降钙素基因多肽对骨代谢的影响

根据骨质疏松的发病机制不同,可分为原发性骨质疏松和继发性骨质疏松,原发性骨质疏松又可分为绝经性骨质疏松和老年性骨质疏松。骨质疏松是由多种发病因素共同作用的结果,在各型骨质疏松中,降钙素均发挥重要的调节作用。近年研究发现某些神经、血管活性肽,如降钙素基因多肽(Calcitonin gene-related peptide,CGRP）,在结构和功能上与降钙素具有一定的相似性。本文将降钙素基因多肽对骨代谢影响的相关研究进展作以下综述。     

降钙素基因相关肽对骨质疏松症骨组织及胃肠作用的研究进展

现代研究发现,腰椎椎体含有丰富的降钙素基因相关肽（calcitonin gene-related peptide,CGRP)阳性神经纤维,CGRP可作用于破骨细胞,抑制骨质吸收;还能作用于成骨细胞,促进骨质形成。CGRP在胃肠道内广泛分布,CGRP与受体结合对胃肠运动起抑制作用,CGRP含量过高可能是骨质疏松症患者便秘并发症的一个病因。对骨质疏松症患者早期进行便秘防治干预,可减轻骨质疏松症患者的痛苦,促进骨质疏松症的康复有重要作用。骨质疏松症患者腰椎骨密度的变化、血浆CGRP的含量与便秘程度的关系还有待于进一步研究。     

Effect of intracisternal and intravenous calcitonin gene-related peptide on experimental cerebral vasospasm in rabbits

Summary We investigated the vasodilatory effect of intracisternal (i.c.) and intravenous (i.v.) administration of calcitonin gene-related peptide (CGRP) on arterial narrowing after experimental subarachnoid hemorrhage (SAH). Forty-one rabbits were divided into five groups: control (normal animals); SAH plus i.c. infusion of vehicle; SAH plus i.c. infusion of CGRP; SAH plus i.v. infusion of vehicle; SAH plus i.v. infusion of CGRP. In all but the control group, either CGRP (100ng/kg/min) or vehicle solution was infused for two hours immediately prior to sacrifice by perfusion-fixation. A morphometric technique was employed to measure the luminal diameter of rabbit basilar arteries two days after SAH. The diameter of the basilar arteries in either the i.c. or i.v. CGRP groups was significantly greater than that of the respective vehicle group (i.c., p < 0.001; i.v., p < 0.01). Although there was no significant difference in systemic arterial blood pressure after infusion between the i.c. vehicle and i.c. CGRP groups, i.v. CGRP caused significant hypotension. Our results suggest that exogenous CGRP has some therapeutic potential for arterial narrowing after SAH not only by intrathecal application, but also by systemic use.     

Human calcitonin gene-related peptide possesses weak inhibitory potency of bone resorptionin vitro

Summary Synthetic human calcitonin gene-related peptide (CGRP) was examined for the action on the bonein vitro. Human CGRP inhibited bone resorption stimulated by both human parathyroid hormone and basal. The mode of inhibitory action of human CGRP seemed to be similar to that of calcitonin and the relative potency of human CGRP to inhibit bone resorption is one five-hundredth of that of human calcitonin. Thus, a novel pharmacological action of CGRP was demonstrated.     

Targeted expression of calcitonin gene-related peptide to osteoblasts increases bone density in mice.

The neuropeptide calcitonin gene-related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin-like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost-CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg- mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg- littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg- mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.     

