肌卫星细胞体外培养及其对胰岛素样生长因子Ⅰ刺激的反应

目的原代分离、纯化、培养大鼠肌卫星细胞，观察体外培养肌卫星细胞的增殖与分化特性及对类胰岛素样生长因子Ⅰ（IGF-Ⅰ）刺激的反应。方法选用Wistar大鼠，切取双下肢及背部肌肉，两步酶消化法分离卫星细胞，不连续密度梯度离心法纯化后体外培养，观察其生长与分化特性，绘制生长曲线及融合率曲线，MTT法测定卫星细胞对不同浓度IGF-Ⅰ刺激的反应，并对其生长分数及融合率给予观察。结果此方法可以获得足量、纯度较高的肌卫星细胞。结论适宜浓度（100ng／ml）的IGF-Ⅰ对肌卫星细胞有较强的促增殖及促分化作用。     

红细胞生成素(EPO)对脑源性神经干细胞增殖与分化的调控作用

目的探讨红细胞生成素(EPO)对大鼠海马神经干细胞(NSCs)体外增殖和分化的影响。方法利用无血清培养、单细胞克隆技术,体外培养SD大鼠海马组织源NSCs,在NSCs增殖和分化过程中添加不同剂量EPO,以免疫细胞化学方法对NSCs及其分化后的细胞进行鉴定。结果与对照组相比,不同接种密度条件下的NSCs加入EPO后,增殖期表达Nestin的细胞增加2～4倍,分化期表达Nestin的细胞数明显减少;表达Tuj1和GFAP的细胞出现早,且Tuj1阳性细胞较对照组明显增加(P<0.05),加入antiEPO后Tuj1阳性细胞较对照组明显减少(P<0.01);相同细胞接种密度下,添加不同剂量EPO后Tuj1阳性细胞数呈剂量依赖性增加。结论EPO可促进大鼠海马源NSCs的增殖,并使其呈剂量依赖性的向神经元分化。     

NO供体对大鼠成骨细胞增殖分化及BMP-2表达的影响

目的:研究NO对大鼠成骨细胞的增殖分化、碱性磷酸酶(ALP)活性及对BMP-2表达的影响。方法:体外分离培养新生SD大鼠颅骨成骨细胞,加入不同浓度的NO供体(NOC-18),不同时间段采用噻唑蓝(MTT)比色法检测成骨细胞增殖,测定细胞ALP活性;应用免疫细胞化学法检测BMP-2的表达。结果:3个浓度组NO对成骨细胞均有显著的增殖作用,3组成骨细胞BMP-2表达均增加,其中以NOC-18 10μmol组对成骨细胞增殖的作用最强。结论:NO对大鼠成骨细胞增殖和分化有双重调节作用,适量低浓度的NO能够刺激成骨细胞增殖分化,增强细胞的骨形成作用,提高成骨细胞ALP活性;高浓度NO对成骨细胞的增殖分化作用弱,NO对成骨细胞的作用与调节和细胞BMP-2的表达有关。     

幼龄大鼠骨骼肌卫星细胞原代培养的实验研究

目的:为改进大鼠骨骼肌卫星细胞的体外培养方法,获取高纯度的肌卫星细胞。方法:选用幼龄SD大鼠,通过两步消化法和简易的两次差速贴壁法得到高纯度的肌卫星细胞。所得细胞通过免疫组织化学方法加以鉴定。结果:骨骼肌卫星细胞纯化率高,细胞生长良好,后期增殖较快。结论:本实验成功建立了骨骼肌卫星细胞的纯化和鉴定方法,可用于骨骼肌细胞增殖和细胞移植的相关研究。     

大鼠肝卵圆细胞体外分离培养和诱导分化为肝细胞的初步研究

目的 探索体外诱导成年Wistar大鼠肝卵圆细胞(HOCs)分化为肝细胞的可行性.方法 雄性Wistar大鼠36只,建立肝卵圆细胞增殖模型,采用两步法灌注和Percoll密度梯度离心法分离纯化HOCs,行体外培养并添加HGF、OSM和FGF4诱导其分化.结果 每只模型大鼠肝脏体外分离可获得约1.34×105/ml HOCs,细胞为圆形、椭圆形或多角形,大小为正常肝细胞1/6～1/3,核质比例大,2周后可呈克隆样增殖生长.所获HOCs胞浆和胞膜表达干细胞标志Thy-1及C-kit,其原始细胞标志AFP呈阳性表达;能稳定传代,在HGF、OSM和FGF4刺激下形态逐渐发生改变,细胞伸展且体积渐增大,贴壁能力减弱,诱导14天后分化细胞Alb表达明显阳性,且随着诱导时间延长阳性率逐渐升高;诱导分化的细胞胞浆G-6-P及PAS染色均呈阳性.结论 HOCs在体外一定条件刺激下可诱导分化为肝细胞.     

