發(fā)布時(shí)間：2018-06-01 15:33

  本文選題：細(xì)胞永生化 + iSCCSs�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景目前在臨床中,由于創(chuàng)傷、腫瘤、退變及先天性等因素導(dǎo)致的骨缺損還很常見,雖然在骨重建中骨科內(nèi)固定和手術(shù)技術(shù)方面取得了很大的進(jìn)步,但通過手術(shù)進(jìn)行骨重建中還是會(huì)遇到很多的挑戰(zhàn),比如自體骨量和供區(qū)的限制等。隨著再生醫(yī)學(xué)和骨組織工程的發(fā)展,給骨重建提供了一種新的選擇。種子細(xì)胞、生物支架材料、生物活性因子是骨組織工程的三要素,其中種子細(xì)胞是骨組織工程的最基本的環(huán)節(jié)。種子細(xì)胞為再生醫(yī)學(xué)和組織工程提供活性成分的主要來源,是其核心部分。干細(xì)胞作為具有多向分化潛能的特點(diǎn),是目前應(yīng)用最廣泛的種子細(xì)胞。顱骨骨縫組織具有很強(qiáng)的增殖能力,其在人生中的前三十年中顱骨的形成過程中起著至關(guān)重要的作用。與一般長骨軟骨下成骨方式不同的是顱骨骨縫組織的成骨主要是通過膜內(nèi)成骨。其成骨原理是在一定的刺激條件下其骨化位點(diǎn)通過重疊的成骨前緣間充質(zhì)干細(xì)胞的聚集、分化而進(jìn)一步成骨。研究表明顱骨骨縫干細(xì)胞具有一定的分化潛能。通過顱骨骨縫組織干細(xì)胞的研究,對(duì)進(jìn)一步研究膜內(nèi)成骨的機(jī)制和特點(diǎn)、探索為再生醫(yī)學(xué)和組織工程良好的種子細(xì)胞具有重要意義,同時(shí)也為進(jìn)一步研究顱骨骨縫早閉等疾病有重要意義。通常情況下正常的組織細(xì)胞,在體外培養(yǎng)一段時(shí)間后,細(xì)胞會(huì)出現(xiàn)生長停滯,即M1期(衰老期),細(xì)胞繼續(xù)增殖后會(huì)進(jìn)入增殖抑制狀態(tài),即M2期(危機(jī)期),隨后細(xì)胞逐漸出現(xiàn)退化、死亡。這些情況同樣會(huì)發(fā)生在干細(xì)胞身上。這為再生醫(yī)學(xué)和組織工程細(xì)胞來源及研究帶來諸多限制,尤其是那些稀少、取材困難的細(xì)胞。通過細(xì)胞永生化可以使細(xì)胞獲得持續(xù)增殖的能力。為再生醫(yī)學(xué)和組織工程提供了足夠的種子細(xì)胞,更有利于進(jìn)行實(shí)驗(yàn)研究。BMPs屬于TGF-β超家族,家族至少有20個(gè)成員,BMPs在大多數(shù)物種均有表達(dá)。BMPs在體內(nèi)具有廣泛的作用,除了廣為所知的誘導(dǎo)成骨分化的能力外,還參與多種組織和器官的形成和修復(fù)。多種BMPs被報(bào)道具有誘導(dǎo)成骨的能力,誘導(dǎo)成骨BMP2、BMP4、BMP6、BMP、BMP9的報(bào)道較多。課題組前期實(shí)驗(yàn)證明,BMP9在眾多BMPs中對(duì)骨間充質(zhì)干細(xì)胞具有最強(qiáng)的成骨誘導(dǎo)能力,同時(shí)還有誘導(dǎo)成軟骨和成脂肪的能力。課題組前期研究亦證實(shí),BMP9對(duì)多種不同部位來源的干細(xì)胞具有較好的多向誘導(dǎo)分化能力。因此,在顱骨骨縫干細(xì)胞研究中,我們?nèi)赃x擇BMP9作為細(xì)胞因子進(jìn)行誘導(dǎo)永生化小鼠顱骨骨縫干細(xì)胞。作為再生醫(yī)學(xué)和組織工程很重要的組成部分,材料科學(xué)的發(fā)展極大的推進(jìn)了再生醫(yī)學(xué)和骨組織工程的發(fā)展,越來越多科學(xué)實(shí)驗(yàn)和臨床應(yīng)用得到了驗(yàn)證和推廣。目前對(duì)于骨組織材料的基本特性是能給細(xì)胞遷移、增殖和分化提供三維結(jié)構(gòu)環(huán)境,在一些承重的組織中,它同樣可以起到暫時(shí)的機(jī)械結(jié)構(gòu)支撐作用。一個(gè)理想的支架材料需要滿足以下特點(diǎn)：①骨組織材料的三維和多孔網(wǎng)狀結(jié)構(gòu)有利于細(xì)胞生長、營養(yǎng)和代謝產(chǎn)物的運(yùn)輸。②表面特性有利于細(xì)胞貼附、遷移、增殖和分化。③具有生物相容性,不會(huì)引起免疫反應(yīng),在細(xì)胞／組織生長過程中其生物降解率可控。④其力學(xué)特性應(yīng)當(dāng)跟移植區(qū)域的組織相當(dāng)⑤應(yīng)當(dāng)具有可塑性。PCCN作為一種新型的支架材料,除了具備以上特點(diǎn)外,還具有溫敏可控塑形的特點(diǎn)。為了驗(yàn)證這種新型支架材料對(duì)骨及軟骨修復(fù)中的作用,我們用PCCN復(fù)合BMP9誘導(dǎo)下的小鼠顱骨骨縫干細(xì)胞體內(nèi)注射的方法來驗(yàn)證新型支架在組織工程中的作用。研究目的：1、運(yùn)用piggyBac轉(zhuǎn)座系統(tǒng)構(gòu)建永生化的小鼠顱骨骨縫干細(xì)胞,并進(jìn)行干細(xì)胞標(biāo)志物鑒定。2、比較SCCSs與iSCCSs細(xì)胞系的增殖率差異。3、iSCCSs的逆轉(zhuǎn)永生化及體內(nèi)成瘤風(fēng)險(xiǎn)研究。4、BMP9誘導(dǎo)永生化小鼠顱骨骨縫干細(xì)胞的體外分化潛能研究。5、BMP9誘導(dǎo)永生化小鼠顱骨骨縫干細(xì)胞并復(fù)合PCCN體內(nèi)分化初步研究。研究方法：1、選取1周大小的無菌CD1小鼠,麻醉起效后將小鼠斷頸處死,無菌操作暴露顱骨骨縫,取顱骨骨縫組織,采用Ⅰ型膠原酶溶解方法使細(xì)胞貼壁獲得原代細(xì)胞。用piggyBac轉(zhuǎn)座系統(tǒng)將永生化基因SV40T抗原轉(zhuǎn)染至原代細(xì)胞中,構(gòu)建永生化小鼠顱骨骨縫干細(xì)胞。2、采用細(xì)胞免疫熒光染色的方法鑒定iSCCSs的間充質(zhì)干細(xì)胞標(biāo)志物。3、通過體外培養(yǎng)觀察、結(jié)晶紫染色、WST-1等方法,比較永生化小鼠顱骨骨縫干細(xì)胞與原代細(xì)胞增殖能力的差異。4、體外通過利用Flip/FRT重組系統(tǒng),將永生化基因SV40T抗原敲除,通過體外培養(yǎng)觀察、結(jié)晶紫染色、WST-1及Touch-down qPCR等方法來鑒定iSCCSs的逆永生化；通過對(duì)裸鼠體內(nèi)注射iSCCSs后活體成像檢測來鑒定其有無體內(nèi)成瘤風(fēng)險(xiǎn)性。