溴化乙锭在毛细管DNA传感器中的应用

目的:构建一种新型毛细管的DNA生物传感器。方法:实验于2004-10/2005-12在四川大学华西基础医学与法医学院生物化学实验室完成。毛细管内壁通过poly-l-lysine将20-mer-ssDNA(探针)固定,与互补靶核苷酸杂交后,通过溴化乙锭染色后检测与0.033 mmol/L靶核苷酸杂交后的荧光强度;同样浓度的靶核苷酸序列连续测定3次,进行重现性比较;及对不同杂交时间的荧光强度进行检测。结果:通过溴化乙锭对毛细管固定探针与互补靶DNA杂交进行染色,用RF5000荧光光度仪测定荧光值,测定组高于试剂空白组,差异有显著性意义[分别为(203.15±19.41),(133.05±11.82)s-1,t=4.1106,P<0.05]。杂交14h的荧光强度明显高于杂交12h,差异有显著性意义[分别为(203.06±17.36),(101.54±25.63)s-1,P<0.01],重现性较好。结论:在毛细管DNA传感器构建中,溴化乙锭可作为荧光标记物对模式寡核苷酸进行检测。     

鼻咽癌癌组织中Midkine基因表达的研究

目的研究Midkine(MK)基因在鼻咽癌(NPC)组织中的表达。方法用Trizol法提取鼻咽癌组织和正常鼻咽组织的RNA,经RT-PCR获得扩增的MK DNA,溴化乙锭琼脂糖凝胶电泳检测PCR产物。结果45例鼻咽癌组织中有37例在447 bp处出现1条MK的特征条带。7例鼻咽部正常组织均表达阴性。24例颈淋巴结转移患者中20例鼻咽癌组织MK mRNA表达阳性(表达率为83.33%),21例无颈淋巴结转移中17例鼻咽癌组织MK mRNA表达阳性(表达率为80.95%)。结论MK mRNA在鼻咽癌组织特异表达,MK有望作为鼻咽癌筛查的指标之一。     

妊娠中肾细胞因子mRNA在人食管癌组织中的表达

目的 研究妊娠中肾细胞因子（midkine, MK)mRNA在食管癌组织中的表达。方法 用Trizol提取食管癌组织和相应癌旁正常组织RNA，经RT－PCR获得扩增的MK cDNA，溴化乙锭琼脂糖凝胶电泳检测PCR产物。结果 6例食管癌组织在450bp处均出现一条MK的特征条带，其中1例低分化食管癌MK条带最深。6例癌旁正常组织均检测不出MK mRNA。此外，2例食管癌组织除显示MK的特征条带外，在280bp处出现一条截短型MK（tMK)区带。结论 MK在食管癌组织特异表达，表达程度可能与细胞分化程度相关。     

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.     

Differential effect of Cyclosporin A and FK506 on SPARC mRNA expression by human gingival fibroblasts.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein that mediates cell-matrix interactions. In adults, its expression is mostly limited to tissue undergoing remodeling. During the development of Cyclosporin A (CsA)-induced gingival overgrowth (GO) a remodeling of the connective compartment occurs. By contrast, clinical trials showed that FK506 is not related to GO. SPARC expression and its involvement in GO is unknown. Our aim was, therefore, to analyze the effect of CsA and FK506 on SPARC gene expression. METHODS: Cultured human gingival fibroblasts were incubated with CsA, FK506 or with their vehicle (VH) for 24, 48 and 72 h. SPARC gene expression was determined by RT-PCR. RESULTS: SPARC mRNA levels tended to increase 72 h after CsA treatment, whilst they are undetectable in FK506-treated fibroblasts, compared to VH. CONCLUSION: This gene expression profile is consistent with the involvement of SPARC in the mechanisms leading to the development of CsA-induced GO. By contrast, the undetectable SPARC mRNA levels in FK506-treated fibroblasts suggest that FK506 may be associated with a role of ECM stabilization, that does not induce GO.     

Differential expression of Ia molecules by human monocytes.

