适用于基因转染的细胞培养板的研制

目的:研制用于基因转染的细胞培养板.方法:用0.2%的明胶稀释LPEI, BPEI, Superfect,Lipofectamine 2000等转染试剂并将它们固定在96孔细胞培养板上.以绿色荧光蛋白(GFP)基因和荧光素酶基因(Luciferase)为报告基因检测培养板的转染效率,用MTT法检测转染24 h后的细胞毒性,将LPEI包被的培养板放置在37℃环境下进行稳定性研究.结果:所有试剂包被的培养板均有一定的基因转染效率,聚合物类试剂的效率高于脂质体类的Lipofectamine 2000.其中,LPEI包被的培养板的转染效率最高、细胞毒性最低.当LPEI的包被量为3.2 μg/孔时,293细胞在转染24 h后的荧光素酶活性达3.0×108 RLU/mg,细胞存活率为85%左右.LPEI包被的培养板在37℃保温14 d后基因转染效率无明显变化.结论:LPEI包被的转染平板具有转染效率高、细胞毒性低、稳定性好的优点.该平板具有操作简便、节省时间的优点,可用于进行大规模的基因转染研究.     

靶向DNA连接酶Ⅳ小分子抑制剂SCR7提升CHO-K1细胞定点整合效率研究

目的 应用DNA连接酶Ⅳ（Lig4）的小分子抑制剂SCR7处理中华仓鼠卵巢（CHO）-K1细胞,验证其能否提高CHO-K1细胞中由CRISPR/Cas9介导的外源基因在ROSA26安全位点的定点整合效率。方法 构建靶向CHO-K1细胞ROSA26位点的特异性pX330-sgRNA-ROSA26质粒以及带启动子失活的GFP报告基因同源打靶载体,摸索并确定SCR7在CHO-K1细胞内作用的适宜浓度及处理时间,通过流式细胞术检测SCR7对CHO-K1细胞定点整合效率,同时评价剂量范围内SCR7对细胞凋亡的影响。结果 在CHO-K1细胞中成功构建靶向ROSA26位点的GFP报告系统,该系统可用于验证CRISPR/Cas9介导的定点整合效率;确定了SCR7对CHO-K1细胞作用的合适剂量和处理时间,在该条件下SCR7对CHO-K1细胞无明显细胞毒性;证实了SCR7在剂量范围内能有效提升外源基因在CHO-K1细胞ROSA26位点的整合效率,且对细胞凋亡无明显影响。结论 SCR7能提升CHO-K1细胞定点整合效率,该发现为应用Lig4小分子抑制剂构建具备高效表达外源基因的重组CHO细胞系奠定了基础。     

穿膜肽TAT的表达与活性的初步研究

目的：构建原核表达载体,获得纯化的融合蛋白TAT-GFP,以便于考查TAT的细胞定位及穿膜功能。方法：将TAT基因与绿色荧光蛋白（GFP）基因串联于原核表达载体pET28a中,通过IPTG诱导融合表达。分离纯化的融合蛋白与BHK-21细胞共孵育一定时间后,激光共聚焦显微镜观察其在细胞内的定位;同时以尾静脉注射方式进行小鼠给药实验,一定时间后麻醉,将主要器官组织取出、固定、冰冻切片,荧光显微镜下观察蛋白的分布情况。结果：DNA测序证明成功构建融合蛋白表达载体pET28a-tat-gfp。经诱导表达、纯化可获得纯度为85%以上的融合蛋白TAT-GFP。细胞实验表明,融合蛋白可迅速透过细胞膜并广泛分布于BHK-21细胞内,其中尤以细胞核内最多,而对照组GFP蛋白则不能穿透细胞膜入胞;动物实验中,给药2h后即可观察到TAT携带GFP到达小鼠的各主要器官和组织,甚至穿透血脑屏障分布于脑组织部分。结论：融合表达的穿膜肽TAT具有一定的携带生物分子（GFP）穿膜功能,本研究为深入了解TAT的性质及其未来应用奠定了一定的基础。     

穿膜肽为载体的分子影像学应用研究进展

由于细胞膜的天然屏障作用,使得许多生物活性分子难以进入细胞发挥作用,这使人们在分子水平上诊治疾病受到了很大的限制.穿膜肽是1988年新发现蛋白转导肽,它可以高效地穿透细胞膜,并且在不依赖受体、能量、温度情况下可携带多种活性物质进入各种细胞.本文就穿膜肽的特点,在功能上转运蛋白、抗体、脂质体、基因以及作为分子成像新途径能结合荧光素,磁共振对比剂等进行细胞内显影作一个综述介绍.     

