【摘要】：第一部分FGF9對(duì)RANKL體外誘導(dǎo)破骨細(xì)胞分化的影響及其機(jī)制探討目的:利用經(jīng)RANKL體外誘導(dǎo)的破骨細(xì)胞,觀察FGF9對(duì)破骨細(xì)胞分化的影響并探討其可能機(jī)制。方法:利用RANKL誘導(dǎo)原代單核細(xì)胞法體外培養(yǎng)破骨細(xì)胞,細(xì)胞因子M-CSF、RANKL的濃度分別為30ng/ml,50ng/ml。采用兩種方法研究FGF9對(duì)破骨細(xì)胞的影響:(1)取4周齡FGF9WT/WT(WT)和FGF9WT/S99N(HE)小鼠各兩只,利用RANKL誘導(dǎo)原代單核細(xì)胞法體外培養(yǎng)破骨細(xì)胞。(2)在誘導(dǎo)過(guò)程中分別加入不同濃度(10ng、20ng、50ng)的FGF9蛋白,將未加入FGF9蛋白的作為對(duì)照組。定期更換培養(yǎng)液及誘導(dǎo)因子和相應(yīng)濃度的FGF9蛋白,直至破骨細(xì)胞分化成熟。行TRAP染色鑒定破骨細(xì)胞。倒置相差顯微鏡下觀察各組破骨細(xì)胞的形態(tài)并拍照。利用Image Pro Plus 6.0軟件測(cè)量每視野破骨細(xì)胞數(shù)量,并利用SPSS 21.0統(tǒng)計(jì)軟件進(jìn)行分析和檢驗(yàn)并繪制柱形圖。通過(guò)Real-Time PCR技術(shù)檢測(cè)FGF9對(duì)破骨細(xì)胞分化相關(guān)基因Trap、Mmp9、Ctsk表達(dá)量的影響。結(jié)果:(1)鏡下觀察可見(jiàn)WT組和HE組,10ng、20ng、50ng組和對(duì)照組均有形態(tài)不規(guī)則、多核的TRAP染色陽(yáng)性的破骨細(xì)胞生成,實(shí)驗(yàn)組(10ng、20ng、50ng組)與對(duì)照組相比,生成的TRAP染色陽(yáng)性的破骨細(xì)胞體積更大,數(shù)量更多,胞漿更為伸展。統(tǒng)計(jì)分析后顯示,WT組的破骨細(xì)胞多于HE組,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05);20ng組生成的破骨細(xì)胞數(shù)目最多,10ng次之,對(duì)照組生成的破骨細(xì)胞數(shù)目最少,實(shí)驗(yàn)組各組與對(duì)照組相比,差異均有統(tǒng)計(jì)學(xué)意義(分別為P0.05,P0.005,P0.05)。(2)破骨細(xì)胞分化相關(guān)基因(Trap、Mmp9、Ctsk)m RNA表達(dá)水平在兩組間比較顯示,與WT組相比,Trap和Mmp9 m RNA表達(dá)水平在HE組均明顯下調(diào)(P0.05,P0.001)。結(jié)論:成纖維細(xì)胞生長(zhǎng)因子9(FGF9)可促進(jìn)破骨前體細(xì)胞向破骨細(xì)胞的分化,并且其促進(jìn)作用與濃度無(wú)相關(guān)性。其作用機(jī)制可能通過(guò)影響破骨細(xì)胞分化相關(guān)基因Trap和Mmp9的轉(zhuǎn)錄水平進(jìn)而影響破骨細(xì)胞的分化。第二部分老年男性血清維生素D與甲狀旁腺激素及骨代謝指標(biāo)的相關(guān)性研究目的:探討老年男性血清維生素D水平,及其與甲狀旁腺激素及骨代謝指標(biāo)的相關(guān)性。方法:收集2010年9月至2013年9月在上海瑞金醫(yī)院老年病科病房住院及門(mén)診患者895例,平均年齡為76歲。測(cè)定其血清25-羥基維生素D[25-(OH)D]、血鈣(Ca)、血磷(P)、甲狀旁腺激素(PTH),I型原膠原分子N-端前肽(PINP)及β-I型膠原C端肽(β-CTX)水平。根據(jù)血清25-(OH)D水平將患者分為維生素D嚴(yán)重缺乏組(25nmol/L),維生素D缺乏組(25-50 nmol/L),維生素D不足組(50-75 nmol/L)和維生素D充足組(75 nmol/L)。結(jié)果:(1)895例老年男性患者年齡60~99歲,平均年齡76歲。血清25-(OH)D平均值為(43.52±21.97)nmol/L。維生素D缺乏者(≤50nmol/L)為592位(67%);維生素D不足者(50-75 nmol/L)為223人(25%);維生素D缺乏或不足者高達(dá)92%;維生素D充足者(75 nmol/L)僅為80人(8%)。(2)不同年齡段血清25-(OH)D的比較顯示,血清25-(OH)D水平隨增齡而逐漸降低。60~69歲組25-(OH)D值最高,為(46.27±20.76)nmol/L,與其它各組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。相關(guān)分析表明,血清25-(OH)D與年齡呈負(fù)相關(guān)(相關(guān)系數(shù)r=-0.088,P=0.008)。(3)甲狀旁腺素(PTH)的平均水平為(55.74±29.06)pg/ml。相關(guān)分析顯示,25-(OH)D與PTH、PINP、β-CTX均呈負(fù)相關(guān)(r值分別為:—0.209,-0.109,-0.122,P均0.05)。血25-(OH)D與Ca呈正相關(guān)(r=0.206,P=0)。血25-(OH)D與BMI、P均無(wú)相關(guān)性(P均0.05)。結(jié)論:老年男性存在嚴(yán)重的維生素D缺乏或不足。血25-(OH)D與PTH、年齡、PINP、β-CTX均呈負(fù)相關(guān)。
[Abstract]:The effect of part FGF9 on the differentiation of osteoclast induced by RANKL in vitro and its mechanism; Objective: To observe the effect of FGF9 on osteoclast differentiation and to explore its possible mechanism by using RANKL induced osteoclasts in vitro. Methods: using RANKL to induce the primary mononuclear cells to cultivate osteoclast, cytokine M-CSF, RANKL concentration in vitro 30ng/ml and 50ng/ml. were used to study the effect of FGF9 on osteoclasts: (1) 4 weeks old FGF9WT/WT (WT) and FGF9WT/S99N (HE) mice were used to induce osteoclasts in vitro by using RANKL to induce primary mononuclear cells. (2) the FGF9 proteins were added to different concentrations (10NG, 20ng, 50NG) in the induction process, and they would not be added to the protein. As a control group, the culture fluid and the inducible factor and the corresponding concentration of FGF9 protein were regularly replaced until the osteoclasts were differentiated and mature. TRAP staining was used to identify the osteoclasts. The morphology of the osteoclasts in each group was observed under the inverted