Human immune response genes can be divided into three distinct loci, each of which codes for three distinct families of Ia molecules: HLA-SB, HLA-DC, and HLA-DR. The tissue distribution and function of only one of these Ia molecules, HLA-DR, has been thoroughly studied. Using monoclonal antibodies, we examined the display of HLA-DR and HLA-DC molecules by adherent, human peripheral blood monocytes. The results of these studies demonstrate that although all human peripheral blood monocytes display easily detectable HLA-DR molecules, only 50% display easily detectable HLA-DC molecules. Separation of peripheral blood monocytes into HLA-DC+ and HLA-DC- cells demonstrates that each population displays an equivalent density of HLA-DR molecules. Therefore, on the basis of differences in their display of these two Ia molecules, adherent peripheral blood monocytes can be divided into two broad populations: HLA-DR+, HLA-DC+, and HLA-DR+, HLA-DC-. Despite the dis-coordinate display of these Ia antigens, the expression of both HLA-DR and HLA-DC can be regulated by a common signal, gamma interferon (IFN-gamma). Incubation of monocytes for 96 h in autologous serum leads to a marked decrease in the expression of both HLA-DR and HLA-DC. Addition of recombinant IFN-gamma to the cultures leads to reexpression of both HLA-DR and HLA-DC to levels comparable to those seen in fresh monocytes. In addition, although IFN-gamma does not modulate all monocyte surface markers, it can be demonstrated to modulate expression of one marker, MAC 120, in a manner similar to that observed for Ia antigens. These studies demonstrate that among human peripheral blood monocytes, the distribution of the Ia molecule, HLA-DC, is not coordinate with that of HLA-DR, although both respond to the same regulatory signal.     

Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-gamma, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-gamma-inducible CXC chemokines--IFN-inducible protein 10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha chemoattractant (I-TAC)--by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MO) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MO, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1beta, TNF-alpha, and CD40 ligand potentiated IP-10 expression from IFN-gamma-stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-gamma induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis.     

Effect of fialuridine on replication of mitochondrial DNA in CEM cells and in human hepatoblastoma cells in culture.

Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU. Total cellular DNA was isolated, normalized for the number of cells, and slot hybridized to a probe specific for mtDNA sequences. Treatment of CEM cells with FIAU did not result in a dose-dependent decrease in the amount of mtDNA. In contrast, dideoxycytidine (ddC) inhibited mtDNA replication by 50% at a concentration of approximately 0.1 microM. After 6 days of incubation, both compounds displayed a 50% toxic dose at a concentration of approximately 2 microM in CEM cells and approximately 34 microM in human hepatoblastoma cells (HepG2). In further experiments, CEM cells were incubated for 15 days in up to 2.5 microM FIAU, and again, no inhibition of mtDNA was observed. Over a 6-day incubation, FIAU, at concentrations of up to 200 microM, also failed to inhibit mtDNA replication in either HepG2 or HepG2 cells which constitutively replicate duck hepatitis B virus. In contrast, ddC inhibited mtDNA replication in these cells with a 50% inhibitory concentration of approximately 0.2 microM over a 6-day incubation. Treatment of cells with either FIAU or ddC resulted in a dose-dependent increase in lactate levels in the cell medium, indicating that any effect of FIAU on mitochondrial function may not be related to inhibition of mtDNA replication on the basis of the in vitro data. Alternative explanations for mitochondrial toxicity are considered.     

