【作者】 庞向华；

【导师】 刘庆思；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2008， 博士

【摘要】 目的:通过体外培养获得成骨细胞,借助抗体芯片技术检测OP患者成骨细胞相关细胞因子的变化,探讨骨质疏松患者成骨细胞相关细胞因子蛋白表达与“肾阴虚”、“肾阳虚”证型的相关联系,从而揭示骨质疏松症中医证型的本质,并为骨质疏松症中医证型的研究提供合理的客观化依据。方法:回顾近年来现代中医对骨质疏松症的研究进展、成骨细胞与骨代谢的关系、骨质疏松症证型的研究进展以及成骨细胞相关细胞因子与骨质疏松症关系的研究进展,并复习蛋白芯片技术和体外培养成骨细胞技术的概况,在此基础上进行相关的实验和临床研究。实验研究:从三组行全髋置换术的患者（肾阳虚组、肾阴虚组、正常对照组）股骨头松质骨中用改良酶消化法分离成骨细胞。通过倒置相差显微镜观察原代细胞的形态,并采用碱性磷酸酶染色和矿化结节染色鉴定成骨细胞。用流式细胞仪检测三组成骨细胞胞内Ca²⁺阳性百分比和荧光值,采用茜素红染色镜下观察三组成骨细胞钙化结节并计数。临床研究;选择肾阴虚型和肾阳虚型OP合并股骨颈或股骨粗隆间骨折的患者各8例,股骨颈或股骨粗隆间骨折患者无OP的患者8例,三组患者均行髋关节置换术。取出术中置换的股骨头,进行成骨细胞培养,经过鉴定,所培养的细胞具有典型的成骨细胞形态和功能。对三组患者的成骨细胞进行细胞因子抗体芯片的检测,从而筛选出肾阴虚组、肾阳虚组与正常组成骨细胞蛋白表达有差异的细胞因子,以及肾阴虚组、肾阳虚组之间蛋白表达有差异的细胞因子。结果:实验研究结果:在经酶消化过滤后,取得的成骨细胞刚接种入培养瓶时,是悬浮的单细胞,呈折光性较高的球形。2小时后开始有成骨细胞贴壁,到20小时后多数成骨细胞已贴壁。于接种两周左右成骨细胞生长满瓶壁,细胞密集,形态单一,呈长梭形,此时生长减慢,开始出现多层生长,并开始出现钙结节。细胞接种后第12天,行碱性磷酸酶染色,成骨细胞胞浆中出现黑色颗粒或块状沉淀,广泛分布于成骨细胞胞浆,成骨细胞纯度高。在抗坏血酸、β-甘油磷酸钠存在下,细胞培养18d可形成矿化结节,茜素红染色成红色。实验结果显示培养获得的原代细胞表现出成骨细胞的各种特征,ALP检测阳性,在含β-甘油磷酸钠和抗坏血酸的培养液中培养14-20d,可形成钙化结节,表明我们培养的细胞是成骨细胞,具有典型成骨细胞的形态和功能,而且纯度较高。流式细胞仪检测结果显示,正常组成骨细胞胞内Ca²⁺阳性百分比和荧光值均明显高于肾阳虚及肾阴虚组,其差异有显著性意义（P＜0.01）,肾阳虚组及肾阴虚组成骨细胞胞内Ca²⁺阳性百分比和荧光值无显著性差异（P＞0.05）。采用茜素红染色镜下观察,正常组成骨细胞钙化结节计数明显高于肾阳虚及肾阴虚组,其差异有显著性意义（P＜0.01）,肾阳虚组及肾阴虚组钙化结节计数无显著性差异（P＞0.05）。临床研究结果:与正常对照组相比,肾阳虚组和肾阴虚组OB的BMP-4、BMP-6、FasFGF-4、FasFGF-6、IGF-1、IL-6、IL-2、OPG等18种细胞因子蛋白表达水平明显上升或下降（P＜0.05,P＜0.01）,肾阴虚组OB的IL-6、TGF-1的蛋白表达水平较肾阳虚组高,差异有显著性（P＜0.05,P＜0.01）。OP患者中医证型的本质可能与OB相关细胞因子的蛋白表达密切相关,我们同时发现,蛋白表达有差异的细胞因子多是与骨代谢有关的,且其表达上升或下降与该细胞因子的功能密切相关。BMP-4、BMP-6、OPG是骨形成指标,其蛋白表达与正常对照组相比是明显上升的,而FasFGF-4、FasFGF-6、IGF-1、IL-6、IL-2等骨吸收指标,其蛋白表达与正常对照组相比是明显下降的。结论:1、改良酶消化法能获得体外培养研究所需要的高纯度成骨细胞。2、多种方法联合应用可作为体外成骨细胞培养的鉴定标准。3、生理状态下成骨细胞胞内Ca²⁺含量高于骨质疏松状态下的成骨细胞,但成骨细胞钙离子的摄取能力与“肾阴虚证”、“肾阳虚证”无明显相关。4、生理状态下的成骨细胞的钙化能力高于骨质疏松状态下的成骨细胞,但成骨细胞的钙化能力与“肾阴虚证”、“肾阳虚证”无明显相关。5、肾阴虚型和肾阳虚型OP患者成骨细胞具有相近的钙化能力和钙离子的摄取能力,骨质疏松证型实质的研究还需从更深层次探讨。6、骨质疏松症肾阴虚与肾阳虚证的物质基础可能是骨代谢相关细胞因子。肾阳虚证OB相关细胞因子的蛋白表达的变化趋势与肾阴虚组是基本一致的。7、成骨细胞IL-6、TGF-1蛋白表达程度的不同可能是OP患者肾阳虚证与肾阴虚证本质上的差异。更多还原

