注射脐带间充质干细胞对荷斯坦公犊生长性能及血清生化指标的影响

发布时间：2018-01-27 13:07

  本文关键词： 荷斯坦公犊 脐带间充质干细胞 生长性能 血清生化　出处：《新疆农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：为了探究注射脐带间充质干细胞对荷斯坦公犊生产性能及血清生化指标的影响,试验一采集健康荷斯坦新生犊牛的脐带组织,通过组织块贴壁法和胰酶消化筛选法体外分离、纯化及培养牛脐带间充质干细胞(UC-MSCs),并观察其生物学特性,绘制第1、5及10代细胞生长曲线,茜素红染色检测其体外诱导成骨分化能力,油红O染色法检测其体外诱导成脂能力。结果表明,获得的细胞经传代培养依然保持良好的形态,增殖能力保持与1-5代一致。体外诱导成骨细胞21 d,细胞培养板中出现骨骼细胞钙化结节,经茜素红染色呈红色结节,证明其具有被诱导分化为骨细胞的能力;体外诱导成脂细胞14 d,细胞培养板中现脂肪滴,证明其具有被诱导分化为脂肪细胞的能力,试验表明牛脐带间充质干细胞能在体外培养、增殖并且具有间充质干细胞的分化能力特性。试验二选取1月龄体重相近的荷斯坦公犊28头,随机分为对照组和试验组,将试验一培养的脐带间充质干细胞以3×105/kg稀释至8 ml颈静脉注射至试验组公犊体内,对照组注射等量生理盐水。试验期154 d。试验组和对照组日粮配方、营养水平和管理条件均相同。试验从公犊1月龄注射,到6月龄结束,每次注射前空腹测体重、体高、体斜长、胸围、管围和腰角宽。颈静脉懫血5 ml,3500r/min离心15 min,收集血清-20°C保存。血清中Ca、P、总蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、甘油三酯(TG)、胆固醇(CHO)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)、肌酸激酶(CK)、生长激素(GH)、瘦素(LEP)、生长抑素(SS)、甲状腺素(T4)、三碘甲腺原氨酸(T3)、类胰岛素生长因子(IGF-1)、血清中血管内皮生长因子(VEGF)、成纤维细胞生长因子(bFGF)、转化生长因子(TGF-α)、骨形态发生蛋白(BMP-7)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白G(IgG)、干扰素γ(IFN-γ)、白细胞介素1(IL-1)、白细胞介素2(IL-2)、白细胞介素6(IL-6)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量均委托北京华英生物有限公司进行测定。试验结果:注射UC-MSCs后,试验组公犊平均体重在4、5、6月龄显著高于对照组(P0.05);试验组公犊平均日增重在3、4月龄显著高于对照组(P0.05);而在6月龄时,极显著高于对照组(P0.01);试验组各项体尺指标在公犊生长发育高峰期时均高于对照组,且差异显著(P0.05);试验组血清中P含量在3、4、6月龄时显著高于对照组(P0.05),试验组血清中Ca含量在5、6月龄时显著高于对照组(P0.05);4月龄时,试验组血清中ALP、LDH、CK均显著高于对照组(P0.05),试验组血清中BUN水平显著低于对照组(P0.05);5月龄时,试验组血清中Alb、ALP水平显著高于对照组(P0.05);6月龄时,试验组血清中TP水平显著高于对照组(P0.05),且两组血清中CK水平差异极显著(P0.01);4月龄时,试验组血清SS水平显著低于对照组(P0.05);6月龄时,试验组血清中T3、T4水平表现差异极显著(P0.01);试验组血清中IGF-1在4月龄时显著高于对照组(P0.05);3月龄时,对照组与试验组血清中VEGF、TGF-α表现显著差异(P0.05);6月龄时,试验组血清中VEGF、bFGF、TGF-α水平均显著高于对照组(P0.05);2、3、4、6月龄时,两组血清中BMP-7出现显著差异(P0.05);试验组血清中IgA在4月龄时极显著高于对照组(P0.01),6月龄时显著高于对照组(P0.05);两组血清中IgG水平在2、5月龄表现差异极显著(P0.01),6月龄时差异显著(P0.05);3、4月龄时,试验组血清中白细胞介素2(IL-2)水平极显著(P0.01)或显著(P0.05)低于对照组;试验组血清中MDA水平在2、3、4、5、6月龄时均极显著低于对照组(P0.01);5月龄时,试验组血清中SOD水平显著高于对照组(P0.05),6月龄时表现极显著(P0.01)。结论:注射UC-MSCs能够显著提高公犊血清中Ca、P、TP、Alb、ALP、LDH的含量,降低血清中BUN、CK水平,从而稳定机体内环境,促进公犊机体蛋白、骨骼的合成和沉积,且显著提高了公犊血清中VEGF、TGF-α、bFGF、BMP-7水平,降低血清中GH、SS、LEP激素水平,提高血清中T4、T3水平,血清中这些生长因子和激素水平与公犊的生长发育密切相关,间接影响其增重和体尺,增加荷斯坦公犊的发育速度,并通过显著提高公犊牛血清中IgA、IgG等免疫因子水平,降低血清中MDA水平,提高SOD活性,增强机体清除自由基的能力,降低血清中炎性因子干扰素γ(IFN-γ)、白细胞介素1(IL-1)、IL-2、白细胞介素6(IL-6)浓度,减少内部炎症发生,从而提高公犊机体免疫力,促进公犊健康生长发育,进而影响其生长性能。
[Abstract]:In order to explore the injection effect of human umbilical cord mesenchymal stem cells on the production performance of Holstein bull calves and serum biochemical indexes, a test collection healthy Holstein newborn calves umbilical cord tissues by tissue adherent method and trypsin digestion method in vitro screening separation, purification and culture of bovine umbilical cord mesenchymal stem cells (UC-MSCs), and to observe its biological the characteristics of 1,5 and the 10 generation, draw the cell growth curve, alizarin red staining was used to detect the in vitro osteogenic ability, detection of adipogenic ability in vitro by oil red O staining. The results showed that the cells obtained by subculture still maintain good shape, proliferation ability is consistent with the 1-5 generation of in vitro induced osteogenesis. 21 d cells, cell culture bone calcification in the plate, with alizarin red staining showed red nodules, were induced to prove its ability to differentiate into bone cells; in vitro induced adipogenic cells 14 d, fine The cell culture plate is fat droplets, prove its ability to differentiate into fat cells, test showed that bovine umbilical cord mesenchymal stem cells can be cultured in vitro, proliferation and differentiation of mesenchymal stem cell properties. Experiment two selected 1 month old Holstein male calves with similar body weight 28, were randomly divided into control group and the test group, will test a culture of umbilical cord mesenchymal stem cells to 3 x 105/kg diluted to 8 ml intravenous injection to experimental group calves in control group normal saline injection. The test period of 154 D. experimental group and control group diet, nutrition level and management conditions were the same from the test. The male calves 1 month old injection, and ended in 6 month old, before each injection measured the weight, body height, body length, chest circumference, circumference and waist width. Zhi jugular vein blood 5 ml 3500r/min centrifugation for 15 min, serum -20 ~ C preservation. The levels of Ca, P, total protein, albumin (TP) (ALB ),灏跨礌姘，

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