降钙素基因相关肽对大鼠成骨细胞增殖和分化的影响

目的 探讨外源性降钙素基因相关肽（CGRP）对大鼠颅盖骨来源成骨细胞的影响，以进一步了解CGRP促进成骨的作用机制。方法 利用二次酶消化法培养的成骨细胞，加入含不同浓度CGRP的条件培养液，24小时后通过MTT比色，^3H-TdR掺入以及细胞内碱性磷酸酶（ALP）含量的测定来观察CGRP对成骨细胞增殖和分化的影响。结果 CGRP可以明显促进成骨细胞增殖，而且作用呈剂量依赖性。CGRP对细胞内ALP含量无明显影响。结论 CGRP能够直接作用于成骨细胞并通过促进其增殖而有利于骨形成。     

失神经支配在大鼠骨折愈合过程中降钙素基因相关肽对骨保护素/破骨细胞分化因子影响的实验研究

目的 通过建立选择性切断大鼠感觉/运动神经联合胫骨骨折的动物模型,研究感觉/运动神经损伤后对骨折愈合的影响,并检测骨折愈合过程中降钙素基因相关肽(calcitonin gene related peptide,CGRP)对骨保护素(osteoprotegerin,OPG)/破骨细胞分化因子(receptor activator nuclear factor kappa B ligand,RANKL)体系的影响,初步探讨周围神经调节骨折愈合的机制.方法 取60只Wistar大鼠随机分为3组:前根(运动神经)切断+胫骨骨折组(anterior rhizotomy group,ART组);后根(感觉神经)切断+胫骨骨折组(posterior rhizotomy group,PRT组);单纯胫骨骨折组(sham operated group,SO组).分别于骨折术后7、14、21、28d处死大鼠,在骨折处上、下5mm部位取骨痂标本.免疫组织化学检测CGRP、OPG、RANKL的表达;采用软件Image-proplus 6.0进行图片分析;采用SPSS16.0进行统计学分析.结果 CGRP在SO组各时间点均呈强阳性表达,术后14 d和21 d,SO组、ART组和PRT组比较差异均有统计学意义(P＜0.05).OPG在SO组术后7d呈强阳性表达,以后逐渐下降但保持较高水平;术后14 d,PRT组和SO组、ART组比较差异有统计学意义(P＜0.05);术后21 d,SO组和PRT组、ART组比较差异有统计学意义(P＜0.05).RANKL在SO组21 d为表达高峰,以后逐渐下降;术后14 d,PRT组和ART组、SO组比较差异有统计学意义(P＜0.05);术后21 d,SO组和ART组、PRT组比较差异有统计学意义(P＜0.05).结论 在骨折愈合过程中,感觉神经纤维对骨折愈合的影响较运动神经纤维显著.CGRP能够调节OPG/RANKL的表达量的比值,从而影响骨折的愈合过程.失神经支配(尤其是感觉神经)导致CGRP对OPG/RANKL表达的调节作用降低,这不利于骨折的愈合.完整的神经支配是正常骨折愈合的必要条件之一.     

烧伤早期营养对大鼠降钙素基因相关肽影响的实验研究

目的观察烧伤早期不同营养途径对肠道的复苏效应,及其与降钙素基因相关肽(cal-citoningene-relatedpeptide,CGRP)的关系方法30%TBSAⅢ度烧伤Wistar大鼠,随机分为灌喂组(EF组,30只)及早期静脉营养组(EPN组,30只).此外,6只大鼠不烧伤,作对照组(C组).伤后6、12、24、48、72h观察大鼠一般情况、肠黏膜血流量、血浆CGRP免疫活性物质浓度、CGRP染色阳性物质在肠道的变化.结果烧伤后大鼠肠黏膜血流量下降,EF组下降程度明显低于EPN组(P＜0.05～0.01);烧伤后血浆CGRP升高,EF组血浆CGRP在伤后72h明显低于EPN组(P＜0.01);肠道CGRP阳性染色物质在烧伤后明显减少(P＜0.05～0.01),EF组在伤后24、48h明显多于EPN组(P＜0.05);肠黏膜血流量下降和肠肌间丛CGRP下降呈正相关(r=0.72,P＜0.05).结论肠道营养在烧伤早期肠道的复苏效应方面较静脉营养优越,伤后肠道CGRP免疫活性物质减少,血浆CGRP免疫活性物质过度增高可能是烧伤后肠道血供减少的重要机制之一,早期肠道营养可能通过调节CGRP而发挥复苏作用.     

