生长因子联合诱导大鼠间充质干细胞向软骨分化的实验研究

目的探索在体外培养条件下，转化生长因子（TGF）-β1、胰岛素样生长因子（IGF）-Ⅰ和骨形成蛋白（BMP）-2诱导间充质干细胞（MSCs）向软骨细胞分化的最佳组合。方法在体外单层培养条件下，利用TGF-β1、IGF-Ⅰ、BMP-2的组合作为诱导因子进行培养，用反转录聚合酶链反应（RT—PCR）检测细胞表达Ⅱ型前胶原mRNA水平，阿新蓝染色检测蛋白聚糖合成情况。结果BMP-2＋IGF-Ⅰ组、TGF-β1组、TGF-β1＋IGF-Ⅰ组、TGF-β1＋BMP-2组、TGF-β1＋IGF-Ⅰ＋BMP-2组Ⅱ型前胶原mRNA水平分别为0．147±0．062、0．478±0．069、0．489±0．022、0．653±0．013、0．807±0．051；而各组阿新蓝染色均阳性。结论TGF-β1、IGF-Ⅰ、BMP-2三者合用时诱导MSCs向软骨分化的效应最大。     

骨形成蛋白在低氧性肺动脉高压发病中的作用

目的观察低氧性肺动脉高压大鼠肺组织中骨形成蛋白2（BMP-2）的变化及BMP在低氧诱导内皮细胞凋亡中的作用，探讨BMP在低氧性肺动脉高压发病中的作用。方法将20只雄性Wistar大鼠随机分为2组，低氧组经常压低氧处理3周，建立大鼠低氧性肺动脉高压模型，采用免疫组织化学染色法观察大鼠肺组织中BMP-2的表达。用图像分析技术检测大鼠肺小动脉形态改变及BMP-2表达强度的变化。建立人脐静脉内皮细胞（HUVEC）低氧培养模型，加入BMP阻断剂Noggin，用流式细胞仪检测细胞凋亡率。结果低氧3周后，大鼠平均肺动脉压（mPAP）为（29．5±0．9）mmHg（1mmHg=0．133kPa），与对照组的（16．3±0．5）mmHg比较明显增加；低氧大鼠肺小动脉管壁增厚、管腔狭窄，表现为管壁厚度占外径的百分比（WT％）和管壁面积占血管总面积的百分比（WA％）明显升高，分别为（27±7）％和（80±8）％，对照组分别为（16±5）％和（54±11）％，两组相比差异有统计学意义（P〈0．01）；低氧组肺小动脉壁BMP-2积分吸光度值（M）为13463±5755，对照组为6124±1199，两组相比差异有统计学意义（P〈0．01），且与WT％和WA％呈明显正相关（r值分别为0．744和0．693，P〈0．01）。低氧诱导内皮细胞凋亡增加，低氧24h时的细胞凋亡率为（14．23±1．01）％，48h时为（25．21±8．58）％；低氧前预先加入Noggin，低氧24h时细胞凋亡率为（11．91±0．57）％，48h时为（15．01±0．15）％，差异有统计学意义（P〈0．01）。结论慢性低氧时BMP-2表达增多；低氧诱导内皮细胞凋亡，BMP阻断剂可抑制低氧诱导的内皮细胞凋亡；BMP在低氧性肺动脉高压发病过程中起一定的作用。     

