本文選題：未羧化骨鈣素 + 氟化鈉��； 參考：《新疆醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分過(guò)量氟對(duì)機(jī)體內(nèi)骨鈣素含量與糖代謝的影響目的:探討不同劑量氟作用于機(jī)體后對(duì)骨鈣素及糖代謝的影響。方法:1.選取確診為氟骨癥的65例患者為氟骨癥組,診斷無(wú)骨病變且排除相關(guān)骨骼疾病的其他65例病人作對(duì)照組。記錄一般情況,計(jì)算體質(zhì)指數(shù)(Body Mass Index,BMI),酶聯(lián)免疫吸附實(shí)驗(yàn)法(Enzyme linked immunosorbent assay,ELISA)測(cè)定血清總骨鈣素(total Osteocalcin,t OCN)、未羧化骨鈣素(uncarboxylated Osteocalcin,ucOCN)、空腹胰島素(Fasting Insulin,FINS)含量,化學(xué)法測(cè)定血糖(Glucose,GLU)、糖化血清蛋白(Glycated serum protein,GSP)含量,計(jì)算胰島素抵抗指數(shù)(Insulin resistence index,HOMA-IR)。2.18-24g的C57雄性小鼠36只分為3組,0 mg/L、100 mg/L、150 mg/LNaF干預(yù),2、4、6、8、10、12 W后檢測(cè)體質(zhì)量。12 W后化學(xué)發(fā)光法檢測(cè)血糖、糖化血紅蛋白(Glycosylated hemoglobin,Hb A1c)含量,ELISA法檢測(cè)t OCN、ucOCN、FINS、胰高血糖素(Glucagon)含量。蘇木精-伊紅染色法(Hematoxylin-Eosin staining,HE)染色觀察胰腺病理學(xué)變化,免疫組化檢測(cè)胰腺骨鈣素(Osteocalcin,OCN)、周期蛋白D2(Cyclin D2)的表達(dá)。結(jié)果:1.氟骨癥組與對(duì)照組比較ucOCN、FINS、GSP明顯增高(P0.05)。氟骨癥組t OCN、HOMA-IR高于對(duì)照組,但無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。氟骨癥組GLU低于對(duì)照組無(wú)統(tǒng)計(jì)學(xué)意義。相關(guān)回歸分析顯示ucOCN與FINS呈正相關(guān)(P0.05)。ucOCN與GLU、HOMA-IR呈負(fù)相關(guān)(P0.05),ucOCN與BMI、t OCN、GSP無(wú)相關(guān)性(P0.05)。2.(1)各染氟組間t OCN、ucOCN含量比較差異均有統(tǒng)計(jì)學(xué)意義(F=32.31、17.41,P均0.05)。其中各染氟組小鼠t OCN、ucOCN均高于對(duì)照組。組間GLU比較差異均有統(tǒng)計(jì)學(xué)意義(F=22.75,P均0.05),其中各染氟組小鼠GLU均高于對(duì)照組。組間Hb A1c、Glucagon、FINS含量比較差異均有統(tǒng)計(jì)學(xué)意義(F=102.40、73.61、5.32,P均0.05),其中各染氟組Hb A1c、FINS均高于對(duì)照組。各染氟組Glucagon含量均低于對(duì)照組。ucOCN與t OCN、FINS呈正相關(guān),與GLU、Hb A1c呈負(fù)相關(guān),與Glucagon無(wú)相關(guān)性。(2)病理學(xué)檢查:150 mg/L染氟組腺泡細(xì)胞形狀偶見(jiàn)不規(guī)則,少量細(xì)胞界限稍模糊,部分胰島細(xì)胞細(xì)胞核變少,分布較稀疏。(3)染氟組小鼠胰腺中OCN表達(dá)呈陰性,Cylin D2的表達(dá)呈陽(yáng)性。結(jié)論1.過(guò)量氟可導(dǎo)致氟骨癥患者與小鼠血清2ucOCN顯著升高,表現(xiàn)為骨組織增殖的骨相損傷。2.過(guò)量氟可導(dǎo)致氟骨癥患者與小鼠糖代謝紊亂,表現(xiàn)為糖代謝異常的非骨相損傷。3.過(guò)量氟作用時(shí),ucOCN與FINS有相關(guān)性。第二部分不同劑量氟對(duì)體外培養(yǎng)胰島β細(xì)胞功能的影響目的:觀察不同劑量氟化鈉(NaF)對(duì)體外培養(yǎng)的小鼠胰島β細(xì)胞增殖活力和胰島素(Insulin,INS)分泌的影響。