微载体技术与普通方法成骨诱导培养人骨髓间充质干细胞的比较

目的：比较微载体法与普通培养瓶法成骨诱导培养人骨髓间充质干细胞，建立一种大量、快速获得骨组织工程种子细胞的方法。 方法：实验于2003—12／2004—10在西南医院中心实验室完成。取在西南医院检查的15名健康自愿者骨髓组织进行梯度离心，采用普通培养法培养。将普通培养瓶培养的第3代人骨髓间充质干细胞分别应用搅拌式生物反应器和微载体成骨诱导培养，分别于培养的第2，4，6，8，10．12，14天检测诱导培养细胞的碱性磷酸酶活性，观察细胞增殖情况，作出生长曲线。比较两种方法细胞增殖速度（收获细胞数除以接种细胞数，为细胞扩增倍数，以之除以培养天数为增殖速度）。 结果：①微载体法成骨诱导培养人骨髓间充质干细胞的增殖动力学并与普通法的比较：培养微载体法接种24h后约87％的人骨髓间充质干细胞细胞贴附于微载体并铺展，3d后生长加速。10—11d后细胞生长达最大值，最终细胞收获密度为接种时的15—20倍，微载体法诱导培养人骨髓间充质干细胞的速度是普通培养法的3—5倍，两者差异有显著性（P〈0．01）。②微载体法成骨诱导培养人骨髓间充质干细胞的碱性磷酸酶活性并与普通成骨诱导培养法比较：两种方法成骨诱导培养人骨髓间充质干细胞的碱性磷酸酶活性均在培养的第12天达最大值。微载体成骨诱导细胞的碱性磷酸酶活性与普通成骨诱导方法差异无显著性（P〉0．05）。 结论：应用微载体技术可以成功地进行人骨髓间充质干细胞的成骨诱导，更快地扩增，利于满足骨组织工程量和质的需要。     

微载体技术培养人骨髓间充质干细胞的成骨诱导实验

目的:以微载体技术成骨诱导培养人骨髓间充质干细胞,建立一种大量、快速获得骨组织工程种子细胞的方法。方法:实验于2002-12/2003-08在第三军医大学西南医院中心实验室完成。将普通培养瓶培养的第3代人骨髓间充质干细胞采用搅拌式生物反应器和微载体成骨诱导培养,观察细胞增殖情况,作出生长曲线。分别于培养的第2,4,6,8,10,12,14d检测诱导细胞的碱性磷酸酶活性,并与普通成骨诱导方法进行对比。最后进行细胞碱性磷酸酶染色、Ⅰ型胶原、骨钙素的免疫组织化学染色,观察培养细胞的成骨细胞表型的表达。结果:微载体法接种24h后约87%的人骨髓间充质干细胞贴附于微载体并铺展,3d后生长加速,约10～11d后细胞生长达最大值,最终细胞收获密度为接种时的约15～20倍。诱导细胞的碱性磷酸酶活性在培养的第12天达最大值,并表达碱性磷酸酶、Ⅰ型胶原、骨钙素等成骨细胞表型。微载体成骨诱导细胞的碱性磷酸酶活性与普通成骨诱导方法差异无显著性意义(P>0.05)。结论:应用微载体可以成功进行人骨髓间充质干细胞的成骨诱导扩增,可以满足骨组织工程量和质的需要。     

