Gli1阳性的间充质干细胞在损伤诱导的异位骨化发生发展中的研究
发布时间：2018-09-08 15:09
【摘要】：【研究背景】异位骨化(Heterotopic Ossification,HO)是指在正常骨骼系统之外(如:肌肉,跟腱等组织)的真正成骨。一般分为两类,第一类是创伤诱导的获得性的异位骨化。常发生于脊髓、神经损伤,大面积烧伤、烫伤,关节置换手术,矫形手术,战时创伤等。对于其发病机理,至今依然并不清楚。临床治疗措施相对单一,如手术切割,放射治疗,和非甾类抗炎药的使用,但是效果,预后都不甚好。当前遇到的最大的困难就是,即使使用物理化学手段将病变骨化区域摘除,只有一定缓解的作用,依然有大部分的患者会出现复发的现象。另外一种则是相对比较严重的,遗传性的异位骨化。常见有骨形态发生蛋白Ⅰ类受体ACVR1功能获得性突变导致的进行性骨化纤维发育不良(Fibrodysplasia Ossificans Progressiva,FOP),以及编码Gα蛋白基因GNAS缺失突变引起的进行性骨发育异常(Progressive Osseous Heteroplasia,POH)。由于其一旦发病,难以逆转,所以当前对其研究较多。据报道,BMP-SMAD信号通路以及BMP-MAPK信号通路调控细胞向成骨分化进而调控异位骨化的进程。但是由于FOP病人一旦接受创伤诱导,将持续性异位成骨,最终失去生命,因而大多数研究都是停留在FOP病人的外周血,新生脱落的组织如乳牙等。总的来说,遗传性的异位骨化研究较为透彻,但是关于获得性的异位骨化,其发病原因多变,机理不明,发生率较高,社会影响较大,且治疗手段缺乏,所以获得性的异位骨化理应引起我们的重视。关于异位骨化的细胞起源,一直都有争议,有些学者认为是由内胚层的细胞,如内皮细胞,通过内皮细胞间质样转化成间质细胞参与异位骨的形成。甚至有人提到是由外胚层细胞发育而来。但是,主流的观点还是认为由中胚层的细胞,如间充质干细胞通过向软骨细胞,成骨细胞分化增殖来参与异位骨的形成。间充质干细胞,当前研究很热的一种成体干细胞,首次被发现于骨髓中一类区别与造血干细胞,但是有向软骨细胞,成骨细胞,成脂细胞的分化潜能,同时又是具有克隆形成能力的贴壁细胞。后来,又相继在胎盘,乳牙,骨,软骨,脂肪,骨骼肌,子宫内膜等组织中被发现。现在被广泛应用于再生医学,甚至是癌症治疗。其最大的特点就是具有较低的免疫原性和多向分化能力。经证实,间充质干细胞在体外可以分化为骨细胞,软骨细胞,脂肪细胞,甚至是心肌细胞,神经元。正由于其与骨骼系统密切相关,并且分布区域符合异位骨化发生的条件,所以被认为是异位骨化真正的细胞来源,我们前期的研究也表明,Glast阳性的干细胞/前体细胞直接参与了异位骨化各个阶段的形成。并且这些Glast阳性的细胞表达间充质干细胞的相对特异的marker,这就在一定程度中佐证了间充质干细胞参与异位骨化的假设。另外,已有报道正式参与异位骨化形成的其他细胞亚群,如:Tie2~+,Mx1~+,Scx~+细胞,也都被证实很有可能是间质来源。最近,有学者证实胶质瘤相关癌基因同源基因1(Glioma-associated oncogene homolog 1,Gli1)蛋白是间充质干细胞的表面分子标志物(marker),而Gli1是Hedgehog(Hh)信号通路中一个至关重要的转录因子,并且,Hh信号通路参与调控正常骨的发育,尤其是软骨内骨化。所以这引起我们极大的兴趣去探究Hh信号通路在HO中到底扮演了什么角色。另外,CD133最近也被作为间质细胞的一个marker,尤其是造血干细胞和肌肉卫星细胞,值得一提的是肌肉卫星细胞,据报道也与异位骨化的形成有着密切的关系。所以本课题组提出假说,在具有FOP表型的Nse-Bmp4转基因鼠中,Gli1~+和CD133~+细胞是否都会直接参与异位骨化的进程,并进一步探究Gli1和CD133分别标记什么样的细胞。这不仅给异位骨化的发病机制研究甚至是治疗带来一线新的希望,另外对于间充质干细胞的在体研究也有着深远的意义。【研究方法】1.从国外引进Nse-BMP4小鼠模型,将其与Gli1-Cre ERT或者CD133-cre ERT小鼠进行交配,筛选出双阳性的子代小鼠(Gli1-cre ERT;Nse-Bmp4)或者(CD133-cre ERT;Nse-bmp4),进而与报告小鼠Zsgreen杂交,从而得到三重转基因小鼠模型(Gli1-cre;Nse-Bmp4;Zsgreen)。简单来说,Zsgreen报告小鼠,在cre重组酶的作用下,能够将Zsgreen前面的Stop基因盒子敲除,从而表达Zsgreen,发绿色荧光。所以在三重转基因小鼠模型中,我们可以直观地通过观察荧光来追踪Gli1或者CD133在异位骨化中的分布情况。2.成年三重转基因鼠(周龄大于一个月),按文献方法(1)腹腔注射他莫昔芬(Tamoxifen)溶液,然后通过向肌肉注射心脏毒素(cardiotoxin)损伤肌肉,在损伤后1周,2周,4周(分别对应着异位骨化早期,中期,后期)取相应时相的小鼠处死,并取其目的组织,即损伤的左后肢。将目的组织(即异位骨区域以及对照组未损伤部位)放入4%多聚甲醛中固定,过夜。第二天,放入20%EDTA溶液中进行脱钙一周,之后取组织进行冰冻切片,进行组织化学分析。3.免疫组化染色,确定Gli1-cre ERT或者CD133-cre ERT标记的细胞是否参与异位骨化的形成,另外通过与间充质干细胞的表面分子标志物,内皮细胞表面分子标志物,骨细胞表面分子标志物,软骨细胞表面分子标志物进行共染来验证其真实身份。首先取不同时相的切片,用胎牛血清封闭40min,加入一抗,过夜。第二天加二抗,核染色。用荧光显微镜观察各个蛋白在组织中的表达情况。【研究结果】1.Gli1或者CD133标记的祖细胞在小鼠体内皆有广泛表达,其中Gli1标记的细胞大多数分布在间质,且参与了正常长骨的形成;而CD133标记的细胞异质性比较大。2.Gli1标记的祖细胞在肌肉组织中包绕着血管,而CD133标记的细胞与血管关系并不紧密。3.Gli1标记的祖细胞参与了各个阶段异位骨化进程,而CD133标记的细胞在HO中几无贡献。4.干细胞微环境参与调控HO的进程。5.部分Gli1-cre ERT标记的祖细胞表达间充质干细胞的marker。Gli1-cre ERT标记的细胞在软骨形成期表达SOX9,在成骨阶段表达ALP,RUNX2。6.Bmp-smad信号通路介导了HO的形成。【结论】1.Gli1标记的间充质干细胞参与了异位骨化的形成,CD133~+细胞则相反。2.干细胞微环境参与调控HO的进程。3.Bmp-Smad信号通路参与了损伤诱导的HO的形成。
[Abstract]:[Background] Heterotopic ossification (HO) refers to the true osteogenesis outside the normal skeletal system (e.g. muscles, Achilles tendons, etc.). It is generally divided into two categories. The first is trauma-induced heterotopic ossification. It often occurs in the spinal cord, nerve injury, extensive burns, scalds, joint replacement surgery, orthopedic surgery, and warfare. The pathogenesis of the disease is still unclear. Clinical treatment is relatively simple, such as surgical excision, radiotherapy, and the use of non-steroidal anti-inflammatory