正常和炎症来源的人牙周膜干细胞内皮向分化能力的比较研究

发布时间：2018-05-23 08:22

本文选题：人牙周膜干细胞 + 慢性牙周炎　；参考：《第四军医大学》2014年硕士论文

【摘要】：牙周病作为口腔常见慢性疾病会导致牙周支持组织的破坏，包括硬组织如牙槽骨的吸收和软组织如牙龈的退缩等，如未及时治疗，将会导致牙齿的松动甚至缺失，严重影响人们的健康生活。目前牙周支持组织的再生和重建仍然是牙周治疗尚未解决的难题，近年来组织工程成为牙周再生研究的热点方向，其中的种子细胞来源及其功能表现更是关键性影响因素。牙周膜干细胞（periodontal ligament stem cells,PDLSCs）由于组织来源相近、高度增殖能力及多向分化能力等特点已成为牙周组织工程的首选种子细胞。但牙周膜干细胞通常需要取健康人因正畸或阻生所拔除的牙齿，来源比较局限，因此炎症组织来源的牙周膜干细胞（inflammatory derivedperiodontal ligament stem cells,iPDLSCs）也已经被分离培养和鉴定，并证明其同样具有高度增殖能力，多向分化能力等种子细胞特征。正常和炎症来源的牙周膜干细胞均能在成骨、成脂微环境中正常分化，但两者的分化能力差异比较目前仍有争议。组织工程三要素还包括细胞因子和支架材料，除此之外，血供也是不容忽视的要素。通过组织工程技术再生的组织只有在血供充足的情况下才能稳定生存。而牙周组织工程所涉及的牙根面部位和需要重建的大量牙槽骨缺损处，必须有血管的形成才能为再生组织提供营养，这是保证牙周再生获得成功不可缺少的一环。前期骨髓来源、脂肪来源、皮肤来源等的间充质干细胞均被证明在成血管培养基诱导下能向内皮细胞分化且具有成血管能力，并且炎症环境可促进脂肪干细胞向内皮细胞分化。因此本实验旨在探索牙周膜干细胞是否具有内皮向分化能力，并同时比较正常和炎症来源的牙周膜干细胞成血管能力的差异，以期为牙周膜干细胞成骨及成血管能力的共同实现提供参考，为牙周组织工程的应用提供依据。研究目的：通过成血管培养条件诱导正常和炎症来源的人牙周膜干细胞，观察其内皮向分化的能力，并且比较其成血管能力的差异，为牙周组织再生的作用机制及应用提供依据。研究方法： 1、牙周膜干细胞的分离、培养和鉴定：临床收集正常和炎症来源的离体牙，通过改良酶组织块消化法分离培养牙周膜细胞，经有限稀释法纯化得到牙周膜干细胞。流式细胞术检测干细胞表面标记物；CCK-8法检测并比较PDLSCs和iPDLSCs的生长曲线及增殖能力；克隆形成实验检测PDLSCs和iPDLSCs自我更新能力并比较克隆形成率的大小。 2、牙周膜干细胞的内皮向诱导：当PDLSCs和iPDLSCs生长达80%-90%汇合时，弃原培养液，加入诱导培养基（含20mL/L胎牛血清，10mL/L青霉素链霉素混合液，25ng/mLVEGF，25ng/mLbFGF的-MEM培养液），常规条件下培养10天。 3、体外检测并比较PDLSCs和iPDLSCs成血管能力：通过免疫荧光、real-timeRT-PCR、Matrigel assay来检测PDLSCs和iPDLSCs内皮细胞标记物的表达及体外管腔形成能力。 4、体内实验检测PDLSCs和iPDLSCs成血管能力：扫描电镜观察PDLSCs和iPDLSCs在HA/β-TCP材料表面的黏附伸展情况，并植入裸鼠皮下，3周后取材，组织学观察其体内成血管能力。 5、统计学分析：用SPSS19.0统计软件对数据（x±s）进行方差分析，两两比较用t检验，检验水准=0.05。研究结果： 1、经培养纯化成功获取PDLSCs和iPDLSCs，显微镜下观察细胞均呈长梭形纺锤状；流式细胞术检测细胞均阳性表达间充质干细胞标记物：CD29、CD90、CD105、CD146，两者表达率无统计学差异，阴性表达造血干细胞标记物：CD34、CD45。CCK-8检测结果显示PDLSCs和iPDLSCs生长曲线均呈“S”型，6天前各时间点，iPDLSCs的数量高于正常PDLSCs，尤其是第3和第4天，具有统计学意义（P＜0.05）。PDLSCs和iPDLSCs均具有一定的克隆形成能力，其中iPDLSCs克隆形成率高于PDLSCs，差异具有统计学意义。 2、PDLSCs和iPDLSCs经成血管诱导10天后，显微镜下观察细胞呈“鹅卵石”样，与内皮细胞形态基本相同。 3、PDLSCs和iPDLSCs内皮向诱导10天后，免疫荧光检测显示两者均阳性表达内皮细胞标记物VEGF、vWF，iPDLSCs表达的阳性率高于PDLSCs；real-timeRT-PCR检测两者诱导后均表达CD31、vWF、VE-cadherin，其中iPDLSCs的CD31、VE-cadherin mRNA表达水平均明显高于PDLSCs，差异有统计学意义（P＜0.05）；诱导后的PDLSCs和iPDLSCs均能在Matrigel基质胶上形成管腔样结构，并相连成网状，通过软件半定量分析，iPDLSCs形成的分支节点数、管腔长度均高于PDLSCs，差异有统计学意义（P＜0.05）。 4、经扫描电镜观察，PDLSCs和iPDLSCs在HA/β-TCP表面均能正常黏附并伸展，植入裸鼠皮下3周后HE染色可见有新生血管生成，其中炎症来源的牙周膜干细胞诱导组形成的血管数量最多，免疫组化染色可见诱导后的PDLSCs和iPDLSCs均阳性表达CD31。结论 PDLSCs和iPDLSCs具有高度增殖能力和自我更新能力，，在成血管诱导培养基作用下均能向内皮细胞分化，而iPDLSCs成血管能力强于PDLSCs。
[Abstract]:Periodontal disease, as a common chronic disease in the mouth, can lead to the destruction of periodontal support tissue, including the absorption of hard tissue such as alveolar bone and the withdrawal of soft tissue such as gingiva. If not treated in time, it will lead to the loosening or even loss of the teeth. The regeneration and reconstruction of periodontal support tissues are still periodontal treatment. In recent years, tissue engineering has become a hot topic in the study of periodontal regeneration. The source and function of the seed cells are the key factors. The periodontal ligament stem cells (PDLSCs) has become a kind of high proliferation and multidirectional differentiation because of the similar tissue sources. Periodontal tissue engineering is the first choice of seed cells. But periodontal stem cells usually need to be extracted from healthy people because of orthodontic or hindrance. The source of inflammatory derivedperiodontal ligament stem cells (iPDLSCs) has also been isolated and identified, and it has been proved to be the same. It is characterized by high proliferation and multidirectional differentiation. Normal and inflammatory periodontal stem cells can differentiate normally in osteogenesis and lipid microenvironments, but differences in differentiation are still controversial. The three elements of tissue engineering include cellular and scaffold materials, in addition to this, blood supply is not allowed. The elements of neglect. The tissue regenerated by tissue engineering can survive only if the blood supply is sufficient. The root surface parts involved in the periodontal tissue engineering and the large number of alveolar bone defects that need to be rebuilt must be formed to provide camping for the regenerated tissue, which ensures the success of periodontal regeneration. A missing link. Mesenchymal stem cells, such as early bone marrow sources, fat sources, and skin sources, have been proved to differentiate into endothelial cells and have angiogenic ability under the induction of vascular media, and the inflammatory environment can promote the differentiation of fat stem cells into endothelial cells. In order to provide reference for the common realization of osteogenesis and vascular ability of periodontal ligament stem cells, the differentiation ability of skin to differentiation and the comparison of the vascular ability of periodontal membrane stem cells between normal and inflammatory sources is expected to provide a basis for the application of periodontal tissue engineering.