SIRT1对肌卫星细胞和骨骼肌再生的影响

肌卫星细胞及其子代成肌前体细胞在骨骼肌再生中起关键作用。衰老引起肌卫星细胞数量减少,骨骼肌的再生修复能力受损,肌细胞凋亡速度加快,最终导致肌肉萎缩、肌肉力量下降。SIRT1是一种NAD+依赖的组蛋白去乙酰化酶,介导能量代谢调节、细胞增殖分化、氧自由基代谢、基因表达等多种生理过程。SIRT1可通过调节肌卫星细胞的活化、增殖、分化,对骨骼肌的再生能力产生影响。运动训练上调SIRT1的活性和表达水平,促进SC的活化增殖,改善骨骼肌再生。     

骨骼肌内源性生肌因子成肌活性随衰老下降

目的:观察和探讨骨骼肌内源性生肌因子(39kDa)成肌活性随衰老而发生的改变。方法:应用等电聚焦结合聚丙稀酰胺凝胶电泳多步功能测试分筛法对青年和老年大鼠骨骼肌内源性39kDa生肌因子进行分离,利用体外培养和体内模型对青年和老年大鼠骨骼肌生肌因子的成肌活性进行比较,采用增殖细胞抗原(PCNA)免疫组织化学技术观察生肌因子对骨骼肌干细胞—卫星细胞的增殖作用。结果:处理组大鼠骨骼肌内源性39kDa生肌因子C2C12细胞数目显著多于对照组,处理组39kDa生肌因子MTT值显著高于对照组(P <0 . 0 5 )。青年和老年生肌因子处理组都有相当一部分C2C12细胞融合形成肌管,但青年大鼠骨骼肌生肌因子融合形成的肌管数目显著多于老年大鼠。体内研究发现:注射39kDa生肌因子的青年大鼠胫骨前肌中有许多PCNA阳性细胞核,但在对照组中未发现PCNA阳性细胞核。然而,在经注射的老年大鼠胫骨前肌中只发现少量PCNA阳性细胞核。PCNA阳性细胞核半定量计数结果表明:经生肌因子注射的青年大鼠胫骨前肌PCNA阳性细胞核显著多于老年大鼠(P <0 .0 5 )。本研究结果提示:39kDa生肌因子能诱导骨骼肌干细胞—卫星细胞的增殖和分化,青年大鼠骨骼肌中39kDa生肌因子的成肌活性高于老年大鼠,骨骼肌39kDa生肌因子成肌活性随衰老而减弱可能是老年个体     

不同浓度葡萄糖对大鼠间皮细胞增殖及细胞外基质产生的影响

观察了不同浓度葡萄糖对膜腹间皮细胞增殖和产生细胞外基质的作用，发现：①不同浓度的高渗葡萄糖可以抑制大鼠腹膜间皮细胞的增殖，引起间皮细胞的死亡，增强层粘连蛋白和Ⅳ型胶原的产生；②葡萄糖浓度与在此环境下培养的活细胞百分比呈负相关，但与层粘连蛋白的产生呈正相关，且活细胞百分比与层粘连蛋白的产生呈负相关。提示：培养液中葡萄糖的浓度在大鼠腹膜间皮细胞的增殖和细胞外基质的产生中起重要作用。     

