幼年成骨细胞体外培养传代衰老与70岁以上老年人成骨细胞的生物学特性比较

目的通过对幼年人成骨细胞（osteoblasts,OB）体外培养传代衰老与70岁以上老年人成骨细胞的生物学特性比较,观察培养代数与年龄的相关性,拟建立幼年人成骨细胞体外培养传代模拟衰老模型。方法选取≤5岁（2例,男1,女1）和≥70岁（14例,男5,女9）两个年龄组的股骨颈松质骨进行成骨细胞体外培养,幼年组细胞自第二代开始每5～10d传代一次,至第17代终止。培养期间观察第2,5,10,15代细胞的形态、增殖能力与碱性磷酸酶（alkaline phosphatase,ALP）活性,以及应用RT-PCR方法检测BGP,IGF-1,OPG,ODF基因mRNA表达,并与体外培养的70岁以上老年人成骨细胞进行比较。结果形态观察提示成骨细胞体外连续传代至第10-15代时,其胞体增大、折光性降低,呈塌陷状,细胞器稀少、糖原增多、溶酶体增多,与70岁以上老年人成骨细胞相似。MTT相对比值随传代次数增加而降低,第10-15代细胞明显低于第2-5代细胞,而与70岁以上老年人成骨细胞相似;ALP活性于10-15代时增高,与70岁以上老年人成骨细胞相似。幼年成骨细胞骨钙素（bone gla-protein／osteocalcin,BGP）、胰岛素样生长因子-1（insulin-like growth factor-1,IGF-1）、破骨细胞分化抑制因子（osteoprotegerin,OPG）、破骨细胞分化因子（osteoclast differentiation factor,ODF）、OPG／ODFmRNA表达随传代次数增加呈降低趋势,第10与15代细胞其表达量低于第2代细胞,也低于70岁以上老年人成骨细胞。结论幼年成骨细胞体外连续传代培养10-15代时,其形态结构、增殖能力与某些基因mRNA表达与体外培养第二代70岁以上老年人成骨细胞有相似性,有希望作为体外培养模拟衰老模型。     

电离辐射致股骨头坏死早期病理特点和机制的研究

Objective To observe the earlier pathological character and mechanism of radiation osteonecrosis in femoral head, in order to provide evidences for the earlier diagnosis and prevention of radiation osteonecrosis of femoral head. Methods Single femoral head of rats were irradiated singly with 30 Gy of 137 Cs γ-ray. The rats were executed after 2, 6 and 12 weeks, then the femurs were stained with HE and histopatholngical changes were observed by light microscope. The bone marrow mesenchymal stem cells (BMSCs) were cultured after 2 weeks and its proliferation and the colony formation were observed. The rats were endo-perfused with microfili contrast medium 12 weeks later, and the 3-dimensional structure of capillaries by Micro-CT was re-estabhshed to detect the pathological changes of capillaries after irradiation. Result The irradiated femur showed deranged cbondrocyte, decreased osteocyte, shrinking nucleus, increased empty bone lacuna and reduced bone trabocnla (P < 0.05). Micro-CT showed the discontinued small vessels and absence(6.65 %) capillaries in irradiated femur were obviously less than those of the unirradiated (12.3 %)(P < 0.001). The proliferation of BMSCs was slowed, the number of colony in irradiated group (10 %) was less than that of control (21 %) (P < 0.001). Conclusions The preliminary histopathological changes of osteoradionecrosis on femoral head could be increased the empty bone lacuna, and the bone lacuna above 30 % was the sign of the earlier period of osteoradionecrosis. The osteonecrosis of femoral head induced by radiation is not only correlated to the damages to the bone, but also to the damages to BMSCs and capillaries.     

大鼠卵巢局部γ射线照射后的骨形态计量学改变

电离辐射对性腺的损伤已为人们所知 ,但对其产生的骨代谢远后效应研究甚少。早在 6 0年代 ,有人报道用 4 4Ｇｙ与30Ｇｙ的Ｘ射线局部照射大鼠卵巢 ,导致雌激素水平显著下降[1 ,2 ] 。笔者曾以 5 0ＧｙＸ射线局部照射大鼠卵巢[3] ,观察到卵巢辐射损伤后的骨吸收明显增强。在此基础上 ,笔者用5 0Ｇｙγ射线局部照射大鼠卵巢 ,90ｄ后进行血清雌二醇测定和骨组织病理学变化的观察 ,并与去势雌性大鼠和假手术大鼠进行了比较。一、材料和方法1 实验动物 :雌性ＳＤ大鼠 ,5月龄 ,体重 2 6 0～ 2 70ｇ ,由上海必凯实验动物公司提供 (清洁级 ,沪医实验…     

