未羧化骨钙素对氟致糖代谢紊乱的调节作用

发布时间：2018-06-16 10:39

本文选题：未羧化骨钙素 + 氟化钠　；参考：《新疆医科大学》2017年硕士论文

【摘要】：第一部分过量氟对机体内骨钙素含量与糖代谢的影响目的:探讨不同剂量氟作用于机体后对骨钙素及糖代谢的影响。方法:1.选取确诊为氟骨症的65例患者为氟骨症组,诊断无骨病变且排除相关骨骼疾病的其他65例病人作对照组。记录一般情况,计算体质指数(Body Mass Index,BMI),酶联免疫吸附实验法(Enzyme linked immunosorbent assay,ELISA)测定血清总骨钙素(total Osteocalcin,t OCN)、未羧化骨钙素(uncarboxylated Osteocalcin,ucOCN)、空腹胰岛素(Fasting Insulin,FINS)含量,化学法测定血糖(Glucose,GLU)、糖化血清蛋白(Glycated serum protein,GSP)含量,计算胰岛素抵抗指数(Insulin resistence index,HOMA-IR)。2.18-24g的C57雄性小鼠36只分为3组,0 mg/L、100 mg/L、150 mg/LNaF干预,2、4、6、8、10、12 W后检测体质量。12 W后化学发光法检测血糖、糖化血红蛋白(Glycosylated hemoglobin,Hb A1c)含量,ELISA法检测t OCN、ucOCN、FINS、胰高血糖素(Glucagon)含量。苏木精-伊红染色法(Hematoxylin-Eosin staining,HE)染色观察胰腺病理学变化,免疫组化检测胰腺骨钙素(Osteocalcin,OCN)、周期蛋白D2(Cyclin D2)的表达。结果:1.氟骨症组与对照组比较ucOCN、FINS、GSP明显增高(P0.05)。氟骨症组t OCN、HOMA-IR高于对照组,但无统计学意义(P0.05)。氟骨症组GLU低于对照组无统计学意义。相关回归分析显示ucOCN与FINS呈正相关(P0.05)。ucOCN与GLU、HOMA-IR呈负相关(P0.05),ucOCN与BMI、t OCN、GSP无相关性(P0.05)。2.(1)各染氟组间t OCN、ucOCN含量比较差异均有统计学意义(F=32.31、17.41,P均0.05)。其中各染氟组小鼠t OCN、ucOCN均高于对照组。组间GLU比较差异均有统计学意义(F=22.75,P均0.05),其中各染氟组小鼠GLU均高于对照组。组间Hb A1c、Glucagon、FINS含量比较差异均有统计学意义(F=102.40、73.61、5.32,P均0.05),其中各染氟组Hb A1c、FINS均高于对照组。各染氟组Glucagon含量均低于对照组。ucOCN与t OCN、FINS呈正相关,与GLU、Hb A1c呈负相关,与Glucagon无相关性。(2)病理学检查:150 mg/L染氟组腺泡细胞形状偶见不规则,少量细胞界限稍模糊,部分胰岛细胞细胞核变少,分布较稀疏。(3)染氟组小鼠胰腺中OCN表达呈阴性,Cylin D2的表达呈阳性。结论1.过量氟可导致氟骨症患者与小鼠血清2ucOCN显著升高,表现为骨组织增殖的骨相损伤。2.过量氟可导致氟骨症患者与小鼠糖代谢紊乱,表现为糖代谢异常的非骨相损伤。3.过量氟作用时,ucOCN与FINS有相关性。第二部分不同剂量氟对体外培养胰岛β细胞功能的影响目的:观察不同剂量氟化钠(NaF)对体外培养的小鼠胰岛β细胞增殖活力和胰岛素(Insulin,INS)分泌的影响。方法:0、0.1、0.5、1.0、2.0、4.0、8.0、16.0 mg/LNaF分别干预小鼠胰岛Beta-TC-6细胞24 h、48 h、72 h、96 h后,HE染色观察细胞形态,噻唑蓝(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl-2-H-Tetrazolium Bromide,MTT)比色法检测细胞增殖活力;ELISA法测定胰岛素分泌变化。结果:0.5 mg/L、1.0 mg/LNaF作用72 h时,可使胰岛β细胞增殖活力和胰岛素分泌较对照组明显增强(P(27)0.05);≥8.0mg/L时随着NaF剂量的增加和作用时间的延长,细胞增殖活力和胰岛素分泌明显减弱(P(27)0.05),且细胞生长逐渐缓慢,数量减少,不易贴壁或融合成片,多呈椭圆或圆形细胞。结论:低剂量NaF促进胰岛β细胞增殖和胰岛素分泌,随着剂量增大和时间延长,过量NaF对细胞增殖活力和胰岛素分泌能力的抑制逐渐增强,呈剂量效应关系。第三部分ucOCN-GPRC6A通路阻断后过量氟对体外培养胰岛β细胞功能的影响目的:阻断(未羧化骨钙素-G蛋白耦联受体C家族6组成员A,uncarboxylated Osteocalcin-G protein coupled receptor family C,Group 6,subtype A,ucOCN-GPRC6A)通路后,观察过量氟对体外培养小鼠胰岛Beta-TC-6细胞INS分泌、增殖能力的影响,探讨氟是否通过ucOCN-GPRC6A通路影响小鼠胰岛β细胞功能。方法:选取ucOCN-GPRC6A通路阻断和未阻断两组小鼠胰岛Beta-TC-6细胞,8 mg/LNaF、150mmol/LucOCN、8 mg/L NaF+150 mmol/LucOCN分别干预48 h,ELISA法测INS含量,提取总RNA、蛋白,RT-PCR、Western blot法检测Cyclin D1、Cyclin D2表达。结果:1.非转染:INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达NaF组较空白组显著降低(P(27)0.05),ucOCN组INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达较空白组升高,NaF+ucOCN组INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达较空白组、ucOCN组降低,较NaF组升高。2.转染:ucOCN组INS分泌水平、Cyclin D1、Cyclin D2m RNA、蛋白的表达较空白无显著差异。NaF+ucOCN组INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达较空白组降低(P(27)0.05),较NaF染毒组无统计学意义。3.两组间比较:非转染ucOCN组较转染ucOCN组INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达显著升高(P(27)0.05);非转染NaF+ucOCN组较转染NaF+ucOCN组INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达显著升高(P(27)0.05);非转染空白组、NaF染毒组与转染空白组、NaF染毒组INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表达无显著差异。结论:1.ucOCN能够减轻过量氟对胰岛细胞的损伤效应。2.氟可通过ucOCN-GPRC6A通路作用于胰岛细胞。