氟化物对成纤维细胞和成骨细胞骨形态发生蛋白-2表达的影响

目的观察氟化物对成纤维细胞和成骨细胞骨形态发生蛋白-2（BMP-2）表达的影响。方法采用体外细胞培养的方法，将细胞分为对照组和6个染氟（F^-，0．1、1．0、100．0、1000．0、10000．0、20000．0μg／L）组，分别在5个染氟时间段（2、4、24、48、72h）收集培养上清液和细胞，应用酶联免疫吸附法（ELISA）和免疫组化方法检测BMP-2蛋白，RT-PCR方法检测染氟48hBMP-2mRNA的表达。结果与对照组比较．成纤维细胞染氟48hBMP-2mRNA表达呈普遍增强趋势，但差异无统计学意义（P〉0．05）；免疫组化检查，可见染氟0．1、1．0、1000．0μg／L组成纤维细胞BMP-2阳性着色较对照组增强；染氟72h，培养上清中BMP-2蛋白在1．0、100．0、20000．0μg／L组分别为（0．11±0．01）、（0．11±0．01）、（0．11±0．01），与对照组（0．07±0．01）比较有明显增高（P〈0．05）。与染氟成纤维细胞比较，染氟成骨细胞BMP-2mRNA和蛋白表达升高出现早．持续时间较长．增强程度更为明显。结论成纤维细胞和成骨细胞在氟化物的刺激下，BMP-2mRNA和蛋白表达有所升高，推测BMP-2可能是氟化物诱导成纤维细胞中成骨细胞核心结合因子α1（cbfa1）和骨钙素（OCN）表达的重要中介环节。     

骨髓源性心肌干细胞移植治疗心肌梗死的实验研究

目的研究骨髓源性心肌干细胞（MCSC）移植对心肌梗死的治疗作用。方法通过单细胞克隆培养技术从雄性SD大鼠骨髓间充质干细胞（MMSC）中筛选MCSC。结扎雌性SD大鼠的左冠状动脉前降支,建立心肌梗死模型,1周后于梗死区边缘移植MMSC和MCSC。移植后4周,用超声心动图检测心功能变化。取心肌组织作冷冻切片,用HE和Masson染色法显示瘢痕区的组织结构变化,通过免疫组织化学染色标记血管内皮生长因子受体-1阳性（VEGFR-1^＋）微血管,用图像分析系统测量瘢痕面积和微血管密度。利用原位荧光杂交标记含有Y染色体的MCSC,并检测心肌特异性肌钙蛋白T（cTnT）的表达。结果筛选的MCSC表达c—kit,心肌早期转录因子Nkx2．5呈低表达。细胞移植后4周,MCSC移植组的左室短轴缩短分数（62．9％±2．2％）和左室射血分数（32．8％±1．1％）高于MMSC移植组（分别为55．7％±1．6％和28．2％±1．6％）和对照组（分别为42．4％±2．1％和23．6％±1．2％）;MCSC组心肌梗死面积比率（8．7％±0．7％）低于MMSC组（12．0％±1．1％）和对照组（16．8％±0．9％）。含有Y染色体的MCSC表达cTnT,与受体心肌相续。MCSC移植组的梗死区周围微血管密度[（101．8±6．2）条／mm^2]大于对照组[（68．4±4．9）条／mm^2],与MMSC组[（97．2±3．2）条／mm^2]比较,差异无统计学意义。结论移植入心肌梗死模型的MCSC能够分化为功能性心肌,明显改善心功能,并诱导血管新生。MCSC的移植治疗效果优于MMSC。     

骨形态发生蛋白在修复长段骨缺损模型中的表达及意义

将32只普通家兔随机分为6组，制成左、右桡骨15mm骨缺损模型，左侧桡骨骨缺损处用自体髂骨骨松质颗粒、骨髓及部分旋前圆肌肌肉联合移植作为实验组，右侧作为对照组，不做任何处理，任其自然生长。于术后1、2、4、6、8周后处死动物取材，标本进行X线检查及骨形态发生蛋白（BMP）免疫组化染色和BMP2的cDNA探针原位杂交并在光镜下观察BMP在修复的长段缺损骨中表达。结果：为实验组在术后1周时与对照组无明显区别，2、4、6、8周相同时间内BMP在修复长段骨缺损模型中的表达强度均强于对照组。认为BMP是骨缺损修复过程中的一种重要生长因子。     