微载体技术培养人骨髓间充质干细胞的成骨诱导实验

目的：以微载体技术成骨诱导培养人骨髓间充质干细胞，建立一种大量、快速获得骨组织工程种子细胞的方法。方法：实验于2002—12／2003—08在第三军医大学西南医院中心实验室完成。将普通培养瓶培养的第3代人骨髓间充质干细胞采用搅拌式生物反应器和微载体成骨诱导培养，观察细胞增殖情况，作出生长曲线。分别于培养的第2，4，6，8，10，12，14d检测诱导细胞的碱性磷酸酶活性，并与普通成骨诱导方法进行对比。最后进行细胞碱性磷酸酶染色、Ⅰ型胶原、骨钙素的免疫组织化学染色，观察培养细胞的成骨细胞表型的表达。结果：微载体法接种24h后约87％的人骨髓间充质干细胞贴附于微载体并铺展，3d后生长加速，约10～11d后细胞生长达最大值，最终细胞收获密度为接种时的约15～20倍。诱导细胞的碱性磷酸酶活性在培养的第12天达最大值，并表达碱性磷酸酶、Ⅰ型胶原、骨钙素等成骨细胞表型。微载体成骨诱导细胞的碱性磷酸酶活性与普通成骨诱导方法差异无显著性意义(P&;gt;0．05)。结论：应用微载体可以成功进行人骨髓间充质干细胞的成骨诱导扩增，可以满足骨组织工程量和质的需要。     

大鼠骨髓间充质干细胞体外向成骨细胞分化培养的生物学特性

背景:骨髓间充质干细胞因取材方便、对机体损伤小,是目前最为合适的骨组织工程种子细胞,但其体外培养的具体方法及生物特性仍无定论.目的:建立一种简单有效的骨髓间充质干细胞的体外培养及成骨诱导方法,并探讨其体外培养的生物学特点.方法:采用贴壁法分离提纯Wistar大鼠骨髓间充质干细胞,传至第3代细胞后,分别在普通培养基(对照组)和成骨诱导培养基(实验组)中培养10 d,绘制细胞生长曲线.于培养第4,7,10,13,16天检测两组细胞碱性磷酸酶活性,并对细胞爬片行Von kossa染色.结果与结论:采用贴壁法获得了均一性较好的骨髓间充质干细胞,对照组细胞生长速度明显快于实验组细胞.对照组细胞碱性磷酸酶活性明显低于实验组(P<0.05).实验组细胞Von kossa染色为阳性,对照组为阴性.说明贴壁筛选法是一种简单有效的骨髓间充质干细胞的分离纯化方法,骨髓间充质干细胞在体外可诱导分化为成骨细胞.     

大鼠骨髓间充质干细胞体外向成骨细胞分化培养的生物学特性

背景：骨髓间充质干细胞因取材方便、对机体损伤小,是目前最为合适的骨组织工程种子细胞,但其体外培养的具体方法及生物特性仍无定论。目的：建立一种简单有效的骨髓间充质干细胞的体外培养及成骨诱导方法,并探讨其体外培养的生物学特点。方法：采用贴壁法分离提纯Wistar大鼠骨髓间充质干细胞,传至第3代细胞后,分别在普通培养基（对照组）和成骨诱导培养基（实验组）中培养10d,绘制细胞生长曲线。于培养第4,7,10,13,16天检测两组细胞碱性磷酸酶活性,并对细胞爬片行Vonkossa染色。结果与结论：采用贴壁法获得了均一性较好的骨髓间充质干细胞,对照组细胞生长速度明显快于实验组细胞。对照组细胞碱性磷酸酶活性明显低于实验组（P〈0.05）。实验组细胞Vonkossa染色为阳性,对照组为阴性。说明贴壁筛选法是一种简单有效的骨髓间充质干细胞的分离纯化方法,骨髓间充质干细胞在体外可诱导分化为成骨细胞。     

大鼠骨髓间充质干细胞成骨诱导分化的超微结构变化

背景:目前对骨髓间充质干细胞成骨诱导分化的超微结构观察的报道甚少.目的:采用全骨髓贴壁法分离培养大鼠骨髓间充质干细胞,成骨诱导并染色鉴定,利用电镜观察诱导前后细胞超微结构变化特点.方法:全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,流式细胞仪检测细胞表面标记物,成骨染色鉴定向成骨方向诱导分化,扫描电镜及透射电镜观察成骨诱导前后细胞超微结构变化.结果与结论:培养的第3代骨髓间充质干细胞纯度高、活力强,成骨诱导后的碱性磷酸酶活性染色、钙化结节染色均呈阳性.扫描电镜及透射电镜观察显示,经向成骨细胞诱导分化后,细胞形态铺展,不规则,其线粒体、粗面内质网、空泡明显增多,表明细胞功能活跃.     

