In vitro suppression of vascular smooth muscle cell proliferation using adenovirus-mediated transfer of the cd gene

Objective : To develop a treatment strategy for restenosis with a replication-deficient recombinant adenovirus (AdCMVCD) containing the cd gent whose gene product phosphorylates the prodrug 5-flurocytosine to form a nucleoside analog that inhibits DNA synthesis. Methods: Cultured primary rabbit smooth muscle cell (SMC) infected with AdCMVCD was incubated in 5- flurocytosine-containing medium and assayed with a Coulter Counter on day 2, 6, 10, and 14 for the establish- ment of proliferation curves. In addition, to observe an inhibitory effect on neighboring SMC, only a portion of the SMC population received the cd transgene. Results : The antiproliferative effect of AdCMVCD was dependent on both concentation of virus and concentration of 5-flurocytosine . Transformed cells demonstrated a bystander effect wherein SMC expressing cd gene exerted an antiproliferative effect on neighboring cells that did not express cd gene . Conclusion:Theresult demonstrates the potential utility of adenovirus-mediated cd gene transfer for the treatment of restenosis after balloon injury.

鸡SLMO2基因CDS区克隆、生物信息学及组织表达分析

为了探究类果蝇慢移基因的氨基酸序列特征,探索其在不同肤色鸡不同组织中mRNA表达变化规律,实验采集不同肤色鸡各组织,PCR克隆获得SLMO2完整CDS区,进行生物信息学分析及组织表达分析。实验得到SLMO2的CDS区序列全长560 bp,其中可编码氨基酸174个。生物信息学研究结果表明,鸡SLMO2蛋白并不具有信号肽和跨膜结构,蛋白主要结构以无规性卷曲的α-螺旋结构结合为主,且蛋白分布在线粒体内膜间隙。通过氨基酸序列对比发现,鸡与爪蟾的亲缘关系较近。qRT-PCR结果表明,SLMO2在背肤组织中相对表达量极显著高于其他组织,且该基因在丝羽乌骨鸡背肤组织中表达量极显著高于“豫粉1号”H系鸡。以上结果为进一步探究鸡SLMO2对黑色素沉积的调控机制提供了理论依据。

CD163基因敲除iPAMs的构建及其感染PRRSV的特征分析

[目的]获得分化抗原163(cluster of differentiation 163,CD163)基因敲除的永生化猪肺泡巨噬细胞系(immortalized porcine alveolar macrophages,iPAMs),并从细胞水平研究CD163蛋白在猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)入侵过程中的作用。[方法]将靶向CD 163基因第7外显子的sgRNA质粒(pX330-sgCD163)转染至iPAMs,转染48 h后通过有限稀释法筛选单克隆细胞并进行基因型鉴定,通过Western blotting和脱靶分析来筛选CD 163基因敲除的iPAMs;通过实时荧光定量PCR、Western blotting、间接免疫荧光试验(indirect immunofluorescence assay,IFA)和半数细胞培养物感染量(50%tissue culture infective dose,TCID 50)等试验分析CD 163基因敲除的iPAMs感染PRRSV的特征。[结果]测序结果表明,100株单克隆细胞中有1株CD 163双等位基因敲除的iPAMs,敲除效率为1%;Western blotting结果显示,CD 163基因敲除的iPAMs中检测不到CD163蛋白的表达,且在预测的脱靶位点未检测到脱靶效应。实时荧光定量PCR结果表明,与野生型iPAMs组相比,CD 163基因敲除的iPAMs组病毒拷贝数极显著降低(P<0.01);Western blotting和IFA结果表明,在接毒24 h后的CD 163基因敲除的iPAMs组中未检测到PRRSV-N蛋白的表达;TCID 50试验结果同样显示,在CD 163基因敲除的iPAMs组中未观察到细胞病变。[结论]本研究利用CRISPR/Cas9编辑系统成功构建了CD 163基因敲除的iPAMs,该细胞系可以完全抵抗PRRSV感染。该细胞系的建立为后续研究PRRSV与CD163蛋白的互作机制提供了新型试验材料。