外源性机械生长因子对大鼠骨骼肌卫星细胞生物学特性的影响

目的:研究机械生长因子(MGF)促离体培养的骨骼肌卫星细胞增殖的最佳浓度,并观察该浓度对细胞生长曲线、总蛋白及凋亡情况的影响。方法:选用雄性SD大鼠1只,无菌条件下取后肢肌肉,采用改进的两步酶消化法结合差速贴壁技术,分离及纯化骨骼肌卫星细胞。取第3代骨骼肌卫星细胞,分别采用终浓度为15、25、50、100、200 ng/ml的MGF进行干预,作为实验组;另以单纯加入100μl生长培养基作为对照组,以加入100μl DMEM作为阴性对照组。同步化后,于培养24 h后加入CCK-8溶液。采用CCK-8比色法检测不同浓度MGF下细胞的增殖情况并筛选出最佳浓度后,绘制生长曲线,同时运用BCA法及Hoechst 33342、PI双染法测定最佳增殖浓度的MGF对卫星细胞蛋白合成及细胞凋亡的影响。免疫细胞化学法鉴定骨骼肌卫星细胞。结果:①终浓度为15、25、50、100 ng/ml的MGF均有促进骨骼肌卫星细胞增殖的作用(P<0.01),其中25 ng/ml MGF促增殖作用最佳。②与对照组相比,MGF组细胞生长曲线左移,倍增时间、平台期均缩短。③与对照组相比,25 ng/ml MGF作用48 h即对骨骼肌卫星细胞有明显的增殖效果(P<0.05);与对照组相比,25 ng/ml MGF作用骨骼肌卫星细胞48 h和72 h时,其总蛋白含量显著增加(P<0.05);与对照组相比,25 ng/ml MGF作用72h、96h,骨骼肌卫星细胞的平均凋亡指数显著减小(P<0.05)。结论:①机械生长因子可促进体外培养的大鼠骨骼肌卫星细胞增殖,最佳浓度为25 ng/ml;②25 ng/ml MGF可以使骨骼肌卫星细胞提前进入平台期,缩短生长周期;③25 ng/ml MGF可以抑制骨骼肌卫星细胞的凋亡。     

大鼠间充质干细胞的体外分离培养及CFSE标记

目的 探讨体外分离、培养及荧光染料CFSE标记大鼠间充质干细胞(MSCs)的方法.方法 Wistar大鼠10只.用密度梯度离心及贴壁法相结合的方法分离纯化大鼠MSCs并进行鉴定.荧光染料CFSE标记MSCs,荧光显微镜检测标记率,CCK-8法检测标记细胞的增殖能力.结果 原代MSCs 72h贴壁,呈梭形,集落样生长;传代后,细胞变为形态均一,排列有序的成纤维细胞样.CFSE标记当天,荧光显微镜下大鼠MSCs呈明亮的绿色荧光,标记率达100%,随着培养时间延长,荧光强度逐渐减弱,标记率随之下降,标记4周后,标记率下降至78%;标记细胞的增殖能力与未标记细胞相比,差异无统计学意义(P＞0.05).结论 利用密度梯度离心法结合贴壁法可以获得比较纯的MSCs;CFSE短期标记MSCs效率很高,且标记细胞的增殖能力不受影响,但荧光强度及标记率随着时间延长而下降.     

施万细胞高效体外传代培养的实验研究

目的探讨获取大量高纯度施万细胞(SC)的体外培养纯化方法。方法取6~7 d SD乳鼠坐骨神经,立体显微镜下机械性剥除神经外膜与束膜,采用低浓度胰酶消化法和差速贴壁法去除成纤维细胞,阿糖胞苷与G enetic in联合应用进行SC纯化,牛脑垂体提取物(BPE)促进SC增殖。结果经形态学及S-100蛋白相关抗原免疫细胞化学染色鉴定证实所获得的SC状态良好,纯度较高,可达96%以上。结论本方法可以体外获取大量、高纯度的SC,为神经组织工程的研究奠定了实验基础。     

Determination of AB-FUBINACA and 5F-NPB-22 in rats exposed to “Bonsai” via inhalation and analysis of seized product

Synthetic cannabinoids (SCs) are the most rapidly growing class of recreational designer drugs. Illicit drug manufacturers began to produce herbal smoking materials under a variety of brands names, e.g. “Spice, K2, Bonsai, Yucatan Fire”. They were appeared on the European market in 2008. In this study, types of SCs in the herbal product sold as “Bonsai” in Turkey were determined and the identification of these substances in biological samples collected from rats depending on the inhalation of different amounts of plant material were aimed.To determine the SC species in the content of the plant product, analysis was performed via gas chromatography-mass spectrometry. Liquid-liquid extraction methods were utilized for blood and organ samples, while solid-phase extraction with β-glucuronidase enzyme treatment was applied for urine sample preparation. The relationship between the amount of burned plant and the amount of SCs accumulated in the blood, urine and organ samples of rats exposed to the plant product by inhalation was examined by liquid chromatography-tandem mass spectrometry.AB-FUBINACA and 5F-NPB-22 were detected in the herbal product. A significant correlation was found between the amount of herbal product inhaled and the prevalence of SCs, especially in lung tissues while no SCs were detected in the blood and urine samples of rats.There is currently no study on biological samples of individuals exposed to herbal products containing SCs by inhalation. Regarding the findings obtained in this study, the overall increase in the amounts of herbal product inhaled was demonstrated to pose a potential risk to humans.     