[Abstract]:Part 1 the effect of excess fluorine on the content of osteocalcin and glucose metabolism in the body: To explore the effect of fluorine on the body's osteocalcin and glucose metabolism after the different doses of fluorine. Methods: 1. to select 65 patients with fluorosalosis as the fluoroslosis group, to diagnose no osteosis and to exclude the related bone diseases in 65 other patients as control group. The Body Mass Index (BMI) and the enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) were used to determine serum total Osteocalcin (total Osteocalcin, t), non carboxylation of osteocalcin, fasting insulin content, and chemical determination of blood glucose. The Glycated serum protein (GSP) content of glycosylated serum protein (GSP) and the insulin resistance index (Insulin resistence index, HOMA-IR).2.18-24g C57 male mice were divided into 3 groups, 0 mg/L, 100 mg/L, 150 intervening. Obin, Hb A1c) content, t OCN, ucOCN, FINS, and glucagon (Glucagon) content by ELISA method. The pathological changes of pancreas were observed by hematoxylin eosin staining (Hematoxylin-Eosin staining, HE) staining. Immunohistochemistry was used to detect the expression of pancreatic osteocalcin and periodic protein. Results: 1. fluorosis group was compared with the control group. OCN, FINS and GSP were significantly higher (P0.05). T OCN in skeletal fluorosis group was higher than that of control group, but there was no statistical significance (P0.05). The GLU in fluorosis group was less than that of the control group. There were significant differences in the content of t OCN and ucOCN between the fluorine groups (F=32.31,17.41, P 0.05). The t OCN and ucOCN in each fluorine dyed mice were higher than those in the control group. The difference of GLU in the groups was statistically significant (F=22.75, P was 0.05), and all of the mice in the fluorine dyed group were all higher than those in the control group. The study significance (F=102.40,73.61,5.32, P 0.05), among which the Hb A1c and FINS in each fluorine dyed group were higher than those in the control group. The Glucagon content in the fluorine dyed group was lower than that of the control group.UcOCN and t OCN, and the FINS was positively correlated with GLU, and there was no correlation with the Hb. (2) pathological examination: 150 of the fluorine group acinar cells were irregular and a small number of cells. The limit was slightly blurred, the cell nuclei of some islet cells were less and the distribution was thinner. (3) the expression of OCN in the pancreas of the mice infected with fluorine was negative, and the expression of Cylin D2 was positive. Conclusion 1. excessive fluoride can lead to a significant increase in the serum 2ucOCN of the patients with skeletal fluorosis and the bone tissue injury of bone tissue, and the excessive fluoride of.2. can lead to the fluorosis and the mice. The effects of ucOCN and FINS on the function of pancreatic islet beta cells in vitro culture in second different doses of fluoride (NaF) were observed to observe the effect of different doses of sodium fluoride (NaF) on the proliferation of pancreatic islet beta cells in vitro and the secretion of insulin (Insulin, INS) in vitro. The effect of the second different