Construction of dual suicide gene therapy system pTRKH2/CD and pTRKH2/UPRT in Bifidobacterium infantis and its characterization

Objective:We recombine the suicide gene CD,UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function.Methods:CD gene,UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I,and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E.coli.The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation.Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing.RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels.The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay.Results:The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2.After dual endonuclease digestion of plasmid purified from the positively transfected E.coli,two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene.The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data.A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobacterium by RT-PCR.A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium.The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells.Conclusion:CD gene and UPRT gene are successfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifidobacterium Infantis.This dual suicide gene therapy system shows a high efficiency for tumor cells killing.

Different effects of a CD14 gene polymorphism on disease outcome in patients with alcoholic liver disease and chronic hepatitis C infection

AIM： Clinical and experimental data suggest that gut-derived endotoxins are an important pathogenic factors for progression of chronic liver disease. Recently, a C-T （-159） polymorphism in the promoter region of the CD14 gene was detected and found to confer increased CD14 expression and to be associated with advanced alcoholic liver damage. Here, we investigated this polymorphism in patients with less advanced alcoholic liver disease （ALD） and chronic hepatitis C virus （HCV） infection. METHODS： CD14 genotyping was performed by PCR-RFLP analysis in （a） 121 HCV patients, （b） 62 patients with alcohol-associated cirrhosis （AIc-Ci）, （c） 118 individuals with heavy alcohol abuse without evidence of advanced liver damage （Alc-w/o Ci）, and （d） 247 healthy controls. Furthermore, serum levels of soluble CD14 （sCD14） and transaminases were determined. RESULTS： The TT genotype was significantly more frequent in Alc-Ci compared to Alc-w/o Ci or controls （40.3% vs 23.7% or 24.0%, respectively）. In Alc-w/o Ci, serum levels of transaminases did not differ significantly between patients with different CD14 genotypes. In HCV patients, TT-homozygotes had significantly higher sCD14 levels and sCD14 serum levels were significantly higher in patients with advanced fibrosis or cirrhosis. However, no association was found between CD14 genotypes and histological staging or grading. CONCLUSION： Considering serum transaminases as surrogate markers for alcoholic liver damage, the CD14 polymorphism seems to exhibit different effects during the course of ALD. Differences in genotype distribution between cirrhotic HCV patients and alcoholics and the known functional impact of this polymorphism on CD14 expression levels further indicate differences in the pathophysiological role of CD14 and CD14-mediated lipopolysaccharides signal transduction with regard to the stage as well as the type of the underlying liver disease.

PEI-PEG as a siRNA Genetic Vector Demonstrating Interference in the Expression of CD44v6 Protein in Gastric Cancer Cells

OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.

EXPRESSION OF INTRON 9 IN CD44 GENE IN CERVICAL CANCER AND CIN AND ITS CLINICAL SIGNIFICANCE

Objective： To detect the retention of intron 9 in CD44 mRNA in cervical cancer tissue, CIN, cervicitis and their exfoliated cells, and to study their clinical significance in diagnosis and treatment of early-stage, non-invasive cervical cancer, Methods： RT-PCR methods were used to detect the retention of intron 9 in CD44 mRNA in 30 cases of cervical cancer tissue, 11 cases of CIN tissue, 30 cases of cervicitis tissue and their exfoliated cells. Results： The retention rate of intron 9 in CD44 gene transcripts were 76.7% in cervical cancer tissue, 89,8% in corresponding exfoliated cells, 70.8% in CIN tissue, and 60.0% in CIN exfoliated cells, but undetected in neither cervicitis tissue nor exfoliated cells. The relative quantity of intron 9 in CD44 gene transcripts was 1.10 ± 0.12 in cervical cancer tissue, 1.21 ± 0.11 in CIN tissue, 1.11 ± 0.19 in cervical cancer exfoliated cells, 1.17 ± 0.12 in CIN exfoliated cells respectively, but undetected in neither cervicitis tissue nor exfoliated cells. The retention rate and relative content of intron 9 in CD44 gene transcripts in cervical cancer and CIN tissue and their exfoliated cells were statistically higher than that in cervicitis and their exfoliated cells （P〈0.005）. There was statistic difference comparing the retention rate of intron 9 in CD44 gene mRNA transcripts （70.8%） with positive rate in cytology （40.0%） in cervical cancer （x^2=5.78, P〈0.05）, but no statistic difference between the retention rate of intron 9 in CD44 gene transcripts （60.0%） with positive rate in cytology （36.4%） in CIN （x^2=0.585, P〉0.05）. Conclusion： Detecting the retention of intron 9 in CD44 mRNA in cervical exfoliated cells was more sensitivity than traditional cytology exam for diagnosing cervical cancer, and the techniques was worth clinical application.

