不同体积分数血清对培养原代破骨细胞噬骨能力的影响

目的:改变培养液中血清的浓度,观察破骨细胞形态结构,并比较不同体积分数血清对培养的原代破骨细胞噬骨能力的影响。方法:实验于2007-03/06在沈阳医学院中心实验室完成。①实验材料:选取清洁级新生24hWistar大鼠6只;胎牛血清(天津灏洋);新鲜成年牛股骨皮质(市售)。②实验过程:取新鲜成年牛股骨皮质,用磨片机磨成厚50μm的1cm×1cm骨片;取新生大鼠四肢长骨,分离破骨细胞,并用不同体积分数血清(分别为0,0.05,0.1,0.15,0.2,0.25)培养原代破骨细胞。③实验评估:第1,6天倒置显微镜下计数,第3天取细胞玻片观察细胞形态结构;扫描电镜观察不同体积分数血清培养的破骨细胞在牛骨皮质上产生骨陷凹面积的差异。结果:①破骨细胞的分离培养结果:分离培养的破骨细胞数量较少,体积大,胞浆丰满,细胞突起延展,细胞核呈圆形或椭圆形,积聚在细胞中央或排列在细胞周边。②不同体积分数血清培养液中骨片上吸收陷窝深度比较:随着培养液中血清体积分数的增加,骨片上陷窝深度逐渐增加,在培养液中血清体积分数为0.15时,骨片上陷窝最深,此后随着培养基血清体积分数的增加,形成骨陷凹的深度无明显增加。结论:血清体积分数为0.15培养基培养的破骨细胞在牛皮质骨片上产生骨陷凹最深,噬骨能力最强。     

两种方法培养大鼠破骨细胞的噬骨能力比较

背景：原代培养的破骨细胞数量少,而诱导培养产生的破骨样细胞数量多,能够满足一些研究骨代谢实验的要求.但是两种细胞在特异酶及噬骨能力方面是否具有相同的效果,到目前为止,还没有详尽的数据来支持.目的：比较大鼠原代分离的破骨细胞及诱导形成的破骨样细胞噬骨能力上的差异.方法：取24 h内新生Wistar大鼠四肢长骨,酶消化法分离培养破骨细胞,将破骨细胞培养过程中消化下来的骨髓单核细胞加入1,25（OH）2 D 3结果与结论：诱导第9天,破骨样细胞数目为破骨细胞数目的11倍,其形态与原代消化获得的细胞形态相同.抗酒石酸酸性磷酸酶染色呈阳性.甲苯胺蓝染色显示两组破骨细胞在骨片上培养时产生骨陷凹面积及深度差异无显著性意义.结果显示破骨样细胞的形态、特异酶及噬骨能力与破骨细胞无差异.诱导生成破骨样细胞.苏木精-伊红染色、抗酒石酸酸性磷酸酶（TRAP）染色鉴定.用甲苯胺蓝染色共培养骨片比较两组破骨细胞噬骨能力.     

