降钙素基因相关肽诱导前软骨干细胞增殖和成骨分化的实验研究

发布时间：2018-02-20 21:22

本文关键词： 降钙素基因相关肽前软骨干细胞细胞表面标记细胞增殖降钙素基因相关肽前软骨干细胞成骨分化降钙素基因相关肽前软骨干细胞成骨分化 Wnt通路　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分CGRP对前软骨干细胞增殖的影响目的体外获取并培养纯化SD大鼠前软骨干细胞(PSCs),为以后的实验提供细胞来源。观察不同浓度梯度的降钙素基因相关肽(CGRP)在体外对前软骨干细胞增殖的影响,为后续试验确定合适的CGRP刺激浓度。方法1.前软骨干细胞的分离、纯化和体外培养及鉴定：取出生24小时内的SD大鼠乳鼠,手术显微镜下环行切取La Croix环,体外培养细胞。磁性细胞分选系统分离纯化得到前软骨干细胞,并且传代、扩增。倒置相差显微镜下观察细胞生长情况和形态变化：2.采用细胞免疫化学方法检测,辣根过氧化物酶标记,DAB显色,检测细胞表面FGFR-3表达情况；3.CCK-8法检测干细胞的增殖：将纯化后的第3代前软骨干细胞置接种于96孔板中培养,加入浓度分别为0、10-8、10-9、10-10mol/L的CGRP刺激,于孵育的第0、3、7、10、14d按CCK-8试剂盒的要求测定吸光度,绘生长曲线图。比较不同浓度梯度的CGRP对前软骨干细胞增殖的影响。结果1.免疫磁珠分离纯化后的前软骨干细胞绝大部分呈三角形或多角形,透明,贴壁生长,折光性良好,核大且胞浆少,细胞形态及大小较为均一。细胞免疫化学检测可见纯化后的PSCs胞浆中FGFR-3抗体染色阳性；2.对纯化后的PCSCs计数进行细胞生长动力学分析后发现,在相同的时间点,不同浓度的CGRP组间比较没有显著的统计学意义。结论1.免疫磁珠分选法可以简便而有效获得纯化的性状稳定的前软骨干细胞,,为以后的实验提供了丰富的干细胞来源；2. 不同浓度的CGRP对体外培养的前软骨干细胞的增殖没有影响。第二部分CGRP对前软骨干细胞成骨分化的影响目的研究降钙素基因相关肽在体外对前软骨干细胞成骨标志性基因的RUNX2、 OPN、BGP及细胞外基质Collagen Ⅰ的表达,以及碱性磷酸酶活性和矿化化结节产生的影响。方法1.分别以10-9mol/L和Omol/L的CGRP刺激在成骨培养基中诱导培养的实验组和对照组的前软骨干细胞；2.培养至第14d,茜素红染色。在倒置显微镜镜下观察实验组和对照组矿化结节形成的情况并拍照。用图像分析软件分析所得图片;3.按碱性磷酸酶检测试剂盒要求,分别在3、7、10、14d时检测并比较对照组和实验组的碱性磷酸酶的活性：4. 分别于成骨诱导培养的第7d、14d离心收集细胞,RT-PCR检测并比较对照组和实验组成骨细胞相关基因RUNX2、OPN、BGP及细胞外基质Collage I基因的表达。结果1.经CGRP刺激的前软骨干细胞,在成骨诱导培养基中培养14d后茜素红染色的结果与对照组相比,橘红色矿化结节明显增多,结节大。定量检测结果显示两组间矿化结节的面积有显著性差异；2. 两组ALP含量测定,在培养第3d时,两组间无显著差异,7-14d时实验组较对照组ALP含量呈极显著增加的趋势;3. RT-PCR检测显示7d和14d时成骨相关基因RUNX2、OPN、BGP及细胞外基质Collagen I基因的表达均显著增加。结论CGRP在体外可上调前软骨干细胞成骨相关基因RUNX2、OPN、BGP及细胞外基质Collagen I基因的表达,使矿化结节和碱性磷酸酶活性增加,诱导其向成骨分化。第三部分Wnt通路在CGRP诱导前软骨干细胞成骨分化过程中的作用目的探讨经典Wnt通路在CGRP诱导前软骨干细胞成骨分化过程中的作用。方法1. 将第3代细胞接种至6孔培养板内分为4组,分别为：Omol/L的CGRP+Omol/L的DKK-1:10-9mol/L的CGRP+0mol/L的DKK-1;0mol/L的CGRP+10-8mol/L的DKK-1;10-9mol/LCGRP+10-8mol/L的DKK-1。成骨诱导培养基诱导培养7d后,提取总蛋白。BCA法测定总蛋白浓度;2. Western blot检测各组的蛋白β-catenin的表达,使用图形软件分析；3. RT-PCR检测各组前软骨干细胞成骨细胞相关基因Runx2、OPN、BGP及细胞外基质Collagen Ⅰ基因的表达的变化。结果CRGP刺激前软骨干细胞后明显促进蛋白β-catenin表达。加入β-catenin抑制剂DKK-1后,β-catenin表达明显降低,同时下调Runx2、OPN、BGP基因表达,但Collagen Ⅰ基因的表达没有明显改变。CGRP可使受DKK-1抑制的蛋白β-catenin表达显著增加。结论CGRP可能部分通过激活经典Wnt/β-catenin通路促进前软骨干细胞向成骨细胞方向分化。
[Abstract]:Partithe effect of CGRP on precartilaginous stem cells proliferation in vitro to acquire and develop the purification of SD rat precartilaginous stem cells (PSCs) and provide a cell source for future experiments. Observation of different concentrations of calcitonin gene related peptide (CGRP) in vitro of precartilaginous stem cell proliferation, to determine the appropriate the concentration of CGRP for the following experiments. 1. separation methods of precartilaginous stem cells, purified and cultured in vitro and were identified: SD rats within 24 hours, under the surgical microscope circular cut La Croix ring, cells were cultured in vitro. The magnetic cell sorting system of purified PSCs, and passage amplification,. And the morphological changes of cells were observed by inverted phase contrast microscope: 2. using immunocytochemistry detection, horseradish peroxidase, DAB staining, detection of cell surface expression of FGFR-3; 3.CCK-8 method Detection of stem cell proliferation: purified after the third generation of the precartilaginous stem cells were seeded in 96 well plate culture, adding concentration of 0,10-8,10-9,10-10mol/L 0,3,7,10,14d in the CGRP stimulation, incubation was determined by CCK-8 kit, to draw a growth curve. To compare the effects of different concentrations of CGRP on precartilaginous stem cell proliferation. The results of precartilaginous stem cells after immunomagnetic separation and purification of 1. most triangular or polygonal, transparent, adherent growth, good refraction, big nucleus and little cytoplasm, cell morphology and size were uniform. Immunohistochemical detection of FGFR-3 showed the purified PSCs antibody positive staining in the cytoplasm; 2. of the purified PCSCs cell count growth dynamics analysis found that, at the same time, is not statistically significant different concentrations of CGRP group. Conclusion 1. immunomagnetic beads The sorting method can be simple and