DMRIE-C、Lipofectamine2000和TransMessenger转染HCV RNA效率比较

目的比较和优化DMRIE-C、Lipofectamine2000和TransMessenger3种转染试剂的转染效率,选择适合建立丙型肝炎病毒（HCV）复制子或感染克隆的转染试剂和条件。方法将含有萤火虫荧光素酶报告基因的HCVRNA,分别通过上述3种转染试剂转染Huh7和Huh7.5.1细胞系,于转染24h后裂解细胞,测定荧光素酶活性。2~3周后,结晶紫染色细胞集落。结果在Huh7和Huh7.5.1细胞中,DMRIE-C比Lipofectamine2000和TransMes-senger的转染效率高。在Huh7细胞中,4μlDMRIE-C转染试剂组的荧光素酶活性比其他组高（P〈0.05）。在Huh7.5.1细胞中,3μlDMRIE-C转染试剂组的荧光素酶活性比其他组高（P〈0.05）。在细胞集落形成实验中,DMRIE-C组的细胞克隆数多于其他两种转染试剂组。结论在Huh7和Huh7.5.1细胞中,DMRIE-C比Lipo-fectamine2000和TransMessenger的转染效率高。当建立HCV复制子或感染克隆试验时,既要选择合适的RNA转染试剂,又要考虑细胞生长状态的影响。     

放射性碘标记RC-160对A549-hSSTR2细胞受体内化及杀伤作用的研究

目的研究^125I-伐普肽（RC-160）对转染人生长抑素2型受体（hSSTR2）基因的肺腺癌A549细胞（A549-hSSTR2）受体内化的规律，以及^125I-RC-160对A549-hSSTR2细胞的杀伤作用。方法采用放射性配基结合分析法，以^125I-RC·160为放射性配基，测定A549-hSSTR2细胞及未转染hSSTR2基因的A549（A549-pc3）细胞与^125I-RC-160在37℃温育不同时间（0．25，0．5，1，4，8，20和24h）的内化率；采用四甲基偶氮唑蓝（MTT）法测不同浓度^125I-RC-160、Na ^131I、RC-160对A549-hSSTR2细胞及A549-pc3细胞作用24，48，72和96h的杀伤作用。结果37℃条件下温育，^125I—RC-160迅速与A549-hSSTR2受体结合并使受体发生内化。在温育1h时，A549-hSSTR2细胞结合（膜结合＋内化）的放射性计数为总计数的（18．2±1．9）％，明显高于A549-pc3组[（5．7±1．4）％，P〈0．01]。1h后，A549-hSSTR2细胞膜受体内化率（内化部分放射性计数与总放射性计数比）随时间的延长继续增加，膜结合率（膜结合部分放射性与总放射性计数比）随时间的延长逐渐减少。至24h时，受体内化率达（13．0±1．1）％，膜结合率为（3．9±2．2）％。^125I-RC-160对A549-hSSTR2细胞的杀伤作用较对A549-pc3细胞明显增强，并呈一定的剂量-效应和时间-效应关系，在96h时，3700kBq／ml ^131I-RC-160对A549-hSSTR2的抑制率达（78．8±5．9）％。结论^125I-RC-160可以使转染hSSTR2基因的细胞膜受体内化；^131 I-RC-160对A549-hSSTR2细胞的杀伤作用较强，这为外源性受体基因介导核素靶向性内照射治疗肿瘤提供了实验依据。     

p53与HBV相互作用对7721细胞凋亡及p21启动子的影响

为了观察HBV与p53是否存在相互作用的关系，以进一步揭示与原发性肝细胞肝癌发生发展的相关机制，选用7721肝癌细胞系（表达野生型p53，HBV阴性）为靶细胞，应用磷酸钙转染法，将pCMVp53单独或者与野生型HBV质粒（pCMVHBVa）、突变型HBV质粒（pCMVHBVb）共转染细胞，用annexin-V-FITC标记及流式细胞仪检测细胞凋亡的变化。各组另与含有p21基因启动子的荧光素酶报告基因质粒p21-luc共转染细胞，通过检测荧光毒酶的表达，了解p21启动子的活化情况。结果表明，单独的pCMVp53质粒转染7721细胞系能够促进p21报告基因的表达，细胞凋亡率升高；pCMVp53与pCMVHBV。共同转染时，对p21基因启动子的激活作用增强、细胞凋亡率明显增加，而与pCMVHBVb共转染时则否。提示HBV在该细胞内的复制能够诱导增强p53的作用并对其下游基因p21的转录有促进作用，导致细胞凋亡的增强。     