drugs, but the effect and prognosis are not very good. The biggest difficulty encountered at present is that even if the area of ossification of the lesion is removed by physicochemical means, only certain relief can be achieved. The other is relatively severe, hereditary heterotopic ossification. Progressive fibrous dysplasia (FOP), caused by a functional acquired mutation of the bone morphogenetic protein class I receptor ACVR1, and the encoding of the G alpha protein, are common. Progressive Osseous Heteroplasia (POH) caused by GNAS deletion mutation has been studied extensively. BMP-SMAD signaling pathway and BMP-MAPK signaling pathway have been reported to regulate the process of osteogenic differentiation and heterotopic ossification in FOP patients. Once induced by trauma, persistent heterotopic osteogenesis will eventually lead to loss of life, so most studies remain in the peripheral blood of patients with FOP, new exfoliated tissues such as deciduous teeth. Acquired heterotopic ossification should be paid more attention to because of its high social impact and lack of treatment. The origin of heterotopic ossification cells has been controversial. Some scholars believe that endothelial cells, such as endothelial cells, are involved in the formation of heterotopic bone through mesenchymal-like transformation of endothelial cells into mesenchymal cells. It has been suggested that ectopic bone is derived from ectodermal cells. However, the mainstream view is that mesodermal cells, such as mesenchymal stem cells, participate in the formation of heterotopic bone by differentiating and proliferating into chondrocytes and osteoblasts. Mesenchymal stem cells, an adult stem cell currently under investigation, are first identified as a class of differences in bone marrow. With hematopoietic stem cells, but to chondrocytes, osteoblasts, adipocytes differentiation potential, but also with the ability to clone adherent cells. Later, and then in the placenta, deciduous teeth, bone, cartilage, fat, skeletal muscle, endometrium and other tissues have been found. Now widely used in regenerative medicine, even cancer treatment. Mesenchymal stem cells (MSCs) have been shown to differentiate into osteocytes, chondrocytes, adipocytes, even cardiomyocytes and neurons in vitro. Because they are closely related to the skeletal system and their distribution areas meet the conditions for heterotopic ossification, MSCs are thought to be able to differentiate into osteocytes, chondrocytes, adipocytes, and even cardiomyocytes and neurons. Our previous studies also showed that Glast-positive stem cells/progenitor cells were directly involved in the formation of ectopic ossification at various stages, and these Glast-positive cells expressed relatively specific markers of mesenchymal stem cells, which to some extent supported the involvement of mesenchymal stem cells in ectopic bone. In addition, other cell subsets officially involved in heterotopic ossification, such as Tie2~+, Mx1~+, and Scx~+ cells, have also been reported to be likely mesenchymal sources. Recently, some scholars have confirmed that glioma-associated oncogene homolog 1 (Gli1) protein is a surface component of mesenchymal stem cells. Gli1 is a key transcription factor in the Hedgehog (Hh) signaling pathway, and the Hh signaling pathway is involved in regulating normal