5、利用腺病毒感染技術(shù),用Ad-BMP2、Ad-BMP4、Ad-BMP6 Ad-BMP7、 Ad-BMP9分別對(duì)iSCCSs進(jìn)行感染,通過ALP染色、ALP活性讀數(shù)、茜素紅染色和Touch-down qPCR的方法比較多種BMP對(duì)iSCCSs的體外誘導(dǎo)成骨能力。6、利用腺病毒感染技術(shù),用Ad-BMP9分別感染實(shí)驗(yàn)室前期已經(jīng)驗(yàn)證過具有較強(qiáng)成骨能力的干細(xì)胞iCALs、iSCAPs、iMEFs及本實(shí)驗(yàn)構(gòu)建的iSCCSs,通過ALP染色及ALP活性讀數(shù)來大致比較各種細(xì)胞系BMP9誘導(dǎo)下的體外成骨能力。7、通過ALP染色和ALP活性讀數(shù)檢測BMP9誘導(dǎo)iSCCSs成骨分化時(shí)間相關(guān)性和劑量相關(guān)性；運(yùn)用免疫熒光染色的方法來鑒定BMP9誘導(dǎo)下的OCN的表達(dá)；運(yùn)用油紅染色鑒定BMP9誘導(dǎo)下iSCCSs的成脂能力；運(yùn)用Alician Blue染色的方法檢測BMP9誘導(dǎo)下iSCCSs的成軟骨能力。應(yīng)用Touch-down qPCR來鑒定相關(guān)成骨、成脂、成軟骨基因的表達(dá)。8、將Ad-BMP9誘導(dǎo)下iSCCSs采用單純細(xì)胞和復(fù)合新型支架材料PCCN于裸鼠皮下注射和肌肉注射。通過Micro-CT掃描來檢測BMP9的體內(nèi)誘導(dǎo)成骨能力和支架材料PCCN在成骨中的作用。通過對(duì)成骨包塊石蠟切片的HE染色、Alician Blue染色、Trichrome染色來檢測BMP9體外誘導(dǎo)iSCCSs的成骨、成脂、成軟骨能力和PCCN的作用。結(jié)果1、成功獲得原代小鼠顱骨骨縫干細(xì)胞,并通過piggyBac轉(zhuǎn)座系統(tǒng)成功構(gòu)建永生化小鼠顱骨骨縫干細(xì)胞。2、對(duì)iSCCSs進(jìn)行干細(xì)胞表面標(biāo)記物免疫熒光染色,結(jié)果顯示CD29(intergin β1)、CD73、CD113 (prom1)、CD40、CD90 (Thy1)、CD117(c-kit)、 CD 166 (ALCAM)、CD105(Endglin)和BMPRⅡ具有表達(dá)。證實(shí)了iSCCSs保持了間充質(zhì)干細(xì)胞的干細(xì)胞特性。3、通過體外培養(yǎng)觀察、結(jié)晶紫染色、WST-1等方法證實(shí)與原代細(xì)胞相比,永生化后的小鼠扁骨骨縫干細(xì)胞具有更強(qiáng)的增殖能力,這種增殖上的差異自培養(yǎng)后第一天即存在,并且在第三、五、七各個(gè)時(shí)間段均存在明顯差異。4、體外通過利用Flip/FRT重組系統(tǒng),將永生化基因SV40T抗原敲除,通過體外培養(yǎng)觀察、結(jié)晶紫染色、WST-1證實(shí)：對(duì)照組(Ad-GFP組)細(xì)胞增殖能力仍然旺盛,而實(shí)驗(yàn)組Ad-Flip組細(xì)胞的增殖速度明顯變慢,在連續(xù)七天內(nèi),增殖率一致維持在一個(gè)較低的水平。Touch-down qPCR結(jié)果SV40T抗原基因在實(shí)驗(yàn)組的表達(dá)明顯低于對(duì)照組。證實(shí)SV40T抗原基因能夠被敲除,細(xì)胞的永生化可以逆轉(zhuǎn)。5、應(yīng)用piggyBac轉(zhuǎn)座系統(tǒng)成功構(gòu)建螢光蟲熒光素酶(Fluc)標(biāo)記的iSCCS細(xì)胞(iSCCSs-Fluc)。裸鼠體內(nèi)注射iSCCSs后活體成像檢測顯示在iSCCSs-Fluc注射后,5天的時(shí)候信號(hào)強(qiáng)烈,10天的時(shí)候信號(hào)較前出現(xiàn)衰減,到第15天的時(shí)候信號(hào)已經(jīng)基本消失。通過大致觀察皮下注射部位亦無腫瘤樣物生成。表明iSCCSs在體內(nèi)具有較強(qiáng)的增殖能力,但是無成瘤的危險(xiǎn)。6、多種BMPs(BMP2、BMP4、BMP6、BMP7、BMP9)誘導(dǎo)iSCCSs。ALP染色、ALP讀數(shù)、茜素紅染色證實(shí)這五種BMP均有骨誘導(dǎo)能力,但BMP2和BMP9的誘導(dǎo)成骨作用更強(qiáng),其中BMP9最強(qiáng)；成骨基因ALP、OPN、OSX、RUNX2的表達(dá)方面證實(shí)對(duì)于BMP9對(duì)iSCCSs的誘導(dǎo)成骨能力最強(qiáng)。7、ALP染色和讀數(shù)顯示,在BMP9的誘導(dǎo)下,與已經(jīng)驗(yàn)證過具有較強(qiáng)成骨能力的干細(xì)胞iCALs、iSCAPs、iMEFs相比,iSCCSs的成骨能力更強(qiáng)。8、BMP9誘導(dǎo)iSCCSs成骨分化具有正向的時(shí)間相關(guān)性和劑量相關(guān)性。9、免疫熒光染色檢測成骨指標(biāo)OCN的表達(dá),證實(shí)BMP9能夠誘導(dǎo)OCN的高表達(dá)。10、油紅O染色(Oil Red O staining)顯示BMP9誘導(dǎo)組的細(xì)胞成空泡樣,成脂肪細(xì)胞樣變,iSCCSs具有成脂肪細(xì)胞的潛能。11、細(xì)胞團(tuán)的Alician Blue染色,BMP9組藍(lán)染,證實(shí)iSCCSs具有成軟骨細(xì)胞的潛能。成脂、成軟骨基因的表達(dá)與對(duì)照組亦有增高。12、Micro-CT結(jié)果顯示：Ad-BMP9誘導(dǎo)下的iSCCSs,無論是單純細(xì)胞注射,還是復(fù)合支架材料,無論是皮下注射,還是肌肉內(nèi)注射,均可見高密度骨質(zhì)形成,其中皮下包塊及肌肉包塊的橫斷掃描均可見骨皮質(zhì)、骨小梁等結(jié)構(gòu)。而GFP組在提高分辨率時(shí)未見明顯骨質(zhì)形成。同時(shí)肌肉包塊在高分辨率(threshold=950)的情況,Ad-BMP9誘導(dǎo)下的細(xì)胞復(fù)合支架材料在骨質(zhì)上仍為高密度狀態(tài),成骨包塊范圍仍很明顯。證實(shí)在血運(yùn)豐富的區(qū)域更有利于細(xì)胞復(fù)合支架材料的成骨作用。13、HE染色示在BMP9誘導(dǎo)的皮下包塊及肌肉包塊均可見成熟基質(zhì)相互連接形成的網(wǎng)狀礦化結(jié)構(gòu),即骨小梁形成的網(wǎng)狀結(jié)構(gòu),提示BMP9在體外能很好的誘導(dǎo)iSCCSs成骨作用。