电离辐射持久提高人乳房瘤细胞脂质体介导基因的产生与表达

目的：研究照射是否增强人乳房瘤细胞脂质体－ＤＮＡ复合物的产生与表达。方法：①用脂质体ＳＶ４０荧光素酶复合物转染次融合培养的ＭＤＡ－ＭＢ２３１和ＭＣＦ－７人乳房瘤细胞,在其转染前或后２４小时或转染后立即在有或无血清的条件下用１３７　Ｃｓγ射线照射细胞至２Ｇｙ或０～１０Ｇｙ（剂量率为１．５６４Ｇｙ／ｍｉｎ）。②用ＢｅｒｔｈｏｌｄＬＢ９５０１照度计测量细胞溶解液的相对光单位（ＲＬＵ）,同时进行台盼蓝拒染试验测定细胞活力,以每个活细胞的ＲＬＵ表示荧光素酶的活性。③按标准的Ｈｉｒｔ操作步骤提取转染细胞的荧…     

抑制整合素连接激酶（ILK）表达对大鼠肾小球系膜细胞细胞间隙连接蛋白43表达的影响

目的研究抑制整合素连接激酶（ILK）表达对大鼠肾小球系膜细胞（RMC）细胞间隙连接蛋白43（Cx43）表达的影响。方法将大鼠肾小球系膜细胞分为RMC组（n-6）、ILK-consiRNA转染组（n=6）和ILK-siRNA转染组（n=6）。合成抑制ILK基因的siRNA，待细胞长至60％融合后，ILK-siRNA转染组和ILK-con siRNA转染组分别用脂质体转染ILK-siRNA或ILK-con siRNA，RMC组仅加入脂质体，24h后收集细胞，提取总蛋白和总RNA，用RT-PCR和Western blotting观察ILK和Cx43 mRNA和蛋白的表达。转染后继续培养24、48、72h，MTT法检测细胞活力。结果与RMC组和ILK-con siRNA转染组比较，ILK-siRNA组ILK mRNA和蛋白的表达均下降30％-50％（P〈0.05，P〈0.01），Cx43 mRNA水平增加30％-40％（P〈0.05），Cx43蛋白水平增加60％-70％（P〈0.01）。应用ILK-siRNA转染系膜细胞后24、48、72h，细胞活力均显著高于RMC组和ILK-con siRNA转染组（P〈0.05，P〈0.01）。结论抑制ILK途径可上调肾小球系膜细胞Cx43的表达，增强细胞活力；Cx43的调节可能是部分通过ILK途径实现的。     

人转化铁受体基因的克隆和表达

目的探讨人转化铁受体基因（hTfR）克隆和高效表达的方法及临床意义。方法用RT-PCR法从人胚肝、肺组织中克隆hTfR基因，构建重组表达质粒pcDNA3-hTfR和pEGFP-C1-hTfR，转染人胚肾上皮HEK293细胞。用Western印迹法检测pcDNA3-hTfR转染HEK293细胞后hTfR蛋白的表达情况；用荧光显微镜和共聚焦荧光显微镜观察重组pEGFP-C1-hTfR质粒转染HEK293细胞后hTfR蛋白的表达水平及亚细胞定位。结果经过DNA测序证实，克隆的hTfR基因为全长cDNA序列（2332bp）。Western印迹法检测到转染细胞能有效表达190×10^3的hTfR蛋白，荧光显微镜和共聚焦荧光显微镜观察到绿色荧光蛋白（GFP）-hTfR融合蛋白主要定位于细胞膜。结论从人胚肝、肺组织中可以成功克隆hTfR基因，该基因转染HEK293细胞后可获得hTfR蛋白的高效表达。hTfR主要定位于细胞膜。     

