17β-雌二醇联合淫羊藿苷对SD大鼠骨髓问充质干细胞成骨分化的影响

发布时间：2018-06-28 00:32

本文选题：17β-雌二醇 + 淫羊藿苷　；参考：《河北北方学院》2017年硕士论文

【摘要】：因创伤、肿瘤、骨质疏松等造成的骨不连、骨缺损等一直是骨科医师需要解决的难题,骨组织工程学的长足发展有望在未来攻克这一问题。任何骨组织工程骨构建均由种子细胞、培养环境与支架材料组成。骨髓间充质干细胞(BMSCs),是来源于中胚层的一类干细胞,其在体外具有较大的可增殖能力和可多方向分化潜能,存在于骨髓内并具有明显的贴壁能力,可分化为成骨、心肌、脂肪等多种细胞。其在骨代谢调节中起着较为关键的作用。机体增加和维持骨量的细胞来源主要为BMSCs分化成的成骨细胞,是人体骨骼维持健康的重要因素。BMSCs比较容易获得,体外扩增后仍无免疫原性,且易转染外缘性基因并能长期传代表达。另外,应用BMSCs进行自体的移植实验和治疗时几乎不受伦理学限制。体外BMSCs的增殖分化可以受多因素的准确调控,因此它一致被视为移植实验和治疗首选的干细胞。绝经后妇女骨质疏松发病率明显升高,绝经后机体内雌激素水平迅速下降是主要原因,激素替代疗法(HRT)是该病最经典的治疗方案,17β-雌二醇能促进BMSCs成骨分化,且体外浓度在10~(-9) mol/L时作用最为显著。随着祖国医学的不断发展进步,由于“补肾壮骨”中药在临床治疗骨质疏松、骨不连等有明显的疗效。淫羊藿苷可提高碱性磷酸酶(ALP)活性,增加钙化结节和碱性磷酸酶阳性克隆数(CFU-FALP)数量,促进成纤维细胞生长因子(b FGF),类胰岛素一号增长因子(IGF-1)的表达,且淫羊藿苷可双向调节对BMSCs的分化,即促进BMSCs成骨性分化的同时又抑制了BMSCs成脂分化。但长期应用雌激素的副作用较为显著以及淫羊藿等中药的作用又相对迟缓,因此本实验通过研究西药成分17β-雌二醇联合中药淫羊藿提取物淫羊藿苷对大鼠骨髓间充质干细胞体外成骨分化诱导的作用是否有协同作用,在降低药量的同时提高骨髓间充质干细胞向成骨细胞的分化效率,为临床用药提供理论依据。本实验所需的BMSCs均提取于3周龄大小的清洁级SD大鼠的长骨骨髓,运用全骨髓贴壁法进行培养。镜下可见初分离的大鼠骨髓悬液细胞密集,12 h后细胞开始贴壁,部分细胞形态伸展、体积增大,贴附于瓶壁,72 h可见部分细胞伸出突起,突起圆钝而光滑,细胞呈梭形、菱形或多边形,核大而饱满,外观似成纤维细胞样。培养14d后,细胞融合成片,分界变得较为模糊,胞体增大,形态则变为短梭形和不规则形,部分细胞聚集成灶,21 d天后观察诱导细胞部分出现散在的矿化结节,其中心透光度较差。既有研究表明:BMSCs可表达CD29、CD50、CD90等间充质细胞系及内皮细胞系的表面抗原,而不表达CD14、CD45等造血细胞系表面抗原。本研究运用流式细胞仪对BMSCs联合表面抗原进行鉴定,鉴定结果显示,细胞表面抗原CD45阳性表达率为0.5%,细胞表面抗原CD29阳性表达率99.6%,细胞表面抗原CD90阳性表达率99.7%。其结果符合大鼠骨髓间充质干细胞特征。取第4代BMSCs,消化后调整细胞密度至1×10~4/ml,接种于25ml培养瓶中,将培养细胞分为对照组(C、组)、17β-雌二醇作用组(A组)、淫羊藿苷组(B组)、联合诱导组(A+B组)。第4代BMSCs培养24h后进行定向诱导,C组加入基础诱导剂(地塞米松1×10~(-8)mol/L、β-甘油磷酸钠1×10~(-2)mol/L、抗坏血酸5×10~(-2)g/L),A组加入基础成骨诱导剂和17β-雌二醇1×10~(-9)mol/L,B组加入基础成骨诱导剂及淫羊藿苷1×10~(-4)mol/L,A+B组加入基础成骨诱导剂、17β-雌二醇1×10~(-9)mol/L及淫羊藿苷1×10~(-4)mol/L。持续诱导21天,隔天半量换液,7天传代。将BMSCs以1×10~4个/ml接种到培养瓶中,按上述实验分组进行成骨诱导,各组细胞于成骨诱导第7天进行ALP活性测定,各组细胞于成骨诱导第14天进行COL-1相对浓度测定,各组细胞于成骨诱导第21天进行BGP浓度测定,各组细胞分别于成骨诱导第21天进行茜素红钙结节染色。结果显示分别检测到ALP、COL-1、BGP相对浓度为联合组大于单因素诱导组,且单因素诱导组大于对照组,钙结节计数为联合组大于各单因素诱导组,各单因素诱导组均大于对照组,差别有统计学意义经本实验,能够说明:第一、17β-雌二醇和中药有效成分淫羊藿苷均可单独诱导BMSCs体外分化为骨样细胞;第二、二者联合诱导的效果优于各自单独诱导。
[Abstract]:Bone nonunion and bone defect caused by trauma, tumor and osteoporosis have always been a difficult problem for Department of orthopedics physicians to solve. The rapid development of bone tissue engineering is expected to overcome this problem in the future. Any bone tissue engineering bone construction is composed of seed cells, culture environment and scaffold materials. Bone marrow mesenchymal stem cells (BMSCs) are the source of bone tissue engineering. A class of stem cells in the mesoderm, which have large proliferative and multidirectional differentiation potential in vitro, exist in the bone marrow and have obvious adhesion to the wall, and can differentiate into bone, myocardium, and fat cells. It plays a key role in the regulation of bone metabolism. The main source of cells to increase and maintain bone mass is the main source. Osteoblast differentiated from BMSCs is an important factor for maintaining the health of human skeleton,.BMSCs is easy to obtain. After amplification in vitro, there is still no immunogenicity, and it is easy to transfect the external gene and can be expressed for a long time. In addition, the use of BMSCs for autologous transplantation and treatment is not subject to ethical restriction. In vitro, the proliferation and differentiation of BMSCs in vitro It can be regulated by multiple factors, so it is considered to be the first choice of stem cells for transplantation experiments and treatment. The incidence of osteoporosis in postmenopausal women is significantly higher, and the rapid decline in estrogen levels in postmenopausal women is the main reason. Hormone replacement therapy (HRT) is the most classical treatment for the disease, and 17 beta estradiol can promote BMSCs osteogenesis. Differentiation, and in vitro concentration in 10~ (-9) mol/L, the effect is most significant. With the continuous development and progress of Chinese medicine, the Chinese medicine of "kidney tonifying and strong bone" has obvious effect on the clinical treatment of osteoporosis and bone nonunion. Icariin can increase the activity of alkaline phosphatase (ALP), increase the number of calcified nodules and alkaline phosphatase positive clones (CFU-FALP). Quantity, promoting the expression of