bone development, especially endochondral ossification. A marker of plasma cells, especially hematopoietic stem cells and muscle satellite cells, is worth mentioning. Muscle satellite cells are also reported to be closely related to the formation of heterotopic ossification. It not only brings a new hope for the pathogenesis of heterotopic ossification, but also has a profound significance for the in vivo study of mesenchymal stem cells. [Methods] 1. Introducing Nse-BMP4 mouse model from abroad and comparing it with Gli1-BMP4 mouse model. Cre ERT or CD133-cre ERT mice were mated to select two-positive offspring (Gli1-cre ERT; Nse-Bmp4) or (CD133-cre ERT; Nse-bmp4) and hybridized with Zsgreen to produce a triple transgenic mouse model (Gli1-cre; Nse-Bmp4; Zsgreen). So we can directly trace the distribution of Gli1 or CD133 in heterotopic ossification by observing the fluorescence in the triple transgenic mice model. 2. Adult triple transgenic mice (aged more than one month) were injected intraperitoneally according to the literature method (1) Tamoxifen solution was injected into the muscles to injure the muscles. At 1, 2, and 4 weeks after injury (corresponding to the early, middle, and late stages of heterotopic ossification), the mice were killed and the target tissues (i.e. the heterotopic bone area and the control group) were taken from the left hind limb. Immunohistochemical staining was used to determine whether the cells labeled with Gli1-cre ERT or CD133-cre ERT were involved in the formation of heterotopic ossification. In addition, the cells were dried and fine with mesenchymal. Cell surface molecular markers, endothelial cell surface molecular markers, osteocyte surface molecular markers, and chondrocyte surface molecular markers were CO-stained to verify their true identity. Gli1 or CD133-labeled progenitor cells were widely expressed in mice. Most of the cells labeled with Gli1 were distributed in the stroma and participated in the formation of normal long bones. The heterogeneity of CD133-labeled progenitor cells was relatively large. 2. Gli1-labeled progenitor cells were wrapped in muscle tissue. CD133-labeled progenitor cells are involved in the process of heterotopic ossification at various stages, but CD133-labeled cells have little contribution to HO. 4. Stem cell microenvironment is involved in the regulation of HO. 5. Some of the progenitor cells labeled with Gli1-cre ERT express marker. Gli1-cre ERT of mesenchymal stem cells. The labeled cells expressed SOX9 during cartilage formation and ALP during osteogenesis. RUNX2.6. Bmp-smad signaling pathway mediated the formation of HO. [Conclusion] 1. Gli1-labeled mesenchymal stem cells participated in the formation of heterotopic ossification, whereas CD133~+ cells participated in the process of HO. 3. Bmp-Smad signaling pathway participated in the injury. Induced HO formation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681
本文编号：2230878
