Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid-mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly increased transfection yields. Expression of human GDNF via an episomal vector could be detected by in situ hybridization and by the measuring of GDNF protein secreted into the culture medium. When compared to mock-transfected controls, VM tissue expressing recombinant GDNF contained significantly higher numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo- and xenotransplantation treatment in Parkinson's disease.     

Chlamydia trachomatis - the agent

Chlamydiae are obligate intracellular bacteria, parasitizing eukaryotic cells. Chlamydia trachomatis, C. psittaci and C. pneumoniae are the three species of chlamydiae pathogenic to humans. C. trachomatis shows a tropism for the genital and conjunctival epithelia and consists of 19 different serovars which are pathogenic predominantly for the urogenital tract.A distinguishing feature of chlamydiae is their transition between the infectious elementary body that enters the host cell and the non-infectious reticulate body that replicates intracellularly within an inclusion that does not fuse with lysosomes. Chlamydiae depend for some functions upon the host cell; in particular, chlamydiae have little capacity for generating energy.The complete sequence of the 1000-kb chromosome of C. trachomatis is known, as are most of the genes located on the 7.5-kb cryptic plasmid. Recently, several concepts about the biology and the metabolic pathways of C. trachomatis have been revised in relation to the genome sequence, and different novel proteins have been described.     

医院内感染耐药菌质粒的调查

目的：了解医院内感染耐药性菌质粒的分布情况。方法：运用质粒图谱,限制性内切酶谱,质粒消除,接合传递等系列补充实验对医院内感染细菌的R－质粒进行综合分析。结果：医院内感染以革兰氏阴性杆菌为主,占79．6％,其中71％的革兰氏阴性杆菌含R－质粒,DNA分子在2．3－23．1KB间,75．2％的铜绿假单胞菌经质粒消除后,对先锋V,庆大霉素,氨卞青霉素和羟卞青霉素由原来的耐药变为敏感,说明这部分细菌的抗药性是由质粒介导的。另外24．8％的菌株虽然质粒被消除了,但抗药性并未发生改变,铜绿假单胞菌20KB的质粒可转化到受体菌中,使受体菌带有铜绿假单胞菌相同的抗药性,表明这一质粒可在不同种属间传递,有成为“流行质粒”的倾向。结论：医院内感染以革兰氏阴性菌为主,质粒在耐药菌株的传播流行中起重要作用。     

结核分支杆菌Mtb8.4抗原原核表达质粒的构建与鉴定

目的构建结核杆菌低分子质量抗原Mtb8.4基因的原核表达重组质粒,为其蛋白质的表达及免疫学研究打下基础.方法 PCR扩增目的基因,克隆到质粒pET-His T7启动子的下游,转化大肠杆菌TOp10F‘,进行重组子的筛选、鉴定.结果 Mtb8.4基因正向插入表达载体中,测序证实具有正确的读码框,基因序列与文献报道一致.结论重组原核表达质粒pET-His-Mtb8.4构建成功.     

载体对癌胚抗原DNA疫苗在小鼠体内表达CEA及诱导体液免疫应答的影响

目的　比较用pcDNA3和pCI-neo做载体构建的癌胚抗原 (CEA)真核表达质粒在小鼠体内表达CEA及诱导体液免疫应答的差别。方法　将两套重组质粒分别肌注BABL/c小鼠 ,ELISA法检测二者在小鼠体内表达CEA及抗 -CEA水平。结果　以pCI -CEA免疫的小鼠体内表达CEA量高于pcDNA3-CEA组 ,二者OD值分别为 0 65和 0 35 ;抗体量的表达前者OD值为 0 89,后者为0 70 ,比较差异有显著性 (P <0 .0 5 )。结论　表达载体的结构对抗原的表达及抗体的产生具有重要作用     

