BMP9诱导永生化小鼠颅骨骨缝干细胞分化的初步研究

发布时间：2018-06-01 15:33

  本文选题：细胞永生化 + iSCCSs　； 参考：《山东大学》2016年博士论文

【摘要】：研究背景目前在临床中,由于创伤、肿瘤、退变及先天性等因素导致的骨缺损还很常见,虽然在骨重建中骨科内固定和手术技术方面取得了很大的进步,但通过手术进行骨重建中还是会遇到很多的挑战,比如自体骨量和供区的限制等。随着再生医学和骨组织工程的发展,给骨重建提供了一种新的选择。种子细胞、生物支架材料、生物活性因子是骨组织工程的三要素,其中种子细胞是骨组织工程的最基本的环节。种子细胞为再生医学和组织工程提供活性成分的主要来源,是其核心部分。干细胞作为具有多向分化潜能的特点,是目前应用最广泛的种子细胞。颅骨骨缝组织具有很强的增殖能力,其在人生中的前三十年中颅骨的形成过程中起着至关重要的作用。与一般长骨软骨下成骨方式不同的是颅骨骨缝组织的成骨主要是通过膜内成骨。其成骨原理是在一定的刺激条件下其骨化位点通过重叠的成骨前缘间充质干细胞的聚集、分化而进一步成骨。研究表明颅骨骨缝干细胞具有一定的分化潜能。通过颅骨骨缝组织干细胞的研究,对进一步研究膜内成骨的机制和特点、探索为再生医学和组织工程良好的种子细胞具有重要意义,同时也为进一步研究颅骨骨缝早闭等疾病有重要意义。通常情况下正常的组织细胞,在体外培养一段时间后,细胞会出现生长停滞,即M1期(衰老期),细胞继续增殖后会进入增殖抑制状态,即M2期(危机期),随后细胞逐渐出现退化、死亡。这些情况同样会发生在干细胞身上。这为再生医学和组织工程细胞来源及研究带来诸多限制,尤其是那些稀少、取材困难的细胞。通过细胞永生化可以使细胞获得持续增殖的能力。为再生医学和组织工程提供了足够的种子细胞,更有利于进行实验研究。BMPs属于TGF-β超家族,家族至少有20个成员,BMPs在大多数物种均有表达。BMPs在体内具有广泛的作用,除了广为所知的诱导成骨分化的能力外,还参与多种组织和器官的形成和修复。多种BMPs被报道具有诱导成骨的能力,诱导成骨BMP2、BMP4、BMP6、BMP、BMP9的报道较多。课题组前期实验证明,BMP9在众多BMPs中对骨间充质干细胞具有最强的成骨诱导能力,同时还有诱导成软骨和成脂肪的能力。课题组前期研究亦证实,BMP9对多种不同部位来源的干细胞具有较好的多向诱导分化能力。因此,在颅骨骨缝干细胞研究中,我们仍选择BMP9作为细胞因子进行诱导永生化小鼠颅骨骨缝干细胞。作为再生医学和组织工程很重要的组成部分,材料科学的发展极大的推进了再生医学和骨组织工程的发展,越来越多科学实验和临床应用得到了验证和推广。目前对于骨组织材料的基本特性是能给细胞迁移、增殖和分化提供三维结构环境,在一些承重的组织中,它同样可以起到暂时的机械结构支撑作用。一个理想的支架材料需要满足以下特点：①骨组织材料的三维和多孔网状结构有利于细胞生长、营养和代谢产物的运输。②表面特性有利于细胞贴附、迁移、增殖和分化。③具有生物相容性,不会引起免疫反应,在细胞／组织生长过程中其生物降解率可控。④其力学特性应当跟移植区域的组织相当⑤应当具有可塑性。PCCN作为一种新型的支架材料,除了具备以上特点外,还具有温敏可控塑形的特点。为了验证这种新型支架材料对骨及软骨修复中的作用,我们用PCCN复合BMP9诱导下的小鼠颅骨骨缝干细胞体内注射的方法来验证新型支架在组织工程中的作用。研究目的：1、运用piggyBac转座系统构建永生化的小鼠颅骨骨缝干细胞,并进行干细胞标志物鉴定。2、比较SCCSs与iSCCSs细胞系的增殖率差异。3、iSCCSs的逆转永生化及体内成瘤风险研究。4、BMP9诱导永生化小鼠颅骨骨缝干细胞的体外分化潜能研究。5、BMP9诱导永生化小鼠颅骨骨缝干细胞并复合PCCN体内分化初步研究。