[Abstract]:[Background] Heterotopic ossification (HO) refers to the true osteogenesis outside the normal skeletal system (e.g. muscles, Achilles tendons, etc.). It is generally divided into two categories. The first is trauma-induced heterotopic ossification. It often occurs in the spinal cord, nerve injury, extensive burns, scalds, joint replacement surgery, orthopedic surgery, and warfare. The pathogenesis of the disease is still unclear. Clinical treatment is relatively simple, such as surgical excision, radiotherapy, and the use of non-steroidal anti-inflammatory drugs, but the effect and prognosis are not very good. The biggest difficulty encountered at present is that even if the area of ossification of the lesion is removed by physicochemical means, only certain relief can be achieved. The other is relatively severe, hereditary heterotopic ossification. Progressive fibrous dysplasia (FOP), caused by a functional acquired mutation of the bone morphogenetic protein class I receptor ACVR1, and the encoding of the G alpha protein, are common. Progressive Osseous Heteroplasia (POH) caused by GNAS deletion mutation has been studied extensively. BMP-SMAD signaling pathway and BMP-MAPK signaling pathway have been reported to regulate the process of osteogenic differentiation and heterotopic ossification in FOP patients. Once induced by trauma, persistent heterotopic osteogenesis will eventually lead to loss of life, so most studies remain in the peripheral blood of patients with FOP, new exfoliated tissues such as deciduous teeth. Acquired heterotopic ossification should be paid more attention to because of its high social impact and lack of treatment. The origin of heterotopic ossification cells has been controversial. Some scholars believe that endothelial cells, such as endothelial cells, are involved in the formation of heterotopic bone through mesenchymal-like transformation of endothelial cells into mesenchymal cells. It has been suggested that ectopic bone is derived from ectodermal cells. However, the mainstream view is that mesodermal cells, such as mesenchymal stem cells, participate in the formation of heterotopic bone by differentiating and proliferating into chondrocytes and osteoblasts. Mesenchymal stem cells, an adult stem cell currently under investigation, are first identified as a class of differences in bone marrow. With hematopoietic stem cells, but to chondrocytes, osteoblasts, adipocytes differentiation potential, but also with the ability to clone adherent cells. Later, and then in the placenta, deciduous teeth, bone, cartilage, fat, skeletal muscle, endometrium and other tissues have been found. Now widely used in regenerative medicine, even cancer treatment. Mesenchymal stem cells (MSCs) have been shown to differentiate into osteocytes, chondrocytes, adipocytes, even cardiomyocytes and neurons in vitro. Because they are closely related to the skeletal system and their distribution areas meet the conditions for heterotopic ossification, MSCs are thought to be able to differentiate into osteocytes, chondrocytes, adipocytes, and even cardiomyocytes and neurons. Our previous studies also showed that Glast-positive stem cells/progenitor cells were directly involved in the formation of ectopic ossification at various stages, and these Glast-positive cells expressed relatively specific markers of mesenchymal stem cells, which to some extent supported the involvement of mesenchymal stem cells in ectopic bone. In addition, other cell subsets officially involved in heterotopic ossification, such as Tie2~+, Mx1~+, and Scx~+ cells, have also been reported to be likely mesenchymal sources. Recently, some scholars have confirmed that glioma-associated oncogene homolog 1 (Gli1) protein is a surface component of