fibroblast growth factor (B FGF) and insulin like growth factor (IGF-1), and icariin can regulate the differentiation of BMSCs by two way, that is to promote the differentiation of BMSCs into osteogenic differentiation while inhibiting the lipid differentiation of BMSCs, but the side effect of the long-term use of estrogen is more significant and the effect of epimedium and other Chinese herbs is also phase In this experiment, the effect of 17 beta estradiol and epimedium extract of Western medicine and epimedium extract of Epimedium extract on bone differentiation induction in vitro of rat bone marrow mesenchymal stem cells was studied. Theoretical basis. The BMSCs needed in this experiment was extracted from the bone marrow of the long bone of the clean grade SD rats of 3 weeks old. It was found that the bone marrow suspension cells of the first separated rats were dense, and the cells began to stick to the wall after 12 h. Some cells were extended, the volume was enlarged, the cells were attached to the bottle wall, and the 72 h visible part of the cells protruded out. The protuberance is blunt and smooth, the cells are spindle shaped, rhombic or polygonal, the nucleus is large and full, and the appearance is like fibroblast like. After 14d, the cells fuse into pieces, the boundary becomes more blurred, the cell body increases, the morphology becomes short shuttle and irregular, some cells gather together, and 21 d days after the observation of the scattered mineralization of the induced cells. BMSCs can express the surface antigens of CD29, CD50, CD90 and other mesenchymal cells and endothelial cells, but not the surface antigens of CD14, CD45 and other hematopoietic cells. This study uses flow cytometry to identify the joint surface antigen of BMSCs, and the identification results show that the cell surface antigen CD45 Yang The expression rate was 0.5%, the positive expression rate of cell surface antigen CD29 was 99.6%, and the positive expression rate of CD90 of cell surface antigen was 99.7%.. The result accorded with the characteristics of rat bone marrow mesenchymal stem cells. Take fourth generation of BMSCs, and adjust the cell density to 1 x 10~4/ml after digestion and inoculated in the 25ml culture bottle. The cultured cells were divided into the control group (C, group), and 17 beta estradiol. Group (group A), icariin group (group B), combined induction group (group A+B). After the fourth generation of BMSCs culture, 24h was directed, and C group added basic inducer (dexamethasone 1 x 10~ (-8) mol/L, beta glycerol sodium 1 x 10~ (-2) mol/L, 5 x of ascorbic acid), and the group joined the basic osteogenic inducer and 17 beta estradiol 1 x. The bone inducer and icariin 1 x 10~ (-4) mol/L, A+B group added basic osteogenic inducer, 17 beta estradiol 1 x 10~ (-9) mol/L and icariin 1 x 10~ (-4) mol/L. continued to be induced for 21 days, half a half amount of liquid and 7 days to be passed. The BMSCs was inoculated into the culture bottle by 1 * 10~4, and the cells were induced by the above-mentioned experiments. The cells in each group were induced by osteogenesis. The activity of ALP was measured at seventh days. The relative concentration of COL-1 was measured at fourteenth days after osteogenesis induction. The cells in each group were measured at twenty-first days of osteogenesis induction. The cells of each group were stained with alizarin red calcium nodules on twenty-first days of osteogenesis induction. The results showed that the relative concentration of ALP, COL-1, and BGP was greater than the single cause, respectively. The single factor induction group was larger than the control group, and the calcium nodule count was larger than the single factor induction group, and the single factor induction group was larger than the control group. The difference has statistical significance. The first, 17 beta estradiol and the effective component of Chinese medicine can induce BMSCs to differentiate into bone like cells in vitro. The combined effect of the second, second methods is better than that of the separate induction.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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