金属硫蛋白在前列腺癌中的表达及临床意义

目的探讨金属硫蛋白(Metallothionein,MT)在前列腺癌组织中的表达及临床意义。方法应用免疫组织化学方法观察不同分级的66例前列腺癌、20例前列腺增生组织MT的表达情况,分析MT与临床病理因素及预后之间的关系。结果20例前列腺增生组织MT的表达率为100%(20/20),66例前列腺癌组织中MT的表达率为59.1%(39/66),两者比较差异有统计学意义(P<0.05)。MT的表达随着前列腺癌组织学分级、临床分期的增加而增加(P<0.05);与患者血清PSA水平、年龄无相关性(P>0.05);MT阳性表达组与阴性表达组之间的生存率差异有统计学意义(P<0.05)。结论MT的表达与前列腺癌发生、发展及预后有一定的关系。     

Effect of gamma irradiation on metallothionein protein expression in Plantago ovata Forsk

Abstract

Purpose: The effect of gamma rays on metallothionein (MT) expression was studied using the medicinal plant Plantago ovata as the test system.

Materials and methods: Western blotting and Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used for this purpose.

Results: Western blot analysis showed significant induction of metallothionein protein following gamma exposure and that induction was highest at 20 Gy gamma dose. At higher gamma doses (100 Gy) MT expression level declined due to degeneration of cells. MALDI-TOF MS analysis indicated the presence of low molecular weight (7–8 kD) MT molecules following the lower radiation doses.

Conclusion: It was concluded from the MALDI-TOF MS result that low gamma exposure leads to expression of MT-like protein. At high doses of gamma ray, MT homologues or MT-like protein were not identified, possibly because they might have precipitated due to uncontrolled cross-linking and non-specific aggregation.     

Acute and chronic swine rete arteriovenous malformation models: effect of ethiodol and glacial acetic acid on penetration, dispersion, and injection force of N-butyl 2-cyanoacrylate

BACKGROUND AND PURPOSE: Liquid embolic agents are increasingly gaining importance in the embolization of cerebral arteriovenous malformations (AVMs). Currently, the most commonly used agent is N-butyl 2-cyanoacrylate (NBCA). Various NBCA mixtures, arterial hypotension, and Valsalva maneuver (increased positive end-expiratory pressure) during the injection of the acrylate have been used to address hemodynamic and architectural variations of an AVM; however, the precise in vivo polymerization, distribution, and kinetics of NBCA mixtures are unknown. We investigated the effect of different acrylate/Lipiodol mixtures and the addition of glacial acetic acid (GAA) on the penetration, dispersion, and injection force of NBCA. METHODS: A swine rete AVM model that has been described elsewhere was used for the embolization. In one subgroup of animals, embolization was performed immediately after construction of the AVM model. In a second subgroup, a chronic AVM model was used. GAA was added to the NBCA mixture to decrease the pH value of the solution and prolong the polymerization time. The addition of GAA allowed us to reduce the amount of Lipiodol, thereby reducing the viscosity of the mixture. A total of 30 swine were used for both the acute (n = 23) and chronic (n = 7) subgroups. The following mixtures of Lipiodol/NBCA and GAA (% vol/%vol + microL) were used for embolization: 80/20 + 0; 50/50 + 0; 50/50 + 5; 50/50 + 10; and 50/50 + 20. A total of six retia per mixture were used for the analysis. Glue injection pressure profiles were recorded in each experiment. High-resolution radiographic images obtained from the harvested retia were used to correlate the dispersion and depth of glue penetration with the AVM hemodynamics. The effect of different amounts of GAA on the glue dispersion and depth of penetration of the mixtures was also studied. RESULTS: Using the same pressure gradients, less viscous NBCA + GAA mixtures led to a deeper nidal penetration. The addition of 20 microL of GAA resulted in a three times higher penetration and dispersion of the NBCA mixture that was more homogenous. CONCLUSION: The viscosity of the liquid embolic agent used is an important limiting factor for an AVM embolization. Reducing the amount of Lipiodol improves nidus penetration. Quicker polymerization can be overcome by adding GAA, which reduces the pH of the mixture.     