大鼠小脑皮质厚度及神经元数量的年龄性变化

在光镜下比较研究了青年组、成年组及老年组大鼠小脑皮质分子层、颗粒层厚度及浦肯野细胞、颗粒细胞数量的年龄性改变,结果发现：（1）随年龄的增加,分子层厚度出现进行性下降,但组间比较无显著性差异（P＞0．05）;颗粒层厚度老年组比成年组明显增厚（P＜0．05）。（2）浦肯野细胞数量在各年龄组的减少均有显著性差异（P＜0．05）柳随增龄浦肯野细胞数量显著下降,颗粒细胞数量老年组比成年组显著下降（P＜0．05）,上述结果提示：颗粒细胞对衰老的敏感性比浦肯野细胞差,不易发生类似浦肯野细胞消逝那样的衰老变化。     

Aging does not attenuate plantaris muscle hypertrophy in male Fischer 344 rats

PURPOSE: The present study examined the effect of extreme old age on the plasticity of the rat plantaris muscle in response to an increase in mechanical load. METHODS: Male Fischer 344 rats, aged 7 months (adult) and 25 months (old) underwent bilateral surgical ablation of the gastrocnemius muscle to functionally overload (OV) the fast-twitch plantaris muscle for 8 wk RESULTS: At 27 months of age, plantaris wet weight and cross-sectional area (CSA) were unaffected by age, but aging decreased peak isometric tension (Po) 27% (P < 0.05). Plantaris muscle myosin heavy chain composition indicated a loss of faster myosin heavy chains (MHC) isoforms with concomitant increases in slower MHC in old rats (P < 0.05). In adult rats, OV increased muscle CSA and Po 72% and 83%, respectively (P < 0.05). Similarly, OV increased CSA and Po 69% and 73%, respectively, in old rats (P < 0.05). Average fiber CSA increased 57% and 68% in adult-OV and old-OV rats, respectively (P < 0.05). CONCLUSION: Collectively, our data indicate that plantaris muscle mass and plasticity in response to increased mechanical load are well conserved in very aged male Fischer 344 rats.     

In vitro comparison of the biological effects of three transfection methods for magnetically labeling mouse embryonic stem cells with ferumoxides.

In vivo MRI of stem cells (SCs) is an emerging application to evaluate the role of cell therapy in restoring the injured myocardium. The high spatial and temporal resolution combined with iron-oxide-based intracellular labeling techniques will provide a sensitive, noninvasive, dual imaging modality for both cells and myocardium. In order to facilitate this novel imaging approach, much effort has been directed towards developing efficient transfection methods. While techniques utilizing poly-L-lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the fundamental biological effects of these methods on mouse embryonic SCs (mESC) have not been investigated systematically. In this study a longitudinal in vitro evaluation of cellular viability, apoptosis, proliferation, and cardiac differentiation of magnetically labeled mESC was conducted. No significant difference was seen in these biological parameters among the three transfection methods. However, cardiac differentiation was most attenuated by ELP, and iron uptake was most effective by PS.     

血管内皮祖细胞对骨质疏松大鼠骨髓间充质干细胞脂肪化的影响

 目的 探讨血管内皮祖细胞(endothelial progenitor cell,EPC)对骨质疏松(OP)大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)自主脂肪化的影响。方法 选6周龄雌性 SD 大鼠,建立OP模型。体外分离、培养大鼠EPC、BMSC。分别取第3、6代BMSC及原代EPC,建立EPC/BMSC 间接共培养体系。实验分组:假手术组BMSC(BMSC sham)、OP组BMSC(BMSC ovx)、OP BMSC /EPC共培养组(BMSC ovx/EPC);MTT法检测细胞增殖情况;油红 O染色检测成脂分化情况,real- time PCR检测成脂分化标志物PPARγ2的mRNA表达水平。结果 BMSC ovx/EPC 组BMSC的增殖能力显著高于BMSC ovx组;BMSC ovx/EPC组自主脂肪细胞分化水平明显低于BMSC ovx组。结论 EPC可通过间接接触提高骨质疏松大鼠BMSC的增殖能力,并能有效抑制OP大鼠BMSC的自主脂肪化。     