研究方法：1、选取1周大小的无菌CD1小鼠,麻醉起效后将小鼠断颈处死,无菌操作暴露颅骨骨缝,取颅骨骨缝组织,采用Ⅰ型胶原酶溶解方法使细胞贴壁获得原代细胞。用piggyBac转座系统将永生化基因SV40T抗原转染至原代细胞中,构建永生化小鼠颅骨骨缝干细胞。2、采用细胞免疫荧光染色的方法鉴定iSCCSs的间充质干细胞标志物。3、通过体外培养观察、结晶紫染色、WST-1等方法,比较永生化小鼠颅骨骨缝干细胞与原代细胞增殖能力的差异。4、体外通过利用Flip/FRT重组系统,将永生化基因SV40T抗原敲除,通过体外培养观察、结晶紫染色、WST-1及Touch-down qPCR等方法来鉴定iSCCSs的逆永生化；通过对裸鼠体内注射iSCCSs后活体成像检测来鉴定其有无体内成瘤风险性。5、利用腺病毒感染技术,用Ad-BMP2、Ad-BMP4、Ad-BMP6 Ad-BMP7、 Ad-BMP9分别对iSCCSs进行感染,通过ALP染色、ALP活性读数、茜素红染色和Touch-down qPCR的方法比较多种BMP对iSCCSs的体外诱导成骨能力。6、利用腺病毒感染技术,用Ad-BMP9分别感染实验室前期已经验证过具有较强成骨能力的干细胞iCALs、iSCAPs、iMEFs及本实验构建的iSCCSs,通过ALP染色及ALP活性读数来大致比较各种细胞系BMP9诱导下的体外成骨能力。7、通过ALP染色和ALP活性读数检测BMP9诱导iSCCSs成骨分化时间相关性和剂量相关性；运用免疫荧光染色的方法来鉴定BMP9诱导下的OCN的表达；运用油红染色鉴定BMP9诱导下iSCCSs的成脂能力；运用Alician Blue染色的方法检测BMP9诱导下iSCCSs的成软骨能力。应用Touch-down qPCR来鉴定相关成骨、成脂、成软骨基因的表达。8、将Ad-BMP9诱导下iSCCSs采用单纯细胞和复合新型支架材料PCCN于裸鼠皮下注射和肌肉注射。通过Micro-CT扫描来检测BMP9的体内诱导成骨能力和支架材料PCCN在成骨中的作用。通过对成骨包块石蜡切片的HE染色、Alician Blue染色、Trichrome染色来检测BMP9体外诱导iSCCSs的成骨、成脂、成软骨能力和PCCN的作用。结果1、成功获得原代小鼠颅骨骨缝干细胞,并通过piggyBac转座系统成功构建永生化小鼠颅骨骨缝干细胞。2、对iSCCSs进行干细胞表面标记物免疫荧光染色,结果显示CD29(intergin β1)、CD73、CD113 (prom1)、CD40、CD90 (Thy1)、CD117(c-kit)、 CD 166 (ALCAM)、CD105(Endglin)和BMPRⅡ具有表达。证实了iSCCSs保持了间充质干细胞的干细胞特性。3、通过体外培养观察、结晶紫染色、WST-1等方法证实与原代细胞相比,永生化后的小鼠扁骨骨缝干细胞具有更强的增殖能力,这种增殖上的差异自培养后第一天即存在,并且在第三、五、七各个时间段均存在明显差异。4、体外通过利用Flip/FRT重组系统,将永生化基因SV40T抗原敲除,通过体外培养观察、结晶紫染色、WST-1证实：对照组(Ad-GFP组)细胞增殖能力仍然旺盛,而实验组Ad-Flip组细胞的增殖速度明显变慢,在连续七天内,增殖率一致维持在一个较低的水平。Touch-down qPCR结果SV40T抗原基因在实验组的表达明显低于对照组。证实SV40T抗原基因能够被敲除,细胞的永生化可以逆转。5、应用piggyBac转座系统成功构建萤光虫荧光素酶(Fluc)标记的iSCCS细胞(iSCCSs-Fluc)。裸鼠体内注射iSCCSs后活体成像检测显示在iSCCSs-Fluc注射后,5天的时候信号强烈,10天的时候信号较前出现衰减,到第15天的时候信号已经基本消失。通过大致观察皮下注射部位亦无肿瘤样物生成。表明iSCCSs在体内具有较强的增殖能力,但是无成瘤的危险。