三碘甲状腺原氨酸对人神经干细胞分化过程中甲状腺激素受体表达的干预效应

背景:三碘甲状腺原氨酸是脑发育临界期的重要调控因素,在中枢神经系统发育过程中可能决定着神经干细胞的世系分化命运。目的:观察三碘甲状腺原氨酸诱导人神经干细胞分化情况以及分化过程中甲状腺激素受体mRNA的表达变化。设计:开放性实验。单位:天津市武警医学院病理教研室,天津医科大学内分泌研究所。材料:实验于2003-01/2005-03在天津医科大学完成。由天津医科大学总医院提供孕10～12周流产胎儿,产妇及家属均签署知情同意书,实验经医学伦理委员会批准。方法:①无菌条件下取人胚胎两侧大脑半球,剥除脑膜,剪碎脑组织,过滤制备细胞悬液。以20μg/L碱性成纤维细胞生长因子 30nmol/L三碘甲状腺原氨酸联合诱导神经干细胞增殖,按1×109L-1密度分别接种于涂有多聚左旋赖氨酸的24孔板和25mL培养瓶中常规培养,培养液为含N2添加剂的DMEM/F12无血清培养基。48h后半量换液,7d后撤除碱性成纤维细胞生长因子,单独用三碘甲状腺原氨酸诱导分化。②培养1,2,3周时取细胞,RT-PCR半定量检测神经干细胞诱导分化不同阶段甲状腺激素受体mRNA表达的变化,免疫细胞化学鉴定分化后的细胞类型。主要观察指标:①三碘甲状腺原氨酸诱导神经干细胞分化前后细胞形态观察及类型鉴定。②神经干细胞分化过程中甲状腺激素受体mRNA表达的变化。结果:①诱导前细胞呈圆形,表面光滑,并逐渐聚集成神经细胞球,Nestin单染及Nestin BrdU双染均呈免疫细胞化学反应阳性。三碘甲状腺原氨酸诱导分化1周时,细胞大多呈单极或双极,有细长突起,神经丝蛋白、胶质纤维酸蛋白、半乳糖脑苷免疫细胞化学染色呈阳性;诱导3周后可见细胞核呈圆形,突起多且粗,细胞整体呈蜘蛛状,髓鞘碱性蛋白阳性率达80%。②甲状腺激素受体α1mRNA在诱导前神经干细胞状态时表达量最高,三碘甲状腺原氨酸诱导后表达量逐渐下降,至2周时达最低,此后表达量有所回升,但仍低于神经干细胞状态(F=32.49,P=0.008)。甲状腺激素受体α2mRNA表达变化趋势与甲状腺激素受体α1相同。甲状腺激素受体β1mRNA在神经干细胞状态时表达量最低,三碘甲状腺原氨酸诱导后表达量逐渐升高,2周时达最高,且超过同时间点甲状腺激素受体α1的表达(t=15.64,P=0.001),至诱导3周时表达水平降至最低。甲状腺激素受体α3mRNA在三碘甲状腺原氨酸诱导后呈下降趋势,2周时接近干细胞状态(F=51.94,P=0.378),此后又降至较低水平。结论:三碘甲状腺原氨酸能诱导神经干细胞分化为神经元、少突胶质细胞和星形胶质细胞,且分化过程中甲状腺激素受体mRNA存在不同时间顺序的表达。     

Regulation of leptin receptor expression in human papillary thyroid cancer cells

Epidemiological studies suggest an important link between obesity and thyroid cancer. The adipose tissue-derived polypeptide leptin acting via leptin receptor may modulate cell migration of thyroid cancer cells. Previously we have demonstrated that leptin receptor is overexpressed in papillary thyroid cancer and is associated with tumor aggressiveness. The present study was undertaken to explore the possible regulatory factors which would influence leptin receptor expression in papillary thyroid cancer cells. We found that DNA methyltransferase inhibitor (5-Aza-2′-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) reduced leptin receptor expression. Conversely, insulin upregulated leptin receptor expression in a time- and dose-dependent manner. Hypoxia-mimicking agent (cobalt chloride) had no effect on leptin receptor expression. Taken together, our study provides evidence that epigenetic events and insulin stimulation take part in regulation of leptin receptor expression in papillary thyroid cancer cells.     

Differential expression of Ia antigens by rheumatoid synovial lining cells.

