CKIP-1负调控间充质干细胞成骨分化研究

发布时间：2018-05-12 07:08

  本文选题：CKIP-1 + 间充质干细胞　； 参考：《第四军医大学》2015年博士论文

【摘要】：研究目的:骨质疏松是代谢性骨病一个典型的疾病,它的发病基础是骨形成与骨吸收的动态平衡被打破。CKIP-1是一个近期广受关注的蛋白,参与机体多项生理活动过程,其中一个重要功能是骨形成的负调控作用,它通过增强泛素连接酶Smurf1中E3的活性来增强其作用。骨髓间充质干细胞是一种具有多项分化潜能的细胞,并且是成骨细胞的前体细胞,对它功能的调控能够影响其成骨分化,从而影响机体的骨平衡代谢。本研究拟利用CKIP-1基因敲除小鼠模型,分别从大体水平观察CKIP-1影响小鼠整体骨质状况,细胞水平影响干细胞生物学特性及其成骨功能及分子水平初步探讨成骨负调控作用的机制,以期从骨代谢成骨过程的负调控蛋白方面,进一步揭示骨代谢的可能过程及机制,为骨质疏松等骨代谢性疾病的研究及诊疗提供实验依据。研究方法:1.通过建立CKIP-1基因敲除小鼠繁育体系,采用PCR方法鉴定小鼠基因型,并测定小鼠从出生第二周至成年后的体重尾长增长变化,观察分析小鼠大体水平发育情况。2.利用Micro-CT、组织学染色等方法,测定WT(CKIP-1+/+)及KO(CKIP-1-/-)型小鼠股骨、椎骨及下颌骨骨质影像学相关参数的差异,观察其在组织学形态、结构及CKIP-1蛋白定位及表达情况。3.分离培养WT(CKIP-1+/+)及KO(CKIP-1-/-)小鼠BMSCs,采用流式细胞术检测表面标记分子表达,多向分化实验测定成脂、成骨分化能力,证实所培养细胞的来源及性质。4.采用MTT及平板克隆实验,检测WT及KO小鼠BMSCs增殖能力差异,流式细胞术检测两者的细胞周期及凋亡情况,探讨CKIP-1缺失是否对干细胞相关的生物学特性产生影响。5.对WT及KO小鼠BMSCs进行成骨、成脂诱导,检测其成骨及成脂能力差异;检测两组细胞碱性磷酸酶活性差异,并利用裸鼠颅骨缺损实验模型,比较两者BMSCs联合纤维蛋白胶修复裸鼠骨缺损修复成骨能力,应用Micro-CT检测新骨形成相对量,HE染色及Masson三色染色比较其新骨结构差别。6.对WT及KO小鼠BMSCs成骨诱导后,应用实时定量PCR方法及Western Blot方法检测成骨相关分子在m RNA及蛋白水平表达变化,以研究CKIP-1影响干细胞成骨发育可能通过哪些分子产生作用。7.采用Western Blot方法检测MAPK通路相关蛋白在WT及KO小鼠BMSCs经成骨诱导后表达的差异,探讨CKIP-1调控成骨过程中产生变化的通路蛋白,及其磷酸化情况,揭示CKIP-1影响成骨作用的可能机制。结果:1.采用鼠尾粗提法结合PCR扩增进行基因型鉴定,KO型小鼠不表达CKIP-1结构中的3号外显子,表达人工添加的neo抗性基因。选取同窝同性别的WT(CKIP+/+)及KO(CKIP-/-)型小鼠用于后续实验,品相良好的杂合子进行繁殖。并且,WT及KO小鼠自出生后至成年过程中,其体重和尾长的增长均符合线性规律,两组并未体现出差异(P0.05)。2.Micro-CT结果显示,在股骨及椎骨中,KO小鼠的骨体积分数及骨小梁数目均比WT小鼠增多(P0.05),其特定骨表面面积及骨小梁间隙小于WT组(P0.05);而在下颌骨中,其上述指标未见显著差异(P0.05)。WT及KO小鼠股骨、椎骨及下颌骨组织的HE染色显示,两者骨质在大体结构无明显差异;免疫荧光染色显示CKIP-1在椎骨、股骨及下颌骨表达较弱,在牙体组织尤其是牙本质中也有表达。KO小鼠各样本均无CKIP-1表达。3.利用WT及KO小鼠股骨密质骨经胶原酶消化,成功分离培养了间充质干细胞,流式细胞术鉴定其大量表达间充质来源细胞表面标记CD90、CD105及sca-1,几乎不表达造血系来源细胞的CD31和CD45,上述分子在WT组及KO组表达均无显著差异(P0.05)。经成骨成脂诱导后,发现KO小鼠BMSCs的成骨成脂能立均显著强于WT组(P0.05)。4.MTT及平板克隆实验结果显示,KO小鼠BMSCs增殖更快,其克隆形成能力更强(P0.05);流式细胞术检测两者细胞周期显示,KO组小鼠细胞的S期细胞数更多(P0.05),增殖旺盛;而两者在凋亡方面无显著差别(P0.05)。5.经成骨诱导后,KO组小鼠BMSCs显示出更强的体外成骨能力,其茜素红染色和ALP染色结果均显著高于WT组(P0.05);且KO组细胞碱性磷酸酶活性较WT组增强(P0.05);裸鼠颅骨缺损实验中,KO组细胞的体内修复骨缺损能力强于WT组细胞,形成的类骨质更多(P0.05)且结构更加成熟。6.在转录水平,成骨诱导后,CKIP-1表达下降,而转录因子Osterix及Runx2,成骨相关因子ALP、Col、BSP及OCN表达显著增强,KO组高于WT组(P0.05)。蛋白水平的检测结果与前述实验基本符合,成骨诱导后,KO组成骨相关因子表达高于WT组(P0.05)。7.对MAPK通路相关蛋白及其磷酸化水平检测结果显示,WT组小鼠表达CKIP-1蛋白,而KO组不表达;经成骨诱导后,KO组表达Smurf1低于WT组(P0.05),而MEKK2表达显著增强(P0.05),JNK、p-JNK、p-c-jun、p38及p-p38表达升高(P0.05)。此过程中未见ERK1/2、p-ERK1/2表达。结论:1.鼠尾粗提法结合PCR鉴定小鼠基因型较为准确、简洁和高效。CKIP-1敲除不影响小鼠的出生及发育成熟,且小鼠的繁殖遵循孟德尔遗传定律,此繁育体系可稳定遗传。2.KO型小鼠在大体骨质方面,较WT小鼠骨质增强,并且在骨组织结构方面显示出更加优化的结构;而在下颌骨中,这种变化并不明显。CKIP-1在牙本质中也有表达,提示其可能与牙齿的形成及结构相关。3.CKIP-1不影响干细胞表面标记分子的表达及干细胞的凋亡;而可以抑制干细胞的克隆形成能力和增殖速率。4.CKIP-1通过抑制干细胞表达成骨相关分子在m RNA及蛋白水平表达,负调控干细胞的成骨发育,此过程可能由于激活了MEKK2,从而引起JNK、c-jun及p38磷酸化实现。综上所述,CKIP-1是一个负调控干细胞成骨过程的重要蛋白,对其影响干细胞的作用及相关机制的探讨,可能为今后骨代谢相关疾病的治疗提供新思路。
[Abstract]:Objective: osteoporosis is a typical disease of metabolic bone disease. It is based on the dynamic balance between bone formation and bone absorption..CKIP-1 is a recent and widely concerned protein. It participates in a number of physiological processes in the body. One of the important functions is the negative regulation of bone formation, which enhances the ubiquitin ligase Sm. Bone marrow mesenchymal stem cells (MSCs) are a kind of cells with multiple differentiation potential and precursor cells of osteoblasts. The regulation of the function of bone marrow mesenchymal stem cells can affect the osteogenic differentiation of bone marrow cells and affect the bone metabolism of the body. This study is to use the CKIP-1 knockout mouse model from the general level to the general level. To observe the effect of CKIP-1 on the overall bone status of mice, the cell level affects the biological characteristics of the stem cells and the mechanism of osteogenesis and molecular level to investigate the negative regulation of osteogenesis, in order to further reveal the possible process and mechanism of bone metabolism from the negative regulatory proteins of bone metabolism to bone metabolic diseases such as osteoporosis and so on. The research and diagnosis and treatment provide the experimental basis. 