肝病患者血清诱导间充质干细胞表达肝细胞特异性标志物的实验研究

目的：观察重型肝病患者血清对人骨髓间充质干细胞（MSCs）的诱导分化作用，探讨人MSCs分离培养、体外扩增的条件和方法。方法：从肋骨分离、培养人骨髓MSCs、体外扩增培养、鉴定，并采用重型肝病患者血清诱导培养，分别在诱导培养0、7、14、21、28天时留取细胞，并采用免疫细胞化学方法检测肝特异性标志物（AFP、Alb、CK-18）、用PAS进行糖原染色实验。结果：诱导后5天、MSCs表现为肝细胞样细胞，随着诱导培养时间的延长，肝特异性标志物逐渐出现和成熟。AFP在7天时表达水平最高，培养14、21、28天时表达逐渐减弱；Alb、CK- 18和糖原随着诱导时间的延长，表达逐渐增强。结论：密度梯度离心，贴壁培养和消化时间控制相结合，是一种较为有效的分离纯化方法。重型肝病患者血清能诱导骨髓MSCs表达肝细胞的特异性标志物。     

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP.     

脑肿瘤干细胞体外分化过程中的回逆现象分析

目的探讨脑肿瘤干细胞（BTSCs）体外分化过程中的回逆现象，为研究其分化抑制机制奠定基础。方法利用CD133免疫磁珠筛选系统，从肿瘤组织中分离获得的CD133^＋细胞（BTSCs）分成4组进行培养：（1）含10％胎牛血清（FCS）；（2）10％FCS＋丙戊酸钠注射液（VPA）；（3）无FCS＋生长因子；（4）无FCS＋生长因子＋VPA。取不同时间点上的细胞，相差显微镜观察其形态变化：流式细胞术检测与分化相关的标志物、细胞周期和DNA倍体变化；利用免疫激光共聚焦分析与分化相关标志物的共表达情况。结果无FCS条件下培养的BTSCs呈悬浮球状生长，高表达CD133和巢蛋白（nestin），不表达胶质纤维酸性蛋白（GFAP）和β-微管蛋白Ⅲ（β-TubulinⅢ）。G0／G1期细胞占大多数，G2／M期细胞接近0％，DNA都是异倍体，对VPA反应不敏感。含FCS培养的原本悬浮的细胞约4h开始贴壁。均呈圆形。此后逐渐向多形性分化，至7d时分化的细胞部分又返回至圆形。至10d-21d时，有的还能重新恢复球形，并呈悬浮生长。培养3d、7d、10d和21d时，CD133、nestin阳性细胞数先降后升，GFAP^＋和β-TubulinⅢ^＋细胞数始终处于较低水平。含FCS培养液中加入VPA。细胞形态上未见上述的回逆现象，CD133和nestin表达的先降后升现象消失，GFAP和β-TubulinⅢ在第7天以后表达明显升高，但极大部分细胞共表达nestin。而神经干细胞（NSCs）在含FCS培养至10d时，即以GFAP和β-TubulinⅢ表达为主，未见CD133^＋细胞。此外，含血清培养时BTSCs仍以异倍体为主。含少量的G2／M期细胞，加VPA诱导后细胞周期和DNA倍体变化不明显。结论BTSCs在含血清条件下培养出现的多向分化表型不稳定，时有去分化所导致的回逆。加入诱导分化剂VPA培养，虽然能阻止回逆现象出现，并有代表星形胶质细胞和神经元标志物表达上升．但因其共表达nestin而仍属于未完全分化细胞，表明BTSCs分化始终处于受抑状态。     