doses of fluoride on the function of pancreatic islet beta cells in vitro Methods: 0,0.1,0.5,1.0,2.0,4.0,8.0,16.0 mg/LNaF interfered with mouse pancreatic islet Beta-TC-6 cells 24 h, 48 h, 72 h and 96 h respectively. HE staining was used to observe cell morphology, thiazolium (4,5-Dimethyl-2-Thiazolyl) -2,5-Diphenyl-2-H-Tetrazolium Bromide, MTT) cell proliferation activity was detected by colorimetric assay, and the changes of insulin secretion were measured by the method of 0.5. Results 0.5 When mg/L, 1 mg/LNaF acted as 72 h, the proliferation activity of islet beta cells and insulin secretion were significantly enhanced (P (27) 0.05). With the increase of NaF dose and the prolongation of the action time, the cell proliferation activity and insulin secretion were obviously weakened (P (27) 0.05) at the time of 8.0mg/L, and the cell growth was gradually slow and the quantity decreased, and it was not easy to adhere to the wall or melt. Conclusion: low dose NaF promotes the proliferation and insulin secretion of islet beta cells. With the increase of dose and prolongation of time, the inhibitory effect of excess NaF on cell proliferation and insulin secretion is gradually increased, and there is a dose effect relationship. After third division of ucOCN-GPRC6A pathway, excessive fluorine is cultured in vitro. The effect of islet beta cell function: To observe the effect of excessive fluoride on the secretion and proliferation of rat pancreatic islet cells in vitro after blocking the 6 group of A, uncarboxylated Osteocalcin-G protein coupled receptor family C, Group 6, Group 6. Whether fluorine could affect the function of pancreatic islet beta cells in mice through ucOCN-GPRC6A pathway. Methods: the ucOCN-GPRC6A pathway was selected to block and not block the islet Beta-TC-6 cells of two groups of mice. 8 mg/LNaF, 150mmol/LucOCN, and 8 mg/L NaF+150 mmol/LucOCN were treated with 48 h respectively, and INS content was measured by ELISA method. Results: 1. non transfection: INS secretion level, Cyclin D1, Cyclin D2m RNA, the expression of protein in NaF group is significantly lower than that in the blank group (P (27) 0.05), ucOCN group INS secretion level, protein expression is higher than that in the blank group. Group NaF increased.2. transfection: INS secretion in group ucOCN, Cyclin D1, Cyclin D2m RNA, and there was no significant difference in the expression of protein in the INS secretion level of.NaF+ucOCN group, and the expression of protein was lower than that of the blank group (27), compared with that of the blank group (0.05). UcOCN group INS secretion level, Cyclin D1, Cyclin D2m RNA, the protein expression increased significantly (P (27) 0.05), non transfected NaF+ucOCN group compared with the INS secretion of NaF+ucOCN group, Cyclin, protein expression significantly increased (27) 0.05); 1, Cyclin D2m RNA, there is no significant difference in the expression of protein. Conclusion: 1.ucOCN can reduce the damage effect of excessive fluoride on islet cells.2. fluorine can act on islet cells through ucOCN-GPRC6A pathway.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R599

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