Association of promoter polymorphism of the CD14 C (-159) T endotoxin receptor gene with chronic hepatitis B

AIM: To investigate whether single-nucleotide polymor- phisms in the promoter regions of endotoxin-responsive genes CD14 C (-159) T is associated with chronic hepatitis B. METHODS: We obtained genomic DNA from 80 patients with established diagnosis of chronic hepatitis B and 126 healthy subjects served as a control population. The CD 14 C (-159) T polymorphism was investigated using an allele specific PCR method. RESULTS: Twenty seven percent of chronic hepatitis B patients and 75% of controls were heterozygous for CT genotype. The difference between the chronic hepatitis B and control groups was statistically significant [P < 0.0001; Odds ratio (OR) = 2.887; 95% CI: 1.609-5.178]. Twenty four point six percent of chronic hepatitis B and patients 12.3% of the control group were heterozygous for TT genotype. The difference between groups was not statistically significant (P = 0.256; OR = 0.658; 95% CI: 0.319-1.358). Forty eight point four percent of chronic hepatitis B patients and 12.7% of control were homozy- gote for CC genotype (P < 0.004; OR = 0.416; 95% CI: 0.229-0.755). The frequency of allele C was 61.9% and allele T was 38.1% in hepatitis B patients group. The frequency of allele C was 55.2% and allele T was 44.8% for the control group (P = 0.179; OR = 1.319; 95% CI: 0.881-1.977). CONCLUSION: The TT heterozygous genotype was not a risk factor for chronic hepatitis B. CC homozygote genotype is protective for hepatitis B. Lack of heterozy- gosis of genotype CT is a risk factor for chronic hepatitis B. Alleles C or T were not risk factors for chronic hepatitis B. These findings show the role of a single-nucleotide polymorphism at CD14/-159 on the development ofchronic hepatitis B. Endotoxin susceptibility may play a role in the pathogenesis of chronic hepatitis B.

Cell Type Specific Expression of CD80 （B7-1） Gene in Murine

The CD80 （B7-1） costimulatory molecule is expressed on the surface of B cells and its expression is transcriptionally upregulated upon stimuli. To identify the region of murine CD80 promoter that direct cell type specific gene expression, four promoters construct of CD80 gene were generated with DNA fragments fused to the GFP reporter gene. In the present study, significant promoter activity was detected with all four promoter constructs only in the murine B lymphocyte. Further, the CD80 promoter region extending from -3,005 bp to ＋273 bp in relation to the previously reported transcription start site, was identified as tissue specific region. Interestingly, the shortest 700 bp （-427/＋273） of promoter fragment was sufficient to direct the CD80 gene expression. The level of CD80 expression was also found to be modulated by exogenous stimuli in B lymphocyte. Additionally, it was demonstrated that the CD80 gene expression is regulated at the level of transcription where the inducible CD80 gene transcript was detected in B lymphocyte with increasing time.