海绵状、泥灰状脱钙骨基质修复骨缺损

目的探讨海绵状脱钙骨基质(DBM)和泥灰状DBM修复骨缺损的能力。方法将兔四肢长骨骨干脱脂、脱钙后制成长度为500～1000 μm的纤维状及直径为200～400μm的颗粒状DBM,并与2%明胶混合分别制成海绵状DBM和泥灰状DBM。在9只兔双侧桡骨中段做一长10mm的骨膜骨缺损,分别植入海绵状DBM和泥灰状DBM及空白对照,每组各6个缺损,术后观察6周,于4、6周时拍摄X线片,并对实验动物的大体标本、骨密度、生物力学、新生骨矿化率及病理组织学改变进行观察。结果术后4、6周X线片显示两实验组骨缺损均修复,骨髓腔完整;空白对照组无一例修复骨缺损。骨密度测量显示海绵状DBM组新生骨骨密度与正常桡骨间差异无显著性(P >0.05),但泥灰状DBM组的新生骨骨密度与正常桡骨间差异有显著性(P< 0.05)。术后6周生物力学测定显示海绵状DBM组新生骨极限压缩强度值与正常桡骨差异无显著性(P >0.05),泥灰状DBM组低于正常桡骨且差异有显著性(P< 0.05)。两实验组新生骨矿化率差异无显著性(P >0.05)。组织学观察显示两实验组中DBM绝大部分被吸收,形成板状骨骨小梁及完整的骨髓腔,塑形完整, 新生骨内可见骨单位及局部尚未完全骨化的新生骨。结论海绵状DBM和泥灰状DBM均具有诱导成骨活性和骨传导能力,使用方便,新生骨塑形完整,生物力学强度高,矿化     

Calcitonin and calcitonin gene-related peptide in the human prostate gland

BACKGROUND: Calcitonin-related peptides have been found in the human prostate, and calcitonin (CT) and calcitonin gene-related peptide (CGRP) have been demonstrated in subpopulations of neuroendocrine (NE) cells. The purpose of this study was to determine the concentrations of CT and CGRP as well as the densities of NE cells in normal prostates, benign prostatic hyperplasia (BPH), and carcinoma of the prostate (CAP). METHODS: In 42 specimens of radical prostatectomy, the number of CT- and CGRP-immunoreactive NE cells in areas of normal and BPH tissue was determined, and compared with CAP tissue using immunocytochemistry. In addition, by radioimmunoassay (RIA), tissue levels of CT and CGRP were analyzed in extracts from areas of normal, BPH, and CAP tissue, as verified by adjacent histologic sections. RESULTS: A significant decrease in CT-immunoreactive NE cells was observed in hyperplastic nodules of BPH in comparison to normal tissue. These findings were in parallel with a significant reduction in tissue CT level in BPH compared to normal tissue. There was also a marked, but statistically nonsignificant, reduction in CT levels in CAP tissue. In contrast, levels of CGRP in BPH and CAP tissue did not show any significant differences compared to normal tissue. CONCLUSIONS: CT and CGRP are present in NE cells of the human prostate. Calcitonin levels are significantly reduced in BPH, in parallel with a decreased number of CT-immunoreactive NE cells, whereas no significant changes in tissue levels of CGRP were observed. The functional significance of these findings is discussed.     

Selective loss of calcitonin gene-related Peptide-expressing primary sensory neurons of the a-cell phenotype in early experimental diabetes

To evaluate the possible role of neuropeptide immunoreactive primary sensory neurons on the development of nociceptive dysfunction in diabetes, the absolute numbers of immunoreactive substance P and calcitonin gene-related peptide (CGRP) dorsal root ganglion (DRG) cell bodies were estimated in diabetic and nondiabetic BALB/C (p75(+/+)) and p75 receptor knockout (p75(-/-)) mice with unilateral sciatic nerve crush. The total numbers of immunoreactive substance P A-cells, substance P B-cells, CGRP A-cells, and CGRP B-cells in L5DRG were estimated using semithick consecutive sections and the optical fractionator. After 4 weeks of streptozotocin-induced diabetes, the number of immunoreactive CGRP A-cells was reduced from 692 +/- 122 to 489 +/- 125 (P = 0.004) in p75(+/+) mice on the noncrushed side. In p75(-/-) mice, there was no such effect of diabetes on the immunoreactive CGRP A-cell number. In p75(+/+) and p75(-/-) mice, there was no effect of diabetes on the immunoreactive CGRP B-cell number, nor was there any effect of diabetes on the immunoreactive substance P B-cell number. Sciatic nerve crush was associated with a substantial loss of L5DRG B-cells in diabetic and nondiabetic p75(+/+) mice and with substantial loss of immunoreactive substance P cells in diabetic p75(+/+) mice. In diabetic and nondiabetic p75(-/-) mice, there was no crush effect on neuropeptide expression. It is concluded that experimental diabetes in the mouse is associated with loss of immunoreactive CGRP primary sensory neurons of the A-cell phenotype, that this loss could play a role for the touch-evoked nociception in the model, and that the neuronal immunoreactive CGRP abnormality possibly is mediated by activation of the p75 neurotrophin receptor.     