effective to obtain precartilaginous stem cells, purified the stability of traits for future experiments provide a rich source of stem cells; no effect of precartilaginous stem cells in 2. different concentrations of CGRP on the proliferation of cultured CGRP. The second part of precartilaginous stem differentiation objective to study the effect of calcitonin gene related peptide in vitro of precartilaginous stem cells of osteoblast marker genes RUNX2, OPN cells, the expression of BGP and Collagen of the extracellular matrix, and the effect of alkaline phosphatase activity and mineralization of nodules produced. Methods 1. respectively by 10-9mol /L and Omol/L CGRP in the stimulation of osteoblast culture medium induced the experimental group and the control group of precartilaginous stem cells; cultured to the 2. 14d, alizarin red staining. The experimental group was observed in the inverted microscope and the control group of mineralized nodule formation and photographed. Using image analysis software A analysis of the picture; 3. by the alkaline phosphatase assay kit, respectively at 3,7,10,14d was detected and compared with control group and experimental group of alkaline phosphatase activity: 7d 4. respectively in osteogenic medium, 14d cells were collected, RT-PCR was detected and compared with the control group and the experimental composition of osteoblast related genes RUNX2, OPN, the expression of BGP and extracellular matrix Collage I gene. Results 1. stimulated by CGRP Precartilage cells in osteogenic medium compared with the control group the cultivation results of alizarin red staining after 14d medium, orange red mineralized nodules increased significantly. Quantitative detection results showed nodules between two groups of mineralization area there was significant difference between the two groups of 2.; Determination of the content of ALP, 3D in culture, no significant differences between the two groups, 7-14d in experimental group than in the control group ALP was significantly increased; 3. RT-PCR showed 7d and 14d into Bone related genes RUNX2, OPN, BGP and the expression of extracellular matrix Collagen I gene increased significantly. Conclusion CGRP in vitro can upregulate precartilaginous stem cells into bone related genes RUNX2, OPN, BGP and the expression of extracellular matrix Collagen I gene, the mineralized nodules and alkaline phosphatase activity increased to induce a the third part of the Wnt pathway. The differentiation of precartilaginous stem cells osteoblast differentiation objective to investigate the role of canonical Wnt pathway in CGRP induced osteogenic differentiation of precartilaginous stem cells into the role in the induction of CGRP. 1. methods of the third generation of cells were inoculated in 6 well plates were divided into 4 groups, respectively: Omol/L CGRP+Omol/L DKK-1:10-9mol/L CGRP+0mol/L DKK-1; 0mol/L CGRP+10-8mol/L DKK-1; 10-9mol/LCGRP+10-8mol/L DKK-1. osteogenic induction culture medium 7d, determination of total protein concentration of total protein extracted by.BCA; 2. Western blot To detect the expression of -catenin protein beta groups, analysis of the use of graphics software; 3. RT-PCR detected precartilaginous stem related genes in Runx2 cells, OPN cells, BGP cells and extracellular matrix Collagen gene expression changes. Results CRGP stimulation of precartilaginous stem cells obviously increased the expression of -catenin protein beta beta -catenin inhibitor DKK-1 added. After the beta -catenin expression was significantly decreased, while the downregulation of Runx2, OPN, BGP gene expression, but the expression of Collagen gene was not significantly changed by.CGRP can make the protein beta -catenin inhibited DKK-1 expression increased significantly. Conclusion CGRP may promote precartilaginous stem cells to differentiate into osteoblasts by activating the classical Wnt/ beta -catenin pathway.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R683

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