Chitosan-based mucoadhesive gel for oral mucosal toluidine blue O delivery: The influence of a non-ionic surfactant

Photodynamic therapy (PDT) has been successfully employed in the treatment of oral cancer. Toluidine blue O (TBO) is a photosensitizer (PS) that has exhibited remarkable photocytotoxicity in a variety of tumour cells; however, its physicochemical properties, as well as the physicochemical properties of oral mucosa, prevent the drug from reaching the target site at a therapeutic concentration.The aim of this study was to evaluate the influence of Tween 80^® (TW), which has shown potential as a penetration enhancer, on the mucosal retention of TBO for the PDT of oral cancer. 4% Chitosan-based mucoadhesive gels (CH gels) containing or not 5%TW were prepared (both containing 1%TBO), and their physicochemical properties (pH, rheology and mucoadhesion), TBO in vitro release profiles and TBO in vitro mucosal retention were evaluated. In vivo mucosal penetration studies of TBO followed by laser exposition were also carried out.The results showed that 4%CH gels containing 5%TW and 1%TBO have adequate mucoadhesive and rheological properties for oral mucosa use, although they present a slightly acid pH. TBO release studies showed that TW reduces TBO release, but it prolongs TBO release and increases TBO retention in the mucosa. In vivo studies showed that 4%CH gels containing 5%TW and 1%TBO cause an increase in the number of apoptotic cell, after laser exposition.In summary, 4%CH gels containing 5%TW may be a promising vehicle to optimize the penetration of TBO in oral mucosa and to improve the PDT response for the treatment of oral cancer.     

Transverse relaxation and magnetization transfer in skeletal muscle: Effect of pH

Exercise increases the intracellular T₂ (T_2,i) of contracting muscles. The mechanism(s) for the T_2,i increase have not been fully described, and may include increased intracellular free water and acidification. These changes may alter chemical exchange processes between intracellular free water and proteins. In this study, the hypotheses were tested that (a) pH changes T_2,i by affecting the rate of magnetization transfer (MT) between free intracellular water and intracellular proteins, and (b) the magnitude of the T_2,i effect depends on acquisition mode (localized or nonlocalized) and echo spacing. Frog gastrocnemius muscles were excised and their intracellular pH was either kept at physiological pH (7.0) or modified to model exercising muscle (pH 6.5). The intracellular transverse relaxation rate (R_2,i = 1/T_2,i) always decreased in the acidic muscles, but the changes were greater when measured using more rapid refocusing rates. The MT rate from the macromolecular proton pool to the free water proton pool, its reverse rate, and the spin‐lattice relaxation rate of water decreased in acidic muscles. It is concluded that intracellular acidification alters the R_2,i of muscle water in a refocusing rate‐dependent manner, and that the R_2,i changes are correlated with changes in the MT rate between macromolecules and free intracellular water. Magn Reson Med, 2009. © 2008 Wiley‐Liss, Inc.     

Magnetisation transfer ratios of contrast-enhancing and nonenhancing lesions in multiple sclerosis

Magnetisation transfer (MT) is a recently introduced technique for assessing the water content of tissues in vivo and its relationship to macromolecules or membranes. It has been suggested that MT could provide indirect evidence of the characteristics of multiple sclerosis (MS) lesions (oedema, demyelination, or gliosis). Our aims were to characterise brain MS lesions and to compare the magnetisation transfer ratio (MTR) values of lesions with different patterns of contrast enhancement. In patients with MS we measured the MTR of 65 gadolinium-enhancing and 292 nonenhancing lesions. Using the equation published by Dousset et al. we studied 29 patients with clinically definite MS and 10 healthy controls. Lesions had significantly lower MT than the normal-appearing white matter of the patients or the normal white matter of healthy controls. There was no difference in the MTR of enhancing and nonenhancing lesions. Enhancement was homogeneous in 45 and ring-like in 20 lesions; MTR values were lower in the latter. These findings are presumably related to the differences in pathological features of enhancing (different amounts of proteins and inflammatory cells, oedema and demyelination) and nonenhancing (gliosis, demyelination and axonal loss) lesions.     