糖尿病骨质疏松大鼠破骨细胞的改变

目的:采用腹腔注射链脲佐菌素 切除双侧卵巢复合方法建立绝经后糖尿病骨质疏松大鼠模型,观察糖尿病骨质疏松大鼠破骨细胞的改变。方法:实验于2003-12/2004-08在河北医科大学第三医院中心实验室完成。①实验材料:选用2.5～3个月龄清洁级雌性Wistar大鼠60只。②实验分组:完全随机设计分为6组:正常对照组、正常假手术组及正常双侧卵巢切除组均为8只;糖尿病对照组、糖尿病假手术组及糖尿病双侧卵巢切除组均为12只。③实验干预:采用链脲佐菌素诱导制备糖尿病大鼠模型1周后,将大鼠麻醉结扎输卵管切除卵巢。假手术组大鼠不作卵巢切除,余操作同去卵巢组。卵巢切除后0,2,4,8周随机选择各组大鼠1只,收集骨髓,进行体外骨髓破骨细胞样细胞的培养。④实验评估:切除双侧卵巢后2,4,8周时测量各组大鼠体质量、血糖;细胞培养7d后,进行细胞固定和抗酒石酸酸性磷酸酶染色(抗酒石酸酸性磷酸酶染色阳性且细胞核≥3个的细胞认定为破骨细胞样细胞);倒置显微镜计数破骨细胞样细胞数量。结果:制成糖尿病模型及切除卵巢后,3组糖尿病大鼠死亡7只,进入结果分析53只。①各组大鼠血糖和体质量的变化:切除卵巢0,2,4,8周,3组糖尿病大鼠血糖均高于正常组(P<0.01),体质量均低于正常组(P<0.01);4周时,正常双侧卵巢切除组体质量高于同期正常对照组和正常假手术组;糖尿病双侧卵巢切除组体质量在2,4,8周时均高于同期糖尿病对照组和糖尿病假手术组(4周P<0.05,8周P<0.01)。②切除卵巢后各组大鼠体外培养的破骨细胞样细胞形成的变化:糖尿病双侧卵巢切除大鼠破骨细胞多于糖尿病大鼠及正常去卵巢大鼠,且破骨细胞数量随着去卵巢的时间和糖尿病病程延长而增加。③骨髓来源的体外破骨细胞样细胞的形成与血糖、糖尿病病程及去卵巢时间的相关性分析:破骨细胞数量与糖尿病病程呈正相关,而与血糖增高的程度无关(r=-0.537;P=0.109)。结论:①卵巢切除对大鼠的血糖无显著影响,对大鼠体质量的影响被糖尿病减弱,在糖尿病早期破骨细胞样细胞的形成已有增加。②破骨细胞形成增加可能是绝经后糖尿病骨质疏松的原因之一。     

体外诱导大鼠破骨样细胞形成及促进骨吸收的实验

目的:通过大鼠四肢长骨骨髓细胞的体外诱导培养,观察1.25-(OH)2D3对破骨样细胞形成的影响。方法:4周龄SD大鼠长骨骨髓细胞悬液接种于预置盖玻片或牙本质片的24孔培养板内,试验组加入诱导剂1.25-(OH)2D3(1×10-8mol/L)而对照组不加,每3d换液1次,培养2周。结果:培养1周左右光镜下可见有多核破骨样细胞形成,胞体大,抗酒石酸酸性磷酸酶(TRAP)染色阳性,牙本质片上有骨吸收陷窝形成。证明所形成多核细胞为破骨样细胞。结论:1.25-(OH)2D3(1×10-8mol/L)可诱导大鼠骨髓破骨样细胞的形成。     

新生兔破骨细胞的体外分离培养与功能鉴定

目的:选择新生兔为实验对象,观察体外培养破骨细胞形态学和生物学特征。方法:实验于2007-03/06在辽宁医学院生理实验室完成。采用改良Chambers法机械分离兔四肢长骨获得新生兔(出生24h内)的破骨细胞,并应用倒置显微镜、光镜观察其形态学特征。结果:①倒置显微镜观察结果:培养1d后,细胞贴壁生长,数量增多,第3天后,可见多核巨细胞,呈油煎蛋、长条形,漏斗形等。②光镜观察结果:苏木精-伊红染色可见破骨细胞胞浆丰满,呈浅粉色,内含许多空泡结构,核3～20个不等,核呈浅蓝色,每个核含一两个核仁,有时周围伸出伪足样胞浆突起;Giemsa染色核集中于细胞中央或散在于细胞周边,呈花篮状,核浅粉色,胞浆着色较浅,其中可见数个大小不等未着色空泡;TRAP染色胞浆呈酒红色颗粒,核蓝色,核仁一两个;甲苯胺蓝染色可见骨吸收陷窝呈蓝紫色,圆形,椭圆形或不规则形,边界清晰,其边缘反光性强。结论:应用改良Chambers法可成功获得新生兔破骨细胞,且具有骨吸收功能。     

