【作者】 张皎；

【导师】 侯加法；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2004， 博士

【摘要】 1．建立有效的鸡胚额骨成骨细胞的培养方法，为笼养蛋鸡骨质疏松症研究提供新的试验手段。主要剔取15日龄鸡胚颅骨，剪碎，采用混合Ⅱ型胶原酶和胰酶的方法，进行两次消化，获取鸡胚额骨原代成骨细胞群。经消化培养的细胞具有明显的成骨细胞样特点：形态学观察显示成纤维母细胞型，有长梭形、三角形、星形；组化染色结果：碱性磷酸酶黑染、基质前体红染；长期培养能形成肉眼可见的白色矿化结节，茜素红染色为红色结节。总之酶混合消化可以得到较纯的成骨细胞，具有明显的成骨细胞特征。 2．用建立的鸡胚额骨成骨细胞的培养方法，体外直接研究IP98对蛋鸡成骨细胞增殖和分化功能的影响。采用混合胶原酶和胰酶、两次消化的方法，获取成骨细胞，经培养传代，取状态良好的第二、三代细胞在96孔板上进行增殖率和碱性磷酸酶活性的测定。发现酶消化法获得的细胞具有明显的成骨细胞的特点：属于成纤维母细胞型、碱性磷酸酶黑染、基质前体红染，长期培养能形成矿化结节。应用IP98后发现10^-4M IP可抑制成骨细胞增殖，而10^-8M对改善成骨细胞的增值和分化有显著功效（p＜0.01），小于10^-10M浓度的IP则对成骨细胞与对照组无差异。酶混合消化可以得到较纯的成骨细胞群，能够满足药物研究所需，应用IP98后试验结果表明，适宜浓度的IP可促进鸡胚额骨成骨细胞增殖合和分化，提高其碱性磷酸酶活性，增强成骨细胞矿化，促使骨形成。 3．500羽58周龄ISA蛋鸡分为5组，A组为对照组，B、C、D、E组在基础日粮上添加15、25、50、100ppm的IP98，持续饲喂70天。每月采血测血钙、血磷、骨钙素、碱性磷酸酶、雌激素，记录体重，测骨矿含量。每天记录产蛋量、蛋重、破壳蛋数。结果表明：C组产蛋率显著高于对照组和其他实验组，D、E组产蛋率显著低于A、B、C组。C组蛋重、破壳率优于其他各组，差异不显著：各组体重无显著差异；C组血磷显著低于其他各组，B组血钙显著低于其他各组，C组碱性磷酸酶、骨钙素均显著高于其他各组；实验各组雌激素水平上升、无显著差异。结论：日粮中添加适量（25ppm）IP98能改善骨代谢、促进生产性能、提高产蛋率。 4．扩增出准确的ISA蛋鸡ICF-Ⅰ基因序列用于多态性分析。收集鸡血红细胞，提取红细胞基因组DNA，以此为模板，根据Nagaraja的方法适当改变PCR反应条笼养蛋鸡骨质疏松的IP98防治机理及骨质疏松与IGF一I的RFLP关系研究件，扩增出IGF一I启动子上游7Kb处长62lbp的基因片断，先凝胶电泳检验其正确性。再将该扩增片断克隆到PMD 18一T载体再进行序列测定，将测序结果和参照基因序列用DNAstar分析其同源性。l%琼脂糖凝胶电泳初步显示扩增出的目的条带为621bp的基因片断，测序报告所得的结果和参照基因有”%的同源性，说明PCR成功扩增出了目的片断，为下一步的试验研究、利用莫定基拙. 5.从分子遗传学角度来探讨ISA笼养蛋鸡胰岛素样生长因子一I限制性片断多态性与骨质疏松的关系。选择高产期ISA蛋鸡90只，应用PCR扩增鸡ICF一I启动子上游7kb处基因片断，用Pstl限制性内切酶消化产生限制性片断长度多态性，分析工GF一I多态性和胫骨、股骨、肚骨骨密度，骨代谢指标骨钙素和碱性磷酸酶之间的关系。结果显示90只蛋鸡PP、PP、PP基因型频率分别为0.256、0.311、0.433，P、p等位基因频率分另，i为0.4111、0.5889。方差分析（ANOVA）显示肚骨骨密度与ICF一I基因多态性相关，其PP基因型骨密度和骨矿含量均显著大于PP基因型（p<0 .05），股骨、胫骨基因型间无显著差异。骨钙素、碱性磷酸酶各基因型间无显著差异，PP基因型体重显著大于Pp基因型;逐步多元回归分析发现，体重与对各基因型的骨质有良好的影响，PP基因型股骨和胫骨骨质与骨钙素显著负相关，PP基因型肚骨。结论:ISA笼养蛋鸡IGF一I基因多态性和肚骨骨密度显著相关。较大体重有矛!」于笼养蛋鸡骨质发展。P等位基因具有一定的优良骨质指针作用，提示从分子水平探讨骨量丢失以及对骨质疏松症基因诊断和杭性基因选育有一定的显著意义。更多还原