rhPTH(1-34)治疗糖皮质激素诱发大鼠骨质疏松症的实验研究

目的 观察重组人甲状旁腺激素(1-34)[recombinant human parathyroid hormone(1-34),rh-PTH(1-34)]对糖皮质激素引起的大鼠继发性骨质疏松症(OP)的治疗作用。方法 应用肌肉注射地塞米松(dexamethasone.DEX)方法,建立模拟糖皮质激素引起的继发性OP大鼠模型。给予皮下注射5、10、20和40μg·kg-1·d-1rhVTH(1-34)治疗8周。观察骨量、骨生物力学、骨形态计量及骨代谢相关血尿生化指标,综合评价PTH对糖皮质激素诱发OP的治疗效果。结果显示肌注地塞米松大鼠的骨量、骨生物力学较对照组显著性下降,表明诱导大鼠骨质疏松模型成立。不同剂量咖治疗均能显著增加模型大鼠的腰椎与股骨骨密度、股骨干重与灰重,提高股骨三点弯曲最大载荷、提高腰椎骨小梁面积百分比及矿化沉积率(P<0.05-0.001),并呈一定剂量效应关系。血钙、磷无明显变化。结论外源性PTH对糖皮质激素诱发的大鼠OP具有显著治疗作用。     

电离辐射后大鼠组织中丙二醛、组织蛋白酶D及甲2巨球蛋自的变化

动物受电离辐射后,蛋白酶活力增高.给与广谱蛋白酶抑制剂甲₂巨球蛋白(a₂M),可减少动物死亡,推测与清除蛋白酶有关.用⁶⁰Co致死剂量照射wistar大鼠,组织激肽释放酶增加,脾脏组织蛋白酶D活性增高.虽血浆a₂M含量与甲巨球蛋白(aM)捕捉胰蛋白酶活力也增加,但脾脏a₂M增加倍数低于肝,肾等组织.脾脏失重明显.提示a₂M治疗急性放射损伤有效,与保护脾脏等敏感组织,减少组织蛋白水解有关.大鼠致死剂量照射后,脂质过氧化产物丙二醛也增加.人放射性皮肤损伤,血浆组织蛋白酶D活力与丙二醛均增高,超氧化物歧化酶活力降低,给与a₂M治疗,可得到纠正.有报道,蛋白酶参与粒细胞氧自由基的释放,清除蛋白酶似可减少体内氧自由基的含量.推测a₂M在减少蛋白酶的同时,也减少了氧自由基的生成.     

辐射防护药“8401”(芥子酸胺盐衍生物)的致畸和诱变试验

于受孕2～16d的ICR小鼠,用不同剂量“8401”(167、250及330mg/kg)腹腔给药,每天1次。实验结果:用药组胚胎吸收率(9.3～13.0％)及肋骨畸形数均较对照组(0.8％)高。Amls试验结果:不同浓度“8401”在各种条件下,都没有诱发细菌回复突变,当浓度达到5mg/皿时,也未见诱变作用。     

雷公藤多甙对体外成骨细胞增殖的影响

目的　观察雷公藤多甙（ＧＴＷ）对体外培养成骨细胞（ＯＢ）增殖的影响，以进一步探讨雷公藤（ＴＷ）致女性骨质疏松的机制。方法　以ＳＤ大鼠ＯＢ增殖为模型，分离新生ＳＤ大鼠头盖骨ＯＢ，传代后加入不同浓度ＧＴＷ，用ＭＴＴ比色分析法检测ＧＴＷ在体外对ＯＢ增殖的作用和效价。结果　培养２ｄ，ＧＴＷ１μｇ／ｍｌ起显著抑制ＯＢ增殖（Ｐ＜０．０１）；培养４ｄ，ＧＴＷ０．１μｇ／ｍｌ起显著抑制ＯＢ增殖（Ｐ＜０．０５）。药物对ＯＢ增殖的抑制程度与其剂量呈正相关（Ｐ＜０．０５）。结论　ＧＴＷ显著抑制ＯＢ增殖且呈剂量依赖性。ＴＷ对ＯＢ的直接抑制作用，是其导致药物性骨质疏松的一个重要原因。     

猪大脑灰质组织提取液促进体外培养大鼠成骨细胞的增殖

背景：有研究表明大鼠脑组织提取液能够促进大鼠成骨细胞和骨髓基质干细胞的增殖。目的：观察跨种属脑组织固有成分对体外培养大鼠成骨细胞增殖的影响。方法：提取猪和大鼠大脑灰质、白质、垂体、肢体肌肉组织,分别制成匀浆提取液,并设空白对照组,各组提取液分别用于培养大鼠成骨细胞。比较各种组织提取液对成骨细胞增殖的影响。结果与结论：与空白对照相比,猪和大鼠的大脑灰质、白质和垂体组织提取液均显著促进了成骨细胞的增殖（P〈0.05）,各组间比较显示猪和大鼠的大脑灰质较白质、垂体和肌肉组织提取液促进成骨细胞增殖的能力更加显著（P〈0.05）。结果证实,猪脑组织提取液能跨种属促进大鼠成骨细胞的增殖,其中以大脑灰质的促进作用最为显著。     