柚皮甙干预鼠颅顶骨破骨细胞的形成及功能

背景：柚皮甙能诱导骨形态发生蛋白2的基因表达,促进成骨细胞系的增殖和分化。体外细胞实验提示柚皮甙有抑制破骨细胞的形成及抗骨质疏松的作用。目的：建立含有柚皮甙的鼠颅顶骨培养模型,观察不同剂量下柚皮甙对破骨细胞增殖分化的影响。方法：实验选取出生4d的SD乳鼠颅顶骨,在分别含有O,1,10,100mg／L的柚皮甙的培养基中培养,于培养的第1,3,7,10天测定柚皮甙对鼠颅顶骨中破骨细胞标志酶抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度的影响。结果与结论：第1天各组间抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度无明显变化,第3,7天各组间抗酒石酸酸性磷酸酶阳性细胞数随柚皮甙质量浓度增加而减少,而培养基中钙离子浓度变化明显增加,到第10天这种变化趋势最为明显。说明柚皮甙能影响鼠颅顶骨中抗酒石酸酸性磷酸酶阳性细胞数量及其功能,并且这种作用成明显的时间剂量相关性。提示柚皮甙不仅能促进成骨细胞的增殖,同时也能减少破骨细胞的分化或加速其凋亡。     

人护骨素转基因抑制破骨细胞功能的研究

背景转基因治疗骨科系统疾病一直是骨科治疗的难点,构建携带有效基因的病毒是目前治疗因破骨细胞功能异常亢进的骨骼系统疾病的研究方向. 目的构建携带人护骨素(human osteoprotegerin,hOPG)基因的重组腺病毒及其在大鼠体内共同表达.设计单一样本研究.单位解放军第三军医大学大坪医院全军战创中心骨创科.材料实验在解放军第三军医大学烧伤研究所移植免疫实验室完成.材料质粒PUC19-hOPG;穿梭质粒pAdTrack-CMV及腺病毒基因组pAdeasy-1,293细胞株;限制性内切酶 EcoRI,EcoRV,BamHI,KpNI,PmeⅠ,PacⅠ,PCR引物.方法将hOPG基因片段插入到含有GFP基因的穿梭质粒pAdTrack-CMV上,与骨架质粒在大肠杆菌BJ5183胞内进行同源重组,经293细胞包装、扩增后得到携带hOPG和GFP基因的重组腺病毒(Adeasy-Adtrack-CMV-hOPG,Ad-hOPG).经大鼠尾静脉注射109 pfu的Ad-hOPG,在不同时相处死大鼠取其心、肝、肾、脾等组织观察重组腺病毒在体内表达特点.主要结局观察①重组质粒转染293细胞后,观察是否有绿色荧光蛋白的表达.②所获得的重组腺病毒基因组是否含有hOPG基因.③重组腺病毒Ad-hOPG所携带的基因是否能在大鼠体内表达蛋白,及表达时象特点.结果成功地构建了高滴度的携带hOPG基因的重组腺病毒,并能在体内大量表达,长达24 d.结论成功的构建了高滴度的Ad-hOPG,并初步取得了hOPG重组腺病毒体内表达特点.     

V-ATPase a3转运系统影响破骨细胞、骨吸收及骨折愈合的分子机制

背景：V-ATPase a3转运系统在破骨细胞对于骨吸收机制中起重要作用。目的：归纳总结V-ATPase a3转运系统在骨折修复中的表达,以及V-ATPase a3转运系统抑制剂对于骨折愈合的影响,进而能更好的指导临床。方法：在国内外期刊数据库中检索近10年内国内外文献,按检索关键词检索相关文献,筛选出符合纳入标准的文献,对其文献进行质量评估后采纳。结果与结论：V-ATPase a3转运系统广泛存在于真核细胞的细胞质膜和细胞器膜上,V-ATPase a3有2个结构域V0和V1,V0结构域是质子转运的通道,V1结构域主要是水解ATP供能。V-ATPase a3转运系统集中存在于破骨细胞皱褶缘上,逆浓度梯度转运H＋,为破骨细胞提供酸性环境,溶解无机物,为水解酶创造微环境降解有机物,参与骨吸收。因而V-ATPase a3转运系统在骨折的修复与重塑中选作研究靶点。     