酒精性肝纤维化基质降解机制的研究

为揭示酒精性肝病（ALD）肝纤维化基质降解的病理机制，28例ALD肝穿刺组织按其纤维化程度分为3组，应用原位杂交技术分别检测各组肝组织内基质金属蛋白酶－1（MMP－1）、基质金属蛋白酶－2（MMP－2）、膜型基质金属蛋白酶－1（MT1－MMP） mRNA和基质金属蛋白酶抑制酶－1（TIMP－1） mRNA的表达。结果发现，MMP－1、MMP－2、MT1－MMP和TIMP－1 mRNA阳性细胞主要位于纤维化的中央静脉、窦周及汇管区等部位周围，且MMP－2和MT1－MMP mRNA的表达细胞有重叠，MMP－2、MT1－MMP和TIMP－1 mRNA表达阳性细胞数随肝纤维化程度的加重而增多，而MMP－1 mRNA表达阳性细胞数减少，且以纤维化中期变化为著；MMP－1、MMP－2、MT1－MMP和TIMP－1 mRNA表达阳性细胞主要为肝窦壁细胞，少数肝细胞亦呈阳性表达，提示MMP－1养活和TIMP－1增多可能是ALD肝纤维化过程中细胞外基质（ECM）沉积、尤其是Ⅰ型胶原过量沉积的病理机制之一；MMP－2和MT1－MMP在基质降解过程中可能有协同作用，其表达增多可能对ALD时中央静脉纤维化、肝窦血管化起一定促进作用；肝窦壁细胞（肝星状细胞等）为肝组织内MMP－1、MMP－2、MT1－MMP和TIMP－1的主要产生细胞。     

Protection from radiation-induced DNA single-strand breaks by induction of nuclear metallothionein

Purpose : To examine the extent to which nuclear metallothionein protects from radiation-induced DNA damage under aerobic and hypoxic conditions. Materials and methods : A semiquantitative fluorescence image analysis method measured the nuclear content of metallothionein (MT) in ME180 and SiHa human squamous cervical carcinoma cell lines under normal growth conditions, and following MT induction by zinc. The extent of initial DNA damage following 60 Co irradiation under aerobic and hypoxic conditions was assessed using the alkaline comet assay. Results : Provided that cells were maintained at 37°C, most of the cellular content of MT was in the nucleus. Incubation at 4°C caused the rapid translocation of MT from the nucleus into the cytoplasm in both cell lines, with no net loss of cellular MT. Baseline nuclear MT levels were about four times greater in ME180 cells, and were much more readily induced by treatment with 100 μ m zinc acetate, compared with SiHa cells. Under aerobic conditions, MT induction by zinc resulted in no protection in either of the cell lines. Under hypoxic conditions, however, the number of DNA single-strand breaks in zinc-treated cells was reduced by ~40% in ME180, but not in SiHa cells, when compared with non-induced controls. Conclusions : Nuclear MT can exert a significant level of protection from radiation by a mechanism that involves competition with oxygen for DNA radical sites and/or scavenging of free radicals. Because increased MT levels have been reported in hypoxic micro-regions of some solid tumours, this protective mechanism might have clinical relevance.     

The relationship of sustained exercise training and bone mineral density in aging male runners

Fifty-six men aged 42–73 years (50.2±10.0 years), who were competitive distance runners 20–25 years previously, were examined for bone mineral density (BMD) to determine the relationship between sustained distance running and BMD. Subjects were classified as being highly trained (HT, n =17), moderately trained (MT, n =29) or untrained (UT, n =10) according to their training in recent years. Subjects in each group were of similar age (HT 46.5±2.01, MT 53.0±1.51, UT 46.7±2.44 years) and lean body mass. Total body weight (kg) and percentage fat, however, were significantly greater ( P <0.05) in the UT group than in either the MT or HT groups (UT 80.6±2.44 kg, 22.0±1.16%; MT 74.9±1.51 kg, 17.5±0.61%; HT 70.5±1.71 kg, 13.5±0.59%). Lumbar vertebrae and hip region BMD (g·cm⁻²) was determined via dual energy X-ray absorptiometry (DEXA). No differences in BMD were found among the three groups in either the lumbar (HT 1.00±0.02, MT 1.02±0.03, UT 1.07±0.04 g·cm⁻²) or the hip regions (HT 0.99±0.03, MT 0.98±0.02, UT 1.06±0.04 g·cm⁻²). Furthermore, none of the groups had BMD that was significantly different from age-matched normative values taken from a reference database. A moderate correlation was found between body weight and BMD when combining all subjects ( r =0.38 for lumbar and r =0.41 for hip). These results indicate that middle-aged to older males who have sustained exercise training in the form of running do not have significantly different lumbar vertebrae or hip region BMD compared to individuals who run less or not at all.