自体红骨髓复合骨膜碎片移植修复骨缺损的实验观察

目的观察自体红骨髓复合骨膜碎片移植修复骨缺损的形态学变化。方法40只健康中国家兔取双侧桡骨中段制作15 mm长的骨缺损模型。随机选择20只行自体红骨髓复合骨膜碎片移植术（移植组）,分别于术后第1、2、4、6、8周行X线摄片后,取出桡骨标本,大体观察、显微镜观察骨缺损区的形态学变化。另20只为对照组,桡骨骨缺损区内不移植任何组织。结果移植组：第1周移植组织增生;第2周移植组织弹性固定桡骨远近两断端;第4周在原始骨小梁间可见大量成纤维细胞;第6周骨小梁密集,成骨细胞排列在骨小梁周围,骨细胞位于骨陷窝内;第8周可见典型内、外环骨板及哈佛系统,骨髓腔形成,腔内可见多种细胞。对照组：第1周两断端边缘有少量新生组织;第2周新生组织覆盖两断端边缘;第4周可见成骨细胞;第6周有骨小梁形成;第8周形成骨密质并封闭缺损区远近两断端。结论自体红骨髓复合骨膜碎片移植可修复桡骨骨缺损,其过程可分为细胞分化增殖期、幼稚骨形成期及骨改造塑形期3个阶段。     

补肾壮骨合剂对骨缺损修复过程中局部BMP-2基因表达的影响

目的探讨补肾壮骨合剂对同种异体骨修复骨缺损中局部骨形态发生蛋白（BMP-2）基因的影响及其机制。方法选择健康普通家兔48只,将其中36只随机分成实验组和对照组,各18只。两组常规饲养。实验组在常规饲养的基础上加喂补肾壮骨合剂[1g／（kg·d）],早、晚各喂养1次。两组均在无菌条件下暴露桡骨中下1／3段,用骨磨钻制成20mm的骨缺损模型。另12只处死后取髂骨制成颗粒状骨植入上述骨缺损处,分别在术后3d及1、2、4、6、8周处死,两组每次分别处死3只,取骨缺损处标本,用PCR法进行BMP-2基因的动态定量分析。结果术后3d及1、2、4、6、8周时,对照组BMP-2基因分别为1453、4972、1230、411、856、902拷贝,实验组分别为2547、5397、3171、501、1981、1978拷贝,对照组在相同时间内明显低于实验组（P均〈0．05）。术后相同时间,对照组骨缺损修复过程中骨板形成较实验组慢。结论补。肾壮骨合剂可明显提高骨缺损处BMP-2的基因表达水平,显著诱导成骨进程,提高骨组织修复质量。     

冠状动脉内移植自体骨髓单个核细胞和内皮祖细胞对小型猪心肌缺血再灌注损伤的疗效比较

目的比较自体骨髓单个核细胞（BM—MNC）和内皮祖细胞（EPC）移植对小型猪心肌缺血再灌注损伤后修复梗死心肌和改善心功能的疗效。方法23头小型猪心肌缺血再灌注损伤模型分为BM—MNC组[（3．54±0．90）×10^8个细膨头,n=9]、EPC组[（1．16±1．07）×10^7个细胞／头,n=7]以及对照组（n=7）,比较细胞移植前以及移植4周时超声心动图、血液动力学和心肌梗死范围的变化。结果与移植前比较,移植4周时BM-MNC组、EPC组左室射血分数（LVEF）分别降低2％[（68±10）％比（66±7）％,P〉0．05]和0[（69±7）％比（69±8）％,P〉0．05],对照组则降低10％[（70±9）％比（59±7）％,P〈0．05],三组间比较差异有统计学意义（P〈0．05）。LVEF、左室收缩末压（LVESP）、心输出量（CO）和左室等容收缩压力最大上升速率（＋dp／dtmax）的变化值在BM—MNC组和EPC组间的差异无统计学意义（P〉0．05）,而显著小于对照组的变化值（P〈0．05）。舒张末压（LVEDP）和等容舒张压力最大下降速率（-dp／dtmax）在细胞移植前后各组变化不明显（P〉0．05）。EPC和BM—MNC移植的心肌梗死面积均小于对照组[心肌梗死面积百分比分别为[（4．1±0．6）％、（8．4±3．8）％和（11．4±3．2）％,均P〈0．05],EPC组较BM-MNC组有减小的趋势,但差异无统计学意义（P=0．067）。结论心肌缺血再灌注损伤后,自体BM—MNC和EPC移植均可明显改善左室收缩功能,这种作用可能通过减小心肌梗死面积实现。移植EPC与BM-MNC改善心功能的疗效相当,但还需进一步评价。     