小鼠骨髓间充质干细胞的分离培养及生物性状研究

目的：分离培养小鼠骨髓间充质干细胞，研究其体外培养的生物特性。方法：获取骨髓间充质干细胞进行培养，倒置相差显微镜观察细胞生长情况。绘制生长曲线，冷冻保存细胞复苏后的生长状况观察。免疫细胞化学法鉴定细胞表面抗原。在地塞米松、β-甘油磷酸钠、抗坏血酸的作用下，进行成骨诱导分化。结果：原代和传代培养的细胞体外培养细胞呈现梭形、多角形外观，具有较强的生长增殖能力，复苏细胞生长状况无明显改变；细胞CD44。CD54抗原有阳性表达，CD34呈阴性表达。分化细胞表现成骨细胞特性，合成碱性磷酸酶(ALP)能力增强，矿化结节逐渐出现。结论：建立稳定可靠的分离培养小鼠骨髓MSCs方法，分离出的MSCs具有干细胞的生物特性，可用于骨组织工程中的种子细胞。     

Cutaneous postural vasoconstriction is modified by exogenous but not endogenous female hormones in young women

Previously reported attenuation of skin postural vasoconstriction in women during the luteal menstrual cycle phase may be due to a progesterone-mediated decrease in myogenic or veno-arteriolar (VAR) mechanisms. Skin perfusion was measured in the shin and foot dorsum by Laser Doppler Fluxometry during leg dependency that increased vascular transmural pressure below (myogenic constriction only) and above (myogenic and VAR) the 25 mmHg threshold for activation of the VAR, and during venous distension to activate the VAR alone (cuff inflation to 50 mmHg). In six young women with normal menstrual cycles, vasoconstrictor responses to all interventions did not differ between days 7–13 (follicular) and 18–23 (luteal) of the normal menstrual cycle when progesterone levels were low and at their peak respectively. In eight women taking combined oral contraceptives (OC) and tested during pill consumption days, reductions in foot skin perfusion were smaller (P = 0.05) than in the luteal phase of the normal cycle for leg dependency below (−36.9 ± 5.2% OC vs. −52.5 ± 7.8% luteal, mean ± S.E.M.) and above (−43.7 ± 3.4% OC vs. −55.1 ± 4.8% luteal) the VAR threshold, and for venous distension (−53.1 ± 2.6% OC vs. 66.4 ± 5.5% luteal). In women with normal menstrual cycles, impaired postural vasoconstriction may be confined to those who experience pre-menstrual symptoms rather than a direct effect of endogenous hormones. Reduced vasoconstriction in the dependent foot during OC use is consistent with the known vasodilator action of exogenous hormones and its long-term effects     

人参皂苷Rg1诱导骨髓间充质干细胞分化为神经样细胞的作用

目的研究人参皂苷Rg1在体外能否诱导Wistar大鼠骨髓间充质干细胞分化为神经元样细胞。方法通过贴壁法分离大鼠骨髓间充质干细胞,体外培养扩增,人参皂苷Rg1诱导分化,光镜下观察细胞形态,免疫细胞化学检测神经元特异性烯醇化酶（NSE）和神经胶质纤维酸性蛋白（GFAP）的表达,RT-PCR检测细胞NGF mRNA的表达。结果大鼠骨髓间充质干细胞可通过贴壁法成功分离并可以在体外大量扩增。人参皂苷Rg1诱导72h后,部分骨髓间充质干细胞（35．57％±3．59％）转变为神经元样细胞,免疫细胞化学染色NSE呈阳性,分化的神经元样细胞可能表达NGF mRNA。结论人参皂苷Rg1可以在体外诱导大鼠骨髓间充质干细胞分化为神经元样细胞,并且可能表达NGF mRNA。     

成年大鼠神经元和星形胶质细胞细胞周期蛋白依赖性激酶抑制因子表达的差异研究

目的比较研究成年大鼠细胞周期蛋白依赖性激酶抑制因子在神经元和星形胶质细胞的表达差异。方法应用免疫荧光和激光扫描共聚焦显微镜观察成年大鼠生理状态下大脑皮层或海马CA1、CA3、DG区神经元和星形胶质细胞细胞周期蛋白依赖性激酶抑制因子(CDKI)p15Ink4b、p21cipl的表达。结果成年大鼠海马区和大脑皮层的神经元有p15Ink4b和p21cipl的表达,细胞核和细胞浆均有表达,且以胞核为主;星形胶质细胞也有上述细胞周期调控蛋白的表达,便细胞数目较少,并且表达这些指标的星形胶质细胞多聚集在海马区。结论成年大鼠大脑皮层和海马区的神经元和星形胶质细胞均表达p15Ink4b和p21cipl,而其在神经元的表达较星形胶质细胞更为普遍。     