大鼠口轮匝肌和腓肠肌乙酰胆碱受体密度及其与罗库溴铵亲和力的比较

目的 比较大鼠口轮匝肌和腓肠肌乙酰胆碱受体密度及其与罗库溴铵的亲和力,探讨口轮匝肌和腓肠肌对罗库溴铵敏感性差异的机制.方法 清洁级成年雄性SD大鼠8只,体重180～220 g,取口轮匝肌和腓肠肌各一大束,每束分离出等粗的6束,其中1束用于测终板表面积,另外5束分别用于测定不同浓度罗库溴铵[0(未加入罗库溴铵)、2.5、5.0、7.5、10.0μg/ml]时乙酰胆碱受体数量,计算乙酰胆碱受体密度和乙酰胆碱受体与罗库溴铵的亲和力.结果 与口轮匝肌比较,腓肠肌乙酰胆碱受体密度降低,乙酰胆碱受体与罗库溴铵亲和力升高(P＜0.05).结论 与口轮匝肌比较,大鼠腓肠肌乙酰胆碱受体密度降低,乙酰胆碱受体与罗库溴铵亲和力升高,这种差异可能是造成口轮匝肌和腓肠肌对罗库溴铵敏感性差异的机制.

Abstract：

Objective To compare the density of acetylcholine receptor (AchR) in orbicular muscle of mouth and gastrocnemius muscle and their affmity with rocuronium, trying to elucidate the mechanism for the difference in the sensitivity of the muscles innervated by facial and peripheral nerve respectively to muscle relaxant.Methods Eight pathogen-free adult male SD rats weighing 180-220 g were used in this study. Muscle strips were isolated from orbicular muscle of mouth and gastrocnemius muscle. Each muscle strip was further divided into 6 smaller and slender strips of same size using dissection microscope. One strip was stained with acetylcholinesterase to measure end-plate surface area (ESA). The other 5 strips were exposed to different concentrations of rocuronium (0, 2.5, 5.0, 7.5, 10.0μg/ml). The mean density of AchR at end-plate was obtained by AchR0/ESA. (AchR0 was defined as the number of AchR per end-plate without being exposed to rocuronium. AchRE was defined as the number of free AchR per end-plate after being exposed to different concentrations of rocuronium. ) The degree of saturation of AchR with different concentrations of rocuronium at each neuromuscular junction was calculated by (AchR0 - AchRE)/AchR0 which reflects the affinity of AchR with the rocuronium in orbicular muscle of mouth and gastrocnemius muscle. Results The density of AchR was significantly lower while the affinity with rocuronium was higher in gastrocnemius muscle than in orbicular muscle of mouth ( P ＜ 0.05). Conclusion The density of AchR is lower and the affinity of AchR at end-plate with rocuronium is significantly greater in gastrocnemius muscle innervated by sciatic nerve than in orbicular muscle of mouth innervated by facial nerve. This may explain the mechanism for different sensitivity of the muscles innervated by facial and peripheral nerves to rocuronium.     

体外培养人成骨细胞随供体的增龄而改变

目的　观察体外培养的不同年龄组人成骨细胞形态、增殖与功能改变。方法　选取≤5岁 ,30～ 4 0岁 ,5 0～ 6 0岁和≥ 70岁四个年龄组的髂骨、股骨近端或股骨颈松质骨进行培养。培养期间应用相差倒置显微镜活体观察与碱性磷酸酶染色观察成骨细胞的生物学特性。一代成骨细胞生长至汇合时消化计数继续培养 ,二代成骨细胞分别于培养 1天 ,5天检测细胞增殖率 (MTT法 ) ,5天时检测碱性磷酸酶 (ALP)活性 ,14天时送电镜室进行透射电镜观察。结果　活体观察提示成骨细胞随增龄其胞体增大、变薄、空泡增多、突起拉长 ,汇合时趋向于长梭形 ,各年龄组成骨细胞碱性磷酸酶染色均呈阳性反应 ;电镜观察提示成骨细胞随增龄变得瘦长、细胞器减少、高尔基体不发达、糖原增多并呈堆积现象、溶酶体增多、出现空泡 ,细胞核内异染色质增多。成骨细胞生长至汇合时的细胞数与MTT相对比值随增龄而降低 (P <0 .0 1) ;ALP活性随增龄呈增高趋势 ,但差异无统计学意义。结论体外培养不同年龄组人成骨细胞的形态与增殖能力随年龄增加而发生衰老改变