FGF9和维生素D对骨质疏松症作用的基础和临床研究

发布时间：2018-07-13 14:11

【摘要】：第一部分FGF9对RANKL体外诱导破骨细胞分化的影响及其机制探讨目的:利用经RANKL体外诱导的破骨细胞,观察FGF9对破骨细胞分化的影响并探讨其可能机制。方法:利用RANKL诱导原代单核细胞法体外培养破骨细胞,细胞因子M-CSF、RANKL的浓度分别为30ng/ml,50ng/ml。采用两种方法研究FGF9对破骨细胞的影响:(1)取4周龄FGF9WT/WT(WT)和FGF9WT/S99N(HE)小鼠各两只,利用RANKL诱导原代单核细胞法体外培养破骨细胞。(2)在诱导过程中分别加入不同浓度(10ng、20ng、50ng)的FGF9蛋白,将未加入FGF9蛋白的作为对照组。定期更换培养液及诱导因子和相应浓度的FGF9蛋白,直至破骨细胞分化成熟。行TRAP染色鉴定破骨细胞。倒置相差显微镜下观察各组破骨细胞的形态并拍照。利用Image Pro Plus 6.0软件测量每视野破骨细胞数量,并利用SPSS 21.0统计软件进行分析和检验并绘制柱形图。通过Real-Time PCR技术检测FGF9对破骨细胞分化相关基因Trap、Mmp9、Ctsk表达量的影响。结果:(1)镜下观察可见WT组和HE组,10ng、20ng、50ng组和对照组均有形态不规则、多核的TRAP染色阳性的破骨细胞生成,实验组(10ng、20ng、50ng组)与对照组相比,生成的TRAP染色阳性的破骨细胞体积更大,数量更多,胞浆更为伸展。统计分析后显示,WT组的破骨细胞多于HE组,且差异具有统计学意义(P0.05);20ng组生成的破骨细胞数目最多,10ng次之,对照组生成的破骨细胞数目最少,实验组各组与对照组相比,差异均有统计学意义(分别为P0.05,P0.005,P0.05)。(2)破骨细胞分化相关基因(Trap、Mmp9、Ctsk)m RNA表达水平在两组间比较显示,与WT组相比,Trap和Mmp9 m RNA表达水平在HE组均明显下调(P0.05,P0.001)。结论:成纤维细胞生长因子9(FGF9)可促进破骨前体细胞向破骨细胞的分化,并且其促进作用与浓度无相关性。其作用机制可能通过影响破骨细胞分化相关基因Trap和Mmp9的转录水平进而影响破骨细胞的分化。第二部分老年男性血清维生素D与甲状旁腺激素及骨代谢指标的相关性研究目的:探讨老年男性血清维生素D水平,及其与甲状旁腺激素及骨代谢指标的相关性。方法:收集2010年9月至2013年9月在上海瑞金医院老年病科病房住院及门诊患者895例,平均年龄为76岁。测定其血清25-羟基维生素D[25-(OH)D]、血钙(Ca)、血磷(P)、甲状旁腺激素(PTH),I型原胶原分子N-端前肽(PINP)及β-I型胶原C端肽(β-CTX)水平。根据血清25-(OH)D水平将患者分为维生素D严重缺乏组(25nmol/L),维生素D缺乏组(25-50 nmol/L),维生素D不足组(50-75 nmol/L)和维生素D充足组(75 nmol/L)。结果:(1)895例老年男性患者年龄60~99岁,平均年龄76岁。血清25-(OH)D平均值为(43.52±21.97)nmol/L。维生素D缺乏者(≤50nmol/L)为592位(67%);维生素D不足者(50-75 nmol/L)为223人(25%);维生素D缺乏或不足者高达92%;维生素D充足者(75 nmol/L)仅为80人(8%)。(2)不同年龄段血清25-(OH)D的比较显示,血清25-(OH)D水平随增龄而逐渐降低。60~69岁组25-(OH)D值最高,为(46.27±20.76)nmol/L,与其它各组比较差异均有统计学意义(P0.05)。相关分析表明,血清25-(OH)D与年龄呈负相关(相关系数r=-0.088,P=0.008)。(3)甲状旁腺素(PTH)的平均水平为(55.74±29.06)pg/ml。相关分析显示,25-(OH)D与PTH、PINP、β-CTX均呈负相关(r值分别为:—0.209,-0.109,-0.122,P均0.05)。血25-(OH)D与Ca呈正相关(r=0.206,P=0)。血25-(OH)D与BMI、P均无相关性(P均0.05)。结论:老年男性存在严重的维生素D缺乏或不足。血25-(OH)D与PTH、年龄、PINP、β-CTX均呈负相关。
[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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580

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