脊髓损伤大鼠胫骨骨痂中降钙素基因相关肽的改变

目的 研究脊髓损伤后大鼠胫骨骨痂中降钙素基因相关肽（ＣＧＲＰ）的变化及骨痂量的改变。方法 将１００只雌性Ｗｉｓｔａｒ大鼠随机分为２组,Ｔ１０－１１脊髓横断＋右胫骨骨折组和单纯右胫骨骨折组各５０只。术后３、７、１４、２１、２８ｄ分批处死,检查血清碱性磷酸酶（ＡＬＰ）,摄右胫骨Ｘ线片行骨痂评分,骨痂行苏木精－伊红（ＨＥ）染色和ＣＧＲＰ免疫组织化学染色。结果 脊髓缶伤组３、７ｄ,ＡＬＰ均显著升高（Ｐ〈０     

脑损伤大鼠胫骨骨痂中降钙素基因相关肽的改变

目的研究脑损伤后大鼠胫骨骨痂中降钙素基因相关肽（CGRP）的变化及骨痂量的改变。方法将100只雄性Wistar大鼠随机分为两组,脑损伤加右胫骨骨折组和单纯右胫骨骨折组各50只。术后3、7、14、21、28 d分批处死,检查血清碱性磷酸酶,摄右胫骨X线片测量骨痂面积,骨痂行苏木精2伊红（HE）染色和降钙素基因相关肽免疫组织化学染色。结果脑损伤加骨折组较单纯骨折组术后3、7 d,碱性磷酸酶均显著升高（P〈0.01）;脑损伤加骨折组较单纯骨折组术后14、21 d,骨痂X线面积大;脑损伤组早期形成大量纤维骨痂和软骨骨痂,骨痂中降钙素基因相关肽免疫阳性神经纤维多,明显增厚的骨膜内层骨祖细胞、幼稚的软骨细胞胞浆内降钙素基因相关肽强阳性表达;骨折愈合快,降钙素基因相关肽表达明显增强。结论脑损伤后骨痂中降钙素基因相关肽有显著改变,并引起骨痂量和质的改变,推测降钙素基因相关肽参与调节骨折愈合过程。     

降钙素基因相关肽与糖尿病肾病

降钙素基因相关肽(CGRP)是目前已知体内最强的舒血管多肽，属于降钙素基因肽超家族，参与许多重要功能的调节，糖尿病肾病(DN)是胰岛素依赖型和非胰岛素依赖型糖尿病的一种常见的微血管并发症，通过对CGRP与DN微血管病变的发病机制之间的研究，可能为DN的诊断和治疗带来新的手段。     

降钙素基因相关肽与糖尿病肾病

降钙素基因相关肽(CGRP)是目前已知体内最强的舒血管多肽,属于降钙素基因肽超家族,参与许多重要功能的调节,糖尿病肾病(DN)是胰岛素依赖型和非胰岛素依赖型糖尿病的一种常见的微血管并发症,通过对CGRP与DN微血管病变的发病机制之间的研究,可能为DN的诊断和治疗带来新的手段。     

大鼠烫伤后肾上腺内含降钙素基因相关肽神经的改变

目的探讨皮肤烫伤早期大鼠肾上腺内降钙素基因相关肽（ＣＧＲＰ）变化规律,为阐明烫伤后肾上腺内分泌功能改变的机理提供资料。方法采用１５％深Ⅱ度大鼠烫伤模型,以免疫组化方法分别检测烫伤后不同时相点肾上腺皮质、髓质ＣＧＲＰ免疫反应阳性神经和细胞分布密度的改变。结果①在肾上腺被膜,皮质,髓质均有含ＣＧＲＰ神经分布,其中髓质更为密集。在髓质可见含ＣＧＲＰ神经与ＣＧＲＰ免疫反应阳性细胞密切接触;②烫伤早期大鼠肾上腺皮质和髓质含ＣＧＲＰ神经的分布密度均下降,但髓质内ＣＧＲＰ免疫反应阳性细胞的分布密度上升。结论降钙素基因相关肽可能是烫伤大鼠肾上腺功能改变的影响因素之一。     