方法:0、0.1、0.5、1.0、2.0、4.0、8.0、16.0 mg/LNaF分別干預(yù)小鼠胰島Beta-TC-6細(xì)胞24 h、48 h、72 h、96 h后,HE染色觀察細(xì)胞形態(tài),噻唑藍(lán)(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl-2-H-Tetrazolium Bromide,MTT)比色法檢測(cè)細(xì)胞增殖活力;ELISA法測(cè)定胰島素分泌變化。結(jié)果:0.5 mg/L、1.0 mg/LNaF作用72 h時(shí),可使胰島β細(xì)胞增殖活力和胰島素分泌較對(duì)照組明顯增強(qiáng)(P(27)0.05);≥8.0mg/L時(shí)隨著NaF劑量的增加和作用時(shí)間的延長(zhǎng),細(xì)胞增殖活力和胰島素分泌明顯減弱(P(27)0.05),且細(xì)胞生長(zhǎng)逐漸緩慢,數(shù)量減少,不易貼壁或融合成片,多呈橢圓或圓形細(xì)胞。結(jié)論:低劑量NaF促進(jìn)胰島β細(xì)胞增殖和胰島素分泌,隨著劑量增大和時(shí)間延長(zhǎng),過(guò)量NaF對(duì)細(xì)胞增殖活力和胰島素分泌能力的抑制逐漸增強(qiáng),呈劑量效應(yīng)關(guān)系。第三部分ucOCN-GPRC6A通路阻斷后過(guò)量氟對(duì)體外培養(yǎng)胰島β細(xì)胞功能的影響目的:阻斷(未羧化骨鈣素-G蛋白耦聯(lián)受體C家族6組成員A,uncarboxylated Osteocalcin-G protein coupled receptor family C,Group 6,subtype A,ucOCN-GPRC6A)通路后,觀察過(guò)量氟對(duì)體外培養(yǎng)小鼠胰島Beta-TC-6細(xì)胞INS分泌、增殖能力的影響,探討氟是否通過(guò)ucOCN-GPRC6A通路影響小鼠胰島β細(xì)胞功能。方法:選取ucOCN-GPRC6A通路阻斷和未阻斷兩組小鼠胰島Beta-TC-6細(xì)胞,8 mg/LNaF、150mmol/LucOCN、8 mg/L NaF+150 mmol/LucOCN分別干預(yù)48 h,ELISA法測(cè)INS含量,提取總RNA、蛋白,RT-PCR、Western blot法檢測(cè)Cyclin D1、Cyclin D2表達(dá)。結(jié)果:1.非轉(zhuǎn)染:INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)NaF組較空白組顯著降低(P(27)0.05),ucOCN組INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)較空白組升高,NaF+ucOCN組INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)較空白組、ucOCN組降低,較NaF組升高。2.轉(zhuǎn)染:ucOCN組INS分泌水平、Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)較空白無(wú)顯著差異。NaF+ucOCN組INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)較空白組降低(P(27)0.05),較NaF染毒組無(wú)統(tǒng)計(jì)學(xué)意義。3.兩組間比較:非轉(zhuǎn)染ucOCN組較轉(zhuǎn)染ucOCN組INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)顯著升高(P(27)0.05);非轉(zhuǎn)染NaF+ucOCN組較轉(zhuǎn)染NaF+ucOCN組INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)顯著升高(P(27)0.05);非轉(zhuǎn)染空白組、NaF染毒組與轉(zhuǎn)染空白組、NaF染毒組INS分泌水平,Cyclin D1、Cyclin D2m RNA、蛋白的表達(dá)無(wú)顯著差異。結(jié)論:1.ucOCN能夠減輕過(guò)量氟對(duì)胰島細(xì)胞的損傷效應(yīng)。2.氟可通過(guò)ucOCN-GPRC6A通路作用于胰島細(xì)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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R599

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