6、多种BMPs(BMP2、BMP4、BMP6、BMP7、BMP9)诱导iSCCSs。ALP染色、ALP读数、茜素红染色证实这五种BMP均有骨诱导能力,但BMP2和BMP9的诱导成骨作用更强,其中BMP9最强；成骨基因ALP、OPN、OSX、RUNX2的表达方面证实对于BMP9对iSCCSs的诱导成骨能力最强。7、ALP染色和读数显示,在BMP9的诱导下,与已经验证过具有较强成骨能力的干细胞iCALs、iSCAPs、iMEFs相比,iSCCSs的成骨能力更强。8、BMP9诱导iSCCSs成骨分化具有正向的时间相关性和剂量相关性。9、免疫荧光染色检测成骨指标OCN的表达,证实BMP9能够诱导OCN的高表达。10、油红O染色(Oil Red O staining)显示BMP9诱导组的细胞成空泡样,成脂肪细胞样变,iSCCSs具有成脂肪细胞的潜能。11、细胞团的Alician Blue染色,BMP9组蓝染,证实iSCCSs具有成软骨细胞的潜能。成脂、成软骨基因的表达与对照组亦有增高。12、Micro-CT结果显示：Ad-BMP9诱导下的iSCCSs,无论是单纯细胞注射,还是复合支架材料,无论是皮下注射,还是肌肉内注射,均可见高密度骨质形成,其中皮下包块及肌肉包块的横断扫描均可见骨皮质、骨小梁等结构。而GFP组在提高分辨率时未见明显骨质形成。同时肌肉包块在高分辨率(threshold=950)的情况,Ad-BMP9诱导下的细胞复合支架材料在骨质上仍为高密度状态,成骨包块范围仍很明显。证实在血运丰富的区域更有利于细胞复合支架材料的成骨作用。13、HE染色示在BMP9诱导的皮下包块及肌肉包块均可见成熟基质相互连接形成的网状矿化结构,即骨小梁形成的网状结构,提示BMP9在体外能很好的诱导iSCCSs成骨作用。在包块的周边细胞分化较好,可见部分分化的脂肪细胞。皮下包块中,通过比较单纯细胞注射和复合PCCN支架材料,复合PCCN支架材料的样本HE染色中,其骨小梁形成的网状结构连接更加紧密、广泛,更具层次性,提示复合PCCN能更有利于成熟骨的形成。而通过肌肉注射复合PCCN与皮下注射复合PCCN相比,其骨小梁形成的网状结构的成熟度更高,这与Micro-CT结果基本一致。提示血运丰富的区域更有利于细胞复合支架材料的成骨作用。14、阿利新蓝(Alicain Blue)染色示成骨组织包块Alicain Blue染色可见网状蓝染结构,提示BMP9具有诱导iSCCSs分化为软骨细胞的作用。15、成骨包块的Trichrome染色可见大量深蓝色组织形成的网状结构,提示有大量胶原纤维和软骨基质形成。进一步证明了BMP9能在体内有效诱导iSCCSs分化为成骨细胞及成软骨细胞。结论：1利用piggyBac转座系统成功构建永生化小鼠颅骨骨缝干细胞,永生化的小鼠颅骨骨缝干细胞能够长时间进行多代培养,并且永生化的小鼠颅骨骨缝干细胞保持了间充质干细胞干性。2永生化的小鼠颅骨骨缝干细胞增殖率明显高于原代细胞,并且其增殖活性可以因永生化基因SV40T抗原被Flip/FRT重组酶敲除而逆转；体内注射正式永生化小鼠颅骨骨缝干细胞无成肿瘤倾向。3 BMP9对永生化小鼠颅骨骨缝细胞的诱导成骨能力在已知具有诱导成骨能力的BMP中是最强的,并且诱导成骨能力与BMP9具有剂量和时间相关性。4体外实验证实,与常见具有成骨分化潜能的细胞相比永生化小鼠颅骨骨缝干细胞具有较强的成骨能力。5体内、体外实验都证实小鼠颅骨骨缝干细胞具有向成骨、成软骨、成脂肪分化的能力。6新型支架材料PCCN能够给细胞黏附、增殖、分化提供较好的三维分层结构,促进BMP9诱导iSCCSs的成骨和成软骨作用。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R683
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本文编号：1964686