婴儿双歧杆菌介导的CD和UPRT联合5-FC基因疗法对黑色素瘤的体外治疗实验研究

目的采用婴儿双歧杆菌为载体,探讨尿嘧啶磷酸核糖转移酶基因(UPRT)对胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因系统抗瘤作用的增强效应。方法构建原核表达质粒pGEX-UPRT,以电穿孔法将该质粒转化入婴儿双歧杆菌,筛选阳性重组菌并鉴定。采用M TT法检测表达的U PRT是否可以与CD产生抗瘤协同作用,并观察细胞形态学改变。结果重组婴儿双歧杆菌可以正确表达UPRT。体外M TT检测显示CD+UPRT组细胞存活率低于对照组(P<0.01),且可使B 16-F 10鼠黑色素瘤细胞对5-FC的杀伤敏感性(IC50=0.015μm o l/mL)提高,是CD组(IC50=0.127μm o l/mL)的8.5倍。形态学观察CD+UPRT组肿瘤细胞显示出明显的损伤性改变,细胞生长受到明显抑制,而UPRT组和CD组变化明显不如前者。结论婴儿双歧杆菌联合转导UPRT基因可明显增强CD/5-FC自杀基因系统对鼠黑色素瘤细胞B 16-F 10的杀伤作用。

5-FC/CD系统对荷大肠癌裸鼠肿瘤的杀伤效应

目的研究ｃｄ基因对大肠肿瘤的特异杀伤作用，探索自杀基因靶向治疗大肠癌的有效途径．方法逆转录病毒感染法将Ｇ１ｃｅａｃｄＮａ及ｐｃｄ２分别转导入高分泌ＣＥＡ的大肠癌细胞Ｌｏｖｏ中，再分别接种到裸鼠皮下．成瘤后腹腔给予５ＦＣ（５００ｍｇ／ｋｇ）治疗，观察肿瘤重量的变化及病理学特点．结果含Ｇ１ｃｅａｃｄＮａ及ｐｃｄ２逆转录病毒载体的病毒滴度分别为：１３×１０７及２１×１０８ＣＦＵ／Ｌ．所有转基因成功并接种动物均成瘤．腹腔给予５ＦＣ治疗后发现，转由ＣＥＡ基因顺式转录调控序列（ＴＲＳ）驱动ｃｄ基因的组织特异性重组逆转录病毒载体Ｇ１ｃｅａｃｄＮａ的肿瘤对５ＦＣ的敏感性明显高于转非ｃｅａ调控ｃｄ基因的肿瘤，治疗结束后肿瘤重量分别为３１ｍｇ±８ｍｇ及１１３ｍｇ±２３ｍｇ（Ｐ＜００１）．结论本实验提示ｃｅａ转录调控序列可控制ｃｄ基因在ＣＥＡ阳性的大肠癌组织中高效表达。

金属抗性促生细菌Burkholderia sp.减缓玉米幼苗镉毒害和阻抗镉转运

为探明金属抗性促生细菌对减缓玉米镉毒害和阻抗镉转运的影响,本研究采用砂培试验向营养液中添加不同浓度重金属镉,研究接种镉抗性促生细菌YM3(Burkholderia sp.)对玉米苗干质量、生理指标、镉含量及金属转运基因表达情况的影响。结果表明,接种菌株YM3能促进玉米生长,且在镉胁迫环境中菌株促生效果更显著,使玉米根、茎、叶干质量分别增加9.09%~40.00%、63.33%~84.41%、48.50%~67.48%;接种处理玉米叶绿素总量增加1.70%~53.17%、根系活力增加7.40%~16.93%;游离脯氨酸降低3.04%~21.82%;当镉浓度为12 mg·L^(-1)时,丙二醛含量降低26.37%;接种菌株显著降低玉米地上部镉含量,使茎、叶镉含量分别降低13.64%~41.84%和17.85%~20.29%;q-PCR对金属转运相关基因HMA2、HMA3和Nramp5进行表达量的检测,结果表明接种菌株对重金属转运相关基因转录水平的表达调控受镉浓度影响。在0 mg·L^(-1)和8 mg·L^(-1)镉处理中,接菌处理HMA2、HMA3和Nramp5表达量在根中均表现为下调,在8 mg·L^(-1)和12 mg·L^(-1)镉处理中,接菌处理HMA2和Nramp5表达量在叶中均表现为下调。接菌处理通过不同程度影响基因HMA2、HMA3和Nramp5表达量来阻控玉米苗对镉的吸收转运。研究表明,在镉胁迫环境下接种镉抗性促生菌株Burkholderia sp.YM3能促进玉米苗生长,减缓玉米苗镉毒害,并阻控镉从根部向地上部转运,这对于玉米安全生产具有重要应用潜力。

