Program for New-Century Talent of the Ministry of Education of China.     

鼠源TRAIL真核表达质粒的构建及其抑瘤效应的初步观察

采用RT-PCR自小鼠脾细胞RNA中扩增出鼠TRAIL基因的全长cDNA，利用DNA重组技术将其插入到真核表达载体pcDNA3.1中获得重组质粒pX1。经酶切鉴定及序列分析，表明成功构建TRAIL的真核表达质粒。通过直接肌肉内注射进行基因转染，pX1具有诱导肝癌细胞凋亡，抑制肝癌生长的作用。表明TRAIL可作为一种直接杀伤机制应用于肝癌的基因治疗。     

Investigation on the Effects of Soluble Programmed Death-1 （sPD-1）Enhancing Anti-tumor Immune Response

Summary: By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor bearing mice and upregulated as compared with muscle tissues in normal mice and H22 hcpatoma cells. PD-L1 and PD-L2 were also expressed on the surface of the activated T cells. The soluble recombinant sPD-1 expressed from the constructed eukaryotic expression vector could enhance the lysis of tumor cells by lymphocytes stimulated specifically with antigen. The expresssion of sPD-1 by local gcne therapy on the inoculation site of H22 hepatoma cells could inhibit the growth of tumor. The results of this study indicate tbat expression of soluble receptor of negative costimulatory molecules could reduce the inhibitory effect on T cells in tumor microenvironment and enhance the eytotoxicity of T cells on tumor cells. This possibly provides a new method of improving efficacy of tumor gene therapy.     

Construction of eukaryotic expressing vector pCH503 of CH50 and its chemotaxis and anti-tumor function by expressionin vivo in mice

Summary An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function byin vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5′-terminal noncoding region and coding region of signal peptide of mouse IFN-7 cDNA at 5′ side and 3′-terminal noncoden region of human FN cDNA at 3′ side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503in vivo and culturedin vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%–60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can expressin vivo in mice. The transfection of pCH503in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor. This project was supported by a grant from the National Natural Science Foundation of China (No. 39870763) and Trans-Century Training Program Foundation for Talents under the Supervision of Ministry of Education of China.     

14-3-3γ表达载体的构建及其对子宫肌瘤细胞增殖和凋亡的影响

目的 ：构建一种稳定、高表达的14-3-3γ基因真核表达质粒载体,并探索其抗子宫肌瘤的作用。方法：提取子宫肌瘤细胞总RNA,反转录后通过RT-PCR扩增14-3-3γ,并将扩增的目的基因片段插入pCMV-N-Flag真核表达质粒,构建重组质粒14-3-3γ-pCMV-N-Flag,重组体经限制性内切酶酶切分析及测序鉴定后,用脂质体转染技术导入子宫肌瘤细胞。应用Western Blot法检测14-3-3γ蛋白的表达,应用CCK-8检测细胞增殖变化,应用Annexin V-FITC/PI双染色法经流式细胞术检测细胞凋亡。结果：①构建的14-3-3γ-pCMV-N-Flag真核表达载体经酶切后电泳和DNA测序,显示载体构建正确;②子宫肌瘤细胞转染重组体后的14-3-3γ表达明显高于转染前（P＜0.05）;③子宫肌瘤细胞转染重组体后,细胞增殖抑制率为52.90%（P＜0.05）;④转染重组体后肌瘤细胞的凋亡率明显高于转染前（P＜0.05）。结论：本研究成功构建14-3-3γ-pCMV-N-Flag真核表达质粒载体,14-3-3γ在子宫肌瘤细胞内高表达后可抑制细胞的增殖和促进细胞凋亡。     

小鼠TPO cDNA转染前后骨髓基质细胞TPOmRNA表达情况的研究

目的研究小鼠促血小板生成素(TPO)cDNA转染骨髓基质细胞(BMSCs)前后BMSCs中TPOmRNA表达的情况.方法以脂质体介导法将TPOcDNA真核表达质粒转染至BMSCs,用RT-PCR方法检测TPOmRNA的表达.结果转染TPOcDNA的BMSCs 其TPOmRNA表达量明显高于空载体组(P< 0.01)及未转染组(P<0.01).结论阳离子脂质体能有效介导TPOcDNA真核表达质粒转染BMSCs,且转染后BMSCs 中TPOmRNA的表达显著上调.