1. through the establishment of the CKIP-1 gene knockout mice breeding system, the PCR method was used to identify the mice genotypes, and the weight tail length of the mice from second weeks to adulthood was measured, and the general level of development of.2. in mice was observed and analyzed by Micro-CT, histological staining and so on. The differences in the parameters of WT (CKIP-1+/+) and KO (CKIP-1-/-) mouse femur, vertebrae and mandible bone imaging were measured, and the histological morphology, structure and the location and expression of CKIP-1 protein were observed by.3. separation and culture of WT (CKIP-1+/+) and KO (CKIP-1-/-) mice BMSCs. Flow cytometry was used to detect the expression of surface labelled molecules and multidirectional differentiation. Determination of lipid formation and osteogenic differentiation, the origin and properties of the cultured cells were confirmed by.4., MTT and flat clones were used to detect the proliferation ability difference of BMSCs in WT and KO mice. Flow cytometry was used to detect the cell cycle and apoptosis of both of them, and the effect of CKIP-1 deletion on the biological characteristics of stem cells was affected by.5. on WT and KO. Rat BMSCs was induced by bone formation and lipid induction. The difference of bone formation and fat formation ability was detected. The difference of alkaline phosphatase activity in two groups was detected. Using the experimental model of skull defect in nude mice, BMSCs combined with fibrin glue was used to repair bone defect repair in nude mice. The relative amount of new bone formation was detected by Micro-CT, HE staining and Masson three were used to detect the bone formation ability of nude mice. Color staining compared its new bone structure difference.6. to WT and KO mice induced by BMSCs osteogenesis, real-time quantitative PCR method and Western Blot method were used to detect the expression changes of bone related molecules in M RNA and protein level, in order to study the effect of CKIP-1 on the possibility of stem cell development by which molecules produced by CKIP-1.7. by Western method The difference in the expression of pathway related protein in WT and KO mouse BMSCs after osteogenic induction was used to explore the pathway proteins which are regulated by CKIP-1 in the process of osteogenesis, and its phosphorylation, and to reveal the possible mechanism of CKIP-1 affecting the osteogenesis. Results: 1. the genotype identification was carried out by the tail roughing method combined with PCR amplification, and the KO type mice did not express the CKIP-1 junction. Exon 3 expressed the artificial Neo resistance gene. The same homozygous WT (CKIP+/+) and KO (CKIP-/-) type mice were selected for subsequent experiments and good heterozygotes were used to reproduce. And the growth of weight and tail length of WT and KO mice from birth to adult were all in line with the linear rule, and the two groups did not show business travel. P0.05.2.Micro-CT results showed that in the femur and vertebrae, the bone volume fraction and the number of bone trabecula increased in KO mice (P0.05), and the specific bone surface area and bone trabecular space were less than WT group (P0.05), but in the mandible, the above indexes were not significant (P0.05).WT and KO mice femur, vertebrae and mandibular tissue HE. The staining showed that there was no significant difference in the gross structure between the two bones. Immunofluorescence staining showed that the expression of CKIP-1 in the