放射敏感性启动子调控CD基因/5-FC自杀系统慢病毒载体的构建及^(125)I诱导表达的研究

合成了含8个CArG元件的放射敏感性启动子E8,将其连接于胞嘧啶脱氨酶(Cytosine Deaminase,CD)基因及GFP报告基因上游,构建了重组慢病毒载体pGC-FU-E8-codA-GFP。与慢病毒包装系统共转染293T细胞包装重组慢病毒颗粒E8-codA-GFP LV,研究重组慢病毒感染EJ细胞在不同剂量125I的电离辐射下绿色荧光表达情况及其将5-FC转化为5-FU的能力。结果显示:构建的含有E8启动子及CD基因的重组慢病毒载体,包装的重组慢病毒滴度为2×108 TU/mL;经125I照射的重组慢病毒感染EJ细胞均可观察到绿色荧光,其细胞上清液均可检测到5-FC转化成的5-FU紫外峰,其中55.5kBq及74.0kBq 125I照射的细胞组绿色荧光明显,148kBq 125I照射的细胞组,其5-FU紫外峰最为明显。以上结果表明:本工作所构建的放射敏感性启动子调控CD基因/5-FC自杀系统重组慢病毒载体具有电离辐射调控作用,可在125I的电离辐射作用下诱导下游基因表达,为放射性核素125I联合CD基因/5-FC自杀系统对肿瘤细胞的治疗作用研究提供了实验基础。

WTP53联合双自杀基因CD和TK抑制结肠癌细胞SW480生长

目的探讨野生型P53(WTP53)基因联合双自杀基因CD、TK对P53突变的SW 480结肠癌细胞的体外生长抑制作用、旁观者效应以及联合基因应用的效果。方法将P53的表达载体pCMV-P53转染SW 480细胞;用含有CD、TK双自杀基因的反转录病毒感染SW 480,得到稳定转染的阳性克隆。RT-PCR鉴定目的基因的表达。绘制细胞生长曲线等方法观察不同混合条件下WTP53、TK、CD系统的联合应用效应和药物相互作用指数。结果SW 480/P53细胞倍增时间明显延长。SW 480/TK-CD细胞对前体药物丙氧鸟苷(GCV)、5-氟胞嘧啶(5-FC)呈剂量依赖性的细胞生长抑制作用。SW 480/P53和SW 480/TK-CD混合细胞的生长抑制作用、旁观者效应和药物相互作用最显著。结论WTP53基因和TK/GCV、CD/5-FC系统对人结肠癌细胞SW 480均具有生长抑制和旁观者效应,联合应用细胞毒性更明显。

CD14基因启动子区多态性与缺血性脑卒中及血脂的相关分析

目的探讨CD14基因启动子-159C／T、-260C／T多态性与缺血性脑卒中的相关性，并对其与血脂、脂蛋白水平的关系进行分析。方法应用PCR-RFLP的方法检测132例缺血性脑卒中患者（缺血性脑卒中组）和145例对照组的CD14基因型；同时按常规方法测定血浆脂质、脂蛋白水平。结果缺血性脑卒中组总胆固醇、甘油三酯、低密度脂蛋白胆回醇水平明显高于对照组（P〈0．05），CD14基因-159C／T多态性在两组人群中的分布差异有显著性意义（P〈0．05），等位基因频率的相对风险分析发现，C等位基因携带者患缺血性脑卒中的风险是T等位基因的1．556倍（OR=1．556，95％CI=1．108—2．184），携带C等位基因的缺血性脑卒中个体血浆低密度脂蛋白胆固醇水平显著高于不携带者（P〈0．05）。结论CD14基因-159C／T多态性与缺血性脑卒中的发病具有相关性，其中C等位基因是缺血性脑卒中的遗传危险因素；CD14基因-159C／T多态性可能通过影响血脂水平而影响缺血性脑卒中的发生。