降钙素基因相关肽对血管内皮细胞体外血管生成的作用及机制研究

 目的 观察降钙素基因相关肽(calcitonin-gene-related peptide,CGRP)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)的成血管作用,初步探讨其在骨组织工程中的应用价值。方法 体外分离获取HUVECs,采用细胞免疫荧光检测其CGRP受体1的表达。体外成管实验检测CGRP的成血管作用,ELISA法检测CGRP直接作用于HUVECs时血管内皮生长因子(vascular endothelial growth factor, VEGF)的分泌水平。Q-PCR检测CGRP刺激HUVECs不同时间点VEGF、VEGF受体1(FLT1)、VEGF 受体2(KDR)及CGRP受体1 mRNA的表达,Western blot检测HUVECs不同时间点FLT1、KDR的蛋白表达。结果 细胞免疫荧光显示HUVECs表达CGRP受体1,体外成管实验显示CGRP有明显的成血管作用。ELISA显示CGRP能明显促进HUVEC分泌VEGF。Q-PCR结果显示不同浓度组CGRP受体1 mRNA的表达较对照组增高,且在第10天最为明显;Q-PCR及Western blot结果显示不同浓度组FLT1、KDR mRNA和蛋白的表达在各时间点较对照组均增高。结论 CGRP能明显促进HUVECs的体外生成血管,可能与其促进VEGF分泌,增强HUVECs的FLT1与KDR表达有关;同时,CGRP受体表达也增加,可进一步增强CGRP的促血管生成作用。     

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.     

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。     

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.     

Contractility of rat gubernacula affected by calcitonin gene-related peptide and beta-agonist

Purpose

The effects of calcitonin gene-related peptide (CGRP), isoprenaline, and guanethidine on the neonatal rat gubernaculum were investigated in organ culture with the aim of seeing whether β-agonists or β-antagonists are involved in gubernacular development during testicular descent.

Methods

A total of 200 gubernacula were studied. The gubernacula (n = 20 for each experiment) from male rat pups were incubated in aerated culture medium with isoprenaline (1 and 10 μmol/L) with/without CGRP (714 nmol/L) or guanethidine (1 and 10 μmol/L) with/without CGRP (714 nmol/L). Cultures were observed for 5 days by video camera to see spontaneous rhythmic contractions, which are known to correlate with normal gubernacular migration in the rodent.

Results

Of gubernacula cultured without CGRP, 60% showed contractions by day 5, and the groups of isoprenaline and 10 μmol/L guanethidine showed similar rates, but only 15% of the 1 μmol/L guanethidine group showed contractility (P < .005). In contrast, of the gubernacula cultured with CGRP, each group showed high contractility from day 2 of the culture, and there was no difference between each group and the CGRP-alone group.

Conclusions

Although the β-blocker showed a weak inhibitory effect on de novo gubernacular contractility, CGRP had a stronger effect on contractility, overriding any possible effects of the β-neuron pathway. The contractility of the rat gubernaculum during testicular descent is primarily mediated by the genitofemoral sensory nerve fibers via release of CGRP, but β-adrenergic nerves also may be involved.     

不同浓度葡萄糖对大鼠骨髓破骨细胞分化的影响

目的观察不同浓度葡萄糖对大鼠骨髓破骨细胞（Osteoclasts OC）分化的影响，探讨糖尿病骨质疏松的发病机制。方法用M—CSF、RANKL诱导大鼠骨髓单个核细胞分化为OC，同时给予不同浓度的葡萄糖（0、5．5、15、25mmol／L）干预，通过观察抗酒石酸酸性磷酸酶（TRAP）染色阳性OC数、TRAP活性测定、OC膜表面RANK mRNA表达量来分析葡萄糖对破骨细胞分化的影响。结果①高浓度葡萄糖组（25mmol/L）培养7d时TRAP染色阳性OC数及TRAP活性测定均高于对照（0mmol／L）组、葡萄糖5．5mmol／L组（P〈0．01）；与对照组比较高浓度葡萄糖组培养3d时TRAP染色阳性OC数明显增多（P〈0．01）。②葡萄糖呈浓度依赖性上调OC膜表面RANK mRNA的表达，其中高浓度葡萄糖组培养的各时间点与其他3组比较差异有统计学意义（P〈0．01，P〈0．05）。结论①高浓度葡萄糖可促进破骨细胞的分化，其促进作用始于诱导分化的早期。②骨髓微环境中高浓度葡萄糖可引起OC分化增多，可能是糖尿病骨质疏松的发病机制之一。     