mesenchymal stem cells. Gli1 is a key transcription factor in the Hedgehog (Hh) signaling pathway, and the Hh signaling pathway is involved in regulating normal bone development, especially endochondral ossification. A marker of plasma cells, especially hematopoietic stem cells and muscle satellite cells, is worth mentioning. Muscle satellite cells are also reported to be closely related to the formation of heterotopic ossification. It not only brings a new hope for the pathogenesis of heterotopic ossification, but also has a profound significance for the in vivo study of mesenchymal stem cells. [Methods] 1. Introducing Nse-BMP4 mouse model from abroad and comparing it with Gli1-BMP4 mouse model. Cre ERT or CD133-cre ERT mice were mated to select two-positive offspring (Gli1-cre ERT; Nse-Bmp4) or (CD133-cre ERT; Nse-bmp4) and hybridized with Zsgreen to produce a triple transgenic mouse model (Gli1-cre; Nse-Bmp4; Zsgreen). So we can directly trace the distribution of Gli1 or CD133 in heterotopic ossification by observing the fluorescence in the triple transgenic mice model. 2. Adult triple transgenic mice (aged more than one month) were injected intraperitoneally according to the literature method (1) Tamoxifen solution was injected into the muscles to injure the muscles. At 1, 2, and 4 weeks after injury (corresponding to the early, middle, and late stages of heterotopic ossification), the mice were killed and the target tissues (i.e. the heterotopic bone area and the control group) were taken from the left hind limb. Immunohistochemical staining was used to determine whether the cells labeled with Gli1-cre ERT or CD133-cre ERT were involved in the formation of heterotopic ossification. In addition, the cells were dried and fine with mesenchymal. Cell surface molecular markers, endothelial cell surface molecular markers, osteocyte surface molecular markers, and chondrocyte surface molecular markers were CO-stained to verify their true identity. Gli1 or CD133-labeled progenitor cells were widely expressed in mice. Most of the cells labeled with Gli1 were distributed in the stroma and participated in the formation of normal long bones. The heterogeneity of CD133-labeled progenitor cells was relatively large. 2. Gli1-labeled progenitor cells were wrapped in muscle tissue. CD133-labeled progenitor cells are involved in the process of heterotopic ossification at various stages, but CD133-labeled cells have little contribution to HO. 4. Stem cell microenvironment is involved in the regulation of HO. 5. Some of the progenitor cells labeled with Gli1-cre ERT express marker. Gli1-cre ERT of mesenchymal stem cells. The labeled cells expressed SOX9 during cartilage formation and ALP during osteogenesis. RUNX2.6. Bmp-smad signaling pathway mediated the formation of HO. [Conclusion] 1. Gli1-labeled mesenchymal stem cells participated in the formation of heterotopic ossification, whereas CD133~+ cells participated in the process of HO. 3. Bmp-Smad signaling pathway participated in the injury. Induced HO formation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681
【参考文献】
相关期刊论文 前1条
1 Md Shaifur Rahman;Naznin Akhtar;Hossen Mohammad Jamil;Rajat Suvra Banik;Sikder M Asaduzzaman;;TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation[J];Bone Research;2015年01期
，本文编号：2230878
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