【摘要】：【研究背景】異位骨化(Heterotopic Ossification,HO)是指在正常骨骼系統(tǒng)之外(如:肌肉,跟腱等組織)的真正成骨。一般分為兩類,第一類是創(chuàng)傷誘導(dǎo)的獲得性的異位骨化。常發(fā)生于脊髓、神經(jīng)損傷,大面積燒傷、燙傷,關(guān)節(jié)置換手術(shù),矯形手術(shù),戰(zhàn)時創(chuàng)傷等。對于其發(fā)病機(jī)理,至今依然并不清楚。臨床治療措施相對單一,如手術(shù)切割,放射治療,和非甾類抗炎藥的使用,但是效果,預(yù)后都不甚好。當(dāng)前遇到的最大的困難就是,即使使用物理化學(xué)手段將病變骨化區(qū)域摘除,只有一定緩解的作用,依然有大部分的患者會出現(xiàn)復(fù)發(fā)的現(xiàn)象。另外一種則是相對比較嚴(yán)重的,遺傳性的異位骨化。常見有骨形態(tài)發(fā)生蛋白Ⅰ類受體ACVR1功能獲得性突變導(dǎo)致的進(jìn)行性骨化纖維發(fā)育不良(Fibrodysplasia Ossificans Progressiva,FOP),以及編碼Gα蛋白基因GNAS缺失突變引起的進(jìn)行性骨發(fā)育異常(Progressive Osseous Heteroplasia,POH)。由于其一旦發(fā)病,難以逆轉(zhuǎn),所以當(dāng)前對其研究較多。據(jù)報道,BMP-SMAD信號通路以及BMP-MAPK信號通路調(diào)控細(xì)胞向成骨分化進(jìn)而調(diào)控異位骨化的進(jìn)程。但是由于FOP病人一旦接受創(chuàng)傷誘導(dǎo),將持續(xù)性異位成骨,最終失去生命,因而大多數(shù)研究都是停留在FOP病人的外周血,新生脫落的組織如乳牙等。總的來說,遺傳性的異位骨化研究較為透徹,但是關(guān)于獲得性的異位骨化,其發(fā)病原因多變,機(jī)理不明,發(fā)生率較高,社會影響較大,且治療手段缺乏,所以獲得性的異位骨化理應(yīng)引起我們的重視。關(guān)于異位骨化的細(xì)胞起源,一直都有爭議,有些學(xué)者認(rèn)為是由內(nèi)胚層的細(xì)胞,如內(nèi)皮細(xì)胞,通過內(nèi)皮細(xì)胞間質(zhì)樣轉(zhuǎn)化成間質(zhì)細(xì)胞參與異位骨的形成。甚至有人提到是由外胚層細(xì)胞發(fā)育而來。但是,主流的觀點還是認(rèn)為由中胚層的細(xì)胞,如間充質(zhì)干細(xì)胞通過向軟骨細(xì)胞,成骨細(xì)胞分化增殖來參與異位骨的形成。間充質(zhì)干細(xì)胞,當(dāng)前研究很熱的一種成體干細(xì)胞,首次被發(fā)現(xiàn)于骨髓中一類區(qū)別與造血干細(xì)胞,但是有向軟骨細(xì)胞,成骨細(xì)胞,成脂細(xì)胞的分化潛能,同時又是具有克隆形成能力的貼壁細(xì)胞。后來,又相繼在胎盤,乳牙,骨,軟骨,脂肪,骨骼肌,子宮內(nèi)膜等組織中被發(fā)現(xiàn)�，F(xiàn)在被廣泛應(yīng)用于再生醫(yī)學(xué),甚至是癌癥治療。其最大的特點就是具有較低的免疫原性和多向分化能力。經(jīng)證實,間充質(zhì)干細(xì)胞在體外可以分化為骨細(xì)胞,軟骨細(xì)胞,脂肪細(xì)胞,甚至是心肌細(xì)胞,神經(jīng)元。正由于其與骨骼系統(tǒng)密切相關(guān),并且分布區(qū)域符合異位骨化發(fā)生的條件,所以被認(rèn)為是異位骨化真正的細(xì)胞來源,我們前期的研究也表明,Glast陽性的干細(xì)胞/前體細(xì)胞直接參與了異位骨化各個階段的形成。并且這些Glast陽性的細(xì)胞表達(dá)間充質(zhì)干細(xì)胞的相對特異的marker,這就在一定程度中佐證了間充質(zhì)干細(xì)胞參與異位骨化的假設(shè)。另外,已有報道正式參與異位骨化形成的其他細(xì)胞亞群,如:Tie2~+,Mx1~+,Scx~+細(xì)胞,也都被證實很有可能是間質(zhì)來源。最近,有學(xué)者證實膠質(zhì)瘤相關(guān)癌基因同源基因1(Glioma-associated oncogene homolog 1,Gli1)蛋白是間充質(zhì)干細(xì)胞的表面分子標(biāo)志物(marker),而Gli1是Hedgehog(Hh)信號通路中一個至關(guān)重要的轉(zhuǎn)錄因子,并且,Hh信號通路參與調(diào)控正常骨的發(fā)育,尤其是軟骨內(nèi)骨化。所以這引起我們極大的興趣去探究Hh信號通路在HO中到底扮演了什么角色。