在包塊的周邊細(xì)胞分化較好,可見部分分化的脂肪細(xì)胞。皮下包塊中,通過比較單純細(xì)胞注射和復(fù)合PCCN支架材料,復(fù)合PCCN支架材料的樣本HE染色中,其骨小梁形成的網(wǎng)狀結(jié)構(gòu)連接更加緊密、廣泛,更具層次性,提示復(fù)合PCCN能更有利于成熟骨的形成。而通過肌肉注射復(fù)合PCCN與皮下注射復(fù)合PCCN相比,其骨小梁形成的網(wǎng)狀結(jié)構(gòu)的成熟度更高,這與Micro-CT結(jié)果基本一致。提示血運(yùn)豐富的區(qū)域更有利于細(xì)胞復(fù)合支架材料的成骨作用。14、阿利新藍(lán)(Alicain Blue)染色示成骨組織包塊Alicain Blue染色可見網(wǎng)狀藍(lán)染結(jié)構(gòu),提示BMP9具有誘導(dǎo)iSCCSs分化為軟骨細(xì)胞的作用。15、成骨包塊的Trichrome染色可見大量深藍(lán)色組織形成的網(wǎng)狀結(jié)構(gòu),提示有大量膠原纖維和軟骨基質(zhì)形成。進(jìn)一步證明了BMP9能在體內(nèi)有效誘導(dǎo)iSCCSs分化為成骨細(xì)胞及成軟骨細(xì)胞。結(jié)論：1利用piggyBac轉(zhuǎn)座系統(tǒng)成功構(gòu)建永生化小鼠顱骨骨縫干細(xì)胞,永生化的小鼠顱骨骨縫干細(xì)胞能夠長時(shí)間進(jìn)行多代培養(yǎng),并且永生化的小鼠顱骨骨縫干細(xì)胞保持了間充質(zhì)干細(xì)胞干性。2永生化的小鼠顱骨骨縫干細(xì)胞增殖率明顯高于原代細(xì)胞,并且其增殖活性可以因永生化基因SV40T抗原被Flip/FRT重組酶敲除而逆轉(zhuǎn)；體內(nèi)注射正式永生化小鼠顱骨骨縫干細(xì)胞無成腫瘤傾向。3 BMP9對(duì)永生化小鼠顱骨骨縫細(xì)胞的誘導(dǎo)成骨能力在已知具有誘導(dǎo)成骨能力的BMP中是最強(qiáng)的,并且誘導(dǎo)成骨能力與BMP9具有劑量和時(shí)間相關(guān)性。4體外實(shí)驗(yàn)證實(shí),與常見具有成骨分化潛能的細(xì)胞相比永生化小鼠顱骨骨縫干細(xì)胞具有較強(qiáng)的成骨能力。5體內(nèi)、體外實(shí)驗(yàn)都證實(shí)小鼠顱骨骨縫干細(xì)胞具有向成骨、成軟骨、成脂肪分化的能力。6新型支架材料PCCN能夠給細(xì)胞黏附、增殖、分化提供較好的三維分層結(jié)構(gòu),促進(jìn)BMP9誘導(dǎo)iSCCSs的成骨和成軟骨作用。
[Abstract]:The research background is currently in the clinic. Bone defects, such as trauma, tumor, degeneration and congenital factors, are common. Although great progress has been made in internal fixation and surgical techniques in the reconstruction of bone, there are many challenges in the reconstruction of bone through surgery, such as autogenous bone mass and limitation of donor sites. The development of regenerative medicine and bone tissue engineering provides a new choice for bone reconstruction. Seed cells, biological scaffolds and bioactive factors are the three elements of bone tissue engineering, in which seed cells are the most basic link in bone tissue engineering. Seed cells provide the main source of active ingredients for regenerative medicine and tissue engineering. Stem cells are the most widely used seed cells. The bone sewn tissue of the skull has a strong ability to proliferate. It plays a vital role in the process of skull formation in the first thirty years of life. The osteogenesis of the tissue is mainly through the osteogenesis in the membrane. Its osteogenesis principle is that the ossification site of the bone is aggregated, differentiated and further osteogenic. The study shows that the stem cells of the skull bone seams have a certain differentiation potential. It is of great significance to study the mechanism and characteristics of the osteogenesis in the membrane. It is of great significance for the good seed cells of regenerative medicine and tissue engineering. It is also of great significance for the further study of the early closed closure of the cranium. Normally, after a period of time in vitro culture, the cells will appear to be stagnant, that is, M1 phase (senescence). The cells