宫颈粘液电导率在月经周期中的变化规律

采用电化学分析方法,在月经周期中连续测定宫颈粘液的电导率,分析电导率变化规律与排卵期的相关性,此法确认的排卵期与实际排卵期一致,由此为自然避孕提供新途径。     

小檗碱对兔颈动脉粥样硬化中内膜增生和巨噬细胞趋化作用的影响

目的观察小檗碱对家兔颈动脉粥样硬化的内膜中膜比和巨噬细胞变化的影响。方法24只雄性日本大耳白兔随机分为3个试验组,正常组每天肌肉注射生理盐水,普通饲料喂养,对照组高脂喂养,1周后行颈动脉内膜空气干燥术并每日肌肉注射生理盐水,小檗碱干预组高脂喂养,1周后行颈动脉内膜空气干燥术并肌肉注射小檗碱,5周时取手术侧的颈动脉做弹力纤维染色,计算内膜中膜比;巨噬细胞免疫组化检测巨噬细胞在颈动脉粥样硬化病变中的变化,计算巨噬细胞的阳性率。结果对照组内膜厚度明显增加,中膜萎缩变薄,经计算I/M为1.20±0.007,小檗碱组的I/M为0.65±0.008。两组间有显著差异(P<0.01);巨噬细胞免疫组化染色对照组内膜下和中膜有大量巨噬细胞,小檗碱干预组内膜和中膜下也可以见有巨噬细胞沉积,通过计算巨噬细胞阳性率,小檗碱干预组的巨噬细胞阳性率明显小于对照组(P<0.01)。结论小檗碱可以降低家兔颈动脉粥样硬化中的血管内膜厚度、减少粥样斑块中的巨噬细胞数目,从而干预颈动脉粥样硬化的形成。     

Influence of stretch-shortening cycle on mechanical behaviour of triceps surae during hopping

Belli , A. & Bosco, A. 1992. Influence of stretch-shortening cycle on mechanical behaviour of triceps surae during hopping. Acta Physiol Scand 144 , 401408. Received 20 March 1 991 , accepted 3 December 1991. ISSN 0001–6772. Laboratories of Physiology, Universities of St Etienne and Lyon, France and Departments of Biology and Physical Activity, University of Jyväskylä, Finland. Six subjects performed a first series of vertical plantar flexions and a second series of vertical rebounds, both involving muscle triceps surae exclusively. Vertical displacements, vertical forces and ankle angles were recorded during the entire work period of 60 seconds per series. In addition, expired gases were collected during the test and recovery for determination of the energy expenditure. Triceps surae was mechanically modelled with a contractile component and with an elastic component. Mechanical behaviour and work of the different muscle components were determined in both series. The net muscular efficiency calculated from the work performed by the centre of gravity was 17.5±3.0% (mean ± SD) in plantar flexions and 29.9 ± 4.8% in vertical rebounds. The net muscle efficiency calculated from the work performed by the contractile component was 17.4 %% 2.9% in plantar flexions and 16.1 ± 1.47; in vertical rebounds. These results suggest that the muscular efficiency differences do not reflect muscle contractile component efficiency but essentially the storage and recoil of elastic energy. This is supported by the relationship (P < 0.01) found in vertical rebounds between the extra work and the elastic component work. A detailed observation of the mechanical behaviour of muscle mechanical components showed that the strategy to maximize the elastic work depends also on the force-velocity characteristics of the movement and that the eccentric-concentric work of the contractile component does not always correspond respectively to the ankle extension-flexion.