New experimental method for the repair of tracheal defects

The use of bovine carotid artery xenografts to repair partial defects of the cervical and thoracic canine trachea as well as the covering of bronchial stumps was observed up to 32 months after surgery. Healing occurred without problems, and scanning electron microscopy revealed subsequent ciliary epithelialization of the entire graft surface (2 X 2-5 X 1.9 cm). Functionally, the grafts replaced the tracheal wall adequately. The use of resorbable suture material facilitates healing and avoids excessive formation of inflammation and granulation tissue observed with nonresorbable suture material of any kind.     

雪旺细胞促进组织工程骨修复大鼠股骨损的实验研究

目的 探讨雪旺细胞(SCs)对β-磷酸三钙/骨髓基质干细胞诱导分化的成骨细胞(β-TCP/BDOB)组织工程骨修复大鼠股骨缺损的影响. 方法 将SD大鼠骨髓基质于细胞(BMSCs)诱导分化为BDOB,并从SD乳鼠中提取SCs.将36只SD大鼠随机分为3组,每组12只,分别将术前预种植SCs的β-TCP/BDOB组织工程骨(A组)、β-TCP/BDOB组织工程骨(B组)、单纯β-TCP材料(C组)植入大鼠体内修复长度为8 mm的股骨缺损.于术后12周行X线摄片、组织学检查和显微CT扫描观察各组股骨缺损部位新生骨质的形成情况. 结果 术后12周X线片和显微CT扫描结果显示:A组大鼠股骨缺损已完全愈合,B组大鼠股骨缺损部分愈合,C组大鼠股骨缺损未愈合.A组、B组、C组的X线影像学评分平均分别为(12.08 ±0.90)、(9.25±1.06)、(6.17±1.12)分,3组比较差异有统计学意义(F=99.553、P=0.000),3组间两两比较差异均有统计学意义(P＜0.05).组织学检查结果显示:A组植入物中有大量骨质形成,广泛分布肥大的软骨细胞与成骨细胞;B组植入物中也可见散在新生骨形成,软骨细胞与成骨细胞散在稀疏分布;C组植入物中新生骨质形成极少,几乎未见软骨细胞或成骨细胞. 结论 SCs可提高β-TCP/BDOB组织工程骨修复大鼠股骨缺损的效果.     

CGRP介导束缚应激大鼠骨代谢的实验研究

目的探讨降钙素基因相关肽(CGRP)在慢性束缚应激大鼠骨退化中的作用。方法建立束缚应激大鼠模型,随机分为正常组(CON);束缚7 d组(A组);束缚14 d组(B组);束缚21 d组(C组),每组10只。观察股骨软骨下骨组织结构、细胞形态变化;用特异性放射免疫分析方法测定各组大鼠外周血、观察各组降钙素基因相关肽(CGRP)的改变。免疫组化染色检测各组软骨下骨组织CGRP的阳性表达。结果应激后C组大鼠细胞存活率下降(P﹤0.01)。C组外周血及软骨下骨CGRP的阳性细胞表达数均低于正常对照组,含量明显降低(P0.01)。结论在长期慢性束缚应激过程中低表达的神经肽导致骨代谢失衡,并显示退化状态。     