continue to proliferate and enter the proliferation inhibition state, that is, the M2 phase (crisis period), and then the cells gradually deteriorate and die. These will also occur in the stem cells. This brings many limitations to the source and research of regenerative medicine and tissue engineering cells, especially those that are scarce and difficult to obtain. The ability to enable cells to continue to proliferate. It provides sufficient seed cells for regenerative medicine and tissue engineering, which is more beneficial to the experimental research that.BMPs belongs to the TGF- beta superfamily, the family has at least 20 members, and BMPs expresses.BMPs in the body with extensive use in most species, in addition to the widely known ability to induce osteogenic differentiation. A variety of BMPs have been reported to have the ability to induce osteogenesis and have been reported to have the ability to induce osteogenesis, inducing osteogenic BMP2, BMP4, BMP6, BMP, and BMP9. The previous study group showed that BMP9 has the strongest osteogenic induction ability in bone mesenchymal stem cells in many BMPs, and it also induces cartilage and adipose formation in the bone mesenchymal stem cells. The previous study also confirmed that BMP9 has a good multi-directional differentiation ability for various stem cells from different sources. Therefore, we still choose BMP9 as a cytokine to induce the stem bone slit cells in immortalized mice in the study of cranial seted stem cells. It is an important group for regenerative medicine and tissue engineering. In part, the development of material science has greatly promoted the development of regenerative medicine and bone tissue engineering, and more and more scientific experiments and clinical applications have been verified and popularized. The basic characteristics of bone tissue materials are to provide three-dimensional structure environment for cell migration, proliferation and differentiation. In some bearing tissues, it is also available. An ideal scaffold material needs to meet the following characteristics: a three-dimensional and porous structure of bone tissue material is beneficial to cell growth, transport of nutrients and metabolites. (2) surface properties are beneficial to cell attachment, migration, proliferation and differentiation. The biodegradation rate is controllable in the process of cell / tissue growth. (4) the mechanical properties of the cells should be equivalent to the tissue in the transplanted region. The plastic.PCCN should be used as a new type of scaffold material. In addition to the above characteristics, it also has the characteristics of temperature sensitive and controllable shape. The role of bone repair, we use PCCN compound BMP9 induced mouse skull bone slit stem cells in vivo injection method to verify the role of the new scaffold in tissue engineering. 