【Abstract】 Objective:To explore the correlation among related cytokine protein expression of osteoblast in patients with osteoporosis（OP）and "Kidney Yang-deficiency","Kidney Yin-deficiency" TCM Syndrome Types,revelate the essence of osteoporosis TCM Syndrome Types and provide objective base for study about osteoporosis TCM Syndrome Types.Method:The lastest progress of modern TCM’s reseach on OP and TCM Syndrome Types of OP was reviewed,the relation of osteoblast（OB）and bone metabolism was summarized,and also reviewed the relation of OP and related cytokine of OB,outlined technology about protein chip and culture of OB in vitro.The study made of part one（experimental study）and part two（clinical study）is as follows. In part one:OB was separated by improved enzymatic digestion from femoral head of three groups patients（Kidney Yang-deficiency group,Kidney Yin-deficiency group,control group）accepted total hip replacement.The morphology of primary osteoblast was observed by inverted microscope,and OB was identified by mineralized nodes with alizarin red staining and the expression of alkaline phosphatase with modified Gomori’s assays staining.Positive rate and fluorescence value of Ca²⁺in OB were tested by flow cytometry in three groups, and mineralized nodes were observed and counted.In part two:Three groups patients were all undergone hip replacement, including 8 paients of femoral neck fracture or intertrochanteric fracture with Osteoporosis of Deficiency in Kidney yang,8 paients of femoral neck fracture or intertrochanteric fracture with osteoporosis of Deficiency in Kidney yin and 8 paients of femoral neck fracture or intertrochanteric fracture without osteoporosis.The femoral heads replaced of three groups were chosen to culture OB,the OB cultured showed typical morphy and function through identification.The three groups OB were tested by antibody chip of cytokine in order to select the cytokines whose protein expression was different among Kidney Yang-deficiency group and control group,Kidney Yin-deficiency group and control group,Kidney Yang-deficiency and Kidney Yin-deficiency group.Results:In part one:After enzymatic digestion,OB gained was single and suspended just at the beginning of inoculation,showed spherical shape with high refractive index.2 hours later,OB began to adhere to the wall of training bottles,20 hours later,most of osteoblasts were adherent to the wall of training bottles.About two weeks after culture,OB growed over the wall of training bottles,they were intensive,single long spindle shape.OB growth slowed down at this time to begin a multi-storey growth,and calcium nodules began to appear.12 days after inoculation,alkaline phosphatase staining of OB was operated,it appeared black or massive precipitation in,osteoblasts cytoplasmic granules,widely distributed in the cytoplasm of OB showed high purity.In the existence of ascorbic acid andβ-Glycerophosphate disodium salt pentahy-drate（β-GP）, cells cultured for 18 days could formed mineralized nodes,alizarin red stained red.The results showed that the cultured cells in the original showed various features of osteoblasts,ALP testing positive,cells cultured for 14-20 days in the culture medium including ascorbic acid andβ-GP could formed calcified knot which confirmed the cells cultured were osteoblasts with typical osteoblast morphology and function,and high purity.Flow cytometry analysis showed that the positive rate and fluorescence value of OB intracellular Ca²⁺in control group were significantly higher than those in kidney-yang deficiency and kidney-yin deficiency group,there were significant differences（P＜0.01）,there was no significant difference（P＞0.05）between kidney-yang deficiency group and kidney-yin deficiency group in the positive rate and fluorescence value of OB intracellular Ca²⁺.It showed that the count of mineralized nodes in control group were significantly higher than those in kidney-yang deficiency and kidney-yin deficiency group,there were significant differences（P＜0.01）,there was no significant difference（P＞0.05）between kidney-yang deficiency group and kidney-yin deficiency group in the count of mineralized nodes. In part two:Compared with normal control group,18 cytokines such as BMP-4, BMP-6,FasFGF-4,FasFGF-6 and IGF-1,IL-6,IL-2 protein expression levels of OB in kidney-yang deficiency group and kidney-yin deficiency group were significantly increase or decrease（P＜0.05,P＜0.01）,and IL-6,TGF-1 protein expression levels of OB in kidney-yin deficiency group were higher than kidney-yang deficiency group,there were significant differences（P＜0.05, P＜0.01）.The essence of OP TCM type may be closely related to cytokines protein expression,we also found that the cytokines which showed difference in protein expression were relevant to bone metabolism,and increase or decrease of the protein expression was closely related to the function of the cytokines.Conclusion:1.Modified enzyme digestion method can gain high-purity OB in keeping with reseach in vitro.2.Various methods can be combined as the identification criteria of osteoblast culture in vitro culture.3.The concentration of OB intracellular Ca²⁺concentration at physiological state is higher than that at osteoporosis state,and there is no significant correlation between calcium intake capacity of OB and "kidney-yang deficiency syndrome","kidney-yin deficiency syndrome".4.The calcification capacity of OB at physiological state is higher than that at osteoporosis state,and there is no significant correlation between calcification capacity of OB and "kidney-yang deficiency syndrome","kidney -yin deficiency syndrome".5.There are similar calcium intake capacity and calcification capacity of OB between "kidney-yang deficiency" OP and "kidney-yin deficiency" OP,the reseach of the OP syndrome type’ essence needs discussion at a deeper level.6.The material basis of OP "kidney-yang deficiency syndrome","kidney-yin deficiency syndrome" may be cytokines about bone metabolism.The trend of changes in protein expression of related cytokines of OB in kidney-yin deficiency group were consistent with which in kidney-yang deficiency group.7.The different degree of OB’IL-6,TGF-1 protein expression may be the real differences of OP "kidney-yang deficiency syndrome" and OP "kidney-yin deficiency syndrome".更多还原