CD、VEGFR-1基因对恶性胶质瘤细胞和血管内皮细胞共培养体系中VEGF表达及细胞增殖的影响

目的研究胞嘧啶脱氨酶(CD)、血管内皮生长因子受体1(VEGFR-1)基因共表达质粒对恶性胶质瘤细胞和血管内皮细胞共培养体系血管内皮生长因子(VEGF)表达及细胞增殖的抑制作用。方法转染组:CD基因、VEGFR-1基因共表达质粒p EGFP-C1-CD-VEGFR-1转染人血管内皮细胞共培养体系中的人恶性胶质瘤细胞,未转染组:未做细胞转染,对照组:转染空载体。酶联免疫吸附测定(ELISA)法检测培养液上清中VEGF含量,原位杂交及免疫组化检测细胞VEGFmRNA及VEGF的表达,通过流式细胞术观察其对细胞活性的影响。结果转染后转染组细胞VEGFmRNA为0. 62±0. 06,VEGF为0. 42±0. 04,较未转染组及对照组显著降低,培养液中VEGF含量为60. 34±31. 22,较未转染组及对照组显著降低,细胞活性受到抑制,流式细胞术发现转染组细胞的G1期细胞比率明显高于未转染组及对照组,细胞出现凋亡。结论 CD、VEGFR-1基因共表达质粒可抑制恶性胶质瘤细胞和血管内皮细胞共培养体系中VEGFmRNA及VEGF的表达,抑制细胞活性、诱导细胞凋亡。

CD自杀基因系统对T淋巴细胞作用的实验研究

去除供者骨髓中的T淋巴细胞可有效防止移植物抗宿主病 (GVHD)的发生 .用含大肠杆菌胞嘧啶脱氨酶 (EC CD)基因的高滴度逆转录病毒 (1 5× 10 5CFU/ml)感染小鼠T淋巴细胞 ,G418筛选 ,获得稳定表达CD基因的T/pCD2 细胞 .经PCR和RT PCR方法检测证明CD基因已成功地导入T淋巴细胞中并有效表达 .分别用不同浓度的 5 FCyt作用于T/pCD2 及T淋巴细胞 ,光镜下观察不同时间细胞数目变化及MTT法检测细胞活性 .结果表明 ,5 FCyt浓度大于 1μmol/L时 ,即对T/pCD2 细胞有显著的杀伤作用 ,而对正常T淋巴细胞基本无毒性 ,且T/pCD2 细胞在加入药物后生存时间 (3～ 5d)明显短于未转染的T淋巴细胞 (大于 14d)

过量表达分泌型MT3对植物镉富集的影响

金属硫蛋白(metallothionein,MT)MT3是人体中参与重金属解毒的主要蛋白,前期研究表明啤酒酵母(Saccharomyces c erevisiae)α因子信号肽(MF-α)介导重组蛋白EGFP分泌到植物体外。但是目前还没有研究报道转基因植物中过量表达分泌型MT3对植物重金属镉(Cd)的富集能力是否有影响。本研究通过人工方法合成MF-α信号肽、增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和MT3的融合基因MF-α-EGFP-MT3,构建该融合基因的植物表达载体pK-35S-MF-α-EGFP-MT3,转化野生型(WT)烟草和天竺葵获得转基因植物。通过电化学方法检测转基因植物MT3转录水平、转基因植物根系分泌液中EGFP-MT3蛋白的水平。用Cd溶液处理转基因植物,通过表型观察和电化学方法检测根、茎和叶中Cd的含量。结果表明,转基因烟草和天竺葵中都有MT3基因的转录;且根系分泌EGFP-MT3蛋白的量大约为0.45~0.68 mg·g-1(以鲜质量计)。100μmol·L-1的Cd溶液处理转基因烟草植株,表型变化分析发现转基因植株受损情况低于WT,近根部叶片叶绿素含量显著高于WT,说明EGFP-MT3的分泌可降低Cd的毒害作用。转基因烟草植株根、茎和叶片对Cd的富集量比WT高约40%。用50μmol·L-1的Cd溶液处理转基因天竺葵植株,结果表明转基因植株根对Cd的富集量比WT高约30%,茎对Cd的富集量比WT高约4倍。以上结果证明过量表达EGFP-MT3可以提高转基因烟草和天竺葵对Cd的富集能力,可能是EGFP-MT3分泌根系表面增加转基因植物根系对Cd的吸附作用,同时在转基因植物组织细胞内积累的EGFP-MT3也可增加植物组织对Cd的富集作用。