降钙素基因相关肽对大鼠心肌细胞L-型钙电流的影响

目的 观察降钙素基因相关肽（CGRP）对大鼠心肌细胞L-型钙电流（ICa-L）的影响。方法 SD大鼠，雌雄不拘，体重260～280g，急性分离其心肌细胞，用全细胞膜片钳方法记录ICa-L。1.依次记录经台氏液灌流（基础值）、含10^-9、10^-8、10^-7、10^-6mol／L CGRP的台氏液灌流和用台氏液洗脱后ICa-L峰值；2．记录含10^-7mol／L的CGRP台氏液灌流前后ICa-L，并绘制Ⅰ-Ⅴ曲线。结果 10^-7、10^-7mol/L CGRP可抑制大鼠心肌细胞ICa-L（P〈0．05），10^-7mol／L CGRP使大鼠心肌细胞ICa-L Ⅰ—Ⅴ曲线明显上移（P〈0．05），其电流峰值仍在0mV。结论 CGRP可抑制大鼠心肌细胞ICa-L。     

Preliminary study on androgen dependence of calcitonin gene-related peptide in rat penis

To study the androgen dependence of the neurotransmitter, calcitonin gene-related peptide (CGRP) in rat penis. METHODS: Forty-four Sprague-Dawley rats were randomly divided into Group A (intact controls), Group B (castrated) and Group C (gavaged with finasteride 4.5 mg.kg(-1).day(-1)). Four and ten weeks later respectively, half of rats in each group were anaesthetized. Blood samples were taken for the measurement of serum testosterone and dihydrotestosterone (DHT) by means of radioimmunoassay. Penile samples were harvested for the investigation of calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers with immunohistochemistry. The computer-assisted imaging analysis system was applied to calculate the area proportion of the CGRP-positive nerve fibers (CGRP-PNF) in each group. RESULTS: 1) Both 4 and 10 weeks later, testosterone and DHT levels in Group B decreased significantly compared with those in Group A, (P <0.05, P <0.01, respectively); DHT level in Group C was also significantly decreased in comparison with that in Group A for both 4- and 10- week animals (P <0.05); 2) There was no significant differences in area proportion of CGRP-PNF among Groups A, B and C 4 weeks after treatments (P >0.05); However, 10 weeks later, the proportion of CGRP-PNF in Groups B and C was significantly less than that in Group A (P <0.01); 3) The proportion of CGRP-PNF of 4-week animals in Groups B and C was significantly higher than that of 10-week animals (P <0.05). CONCLUSION: The expression of neurotransmitter, CGRP may depend on androgens, including testosterone and DHT in rat penis.     

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。     

Role of stromal cells in osteoclast differentiation in bone marrow

Bone marrow stromal cells have been considered to play an important role in osteoclast differentiation. However, the interaction of these cells in vivo has not been clearly demonstrated. To clarify this, we examined the distribution of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) activities as markers of osteoblastic and osteoclastic cells, respectively. Rat tibiae were fixed and embedded in Technovit 8100 or paraffin. ALPase and TRAPase activities were detected simultaneously on a plastic section by the azo-dye method. ALPase activity was detected on the plasma membranes of osteoblasts and some bone marrow fibroblastic stromal cells. These ALPase-positive cells were connected to each other by cytoplasmic processes, forming a cellular network in bone marrow. The ALPase activity of fibroblastic stromal cells tended to be stronger in those cells close to the bone surface than in the cells in the center of bone marrow. Reticular fibers in bone marrow were found to form a network. The ALPase-positive fibroblastic stromal cells may be reticular cells, because the localization of those cells was in accord with the localization of reticular fibers. The TRAPase-positive mononuclear cells and osteoclasts were mostly observed to be associated with the intensely ALPase-positive fibroblastic stromal cells. Immunoreactivity of osteoclast differentiation factor (ODF) was found in the fibroblastic stromal cells. These findings suggest that the network of ALPase-positive fibroblastic stromal cells in bone marrow serves as a guide for the migration of osteoclast precursor cells toward the bone surface, and may control the differentiation and activity of osteoclasts.