發(fā)布時(shí)間：2018-05-12 07:08

  本文選題：CKIP-1 + 間充質(zhì)干細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2015年博士論文

【摘要】：研究目的:骨質(zhì)疏松是代謝性骨病一個(gè)典型的疾病,它的發(fā)病基礎(chǔ)是骨形成與骨吸收的動(dòng)態(tài)平衡被打破。CKIP-1是一個(gè)近期廣受關(guān)注的蛋白,參與機(jī)體多項(xiàng)生理活動(dòng)過(guò)程,其中一個(gè)重要功能是骨形成的負(fù)調(diào)控作用,它通過(guò)增強(qiáng)泛素連接酶Smurf1中E3的活性來(lái)增強(qiáng)其作用。骨髓間充質(zhì)干細(xì)胞是一種具有多項(xiàng)分化潛能的細(xì)胞,并且是成骨細(xì)胞的前體細(xì)胞,對(duì)它功能的調(diào)控能夠影響其成骨分化,從而影響機(jī)體的骨平衡代謝。本研究擬利用CKIP-1基因敲除小鼠模型,分別從大體水平觀察CKIP-1影響小鼠整體骨質(zhì)狀況,細(xì)胞水平影響干細(xì)胞生物學(xué)特性及其成骨功能及分子水平初步探討成骨負(fù)調(diào)控作用的機(jī)制,以期從骨代謝成骨過(guò)程的負(fù)調(diào)控蛋白方面,進(jìn)一步揭示骨代謝的可能過(guò)程及機(jī)制,為骨質(zhì)疏松等骨代謝性疾病的研究及診療提供實(shí)驗(yàn)依據(jù)。研究方法:1.通過(guò)建立CKIP-1基因敲除小鼠繁育體系,采用PCR方法鑒定小鼠基因型,并測(cè)定小鼠從出生第二周至成年后的體重尾長(zhǎng)增長(zhǎng)變化,觀察分析小鼠大體水平發(fā)育情況。2.利用Micro-CT、組織學(xué)染色等方法,測(cè)定WT(CKIP-1+/+)及KO(CKIP-1-/-)型小鼠股骨、椎骨及下頜骨骨質(zhì)影像學(xué)相關(guān)參數(shù)的差異,觀察其在組織學(xué)形態(tài)、結(jié)構(gòu)及CKIP-1蛋白定位及表達(dá)情況。3.分離培養(yǎng)WT(CKIP-1+/+)及KO(CKIP-1-/-)小鼠BMSCs,采用流式細(xì)胞術(shù)檢測(cè)表面標(biāo)記分子表達(dá),多向分化實(shí)驗(yàn)測(cè)定成脂、成骨分化能力,證實(shí)所培養(yǎng)細(xì)胞的來(lái)源及性質(zhì)。4.采用MTT及平板克隆實(shí)驗(yàn),檢測(cè)WT及KO小鼠BMSCs增殖能力差異,流式細(xì)胞術(shù)檢測(cè)兩者的細(xì)胞周期及凋亡情況,探討CKIP-1缺失是否對(duì)干細(xì)胞相關(guān)的生物學(xué)特性產(chǎn)生影響。5.對(duì)WT及KO小鼠BMSCs進(jìn)行成骨、成脂誘導(dǎo),檢測(cè)其成骨及成脂能力差異;檢測(cè)兩組細(xì)胞堿性磷酸酶活性差異,并利用裸鼠顱骨缺損實(shí)驗(yàn)?zāi)Ｐ?比較兩者BMSCs聯(lián)合纖維蛋白膠修復(fù)裸鼠骨缺損修復(fù)成骨能力,應(yīng)用Micro-CT檢測(cè)新骨形成相對(duì)量,HE染色及Masson三色染色比較其新骨結(jié)構(gòu)差別。6.對(duì)WT及KO小鼠BMSCs成骨誘導(dǎo)后,應(yīng)用實(shí)時(shí)定量PCR方法及Western Blot方法檢測(cè)成骨相關(guān)分子在m RNA及蛋白水平表達(dá)變化,以研究CKIP-1影響干細(xì)胞成骨發(fā)育可能通過(guò)哪些分子產(chǎn)生作用。7.采用Western Blot方法檢測(cè)MAPK通路相關(guān)蛋白在WT及KO小鼠BMSCs經(jīng)成骨誘導(dǎo)后表達(dá)的差異,探討CKIP-1調(diào)控成骨過(guò)程中產(chǎn)生變化的通路蛋白,及其磷酸化情況,揭示CKIP-1影響成骨作用的可能機(jī)制。結(jié)果:1.采用鼠尾粗提法結(jié)合PCR擴(kuò)增進(jìn)行基因型鑒定,KO型小鼠不表達(dá)CKIP-1結(jié)構(gòu)中的3號(hào)外顯子,表達(dá)人工添加的neo抗性基因。選取同窩同性別的WT(CKIP+/+)及KO(CKIP-/-)型小鼠用于后續(xù)實(shí)驗(yàn),品相良好的雜合子進(jìn)行繁殖。并且,WT及KO小鼠自出生后至成年過(guò)程中,其體重和尾長(zhǎng)的增長(zhǎng)均符合線性規(guī)律,兩組并未體現(xiàn)出差異(P0.05)。