【Abstract】 1.The more effective method of isolating the osteoblastic cells from calvariae of embryonic chicken was built in order to supply the further way to study the osteoporosis of cage laying hens. The osteoblastic cells was harvested by sequential enzyme digestion(0.2% cllogenase II and 0.25% trypsin) from bone chips isolated from calvariae of about 15-day embryonic chicken .The good second passage culture is used for identify. The cells were a classic characteristic of osteoblast: be fibroblastic, shape of shuttle, triangle and stellate; Black deposits of AKP, red deposits of promatrix were showed by cytochemical staining and form mineralized node for long culture. Osteoblastic cells could be harvested from the mixed sequential digestion method.2. To study the effect of different concentration of 1P98 on the osteoblastic cells about proliferation and liveness of AKP based on the method of isolating the osteoblastic cells from calvariae of embryonic chicken. The osteoblastic cells was harvested by sequential enzyme digestion (0.2% cllogenase II and 0.25% trypsin) from bone chips isolated from calvariae of embryonic chicken, Through identified, cells were of characteristic by osteoblast. The good second or third passage culture is used for research , which were seeded in 96-well plates at the density of 1.5 x104, which are cultured with 10-4-10-10M IP98 culture medium for 72h. Then MTT and PNPP method were made to assay the proliferation of and activity of AKP of osteoblastic cells. 10-4M IP98 distinctly inhibited the osteoblastic function, but the 10-6M IP98 obviously improved their function, the other concentratin of IP98 has no effect on the osteoblastic cells. The sequential digestion method could obtain pure chicken osteoblastic cells. The appropriate concentration of IP98 may promote the chicken osteoblastic cell’s function, namely, accelerating proliferation and differentiation.3. 500 ISA cage layers were divided into 5 groups. A group is control, IP98 was addto B, C, D, E groups at the dosage of 15ppm, 25ppm,50ppm, 100ppm respectively. The test was carried out for 70 days. Once each month, hen blood was collected to check the Ca, P, BGP, AKP, E2 and the hens were weighted, bone mineral content was tested. Each day, the number of eggs and damaged eggs and the weight of eggs were noted. The egg laying rate of C group was significantly higher than that of control and the other groups, the laying rate of D, E group were apparently lower than that of A, B, C groups. The mean egg weight and shell quality of C group were best. The body weight of C group is lightest, but these were not significant; the serous level of phosphor of C group and calcium of B group was significantly lower. The serous level of AKP and BGP of C group were significantly higher. There were no changes observed in serous level of E2 of all experiment groups. The study demonstrated that the appropriate dosage of IP98 may improve and the production of cage layer during the late cycle and bone metabolism.4. In order to analysis the restrictive fragment length polymorphism of insulin-like growth factor- I of ISA cage layer hens, the sequence of IGF- I gene is amplified exactly by polymerase chain reaction (PCR). Red cells were collected from the hens and the genetic DNA were extracted, as the template, the 621bp fragment located about 7Kb upstream of IGF- I promoter sequence was amplified by the improved Nagaraja’s method, its specificity was checked firstly by gel electrophoresis, and was cloned in pMD18-T vector, then sequenced. The sequencing data was compared with the previously published with the DNAstar software. It showed the 621bp gene fragment by 1% gel electrophoresis, there was 99% homologous between amplified gene and standard one. It is successfully amplify the objective gene, and supply the basis of the study of PCR-RFLP of IGF- I .5. To study the association between restrictive fragment length polymorphism (RFLP) and osteoporosis of ISA cage layer hens. Choos更多还原