Effects of training on functional performance in 65, 75 and 85 year-old women: experiences deriving from community based studies in Odense,Denmark

The aim of the Odense training studies is to elucidate if regular physical training influences the expected decline in physical functional ability in order to assess capacity for postponing dependence in old age. All participants were healthy community-dwelling women representing three different age-cohorts of 65, 75 and 85 year-old subjects. The 65 and 85 year-old participants of the training group took part in physical class-based exercises for eight months with one session of 60 min a week, whereas the 75 year-old women trained twice a week over eight months. The multicomponent training consisted of various exercises typically involving body awareness, rhythm, aerobic performance (walking), muscle strength and muscle endurance, flexibility, reaction and balance exercises. Physical ability was measured as physical performance test (PPT), isometric muscle strength of the trunk, hip and leg, aerobic capacity and walking speed. This shows that regular training can significantly improve physical ability of elderly women with regard to PPT, maximal oxygen uptake and maximal walking speed, suggesting that both young-old and old-old women are able to benefit from regular tailored exercise training. Thus, physical training of old community-dwelling women appears to represent a prophylactic remedy that merits further research aimed at evaluating the preliminary findings of the present studies in larger, less selective groups of participants.     

Diagnostic sonographic changes in the kidneys of 20 infants with congenital nephrotic syndrome of the Finnish type

A renal ultrasonographic study was performed on 20 infants with congenital nephrotic syndrome of the Finnish type. Follow-up ultrasonographic data were obtained on 13 of these patients. The renal lengths increased after 2 months of age. Later, at the age of approximately 18 months, the renal size had decreased again, and was within 2 SD of the predicted mean value. During the first 2 months of life the renal cortex was hyperechogenic and corticomedullary differentiation was clearly visible. Between 2 and 12 months of age the renal cortex was still hyperechogenic in all kidneys, but corticomedullary differentiation had disappeared from 10 of the 26 kidneys examined. After 12 months of age the kidney parenchyma was hyperechogenic and most kidneys had lost corticomedullary differentiation. Thus, the sonographic follow-up is one of the main diagnostic indices f congenital nephrotic syndrome. Correspondence to: O. Koskimies     

Regioisomeric differentiation of the alkyl-substituted synthetic cannabinoids JWH-122 and JWH-210 by GC-EI-MS/MS

Synthetic cannabinoids (SCs) are known to have structural or positional isomers. While regulations on synthetic drugs like synthetic cathinones and SCs have been placed worldwide for the ever-growing variety of new designer drugs, laws may not necessarily be applicable to their isomers. Toxicological differences may also exist among isomers for which most new designer drugs are still uninvestigated; thus, isomer differentiation becomes of forensic importance. The aim of this study was to differentiate the regioisomers of alkyl-substituted naphthoylindole-type SCs JWH-122 and JWH-210. Reference standards of the two drugs and their regioisomers were analyzed by gas chromatography–electron ionization-mass spectrometry (GC–EI-MS) first in full scan mode. Isomers that produced identical EI spectra were further analyzed by GC-tandem mass spectrometry (MS/MS) by selecting appropriate precursor ions. For JWH-210, comparison of the product ion spectra and the relative ion intensity ratios obtained from precursor ions at m/z 312 and 183 enabled differentiation between all seven regioisomers. Complete isomeric differentiation by MS/MS analysis was not attainable for JWH-122; however, combining chromatographic results with MS/MS analysis results enabled differentiation for all isomers. Two basic fragmentation pathways were speculated for both SCs; for JWH-210, fragmentation pathway tendencies differed among the isomers, resulting in their distinguishability. Our results demonstrated that the difference between the methyl (JWH-122) and ethyl (JWH-210) group substituents contributed to fragmentation pathway tendency differences and further distinguishability between the regioisomers. Functional group differences, especially their stereochemistries, were indicated to be critical factors in positional isomer differentiation by GC-MS/MS.