Objective. To evaluate the role of glucose transporter-l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

Objective. To evaluate the role of glucose transporter-l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

人骨髓间充质干细胞的获取与体外培养的研究 : 作为喉、气管软骨组织工程重建种子细胞的可能性

Objective To observe the feasibility of using bone marrow-derived mesenchymal stem cells (MSCs) as seed cells for tissue-engineering in Otolaryngology. Method MSCs were isolated from bone marrow of human rib and purified by centrifuge and cultured in vitro. The proliferation and growth characteristics of MSCs were observed in primary and passage culture. Result Human bone marrow-derived MSCs showed active proliferation capacity in vitro in primary and passage cultures. Conclusion Human bone marrow-derived MSCs have relatively young biologic age and they can be used as the seed cells for tissue engineering.

人VEGF165基因在狗骨髓基质细胞中的表达

目的:检测转染pcDNA3-hVEGFi65狗骨髓基质细胞的VEGF165mRNA表达.方法:用脂质体介导转染狗骨髓基质细胞,用免疫组化及RT-PCR检测VEGF mRNA的表达.结果:重组质粒pcDNA3-hVEGFi65转染骨髓基质细胞后,免疫组化及RT-PCR检测有VEGF mRNA的表达.结论:采用脂质体介导的方法可将hVEGFi65基因转染至狗骨髓基质细胞并表达外源性基因.

氟化物致体外培养大鼠心肌细胞的形态损伤

应用原代培养的心肌细胞作为模型,研究氟化物毒性作用对体外培养心肌细胞的形态损伤,近年来文献中少见报道.用扫描电镜观察培养的细胞,视野广,可观察到细胞簇的全貌及细胞表面的超微改变.笔者通过扫描电镜观察在氟化物作用下体外培养心肌细胞的形态学改变,探讨了氟化物致心肌细胞形态损伤的可能机理.

EpCAM特异性免疫效应细胞对EpCAMhigh腺癌细胞的体外杀伤实验研究

背景：上皮细胞粘附分子(EpCAM CD326)高表达于多种实体腺癌细胞表面，包括结肠癌、胃癌、乳腺癌等。EpCAM在胃肠道肿瘤细胞>95%，与肿瘤细胞的增殖及肿瘤预后有密切关系。近年来， EpCAM已经成为肿瘤免疫治疗的一个新靶点，培养EpCAM特异性免疫效应细胞(Ep-effector)制备新高效特异性杀伤细胞。目的：探讨体外制备的EpCAM抗原特异性免疫效应细胞的特异性杀伤功能。并比较其与腺癌细胞裂解物特异性免疫效应细胞、CIK细胞杀伤功能的差异。方法：采集健康成人外周血，分离淋巴细胞及imDCs。体外诱导淋巴细胞中T细胞转化为CIK细胞并扩增。本实验首次以纯化EpCAM多肽抗原负载imDCs生成EpCAM-mDCs(Ep-mDCs)，Ep-mDCs与自体CIK细胞体外共培养，制备成纯化EpCAM抗原特异性DC-CIK-CTL效应细胞(Ep-effector)。相似的方法，制备LS174-T腺癌细胞全抗原特异性DC-CIK-CTL效应细胞(LS-effector)。设单纯CIK组、LS-effector组及Ep-effector组，选择EpCAMhighLS174-T结肠腺癌细胞及EpCAMlowPaCa-2胰腺癌细胞作为靶细胞。细胞毒试验(CCK-8法)检测不同效靶比时各组效应细胞对EpCAMhigh的LS174-T及EpCAMlow的PaCa-2的杀伤效率。结果：体外制备LS-mDCs及Ep-mDCs表面分子表达量无差异(P>0.05)。随效靶比(E∶T)增高，各组效应细胞对LS174-T及PaCa-2的杀伤效率均升高。Ep-effector组对LS174-T的杀伤效率高于LS-effector组及单纯CIK组，LS-effector组与单纯CIK组无统计学差异；对PaCa-2的杀伤效率，两两组间比较无统计学差异(P>0.05)。结论：本实验首次应用EpCAM负载人外周血来源DC细胞制备Ep-mDCs，并证实其拥有激活T细胞生成特异性CTL的功能，由其制备的效应细胞(Ep-effector)对EpCAMhigh腺癌细胞具有更高的特异性杀伤活性。

To study the underlying mechanism of vasovasotomy infertility, we set up the rabbit model and observed the levels of testosterone, IL-1 activity and TNFα, which represent the functions of monocytes and macrophages on the animal model of vasovasostomy, and probed into the in vitro effect of IL-1β on the production of testosterone by Leydig cells. Rabbit model was set up and divided into vasovasostomy fertility group (VFG) and vasovasostomy infertility group (VIG), and set up peer group of Vasectomy group (VG) and sham operation group (SOG) as controls. The results were as follows: The sperm density and total number is highest in SOG, with that in VFG markedly lower than that in SOG, but significantly higher than that in VIG.　　1. VFG showed significant higher level of testosterone (Ts) than SOG, VG and VIG; Ts of VIG was significantly lower than SOG or VFG. In the vasovasostomy groups, sperm density is positively correlated to the level of testosterone. 　　2. The level of serum cortisol in VIG significantly higher than that in VFG, SOG, and VG. The level of serum cortisol negatively correlated to serum testosterone.3. Serum estradiol(E2) was similar among all the groups (P＞0.05). E2/T ratio is significantly different among the groups, with that in VIG highest and that in VFG lowest.4. Serum IL-1 activity in VIG was significantly higher than that in SOG and VFG(P＜0.05).　　5. Serum TNFα and serum IL-1 activity share similar change, with TNFα in VG significantly higher than that in SOG(P＜0.01), VIG was the highest among the four groups(P＜0.01).Correlation test showed that serum IL-1 activity and TNFα level was negatively correlated to testosterone level; positively correlated to cortisol level and E2/T ratio.6. Immuno-histo-chemistry of CD68 and TNFα in rabbits with vasovasostomy showed that in the testis tissue of SOG, interstitial tissue was normal with no filtration of inflammation cells including CD68 cells. In VFG and VIG, the tubules with resumed sperm production showed negative staining, and atrophic tubules and their surroundings were expression positive of CD68, with that markedly deeper in VIG than that in VFG. TNFα positive expressed granules was found only in atrophic convolted seminiferous tubules and their surroundings, with that deeper in VIG than that in VFG. 7. Culture Leydig cells were put in the following holes:(1) Control; (2) hCG; (3) IL-1; (4)IL-1 β+hCG; (5) IL-1 β+IL-1Ra; and (6) hCG+IL-1β+IL-1Ra. Determined the levels of testosterone by immuno-radiation technique.(1) IL-1 β showed no obvious effect on the Leydig cultured in vitro, but depressed the induction of Leydig cells by hCG to synthesize testosterone; (2) IL-1Ra blocked the depression of IL-1β Leydig cells' bio-synthesis of testosterone induced by hCG.In summary, one of the obvious changes in animals with vasovasotomy is low density of sperms, which is related to the lowering of testosterone. Low level of testosterone is related to the activation of monocytes and macrophages to produce IL-1 and TNFα, which negatively regulate the production of testosterone.