骨髓间充质干细胞诱导内皮细胞与自体骨髓间充质干细胞共培养后的成骨特性

目的：观察由骨髓间充质干细胞诱导的内皮细胞与自体骨髓间充质干细胞共培养时对骨髓间充质干细胞成骨作用的影响。 方法：实验于2005—03／11在复旦大学附属中山医院外科中心实验室完成。①使用密度梯度离心法分离兔骨髓间充质干细胞。②取传两三代细胞分组分别进行如下操作：骨髓间充质干细胞组仅应用基础培养液（DMEM—LG培养基、体积分数为0．1的胎牛血清）常规培养；骨髓间充质干细胞成骨诱导组在基础培养液中加入成骨诱导剂；内皮细胞诱导组在内皮细胞生长培养基作用下将骨髓间充质干细胞诱导为内皮细胞；诱导的骨髓间充质干细胞与内皮细胞联合培养组将骨髓间充质干细胞和诱导而来的内皮细胞按1：1的比例接种于培养板中，加入成骨诱导培养液。③通过干细胞形态、免疫荧光、细胞增殖、碱性磷酸酶活性、骨钙素含量等从酶学、组织学、生化等不同方面观察诱导的内皮细胞对骨髓间充质干细胞成骨活性及细胞生长情况的影响。 结果：①免疫细胞荧光染色结果：内皮细胞诱导组培养诱导的细胞成为内皮细胞。②倒置相差显微镜、苏木精一伊红染色结果：均显示联合培养组骨髓间充质干细胞和诱导的内皮细胞混合生长良好。③四甲基偶氮唑盐法检测结果：各组细胞增殖差异无显著性（P〉0．05）。④碱性磷酸酶活性：除内皮细胞诱导组外，其他3组细胞的活性均随时间的延长而逐渐增高，联合培养组增长最明显，第12天时联合培养组细胞碱性磷酸酶活性已达到（618．46&;#177;28．34）nkat／L，高于其他各组（P〈0．05）。⑤骨钙素检测结果：在培养的第7天、第14天，联合培养组的骨钙素分泌量分别为2．03，3．17μg／L，明显高于其他各组（P〈0．01）。 结论：由骨髓间充质干细胞诱导的内皮细胞能够增强骨髓间充质干细胞的成骨活性，提高骨髓间充质干细胞的增殖能力。     

骨髓间充质干细胞诱导内皮细胞与自体骨髓间充质干细胞共培养后的成骨特性

目的:观察由骨髓间充质干细胞诱导的内皮细胞与自体骨髓间充质干细胞共培养时对骨髓间充质干细胞成骨作用的影响。方法:实验于2005-03/11在复旦大学附属中山医院外科中心实验室完成。①使用密度梯度离心法分离兔骨髓间充质干细胞。②取传两三代细胞分组分别进行如下操作:骨髓间充质干细胞组仅应用基础培养液(DMEM-LG培养基、体积分数为0.1的胎牛血清)常规培养;骨髓间充质干细胞成骨诱导组在基础培养液中加入成骨诱导剂;内皮细胞诱导组在内皮细胞生长培养基作用下将骨髓间充质干细胞诱导为内皮细胞;诱导的骨髓间充质干细胞与内皮细胞联合培养组将骨髓间充质干细胞和诱导而来的内皮细胞按1∶1的比例接种于培养板中,加入成骨诱导培养液。③通过干细胞形态、免疫荧光、细胞增殖、碱性磷酸酶活性、骨钙素含量等从酶学、组织学、生化等不同方面观察诱导的内皮细胞对骨髓间充质干细胞成骨活性及细胞生长情况的影响。结果:①免疫细胞荧光染色结果:内皮细胞诱导组培养诱导的细胞成为内皮细胞。②倒置相差显微镜、苏木精-伊红染色结果:均显示联合培养组骨髓间充质干细胞和诱导的内皮细胞混合生长良好。③四甲基偶氮唑盐法检测结果:各组细胞增殖差异无显著性(P>0.05)。④碱性磷酸酶活性:除内皮细胞诱导组外,其他3组细胞的活性均随时间的延长而逐渐增高,联合培养组增长最明显,第12天时联合培养组细胞碱性磷酸酶活性已达到(618.46±28.34)nkat/L,高于其他各组(P<0.05)。⑤骨钙素检测结果:在培养的第7天、第14天,联合培养组的骨钙素分泌量分别为2.03,3.17μg/L,明显高于其他各组(P<0.01)。结论:由骨髓间充质干细胞诱导的内皮细胞能够增强骨髓间充质干细胞的成骨活性,提高骨髓间充质干细胞的增殖能力。     