质粒载体介导CD基因对喉癌细胞的杀伤作用研究(英文)

目的构建含酵母菌自杀基因CD的质粒表达载体pcDNA3.1()CMV.CD,并利用该载体进行喉癌Hep2细胞株转染研究,体外实验观察前体药物5FC对稳定表达CD基因的喉癌Hep2细胞株的杀伤作用。方法通过RTPCR从酵母菌RNA内扩增出CD基因全长CDS序列,将其定向克隆到质粒表达载体pcDNA3.1()CMV中,重组体质粒经XhoI/HindⅢ双酶切鉴定,并对重组体中的CD基因片段进行序列分析,将鉴定好的阳性重组质粒pcDNA3.1()CMV.CD运用电穿孔法转入喉癌Hep2细胞中。经300～600μg/mlG418正筛选14d和10μg/ml前体药物5FC负筛选,获得稳定表达CD基因的喉癌Hep2细胞株,提取该细胞株细胞的总RNA,经RTPCR鉴定CD基因的表达。MTT法观察不同浓度5FC对稳定表达CD基因的喉癌Hep2细胞及转染空白载体pcDNA3.1()CMV的对照组喉癌Hep2细胞的杀伤作用。结果阳性重组质粒pcDNA3.1()CMV.CD经XhoI/HindⅢ双酶切后获得5353bp的片段及496bp的插入片段,DNA自动序列分析证明重组体质粒含完整的477bp长的CD基因CDS序列。RTPCR从转染细胞总RNA中扩出453bp的预期片段。当添加不同浓度的5FC时,表达CD基因的Hep2细胞不同程度地被杀死,而对照组的细胞几乎未受到影响,两组细胞的相对生存率具有显著差异性(P<0.05)。结论成功构建了质粒表达载体pcDNA3.1()CMV.CD,建立了稳定表达酵母菌CD基因的喉癌表达CD基因的Hep2细胞株,表达CD基因的Hep2细胞可以被5FC杀死。

重组腺病毒rAdCDglyES对小鼠乳腺癌MA737放射效应的影响

目的:研究含CDgly ES双功能融合基因的重组腺病毒(rAdCDglyES)对肿瘤的直接、间接杀伤作用及其对放射的增敏作用。方法:建立MA737乳腺癌津白Ⅱ号小鼠荷瘤模型,按分组要求给予不同处理,设定多个观察指标对各组结果进行比较。结果:按分组要求处理后第25天,第1组瘤体达2.5cm×2.3cm;第6组肿瘤为1.4cm×1.2cm。第6组的中位生存期明显长于其余各组,P<0.01。第1组的凋亡指数较低,平均为2.61‰,而第6组的凋亡指数较高,平均为6.84‰,两组比较差异有统计学意义。第1组的微血管密度(MVD)值较高,为145.81,第6组的MVD值较低,为121.23,P<0.05。结论:含双功能融合基因的重组腺病毒在局部对肿瘤细胞具有直接杀伤作用,亦可抑制肿瘤新生血管的形成,对肿瘤细胞显示间接抑制、杀伤作用;显示该重组腺病毒能够和射线发生协同作用,增加局部放射对肿瘤的抑制作用。