1, the use of piggyBac transposing system Gou Jianyong biochemical mouse skull bone sewn stem cells, and enter the stem cell markers to identify.2, compare SCCSs and iSCCSs The difference of proliferation rate of cell lines.3, iSCCSs reversal immortalization and in vivo tumorigenesis risk study of.4, BMP9 induced in vitro differentiation potential of bone slit stem cells in immortalized mice;.5, BMP9 induced calvarial bone sewn stem cells in immortalized mice and the differentiation of PCCN in vivo. Research methods: 1, selected 1 weeks aseptic CD1 mice, anesthesia, and anesthesia. After drunken effect, the mice were executed with broken neck, and the bone suture of the skull was exposed in aseptic operation. The bone seture of the skull was taken to obtain the primary cells. The immortalized SV40T antigen was transfected into the primary cells by the type I collagenase method, and the bone sewn stem cells.2 of the immortalized mice were constructed, and the cell immunofluorescence was used. The method of light staining was used to identify the marker of mesenchymal stem cells (.3) of iSCCSs. Through in vitro culture observation, crystal violet staining, WST-1 and other methods, the difference of the proliferation ability of immortalized mouse skull bone seture stem cells and primary cells was compared with that of immortalized mice. The immortalized gene SV40T antigen was knocked out by using Flip/FRT recombinant system in vitro and observed through in vitro culture observation. To identify the inverse immortalization of iSCCSs by means of crystal violet staining, WST-1 and Touch-down qPCR, and to identify the risk.5 of the tumor in vivo by the imaging detection of iSCCSs after iSCCSs injection in nude mice, using adenovirus infection technology, Ad-BMP2, Ad-BMP4, Ad-BMP6 Ad-BMP7, Ad-BMP9, respectively. Sex reading, alizarin red staining and Touch-down qPCR methods compared multiple BMP induced osteogenesis to iSCCSs in vitro.6. Using adenovirus infection techniques, Ad-BMP9 infection in the early stage of the laboratory has proved iCALs, iSCAPs, iMEFs, and iSCCSs, which have strong osteogenesis ability, and this experiment was constructed by ALP dyeing and ALP activity. The osteogenesis ability of.7 induced by various cell lines BMP9 was roughly compared. The time correlation and dose dependence of BMP9 induced iSCCSs osteogenesis were detected by ALP staining and ALP activity reading. The expression of OCN under BMP9 induced by BMP9 was identified by immunofluorescence staining, and oil red staining was used to identify the lipid formation of iSCCSs in BMP9 induced iSCCSs. Ability; using Alician Blue staining method to detect the chondrogenic ability of iSCCSs induced by BMP9. Using Touch-down qPCR to identify the related osteogenic, lipid, chondrogenic gene expression.8, Ad-BMP9 induced iSCCSs using simple cells and compound new scaffold material PCCN in nude mice and intramuscular injection. Through Micro-CT