姜黄素调控大鼠骨髓间充质干细胞的成骨分化

背景:姜黄素能明显降低破骨细胞的骨重吸收,诱导破骨细胞凋亡,抑制破骨细胞形成。目的:观察不同浓度姜黄素对大鼠骨髓间充质干细胞成骨分化的影响。方法:采用贴壁法分离培养大鼠骨髓间充质干细胞,传至第3代后采用成骨培养基进行成骨诱导,在诱导前3d于诱导培养基中分别加入0(对照),10,15μmol/L姜黄素。结果与结论:接种培养7d,倒置显微镜下可见分散的骨髓间充质干细胞小集落,集落细胞呈放射状生长。随培养时间延长,细胞集落增大,细胞形态为长梭形。加入成骨培养基7d后细胞形态逐渐由长梭形变为多角形,随成骨诱导时间延长形态变化更加明显。加入不同浓度姜黄素后骨髓间充质干细胞增殖无变化。姜黄素呈剂量依赖性促进骨髓间充质干细胞碱性磷酸酶活性及Runx-2、血红素单加氧酶1的表达。提示姜黄素能有效促进大鼠骨髓间充质干细胞早期成骨分化,其机制可能与提高干细胞血红素单加氧酶1的表达有关。     

人脐血中分离骨髓间充质干细胞成骨诱导前后的生物学特性

目的观察诱导因素对骨髓间充质干细胞向成骨方向分化的影响,探索骨组织工程的种子细胞来源。方法使用密度梯度离心法分离来源于人脐血的骨髓间充质干细胞进行培养,保留已贴壁细胞传代,观察在加有地塞米松、维生素C、β-甘油磷酸钠的培养液中骨髓间充质干细胞的生长及分化情况。结果分离得到的骨髓间充质干细胞呈成纤维样表现,诱导条件下第2代细胞碱性磷酸酶活性增高,12d达到最高,为(33.10±0.54)U/ml,P<0.001,t=-48.32,且出现矿化结节。结论人脐血来源的骨髓间充质干细胞在诱导培养液作用下具有一定的成骨能力,可作为骨组织工程种子细胞。     

Measurement of L-carnitine and acylcarnitines in body fluids and tissues in children and in adults

We have developed a reliable and validated radio-enzymatic method for the assay of L-carnitine and acylcarnitines, using a modification of existing methods. The sensitivity of the assay is 10 mumol/l using 10 microliters of plasma or urine. It is also suitable for measurements of carnitine in a 10 mg sample of liver or muscle obtained by percutaneous biopsy. The use of N-ethylmaleimide in the reaction mixture together with an excess of [1-14C]acetyl CoA ensures that the reaction proceeds to completion and a linear response is obtained. Using this method control ranges have been established for plasma and urine carnitine concentrations in healthy children and adults, and for the carnitine content of liver and muscle in adults. No significant difference was found between fasting and post-prandial plasma carnitine levels. An age-related increase was found in urinary total carnitine and acylcarnitine concentration throughout childhood. These data provide a reliable basis for studies of patients with abnormal carnitine and acylcarnitine metabolism, distribution and excretion.     

Rosamicin in Urethral and Vaginal Secretions and Tissues in Dogs and Rats

In animal studies we investigated the distribution of rosamicin in plasma and urethral and vaginal tissues in rats as well as in urethral and vaginal secretions in dogs. We found concentration ratios between urethral secretion and plasma of 1.9 and between vaginal secretion and plasma of 2.4. The rosamicin concentrations in urethral and vaginal tissue significantly exceeded the levels of all other tissues investigated. Because rosamicin could be valuable for the treatment of bacterial urethritis and the colonization of the vaginal introitus with fecal bacteria in women, it should be investigated clinically in this respect.     

