本文關(guān)鍵詞： 荷斯坦公犢 臍帶間充質(zhì)干細(xì)胞 生長性能 血清生化　出處：《新疆農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：為了探究注射臍帶間充質(zhì)干細(xì)胞對荷斯坦公犢生產(chǎn)性能及血清生化指標(biāo)的影響,試驗一采集健康荷斯坦新生犢牛的臍帶組織,通過組織塊貼壁法和胰酶消化篩選法體外分離、純化及培養(yǎng)牛臍帶間充質(zhì)干細(xì)胞(UC-MSCs),并觀察其生物學(xué)特性,繪制第1、5及10代細(xì)胞生長曲線,茜素紅染色檢測其體外誘導(dǎo)成骨分化能力,油紅O染色法檢測其體外誘導(dǎo)成脂能力。結(jié)果表明,獲得的細(xì)胞經(jīng)傳代培養(yǎng)依然保持良好的形態(tài),增殖能力保持與1-5代一致。體外誘導(dǎo)成骨細(xì)胞21 d,細(xì)胞培養(yǎng)板中出現(xiàn)骨骼細(xì)胞鈣化結(jié)節(jié),經(jīng)茜素紅染色呈紅色結(jié)節(jié),證明其具有被誘導(dǎo)分化為骨細(xì)胞的能力;體外誘導(dǎo)成脂細(xì)胞14 d,細(xì)胞培養(yǎng)板中現(xiàn)脂肪滴,證明其具有被誘導(dǎo)分化為脂肪細(xì)胞的能力,試驗表明牛臍帶間充質(zhì)干細(xì)胞能在體外培養(yǎng)、增殖并且具有間充質(zhì)干細(xì)胞的分化能力特性。試驗二選取1月齡體重相近的荷斯坦公犢28頭,隨機分為對照組和試驗組,將試驗一培養(yǎng)的臍帶間充質(zhì)干細(xì)胞以3×105/kg稀釋至8 ml頸靜脈注射至試驗組公犢體內(nèi),對照組注射等量生理鹽水。試驗期154 d。試驗組和對照組日糧配方、營養(yǎng)水平和管理條件均相同。試驗從公犢1月齡注射,到6月齡結(jié)束,每次注射前空腹測體重、體高、體斜長、胸圍、管圍和腰角寬。頸靜脈懫血5 ml,3500r/min離心15 min,收集血清-20°C保存。血清中Ca、P、總蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、甘油三酯(TG)、膽固醇(CHO)、堿性磷酸酶(ALP)、乳酸脫氫酶(LDH)、肌酸激酶(CK)、生長激素(GH)、瘦素(LEP)、生長抑素(SS)、甲狀腺素(T4)、三碘甲腺原氨酸(T3)、類胰島素生長因子(IGF-1)、血清中血管內(nèi)皮生長因子(VEGF)、成纖維細(xì)胞生長因子(bFGF)、轉(zhuǎn)化生長因子(TGF-α)、骨形態(tài)發(fā)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)、干擾素γ(IFN-γ)、白細(xì)胞介素1(IL-1)、白細(xì)胞介素2(IL-2)、白細(xì)胞介素6(IL-6)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量均委托北京華英生物有限公司進行測定。試驗結(jié)果:注射UC-MSCs后,試驗組公犢平均體重在4、5、6月齡顯著高于對照組(P0.05);試驗組公犢平均日增重在3、4月齡顯著高于對照組(P0.05);而在6月齡時,極顯著高于對照組(P0.01);試驗組各項體尺指標(biāo)在公犢生長發(fā)育高峰期時均高于對照組,且差異顯著(P0.05);試驗組血清中P含量在3、4、6月齡時顯著高于對照組(P0.05),試驗組血清中Ca含量在5、6月齡時顯著高于對照組(P0.05);4月齡時,試驗組血清中ALP、LDH、CK均顯著高于對照組(P0.05),試驗組血清中BUN水平顯著低于對照組(P0.05);5月齡時,試驗組血清中Alb、ALP水平顯著高于對照組(P0.05);6月齡時,試驗組血清中TP水平顯著高于對照組(P0.05),且兩組血清中CK水平差異極顯著(P0.01);4月齡時,試驗組血清SS水平顯著低于對照組(P0.05);6月齡時,試驗組血清中T3、T4水平表現(xiàn)差異極顯著(P0.01);試驗組血清中IGF-1在4月齡時顯著高于對照組(P0.05);3月齡時,對照組與試驗組血清中VEGF、TGF-α表現(xiàn)顯著差異(P0.05);6月齡時,試驗組血清中VEGF、bFGF、TGF-α水平均顯著高于對照組(P0.05);2、3、4、6月齡時,兩組血清中BMP-7出現(xiàn)顯著差異(P0.05);試驗組血清中IgA在4月齡時極顯著高于對照組(P0.01),6月齡時顯著高于對照組(P0.05);兩組血清中IgG水平在2、5月齡表現(xiàn)差異極顯著(P0.01),6月齡時差異顯著(P0.05);3、4月齡時,試驗組血清中白細(xì)胞介素2(IL-2)水平極顯著(P0.01)或顯著(P0.05)低于對照組;試驗組血清中MDA水平在2、3、4、5、6月齡時均極顯著低于對照組(P0.01);5月齡時,試驗組血清中SOD水平顯著高于對照組(P0.05),6月齡時表現(xiàn)極顯著(P0.01)。結(jié)論:注射UC-MSCs能夠顯著提高公犢血清中Ca、P、TP、Alb、ALP、LDH的含量,降低血清中BUN、CK水平,從而穩(wěn)定機體內(nèi)環(huán)境,促進公犢機體蛋白、骨骼的合成和沉積,且顯著提高了公犢血清中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,血清中這些生長因子和激素水平與公犢的生長發(fā)育密切相關(guān),間接影響其增重和體尺,增加荷斯坦公犢的發(fā)育速度,并通過顯著提高公犢牛血清中IgA、IgG等免疫因子水平,降低血清中MDA水平,提高SOD活性,增強機體清除自由基的能力,降低血清中炎性因子干擾素γ(IFN-γ)、白細(xì)胞介素1(IL-1)、IL-2、白細(xì)胞介素6(IL-6)濃度,減少內(nèi)部炎癥發(fā)生,從而提高公犢機體免疫力,促進公犢健康生長發(fā)育,進而影響其生長性能。
[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 ),灝跨礌姘，

本文編號：1468539