失重状态下共育大鼠成骨与破骨细胞中护骨素表达的变化

目的：通过观察失重状态下共育的大鼠成骨与破骨细胞中护骨素的表达及其变化特点，探讨失重状态下骨结构的生物学变化。方法：实验于2004—07／12在哈尔滨医科大学附属二院科研中心完成。体外分离培养1周龄15只Wistar大鼠的颅骨成骨细胞及四肢骨破骨细胞，随机分为4组：成骨细胞+破骨细胞共育组，成骨细胞组，失重下成骨细胞+破骨细胞共育组，失重下成骨细胞组。前两组为空白对照。后两组均在回转加速器中48h。倒置相差显微镜下观察鉴定成骨细胞、破骨细胞，采用多聚酶链反应方法测定各组护骨紊的表达。结果:①护骨紊在各种情况下的表达：总RNA电泳条带28s。18s，5s，表明RNA完整。吸光度比A260／A280在1．8～2．0，失重共育组护骨紊较非失重共育组明显低表达，失重单纯成骨细胞组护骨素表达较失重共育组明显低表达(P&;lt;0．05)。单纯成骨细胞组明显低于失重单纯骨细胞组(P&;lt;0．05)。②5天后成骨细胞与破骨细胞在倒置相差显微镜下观察：成骨细胞呈多角形、三角形，胞浆丰富，邻近细胞中突起与之相联，胞核大，圆形，含一两个核仁；破骨细胞细胞核核大，4～6个，胞浆伸出丝状或片状伪足。结论：失重状态下共育成骨与破骨细胞中护骨素低表达，成骨作用减弱，说明力学作用参与了成骨细胞与破骨细胞之间的信号传递。     

凋亡破骨细胞的拉伸应变

背景：力学应变在骨重建中起重要作用。然而,力学应变是否影响破骨细胞的凋亡仍不清楚。目的：观察力学应变对体外培养破骨细胞凋亡的影响。方法：对小鼠单核细胞RAW264．7采用巨噬细胞集落刺激因子和破骨细胞分化因子诱导,抗酒石酸酸性磷酸酶染色和骨吸收实验确定成功诱导出了破骨细胞。实验共分为3组,对诱导的破骨细胞分别施加0（对照组）,2500和5000υε的基底拉伸应变3d,1h／次,1次／d。结果与结论：与对照组相比,生理强度载荷2500υε阻止了破骨细胞的凋亡和线粒体膜电位的下降。但是,病理强度载荷5000υε对破骨细胞的凋亡和线粒体膜电位没有影响。     

Regulation and enzymatic basis of bone resorption by human osteoclasts

Although much has been learned recently of the mechanisms that regulate osteoclastic differentiation, much less is known of the means through which their resorptive activity is controlled. This is especially so for human osteoclasts. We have recently developed an assay that allows us to measure resorptive activity while minimizing confounding effects on differentiation by optimizing osteoclastogenesis, so that measurable resorption occurs over a short period, and by relating resorption in each culture during the test period to the resorption that had occurred in the same culture in a prior control period. In the present study, we found that RANKL (receptor activator of nuclear factor kappaB ligand) strongly stimulated the release of CTX-I (C-terminal telopeptide degradation product of type I collagen) by osteoclasts over a similar range to that over which it induces osteoclastic differentiation, consistent with a distinct action on osteoclastic function. CT (calcitonin) dose-dependently inhibited bone resorption, whereas PTH (parathyroid hormone), IL (interleukin)-1, TNF-alpha (tumour necrosis factor-alpha), IL-6, IL-8, VEGF (vascular endothelial growth factor), MCP-1 (monocyte chemoattractant protein-1), MIP-1gamma (macrophage inflammatory protein-1gamma), IFN (interferon)-gamma and dibutyryl cGMP had no significant effect. Ca(2+), cyclosporin A, IFN-beta and dibutyryl cAMP all strongly suppressed resorption. Bone resorption was also strongly suppressed by alendronate, the cysteine protease inhibitor E64 and the cathepsin K inhibitor MV061194. Inhibitors of MMPs (matrix metalloproteinases) had no effect on CTX-I release. Moreover, the release of the MMP-derived collagen fragment ICTP (C-terminal cross-linked telopeptide of type I collagen) represented less that 0.01% of the quantity of CTX-I released in our cultures. This suggests that MMPs make, at most, a very small contribution to the bone-resorptive activity of osteoclasts.     

Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro.

Bisphosphonates are useful in treatment of disorders with increased osteoclastic activity, but the mechanism by which bisphosphonates act is unknown. We used cultures of chicken osteoclasts to address this issue, and found that 1-hydroxyethylidenediphosphonic acid (EHDP), dichloromethylidenediphosphonic acid (Cl2MDP), or 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid (APD) all cause direct dose-dependent suppression of osteoclastic activity. Effects are mediated by bone-bound drugs, with 50% reduction of bone degradation occurring at 500 nM to 5 microM of the different agents. Osteoclastic bone-binding capacity decreased by 30-40% after 72 h of bisphosphonate treatment, despite maintenance of cell viability. Significant inhibition of bone resorption in each case is seen only after 24-72 h of treatment. Osteoclast activity depends on ATP-dependent proton transport. Using acridine orange as an indicator, we found that EHDP reduces proton accumulation by osteoclasts. However, inside-out plasma membrane vesicles from osteoclasts transport H+ normally in response to ATP in high concentrations of EHDP, Cl2MDP, or APD. This suggests that the bisphosphonates act as metabolic inhibitors. Bisphosphonates reduce osteoclastic protein synthesis, supporting this hypothesis. Furthermore, [3H]leucine incorporation by the fibroblast, which does not resorb bone, is also diminished by EHDP, Cl2MDP and APD except when co-cultured with bisphosphonate-binding bone particles. Thus, the resorption-antagonizing capacities of EHDP, Cl2MDP and APD reflect metabolic inhibition, with selectivity for the osteoclast resulting from high affinity binding to bone mineral.     

番茄红素对活性氧族介导破骨细胞骨吸收功能的影响

背景：研究表明，番茄红素能够促进成骨细胞的增殖和生长，增加其矿化能力。目的：实验拟验证抗氧化剂番茄红素影响活性氧族介导破骨细胞骨吸收功能的作用途径。设计、时间及地点：随机分组设计，对比观察，实验于2005-01／2006-12在山东省骨科研究所完成。材料：24h内出生的清洁级Wistar大鼠幼鼠30只，用于破骨细胞的培养；番茄红素由Sigma公司提供。方法：将24孔细胞培养板内细胞分为5组。空白对照组：所用培养液不含番茄红素：10^-7mol／L番茄红素组、10^-6mol／L番茄红素组、10^-5mol／L番茄红素组和10^-5mol／L维生素C组培养孔内预先置入盖玻片或薄骨片，对种植其上的破骨细胞分别用含有不同浓度番茄红素或维生素c的d．MEM培养基进行培养。主要观察指标：在分离培养的细胞玻璃爬片或骨片进行抗酒石酸酸性磷酸酶染色、四唑氮蓝染色，最后对骨片进行扫描电镜照像并用image-proplus5．0图像分析软件分析骨吸收陷窝的数目和面积。结果：①抗酒石酸酸性磷酸酶染色阳性多核细胞镜下胞浆酸性磷酸酶活性部位呈紫红色，细胞核染色阴性或淡染，伪足清晰。②四唑氮蓝染色结果提示，10^-5mol／L的番茄红素明显抑制了破骨细胞产生活性氧族的能力。⑨扫描电镜结果显示，破骨细胞在骨片上形成形态不一的骨陷凹，10^-7mol／L的番茄红素部分抑制了骨吸收陷凹面积的增加，而10^-5mol／L的刮茄红素则几乎完全抑制了骨吸收陷凹的形成。结论：番茄红素在体外可以通过抑制破骨细胞产生活性氧族来抑制其骨吸收功能。     

Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts

A monocyte-derived factor with IL-1-like properties has recently been shown to cause resorption of bone in organ culture. We have investigated the action of IL-1 on disaggregated populations of osteoclasts, incubated alone or in the presence of osteoblastic cells, in an attempt to identify the target cell for IL-1 in bone, and to elucidate the mechanism by which IL-1 induces osteoclastic resorption. Osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Under these conditions, the majority of osteoclasts form distinctive excavations in the bone surface within 24 h, the volume of which can be quantified by computer-assisted morphometric and stereophotogrammetic techniques. IL-1 had no effect on bone resorption by osteoclasts alone, but when incubated in the presence of calvarial cells or cloned osteosarcoma cells, it induced a 3.8 (+/- 0.38)-fold increase in osteoclastic bone resorption, with significant enhancement at concentrations of greater than or equal to 30 pg/ml. The osteoblastic populations themselves did not resorb bone. The mechanism by which osteoblastic cells stimulate osteoclasts did not appear to depend upon PG synthesis; nor could we detect a diffusible substance in the medium of stimulated cocultures. These results indicate that IL-1 stimulates bone resorption through a primary action on osteoblasts, which are induced by IL-1 to transmit a short-range signal that stimulates osteoclastic bone resorption.     

失重状态下共育大鼠成骨与破骨细胞中护骨素表达的变化

目的:通过观察失重状态下共育的大鼠成骨与破骨细胞中护骨素的表达及其变化特点,探讨失重状态下骨结构的生物学变化。方法:实验于2004-07/12在哈尔滨医科大学附属二院科研中心完成。体外分离培养1周龄15只Wistar大鼠的颅骨成骨细胞及四肢骨破骨细胞,随机分为4组:成骨细胞+破骨细胞共育组,成骨细胞组,失重下成骨细胞+破骨细胞共育组,失重下成骨细胞组。前两组为空白对照。后两组均在回转加速器中48h。倒置相差显微镜下观察鉴定成骨细胞、破骨细胞,采用多聚酶链反应方法测定各组护骨素的表达。结果:①护骨素在各种情况下的表达:总RNA电泳条带28s,18s,5s,表明RNA完整。吸光度比A260/A280在1.8～2.0,失重共育组护骨素较非失重共育组明显低表达,失重单纯成骨细胞组护骨素表达较失重共育组明显低表达(P<0.05)。单纯成骨细胞组明显低于失重单纯骨细胞组(P<0.05)。②5天后成骨细胞与破骨细胞在倒置相差显微镜下观察:成骨细胞呈多角形、三角形,胞浆丰富,邻近细胞中突起与之相联,胞核大,圆形,含一两个核仁;破骨细胞细胞核核大,4～6个,胞浆伸出丝状或片状伪足。结论:失重状态下共育成骨与破骨细胞中护骨素低表达,成骨作用减弱,说明力学作用参与了成骨细胞与破骨细胞之间的信号传递。     

Formation, mineralization, and resorption of bone in hypophosphatemic rats

Quantitative morphologic methods were used to measure the effects of feeding a low phosphorus diet to intact and thyroparathyroidectomized rats on several processes of bone mineralization and turnover. In severely hypophosphatemic animals, the matrix formation rate was decreased, the osteoid maturation rate was decreased, which indicated a delay in the onset of mineralization, the initial rate of mineralization was decreased, and the endosteal osteoclastic bone resorption rate was increased. In moderately hypophosphatemic animals, there was a substantial increase in bone resorption but no change in formation or in mineralization. The increase in endosteal bone resorption was due to an increase in the linear rate of bone resorption and particularly to an increase in the length of the endosteal resorbing surface. The magnitude of the increase in bone resorption was similar in thyroparathyroidectomized and intact rats indicating that neither parathyroid hormone nor calcitonin is involved in this change. This, together with the finding that there was a strong negative correlation (r = -0.99) between the per cent endosteal resorbing surface and the serum phosphorus, supports the view that the increased resorption was due to hypophosphatemia. This inverse relationship between endosteal resorbing surface and serum phosphorus appeared to hold for values of serum phosphorus above normal. The resorptive response to hypophosphatemia, as previously shown for the resorptive response to excess endogenous parathyroid hormone, was partially inhibited by vitamin D deficiency. Increased resorption occurred at levels of serum phosphorus where no changes were observed in bone formation, mineralization, or growth, suggesting that this resorptive response functions as a homeostatic mechanism to maintain serum and intracellular phosphorus concentrations.     