BMP-2在骨缺损修复过程中的动态定量分析

目的 探讨骨缺损修复过程中骨形态发生蛋白-2(BMP-2)的动态定量变化及意义.方法 家兔30只,取右桡骨用骨磨钻制成长20mm的骨缺损,取自体髂骨制成直径1～2mm颗粒状移植于骨缺损处.术后第1、2.4、6、8周各处死6只取材,行BMP-2的RT-PCR检测.结果 术后第1、2.4、6、8周时,BMP-2的相对表达量为1.00、2.19、3.20、2.29、1.42.结论 BMP-2的动态定量变化可反映骨缺损的修复过程,为临床治疗提供理论依据.     

褪黑素对支气管哮喘小鼠胶原沉积及基质金属蛋白酶9和基质金属蛋白酶组织抑制剂1 mRNA与蛋白表达的动态调节

目的应用小鼠慢性支气管哮喘（简称哮喘）模型，观察褪黑素对慢性哮喘小鼠肺组织中胶原沉积的影响及其机制。方法96只SPF级雄性BALB／c小鼠按随机数字表法分为5组。对照组（21只）腹腔注射生理盐水；哮喘组（22只）腹腔注射生理盐水；褪黑素组（23只）腹腔注射褪黑素；地塞米松组（23只）腹腔注射地塞米松；褪黑素拮抗组（7只）腹腔注射褪黑素拮抗剂2-苯基-N-乙酰色胺。根据实验要求每组采用卵白蛋白致敏并反复雾化吸入2、4、8周时再分为2、4、8周亚组。对照组（每组均为7只）；哮喘组（分别为7、7、8只）；褪黑素组（分别为7、8、8只）；地塞米松组（分别为7、8、8只）；而褪黑素拮抗组只做2周组（7只）。实验造成慢性哮喘模型。用Masson三原色法染胶原纤维；用免疫组化方法标记蛋白；用MetaMorph软件测量单位长度基底膜周径上的胶原面积（Wcol／Pbm）和单位面积上基质金属蛋白酶9（MMP-9）及基质金属蛋白酶组织抑制剂1（TIMP-1）免疫组化阳性面积（PA／UA）；用半定量逆转录-聚合酶链反应（RT-PCR）法测定肺组织相应蛋白mRNA水平。结果褪黑素组2、4、8周WcoL／Pbm分别为（11．8±1．3）、（12．3±1．1）、（12．7±1．4）μm^2／μm，哮喘2、4、8周组分别为（14．5±1．5）、（15．8±1．8）、（16．2±1．4）μm^2／μm，两组比较差异有统计学意义（td值分别为3．89、5．96、5．50，P均〈0．01）；褪黑素组2、4、8周MMP-9的PA／UA像素分别为（9．7±4．9）、（14．8±4．9）、（11．0±6．8）万，哮喘2、4、8周组分别为（15．7±6．1）、（26．2±6．9）、（24．6±6．0）万，两组比较差异有统计学意义（td值分别为3．00、4．83、5．50，P均〈0．01）；褪黑素组2、4、8周MMP-9mRNA水平表达分别为0．80±0．40、0．68±0．15、0．67±0．24，哮喘2、4、8周组分别为1．48±0.29、1．40±0.50、1.20±0．40，两组比较差异有统计学意义（td值分别为3．92、4．50、3．29，P均〈0．01）；地塞米松组2、4、8周Wcol／Pbm（11．6±1．3、12．3±1．0、13．0±1．7）μm^2／μm、MMP-9蛋白[（12．5±5．6）、（14．0±4．7、13．6±4．8）万]和mRNA水平（0．69±0．11、0．61±0．16、1．10±0．40）均较哮喘2、4、8周组降低。褪黑素拮抗2周组与哮喘2周组以上各指标比较差异均无统计学意义（td值=0．96，P〉0．05）。结论早期使用褪黑素可抑制胶原沉积，其作用与地塞米松相当。褪黑素可能通过MMP-9介导的途径抑制哮喘气道重塑。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.