另外,CD133最近也被作為間質(zhì)細(xì)胞的一個marker,尤其是造血干細(xì)胞和肌肉衛(wèi)星細(xì)胞,值得一提的是肌肉衛(wèi)星細(xì)胞,據(jù)報道也與異位骨化的形成有著密切的關(guān)系。所以本課題組提出假說,在具有FOP表型的Nse-Bmp4轉(zhuǎn)基因鼠中,Gli1~+和CD133~+細(xì)胞是否都會直接參與異位骨化的進(jìn)程,并進(jìn)一步探究Gli1和CD133分別標(biāo)記什么樣的細(xì)胞。這不僅給異位骨化的發(fā)病機(jī)制研究甚至是治療帶來一線新的希望,另外對于間充質(zhì)干細(xì)胞的在體研究也有著深遠(yuǎn)的意義。【研究方法】1.從國外引進(jìn)Nse-BMP4小鼠模型,將其與Gli1-Cre ERT或者CD133-cre ERT小鼠進(jìn)行交配,篩選出雙陽性的子代小鼠(Gli1-cre ERT;Nse-Bmp4)或者(CD133-cre ERT;Nse-bmp4),進(jìn)而與報告小鼠Zsgreen雜交,從而得到三重轉(zhuǎn)基因小鼠模型(Gli1-cre;Nse-Bmp4;Zsgreen)。簡單來說,Zsgreen報告小鼠,在cre重組酶的作用下,能夠?qū)sgreen前面的Stop基因盒子敲除,從而表達(dá)Zsgreen,發(fā)綠色熒光。所以在三重轉(zhuǎn)基因小鼠模型中,我們可以直觀地通過觀察熒光來追蹤Gli1或者CD133在異位骨化中的分布情況。2.成年三重轉(zhuǎn)基因鼠(周齡大于一個月),按文獻(xiàn)方法(1)腹腔注射他莫昔芬(Tamoxifen)溶液,然后通過向肌肉注射心臟毒素(cardiotoxin)損傷肌肉,在損傷后1周,2周,4周(分別對應(yīng)著異位骨化早期,中期,后期)取相應(yīng)時相的小鼠處死,并取其目的組織,即損傷的左后肢。將目的組織(即異位骨區(qū)域以及對照組未損傷部位)放入4%多聚甲醛中固定,過夜。第二天,放入20%EDTA溶液中進(jìn)行脫鈣一周,之后取組織進(jìn)行冰凍切片,進(jìn)行組織化學(xué)分析。3.免疫組化染色,確定Gli1-cre ERT或者CD133-cre ERT標(biāo)記的細(xì)胞是否參與異位骨化的形成,另外通過與間充質(zhì)干細(xì)胞的表面分子標(biāo)志物,內(nèi)皮細(xì)胞表面分子標(biāo)志物,骨細(xì)胞表面分子標(biāo)志物,軟骨細(xì)胞表面分子標(biāo)志物進(jìn)行共染來驗證其真實身份。首先取不同時相的切片,用胎牛血清封閉40min,加入一抗,過夜。第二天加二抗,核染色。用熒光顯微鏡觀察各個蛋白在組織中的表達(dá)情況。【研究結(jié)果】1.Gli1或者CD133標(biāo)記的祖細(xì)胞在小鼠體內(nèi)皆有廣泛表達(dá),其中Gli1標(biāo)記的細(xì)胞大多數(shù)分布在間質(zhì),且參與了正常長骨的形成;而CD133標(biāo)記的細(xì)胞異質(zhì)性比較大。2.Gli1標(biāo)記的祖細(xì)胞在肌肉組織中包繞著血管,而CD133標(biāo)記的細(xì)胞與血管關(guān)系并不緊密。3.Gli1標(biāo)記的祖細(xì)胞參與了各個階段異位骨化進(jìn)程,而CD133標(biāo)記的細(xì)胞在HO中幾無貢獻(xiàn)。4.干細(xì)胞微環(huán)境參與調(diào)控HO的進(jìn)程。5.部分Gli1-cre ERT標(biāo)記的祖細(xì)胞表達(dá)間充質(zhì)干細(xì)胞的marker。Gli1-cre ERT標(biāo)記的細(xì)胞在軟骨形成期表達(dá)SOX9,在成骨階段表達(dá)ALP,RUNX2。6.Bmp-smad信號通路介導(dǎo)了HO的形成。【結(jié)論】1.Gli1標(biāo)記的間充質(zhì)干細(xì)胞參與了異位骨化的形成,CD133~+細(xì)胞則相反。2.干細(xì)胞微環(huán)境參與調(diào)控HO的進(jìn)程。3.Bmp-Smad信號通路參與了損傷誘導(dǎo)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R681

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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

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本文編號：2230878