phase contrast microscope and photographed. The number of osteoclasts in each field was measured with the Image Pro Plus 6 software, and the SPSS 21 system was used. The effects of FGF9 on the expression of osteoclast differentiation related genes, Trap, Mmp9, Ctsk were detected by Real-Time PCR. Results: (1) the observation of WT group and HE group, 10NG, 20ng, 50NG group and control group were irregular, multi nucleus TRAP stained osteoclast formation, experimental group (10NG, 20ng, 50NG) compared with the control group, the generated osteoclasts with positive TRAP staining were larger, more and more extensional. The statistical analysis showed that the osteoclasts in the WT group were more than the HE group, and the difference was statistically significant (P0.05); the number of osteoclasts produced in the 20ng group was the largest, and the number of osteoclasts produced in the control group was the osteoclast generated in the control group. The difference between the experimental group and the control group was statistically significant (P0.05, P0.005, P0.05). (2) the expression level of the osteoclast differentiation related genes (Trap, Mmp9, Ctsk) m RNA expression in the two groups showed that the expression level of Trap and Mmp9 m decreased significantly compared with the WT group. Conclusion: fibroblasts Growth factor 9 (FGF9) can promote the differentiation of osteoclast to osteoclast, and its promoting effect is not related to concentration. Its mechanism may affect the differentiation of osteoclast by affecting the transcriptional level of Trap and Mmp9 of osteoclast differentiation related genes. The second part of old male serum vitamin D and parathyroid hormone Objective to study the correlation of bone metabolism index. Objective: To investigate the serum vitamin D level and its correlation with parathyroid hormone and bone metabolism index. Methods: 895 cases of hospitalized and outpatient patients in the geriatrics ward of Shanghai Ruijin Hospital from September 2010 to September 2013 were collected and the average age was 76 years. The serum 25- hydroxyl groups were measured. D[25- (OH) D], blood calcium (Ca), blood phosphorus (P), parathyroid hormone (PTH), I type procollagen N- end propeptide (PINP) and beta -I collagen C end peptide (beta -CTX) level. Group (75 nmol/L). Results: (1) the age of 895 elderly men was 60~99 years old, with an average age of 76 years old. The average value of 25- (OH) D (43.52 + 21.97) nmol/L. vitamin D deficiency (< 50nmol/L) was 592 (67%); vitamin D deficiency (50-75 nmol/L) was 223 (25%); vitamin D deficiency or deficiency was up to 92%; vitamin D sufficient persons (75) were only 8. 0 people (8%). (2) the comparison of serum 25- (OH) D in different age groups showed that serum 25- (OH) D level gradually decreased with age and gradually decreased 25- (OH) D value of.60~69 year group, which was (46.27 + 20.76) nmol/L, and there was significant difference from other groups (P0.05). Correlation analysis showed that serum levels were negatively correlated with age (correlation coefficient, 3). The average level of parathyroid hormone (PTH) was (55.74 + 29.06) pg/ml. correlation analysis showed that 25- (OH) D had a negative correlation with PTH, PINP, and beta -CTX (R values were respectively 0.209, -0.109, -0.122, P are 0.05). Deficiency or insufficiency. Blood 25- (OH) D was negatively correlated with PTH, age, PINP and beta -CTX.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R580

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