大鼠pEGFP-PLZF真核载体的构建及在精原细胞中的表达

目的:构建真核表达重组质粒pEGFP-N1-PLZF,并了解其在大鼠精原细胞中的融合蛋白表达情况.方法:参考分子克隆技术,采用RT-PCR的方法从大鼠睾丸组织中扩增早幼粒细胞白血病锌指蛋白(PLZF),将该基因连接克隆到含有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1上,并用双酶切、测序进行鉴定;将重组质粒用脂质体转染的方法导入精原细胞中,观察有无荧光的表达及用Western blot检测蛋白表达情况.结果:实验从大鼠睾丸组织中获取了编码plzf基因的全序列cDNA,产生了2 kb的目的插入片段,与pEGFP-N1载体连接后经酶切电泳鉴定及DNA测序证实序列正确;重组质粒转染精原细胞24 h后在荧光显微镜下观察到绿色荧光,并用Western 1blot:检测到106 kD目的蛋白的表达.结论:新构建的重组质粒pEGFP-N1-PLZF通过鉴定,结构正确.转染到精原细胞后能在其中表达,发挥功能,为后续研究奠定了基础,对研究PLZF调控精原细胞增殖和分化机制具有重要的意义.     

弓形虫重组耻垢分枝杆菌M.S-P30-ROP2载体活疫苗构建及鉴定

目的构建和鉴定弓形虫1330-ROP2复合基因重组耻垢分枝杆菌M．s载体活疫苗。方法用亚克隆技术把P30-ROP2复合基因克隆入大肠杆菌／分枝杆菌穿梭质粒pSTM3，构建重组质粒pSTM3-P30-ROP2，电穿孔法导入耻垢分枝杆菌中，潮霉素筛选、PCR法鉴定阳性转化子，温度诱导表达鉴定。结果获得pSTM3-P30-ROP2重组穿梭表达载体，SDS—PAGE结果显示P30-ROP2复合基因表迭蛋白产物分子量约为66kDa。结论成功构建弓形虫复合抗原基因P30-ROP2重组耻垢分枝杆菌疫苗，为弓形虫病的防治提供了-种新方法。     

致密斑细胞COX-2的表达及AP-1、NFκB信号通路

目的评估低盐(LS)培养对小鼠致密斑(MMDD1)细胞环氧化酶-2(COX-2)表达及核因子-κB(NF-κB)和活化蛋白-1(AP-1)活性的影响。方法经脂质体转染含NF-κB或AP-1的报告质粒,采用瞬时表达方法检测正常盐(NS)与LS培养对NF-κB和AP-1转录活性的影响。采用RT-PCR检测MMDD1细胞COX-2表达的变化,Westernblot方法检测细胞内p-p38MAPK、p-p44/42、c-Jun、c-Fos和COX-2蛋白的表达。结果LS培养促进了MMDD1细胞COX-2mRNA和蛋白表达(P<0.01)。LS培养后,p38和p44/42的磷酸化程度显著上调(P<0.01),180min后达到高峰。p38抑制剂SB-203580、p44/42抑制剂PD-98059可降低LS诱导的COX-2表达(P<0.01)。LS培养促进了c-Jun、c-Fos蛋白表达(P<0.01),激活了AP-1和NF-κB的转录活性(P<0.01)。25μmol/LNF-κB抑制剂PDTC和20μmol/LAP-1抑制剂curcumin下调了LS诱导的NF-κB、AP-1活性(P<0.01)。25μmol/LPDTC、20μmol/Lcurcumin降低了LS诱导的COX-2mRNA和蛋白表达(P<0.01)。结论LS培养可促进MMDD1细胞COX-2的表达,其作用可能与促进p38MAPK、p44/42激酶的磷酸化,增加NF-κB和AP-1的活性有关。     

肺炎克雷伯杆菌产β-内酰胺酶耐药性质粒的提取及鉴定

目的探讨耐药性质粒在肺炎克雷伯杆菌中的耐药机制。方法采用琼脂稀释法测定临床分离的31株肺炎克雷伯杆菌对阿奇霉素、氨苄西林和头孢菌素类抗生素的耐药性，采用质粒DNA小量制备的碱裂解法对耐药菌株进行质粒提取，通过转化试验对质粒的耐药性进行鉴定。结果31株肺炎克雷伯杆菌对阿奇霉素、氨苄西林和头孢菌素类抗生素有不同程度的耐药现象。其中从两株耐药性肺炎克雷伯菌中分离出两种质粒，经转化后鉴定为耐药性质粒，大小分别为2．3kb和8．2kb。结论肺炎克雷伯杆菌的临床分离株的耐药性部分决定于耐药性质粒的存在。