2.Micro-CT結(jié)果顯示,在股骨及椎骨中,KO小鼠的骨體積分?jǐn)?shù)及骨小梁數(shù)目均比WT小鼠增多(P0.05),其特定骨表面面積及骨小梁間隙小于WT組(P0.05);而在下頜骨中,其上述指標(biāo)未見(jiàn)顯著差異(P0.05)。WT及KO小鼠股骨、椎骨及下頜骨組織的HE染色顯示,兩者骨質(zhì)在大體結(jié)構(gòu)無(wú)明顯差異;免疫熒光染色顯示CKIP-1在椎骨、股骨及下頜骨表達(dá)較弱,在牙體組織尤其是牙本質(zhì)中也有表達(dá)。KO小鼠各樣本均無(wú)CKIP-1表達(dá)。3.利用WT及KO小鼠股骨密質(zhì)骨經(jīng)膠原酶消化,成功分離培養(yǎng)了間充質(zhì)干細(xì)胞,流式細(xì)胞術(shù)鑒定其大量表達(dá)間充質(zhì)來(lái)源細(xì)胞表面標(biāo)記CD90、CD105及sca-1,幾乎不表達(dá)造血系來(lái)源細(xì)胞的CD31和CD45,上述分子在WT組及KO組表達(dá)均無(wú)顯著差異(P0.05)。經(jīng)成骨成脂誘導(dǎo)后,發(fā)現(xiàn)KO小鼠BMSCs的成骨成脂能立均顯著強(qiáng)于WT組(P0.05)。4.MTT及平板克隆實(shí)驗(yàn)結(jié)果顯示,KO小鼠BMSCs增殖更快,其克隆形成能力更強(qiáng)(P0.05);流式細(xì)胞術(shù)檢測(cè)兩者細(xì)胞周期顯示,KO組小鼠細(xì)胞的S期細(xì)胞數(shù)更多(P0.05),增殖旺盛;而兩者在凋亡方面無(wú)顯著差別(P0.05)。5.經(jīng)成骨誘導(dǎo)后,KO組小鼠BMSCs顯示出更強(qiáng)的體外成骨能力,其茜素紅染色和ALP染色結(jié)果均顯著高于WT組(P0.05);且KO組細(xì)胞堿性磷酸酶活性較WT組增強(qiáng)(P0.05);裸鼠顱骨缺損實(shí)驗(yàn)中,KO組細(xì)胞的體內(nèi)修復(fù)骨缺損能力強(qiáng)于WT組細(xì)胞,形成的類骨質(zhì)更多(P0.05)且結(jié)構(gòu)更加成熟。6.在轉(zhuǎn)錄水平,成骨誘導(dǎo)后,CKIP-1表達(dá)下降,而轉(zhuǎn)錄因子Osterix及Runx2,成骨相關(guān)因子ALP、Col、BSP及OCN表達(dá)顯著增強(qiáng),KO組高于WT組(P0.05)。蛋白水平的檢測(cè)結(jié)果與前述實(shí)驗(yàn)基本符合,成骨誘導(dǎo)后,KO組成骨相關(guān)因子表達(dá)高于WT組(P0.05)。7.對(duì)MAPK通路相關(guān)蛋白及其磷酸化水平檢測(cè)結(jié)果顯示,WT組小鼠表達(dá)CKIP-1蛋白,而KO組不表達(dá);經(jīng)成骨誘導(dǎo)后,KO組表達(dá)Smurf1低于WT組(P0.05),而MEKK2表達(dá)顯著增強(qiáng)(P0.05),JNK、p-JNK、p-c-jun、p38及p-p38表達(dá)升高(P0.05)。此過(guò)程中未見(jiàn)ERK1/2、p-ERK1/2表達(dá)。結(jié)論:1.鼠尾粗提法結(jié)合PCR鑒定小鼠基因型較為準(zhǔn)確、簡(jiǎn)潔和高效。CKIP-1敲除不影響小鼠的出生及發(fā)育成熟,且小鼠的繁殖遵循孟德?tīng)栠z傳定律,此繁育體系可穩(wěn)定遺傳。2.KO型小鼠在大體骨質(zhì)方面,較WT小鼠骨質(zhì)增強(qiáng),并且在骨組織結(jié)構(gòu)方面顯示出更加優(yōu)化的結(jié)構(gòu);而在下頜骨中,這種變化并不明顯。CKIP-1在牙本質(zhì)中也有表達(dá),提示其可能與牙齒的形成及結(jié)構(gòu)相關(guān)。3.CKIP-1不影響干細(xì)胞表面標(biāo)記分子的表達(dá)及干細(xì)胞的凋亡;而可以抑制干細(xì)胞的克隆形成能力和增殖速率。4.CKIP-1通過(guò)抑制干細(xì)胞表達(dá)成骨相關(guān)分子在m RNA及蛋白水平表達(dá),負(fù)調(diào)控干細(xì)胞的成骨發(fā)育,此過(guò)程可能由于激活了MEKK2,從而引起JNK、c-jun及p38磷酸化實(shí)現(xiàn)。綜上所述,CKIP-1是一個(gè)負(fù)調(diào)控干細(xì)胞成骨過(guò)程的重要蛋白,對(duì)其影響干細(xì)胞的作用及相關(guān)機(jī)制的探討,可能為今后骨代謝相關(guān)疾病的治療提供新思路。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580
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本文編號(hào)：1877622