scan The osteogenesis ability of BMP9 in vivo and the role of scaffold material PCCN in osteogenesis were detected by HE staining of paraffin section, Alician Blue staining, and Trichrome staining to detect the osteogenesis, fat formation, chondrogenesis and PCCN effect of iSCCSs in BMP9 in vitro. Results 1, the original mouse skull bone sewn stem cells were successfully obtained. Through the piggyBac transposing system,.2 was successfully constructed in the immortalized mouse skull bone sewn stem cells, and the immunofluorescence staining on the surface markers of the stem cells was performed on iSCCSs. The results showed that CD29 (intergin beta 1), CD73, CD113 (prom1), CD40, CD90 (Thy1), CD117, 166, and II were expressed. The stem cell characteristics of stem cells.3, through in vitro culture observation, crystal violet staining, WST-1 and other methods proved that the immortalized mouse bone slit stem cells have a stronger proliferation ability compared with the original cells. The difference in proliferation is found on the first day after culture, and there is a significant difference between third, fifth and seven periods. In vitro, the immortalized gene SV40T antigen was knocked out by Flip/FRT recombinant system, and by culture observation in vitro and crystal violet staining. WST-1 confirmed that the proliferation ability of the control group (group Ad-GFP) was still strong, while the proliferation rate of the Ad-Flip group in the experimental group was significantly slower, and the proliferation rate was kept in a lower water in seven days. The expression of SV40T antigen gene in the flat.Touch-down qPCR was significantly lower in the experimental group than in the control group. It was proved that the SV40T antigen gene could be knocked out, the cell immortalization could reverse.5, and the piggyBac transposing system was used to successfully construct the iSCCS cells labeled with fluorescein luciferase (Fluc). The living body imaging of the nude mice after iSCCSs was detected. The test showed that the signal was strong at 5 days after the injection of iSCCSs-Fluc, and the signal was attenuated at the 10 day, and the signal had basically disappeared at the time of fifteenth days. No tumor samples were produced by the general observation of the subcutaneous injection site. It showed that iSCCSs had strong proliferative energy in the body, but there was no risk of.6, a variety of BMPs (BMP2, BMP). 4, BMP6, BMP7, BMP9) induced iSCCSs.ALP staining, ALP reading, alizarin red staining proved that the five kinds of BMP have bone induction ability, but BMP2 and BMP9 have stronger osteogenesis, among which BMP9 is the strongest, and the expression aspect of osteogenesis gene ALP, OPN, OSX, proves to be the strongest in induction of osteogenesis. Under the induction, the osteogenesis of iSCCSs was stronger.8 than that of iCALs, iSCAPs, iMEFs, which had strong osteogenesis