Cardiovascular action of calcitonin gene-related peptide in humans

Summary Calcitonin gene-related peptide (CGRP) has been localized in cardiac nerve fibers and blood vessels from which it may be released as neurotransmitter or neuromodulator. Acute cardiovascular effects of i.v. administered CGRP have been studied in human subjects. CGRP (25.3 nmol) caused a mean maximal increase of the heart rate of 41 beats per min (P<0.01) and lowered arterial systolic and diastolic pressures by 26 mm Hg and 20 mm Hg, respectively (P<0.01) (n=6 subjects). These effects were associated with facial flushing, and a rise of plasma levels of norepinephrine and epinephrine of 257 pg/ml and 9 pg/ml, respectively (P<0.01). Administration of equimolar amounts of human calcitonin caused no cardiovascular effects except for minor facial flushing. Serum calcium was marginally lowered with both CGRP (0.2 mg/100 ml) and calcitonin (0.4 mg/100 ml) (P<0.05). Further-more, CGRP (12.7 nmol) reduced the preejection period and duration of the electromechanical systole by 26 msec and 66 msec, respectively (P<0.001 andP<0.01), presumably acting as positive inotropic agent. Labetalol, blocking adrenergic receptors, obliterated these inotropic effects, whereas the positve chronotropic and hypotensive actions of CGRP remained unchanged.     

Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects

Demineralized bone matrix commonly is used to enhance and to facilitate bone grafting after skeletal injury or disease; however, the biologic bases for its bone-inducing abilities remain obscure. We have taken advantage of a mouse model of cortical bone defect healing to elucidate its mechanisms of action in vivo. Demineralized bone matrix combined with hyaluronan improved skeletal healing by inducing early deposition of an osteoid matrix. Demineralized bone matrix combined with hyaluronan might accelerate bone formation because it serves as a scaffold on which osteoprogenitor cells attach. We tested this possibility by comparing demineralized bone matrix combined with hyaluronan with heat-inactivated demineralized bone matrix combined with hyaluronan and found that the intact material was superior in terms of its ability to stimulate new bone formation. We also compared the bone inducing capacity of demineralized bone matrix combined with hyaluronan with a synthetic collagen sponge and found that not only the synthetic collagen scaffold delayed bone healing but also impaired bony bridging at later stages of repair. Another important property of demineralized bone matrix combined with hyaluronan was its ability to become actively degraded by osteoclasts during healing. Therefore, demineralized bone matrix combined with hyaluronan may not only attract osteoblasts and stimulate their differentiation, but also induce bone matrix resorption, which is a critically important regulator of bone formation and mineralization.     

降钙素基因相关肽对大鼠骨髓单核巨噬细胞破骨分化作用的实验研究

目的 通过体外实验研究外源性降钙素基因相关肽（calcitonin gene-related peptide,CGRP)对大鼠骨髓单核巨噬细胞 (BMMs)破骨分化能力的影响,为CGRP在抗骨质疏松治疗方面的应用提供理论依据。方法 采用差速贴壁的方法分离出SD大鼠髓腔内单核巨噬细胞,原代培养并传代,在培养体系中添加含不同浓度CGRP (实验组：10–7 mol/L、10–8 mol/L、10–9 mol/ L;对照组：无CGRP)的破骨诱导液,对前体细胞进行破骨诱导7天后进行相关实验检测,分别采用抗酒石酸酸性磷酸酶染色(TRAP染色）观察破骨细胞的生成能力;用RT-PCR方法检测破骨细胞系特征性基因（RANK、TRAP、NFATc1); Western-blot检测破骨特征性TRAP和RANK蛋白的表达;骨磨片甲苯胺蓝染色检测诱导的破骨细胞的骨吸收功能。结果TRAP染色显示 CGRP各浓度组镜下成熟破骨细胞的个数显著低于对照组,有统计学差异(P <0.05)并随CGRP浓度的增高成熟破骨细胞数减少,CGRP组间亦差异明显(P <0. 05);RT-PCR检测破骨特征性RANK、TRAP、NFATc1 mRNA和Westem-blot测破骨特征性TRAP、RANK蛋白的表达各CGRP组均低于对照组(P < 0. 05);骨磨片破骨细胞甲苯胺蓝染色后单倍视野下CGRP组破骨陷窝数量也明显少于对照组(P < 0. 05),且与药物组浓度与陷窝数量呈负相关性。结论 CGRP能够抑制BMMs向破骨方向的分化,为CGRP抗骨质疏松的治疗提供理论支持。     