vertebrae, the femur and the mandible was weak, and there was no CKIP-1 expression in the samples of.KO mice in the dentin, especially in the dentin, and the WT and KO mice were successfully isolated and cultured with collagenase, and the mesenchyme was successfully isolated and cultured. Stem cells, flow cytometry identified a large number of expression of mesenchymal stem cell surface markers CD90, CD105 and Sca-1, almost no expression of CD31 and CD45 in the hematopoietic source cells, the expression of these molecules in the WT and KO groups was not significantly different (P0.05). After the induction of osteogenesis, it was found that the osteogenic energy of BMSCs in KO mice was significantly stronger than that in WT group (P0.0). 5) the results of.4.MTT and plate cloning showed that the proliferation of BMSCs in KO mice was faster and its clone formation was stronger (P0.05). Flow cytometry showed that the cell cycle of the two cells in the KO group was more (P0.05), and the proliferation was exuberant, but there was no significant difference in apoptosis between the two groups (P0.05).5. after osteogenesis, BMSCs display of KO group mice The results of alizarin red staining and ALP staining were significantly higher than that in WT group (P0.05), and the activity of alkaline phosphatase in group KO was stronger than that in WT group (P0.05). In the nude mouse skull defect experiment, the ability of repairing bone defect in the body of the group KO was stronger than that of the WT group cells, and the formation of the osteoid was more (P0.05) and the structure was more mature.6. in the nude mice. Transcriptional level, after osteogenesis, CKIP-1 expression decreased, transcription factor Osterix and Runx2, bone related factors ALP, Col, BSP and OCN were significantly enhanced, KO group was higher than group WT (P0.05). Protein level detection results were basically consistent with the previous experiments. The results of protein and phosphorylation showed that the CKIP-1 protein was expressed in the WT group and the KO group was not expressed. After the induction of bone formation, the expression of Smurf1 in the KO group was lower than that of the WT group (P0.05), and the expression of MEKK2 was significantly enhanced (P0.05), JNK, p-JNK, p-c-jun, expression and expression. Conclusion: 1. rat tail coarse formulation PCR identification of mice is more accurate. Simple and efficient.CKIP-1 knockout does not affect the birth and maturation of mice, and the propagation of mice follows the Mendel's law of inheritance. This breeding system can stabilize genetic.2.KO mice in gross bone, strengthen bone in WT mice, and show more optimization in bone tissue structure. In the mandible, this change does not clearly show that.CKIP-1 is also expressed in dentin, suggesting that.3.CKIP-1 may not affect the expression of the surface marker molecules of the stem cells and the apoptosis of stem cells in relation to the formation and structure of the teeth, but can inhibit the cloning and proliferation of stem cells by inhibiting the stem cells by inhibiting the stem cells. The expression of bone related molecules at m RNA and protein levels negatively regulates the osteogenesis of stem cells. This process may be due to the activation of MEKK2, resulting in the realization of JNK, c-jun and p38 phosphorylation. To sum up, CKIP-1 is an important protein in the osteogenesis process of a negative regulated stem cell, and the effect of the stem cells and related mechanisms on it can be discussed. It can provide new ideas for the treatment of bone metabolism related diseases in the future.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R580
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本文编号：1877622