ability, and BMP9 induced iSCCSs osteogenesis to have a positive time correlation and dose dependent.9. The expression of OCN in the bone index of the bone index was detected by immunofluorescence staining, and it was proved that BMP9 could induce OCN high expression.10. The staining (Oil Red O staining) showed that the cells in the BMP9 induced group were vacuolated, adipocyte like, iSCCSs had the potential of adipocyte potential, Alician Blue staining of the cell group, and the blue staining of BMP9 group, confirmed that iSCCSs had the potential of chondrocytes. Ad-BMP9 induced iSCCSs, both simple cell injection and composite scaffold material, both subcutaneous injection and intramuscular injection, showed high density bone formation. The subcutaneous mass and the transection of the muscle mass could see the structure of the bone cortex and trabecular bone. The GFP group had no obvious bone formation when the resolution was raised. When the muscle mass was in high resolution (threshold=950), the Ad-BMP9 induced cell composite scaffold remained high density on the bone, and the scope of the osteoblast was still obvious. It was proved that the blood rich area was more beneficial to the osteogenesis of the cell composite scaffold.13, and HE staining was shown in the subcutaneous mass and muscle package induced by BMP9. The reticular mineralization structure formed by the interconnected matured matrix, that is, the net structure formed by the trabecular bone, suggests that the BMP9 can induce iSCCSs osteogenesis in vitro. The cells in the periphery of the block are well differentiated and partially differentiated adipocytes. In the subcutaneous mass, a comparison of simple cell injection and compound PCCN scaffolds is made in subcutaneous mass. In the sample HE staining of the composite PCCN scaffold, the net structure of the trabecular bone formed by the bone trabecula is more closely, extensive and more hierarchical, suggesting that the compound PCCN can be more beneficial to the formation of mature bone. The reticular formation of the trabecular bone formed by intramuscular injection of PCCN and subcutaneous injection of PCCN is higher than that of Micro-CT. The results are basically the same. It is suggested that the rich area of blood is more beneficial to the osteogenesis of cell composite scaffold.14. Alicain Blue staining shows that Alicain Blue staining of osteoblast tissue is a visible reticular blue staining structure, suggesting that BMP9 has the effect of inducing iSCCSs to differentiate into cartilage cells, and the Trichrome staining of the osteogenic mass is visible. The net structure of deep blue tissue indicates the formation of a large amount of collagen fibers and cartilage matrix. It further proves that BMP9 can be found in vivo.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R318.08;R683
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本文編號(hào)：1964686