磷酸钙骨水泥人工骨的制备及修复兔桡骨大段骨缺损的实验研究

目的制备水泥型羟基磷灰石(CPC)人工骨,并研究CPC人工骨修复兔桡骨大段骨缺损的成骨效果.方法高温法制备出磷酸四钙,然后在模拟体内环境下将其与无水磷酸氢钙发生水化固化反应,合成水泥型羟基磷灰石人工骨,将自制的CPC人工骨植入新西兰兔桡骨大段骨缺损模型中,术后4、8、12、16周取材,采用HE染色和Masson三色染色分析评价骨缺损修复效果.结果 CPC植入后4周有新生软骨形成及未成熟的骨组织,可见大量的软骨细胞、成骨细胞、胶原组织及较多的小血管.8～16周新骨继续生成,人工骨逐渐改建为成熟的板层骨、骨小梁和髓腔结构.结论 CPC人工骨制备简单,有良好的生物相容性和成骨效果,可能是一种较理想的骨移植替代材料.     

创伤性小腿骨与软组织缺损的显微外科修复

随着现代工业、交通业的不断发展,创伤暴力能量不断增强,严重创伤病例逐年增多,尤其是小腿部的创伤高居全身创伤首位.由于解剖学原因,胫前缺乏肌肉组织覆盖,加之小腿自身血供的因素,容易发生骨外露、骨缺损、骨不连,且处理困难.2000年6月至2007年6月,我院运用显微外科方法治疗274例小腿创伤性骨与软组织缺损患者,且获得随访,取得良好效果,现报告如下.     

Binding of calcitonin and calcitonin gene-related peptide to calvarial cells and renal cortical membranes

Binding of calcitonin (CT) and calcitonin gene-related peptide (CGRP) to rat hemicalvariae and renal membranes was examined in an effort to determine whether CT and CGRP interact with the same bone cell binding site, and to see whether the binding pattern was similar for bone and renal cortex. Specific binding of 125I-salmon CT to rat calvariae was inhibited by unlabeled salmon, porcine, or human CT, but not by rat CGRP. Binding of 125I-rat CGRP to calvariae was inhibited by CGRP and high doses of salmon CT, but not by human or porcine CT. Binding of 125I-salmon CT to renal membranes was inhibited by unlabeled salmon CT or rat CGRP, but no specific binding of 125I-rat CGRP could be detected. The results suggest that separate bone cell receptors for CT and CGRP exist and that CGRP can interact with renal receptors for CT.     

Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development

Amylin and calcitonin gene-related peptide (CGRP) are homologous 37 amino acid peptides that are found in the circulation. Both peptides belong to the calcitonin family. Similar to calcitonin, amylin and CGRP inhibit osteoclast activity, although they are much less potent than calcitonin. Calcitonin is known to act on the latter stages of osteoclast development, inhibiting the fusion of committed preosteoclasts to form mature multinucleated cells; however, whether or not calcitonin acts earlier in the formation of the precursor osteoclasts is controversial. The question of osteoclast development has never been examined with respect to amylin and CGRP. These issues are addressed in the present study. We studied the effects of calcitonin (salmon and rat), amylin (human and rat), and CGRP (human and rat) in mouse bone marrow cultures stimulated to generate osteoclasts using 1alpha,25-dihydroxyvitamin D3. Calcitonin dose-dependently decreased the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as TRAP-positive mono-/binucleated cells at concentrations >10(-13) mol/L. Amylin and CGRP showed similar effects at concentrations >10(-9) mol/L. In addition, calcitonin substantially reduced the ratio of TRAP-positive multinucleated to mono-binucleated cells, indicating an effect on fusion of osteoclast precursors. The present data establish that this family of peptides not only acts on mature osteoclasts but also inhibits their development in bone marrow cultures. This activity is shared by amylin and CGRP. The much greater potency of calcitonin than amylin and CGRP is consistent with the action of these peptides being mediated by calcitonin receptors.