黄芪对新生鼠乏氧缺血脑损伤后海马区神经的保护及学习记忆能力的干预

背景：黄芪可通过减轻兴奋性氨基酸的释放及在细胞间的堆积、缓解Ca^2＋超载、抗氧化等途径抑制凋亡的发生。目的：将黄芪用于未成熟脑缺氧缺血脑损伤的治疗，一方面检测其对海马缺氧缺血后半胱氨酸天冬氨酸蛋白酶-3mRNA表达水平的影响，另一方面通过迷宫实验观察黄芪对缺氧缺血脑损伤的成熟鼠学习记忆能力的干预。设计：随机对照实验。单位：东南大学临床医学院／附属中大医院儿科，基础医学院病理科。材料：实验于2002-10／2003-06在东南大学临床医学院实验中心完成。选取出生7d的同窝SD大鼠114只，随机分成3组：假手术组18只，模型组48只，黄芪治疗组48其。黄芪注射液由成都地奥九泓制药厂生产，规格为10mL／支，含生药20g。方法：模型组与黄芪治疗组建立缺氧缺血脑损伤模型。假手术组不造模。黄芪治疗组于造模后即刻及每天同一时间腹腔注射0．08mL黄芪注射液，7d后停药。模型组于同时间腹腔注射等量生理盐水。假手术组不给药。黄芪治疗组及模型组在缺氧缺血后24h，5d断头取脑，假手术组于假手术后24h断头取脑。各组海马区脑损伤行组织病理学检测，半胱氨酸天冬氨酸蛋白酶-3mRNA的表达采用半定量反转录-聚合酶反应方法进行检查，成年90d龄的大鼠进行三等分迷宫测试其学习记忆能力。3个实验各自独立。主要观察指标：①各组海马区脑损伤组织病理学检测。②各组结扎侧海马半胱氨酸天冬氨酸蚤白酶-3mRNA的表达。③三等分迷宫试验结果。结果：实验纳入大鼠114只，全部进入结果分析。①各组海马区脑损伤组织病理学检测：假手术组双侧海马区组织无水肿、坏死，神经细胞形态正常。神经细胞数为（87．7&;#177;0．6）&;#215;10^3／高倍视野。模型组24h时结扎侧海马区水肿，细胞周围间隙增宽，神经细胞数减少为（68．8&;#177;3．0）&;#215;10^3／高倍视野，与假手术组比较差异显著（P〈0．01）；5d时结扎侧海马体积缩小，锥状细胞层紊乱，神经细胞稀少至（48．7&;#177;2．2）&;#215;10^3／高倍视野，与假手术组及同侧24h时比较均有显著差异（P〈0．01）。黄芪治疗组24h时结扎侧海马区组织水肿较模型组明显减轻，5d时可观察到完整的海马形态，此两时间点神经细胞死亡率均较模型组明显减低（P〈0．01）。②各组结扎侧海马半胱氨酸天冬氨酸蛋白酶-3mRNA的表达：假手术组以低水平表达，吸光度值为0．220&;#177;0．009。模型组于缺氧缺血后逐渐升高，6h时比假手术组升高11％，至24h时mRNA水平达高峰，较假手术组约升高260％（P〈0．01），高峰持续至48h后下降，5d和7d时恢复基础水平。黄芪治疗组变化趋势与模型组相似，但在24，48h两时间点时峰值降低了44％-46％，与模型组比较差异显著（P〈0．01）。③三等分迷宫试验结果：与模型组比较，黄芪治疗组达到学会标准所需训练次数明显减少[（4517&;#177;2．7），（16.1&;#177;2.5）次，P〈0．01]，缺氧缺血24h后记忆保持率显著提高[（48．3&;#177;11．7），（80．0&;#177;9．0）％，P〈0．01]。结论：黄芪可以有效抑制未成熟脑缺氧缺血损伤后海马区神经细胞的凋亡．提高神经细胞存活率，此种保护作用与抑制半胱氨酸天冬氨酸蛋白酶-3的表达有关。同时黄芪能够明显改善未成熟脑缺氧缺血损伤后的学习记忆能力。