The differential expression of Ia antigens was studied in freshly isolated rheumatoid nonlymphoid synovial lining cells (SLC) and rheumatoid synovial fibroblast cell lines cultured in the presence of Interferon-gamma, using a large panel of anti-Ia reagents with monomorphic or polymorphic specificities. All the HLA-DR or -DQ specificities detectable on the corresponding peripheral blood B cells were also expressed in freshly isolated SLC. However, in all instances, the number of DR-positive SLC exceeded the percentage of cells expressing DQ antigens. In addition, the epitope expression of Ia antigens varied within the DR or DQ populations of Ia molecules as revealed by polymorphic reagents. Double-label experiments or using the ingestion of Latex particles as a marker demonstrated that the synovial macrophages (type I SLC) primarily bear the DR+DQ+ phenotype, while there is an additional population of nonphagocytic SLC (previously termed type II SLC) that has a DR+ and monocyte marker negative phenotype but did not have detectable levels of DQ antigens as analyzed by both fluorescence microscopy and cell sorter analysis. This latter population frequently had a morphology showing dendritic processes and rapidly lost the expression of Ia antigens upon culture. Cells with a similar, primarily DR+ phenotype were readily obtained in synovial fibroblast cultures after treatment with Interferon-gamma. These data suggest that there are two populations of Ia+ synovial lining cells: the synovial macrophages (type I cells) with the DR+DQ+ phenotype, and cells probably related to fibroblasts with a DR+ phenotype without detectable DQ antigens (type II cells). The fact that the latter phenotype could be induced by Interferon-gamma treatment of cultured synovial fibroblasts suggests that this mediator may have a similar role in vivo in the activation of certain synovial cell populations.     

Evidence of active efflux in resistance to ciprofloxacin and to ethidium bromide by Mycoplasma hominis

The uptake of fluoroquinolones was characterized for the fluoroquinolone-susceptible strain PG21 of Mycoplasma hominis. Accumulation of fluoroquinolones appeared to occur by passive diffusion. Addition of arginine as the energizer significantly reduced the uptake of fluoroquinolones, suggesting the presence of an energy-dependent efflux process. Reserpine and orthovanadate, two multidrug pump inhibitors, increased significantly the ciprofloxacin (CIP) uptake. In contrast, such a strong effect was not observed for moxifloxacin and pefloxacin uptakes. Two ethidium bromide (EtBr)-resistant strains, selected in vitro, showed a resistance profile compatible with a multidrug-resistant phenotype, with increased MICs for the hydrophilic fluoroquinolones, CIP and norfloxacin, EtBr, and acriflavine. Taking the EtBr-resistant strain RB1La as a model, a significant decrease of the CIP and EtBr uptakes was observed compared to the reference strain PG21. In the presence of reserpine and orthovanadate, both inhibitors of ATP-dependent efflux pumps, the CIP uptake increased significantly, reaching approximately the same level as that of the susceptible strain. Similar results were obtained with EtBr uptake and efflux experiments. Our data suggest the presence of an active efflux system, possibly an ABC-type efflux pump, implicated in the resistance to CIP and unrelated compounds like EtBr in the human mycoplasma M. hominis.     

SCID mice containing muscle with human mitochondrial DNA mutations. An animal model for mitochondrial DNA defects.

Defects of the mitochondrial genome are important causes of disease. Despite major advances in our investigation of patients, there is no effective therapy. Progress in this area is limited by the absence of any animal models in which we can evaluate treatment. To develop such a model we have injected human myoblasts into the tibialis anterior of SCID mice after inducing necrosis. After injection of normal human myoblasts, regenerating fibers expressed human beta-spectrin, confirming they were derived from fusion of human myoblasts. The stability of the muscle fibers was inferred by demonstrating the formation of motor end plates on the regenerating fibers. In addition, we show the presence of human cytochrome c oxidase subunit II, which is encoded by the mitochondrial genome, in the regenerated fibers. After injection of human myoblasts containing either the A8344G or the T8993C heteroplasmic mitochondrial DNA mutations, human beta-spectrin positive fibers were found to contain the mutation at a similar level to the injected myoblasts. These studies highlight the potential value of this model for the study of mitochondrial DNA defects.