Similarities and differences in clients treated and in medications prescribed by APRNs and psychiatrists in a CMHC

This study is part of a larger study comparing prescribing practices of psychiatrists and advanced practice psychiatric nurses (APRNs) using the following three groups of patients: patients treated by psychiatrists, those treated by APRNs, and those treated by both APRNs and psychiatrists at different times in 1 year. Demographics for 5507 patients were examined. A subsample of APRNs and psychiatrists prescribed similar total numbers of medications. Psychiatrists prescribed more types of antidepressant medications other than the SSRI antidepressants, and they prescribed more than twice the number of benzodiazepines. APRNs prescribed more SSRIs and spent more time with clients during medication visits.     

Postural strategies and falls in elderly and in parkinsonism]

OBJECTIVE: To use a posture analysis to show the evolution of postural pattern connected with falls.MATERIAL AND METHOD: It is a prospective study on two groups of 16 persons of more than 60 years. A group concerns 16 small disability off drug parkinsonian patients, a group concerns 16 healthy witnesses. All the persons benefited from a posture recording by means of a force platform and were followed during 1 year. RESULTS: Data analysis underlines three groups of persons corresponding to three postural patterns, independently of the presence of Parkinson disease. A group (n = 18) did not contain fallers, the second (n = 10 ) contained 20% of fallers, the third (n = 4) contained 100% of fallers. Differences between the groups were identified on 16 posturographic parameters. DISCUSSION: A group has a good functional value and one does not record any fall. Its characteristics, which correspond to a category of persons who compensate well for the phenomena of ageing, are found in the literature. A group has an intermediate functional value and regrets 20% of fallers. Kinetic profile reveals a tendency to the stiffness of the posture. This group is going to operate rather ankle strategies. A group has an inferior functional value and regrets 100% of fallers. Kinetic profile seems disrupted and not to be able to adapt itself in a satisfactory way to the situation otherwise than by stereotypical reactions. This group is going to operate systematically much less stabilizing hip strategies. CONCLUSION: A close determinism between physiological neuromotor ageing and Parkinson disease does exist. We showed with a prospective follow-up, the arisen of fall and showed the evolution of postural patterns related to fall. It appears as well that evolution mainly follows three stages leading from a small risk of fall gait pattern to a major risk of fall gait pattern.     

Racial differences in oxidative stress and inflammation: in vitro and in vivo

African American race is an independent risk factor for enhanced oxidative stress and inflammation. We sought to examine whether oxidative-stress and inflammatory markers that are typically measured in humans also differ by race in cell culture. We compared levels between African American and Caucasian young adults and then separately in human umbilical vein endothelial cells (HUVECs) from both races. We found heightened oxidative stress and inflammation in the African Americans both in vitro and in vivo. African American HUVECs showed higher nitric oxide (NO) levels (10.8 ± 0.4 vs. 8.8 ± 0.7 μmol/L/mg, p = 0.03), Interleukin-6 (IL-6) levels (61.7 ± 4.2 vs. 23.9 ± 9.0 pg/mg, p = 0.02), and lower superoxide dismutase activity (15.6 ± 3.3 vs. 25.4 ± 2.8 U/mg, p = 0.04), and also higher protein expression (p < 0.05) of NADPH oxidase subunit p47phox, isoforms NOX2 and NOX4, endothelial nitric oxide synthase (NOS), inducible NOS, as well as IL-6. African American adults had higher plasma protein carbonyls (1.1 ± 0.1 vs. 0.8 ± 0.1 nmol/mg, p = 0.01) and antioxidant capacity (2.3 ± 0.2 vs. 1.1 ± 0.3 mM, p = 0.01). These preliminary translational data demonstrate a racial difference in HUVECs much like that in humans, but should be interpreted with caution given its preliminary nature. It is known that racial differences exist in how humans respond to development and progression of disease, therefore these data suggest that ethnicity of cell model may be important to consider with in vitro clinical research.