The purpose of the study is:
Human periodontal ligament stem cells were induced by vascular culture, and the ability of endothelial differentiation was observed and the difference of their vascular ability was compared, which provided the basis for the mechanism and application of periodontal tissue regeneration.
Research methods:
1, the separation, culture and identification of periodontal membrane stem cells: the isolated teeth of normal and inflammatory sources were collected, the periodontal ligament cells were isolated and cultured by modified enzyme tissue block digestion, and the periodontal membrane stem cells were purified by the finite dilution method. Flow cytometry was used to detect the surface markers of the stem cells. The CCK-8 method was used to detect and compare the growth of PDLSCs and iPDLSCs. Long curve and proliferation ability; clone formation test to detect PDLSCs and iPDLSCs self-renewal ability and compare the size of colony formation rate.
2, the endothelium of the periodontal ligament stem cells was induced. When the growth of PDLSCs and iPDLSCs reached 80%-90%, the culture medium was abandoned, and the inducible medium (including 20mL/L fetal bovine serum, 10mL/L penicillin streptomycin mixture, 25ng/mLVEGF, 25ng/mLbFGF -MEM Culture) was cultured for 10 days under conventional conditions.
3, in vitro and in vitro detection and comparison of the vascular ability of PDLSCs and iPDLSCs: the expression of PDLSCs and iPDLSCs endothelial cell markers and the capacity of the endothelium in vitro were detected by immunofluorescence, real-timeRT-PCR, and Matrigel assay.
4, in vivo, the ability of PDLSCs and iPDLSCs was detected in vivo: scanning electron microscopy was used to observe the adhesion and extension of PDLSCs and iPDLSCs on the surface of HA/ beta -TCP material, and implanted subcutaneously in nude mice, then harvested after 3 weeks, and observed the vascular capacity in the body histologically.
5, statistical analysis: using SPSS19.0 statistical software to analyze variance (x + s), and 22 compare t test to test the level of =0.05..
The results of the study:
1, PDLSCs and iPDLSCs were successfully obtained by culture and purification. The spindle shaped spindle shaped cells were observed under microscope. Flow cytometry was used to detect the positive expression of mesenchymal stem cell markers: CD29, CD90, CD105, CD146. The expression rate of the two cells was not statistically different, and the negative expression of blood stem cell markers: CD34, CD45.CCK-8 detection results showed PD The growth curve of LSCs and iPDLSCs all showed "S" type. The number of iPDLSCs was higher than normal PDLSCs at every time 6 days ago, especially in third and fourth days. There were statistical significance (P < 0.05).PDLSCs and iPDLSCs had certain clone formation ability, and the formation rate of iPDLSCs clones was higher than that of PDLSCs, and the difference was statistically significant.
2, after 10 days of induction by PDLSCs and iPDLSCs, the cells were observed as "cobblestone" under microscope, and the morphology of endothelial cells was basically the same.
3, 10 days after the induction of PDLSCs and iPDLSCs endothelial cells, the immunofluorescence detection showed that both the positive expression of endothelial cell markers VEGF, vWF, iPDLSCs expression was higher than PDLSCs; real-timeRT-PCR detection both expressed CD31, vWF, VE-cadherin, and iPDLSCs CD31. The difference has statistical significance (P < 0.05). The induced PDLSCs and iPDLSCs can form a lumen like structure on the Matrigel matrix and connect into a network. By semi quantitative analysis of the software, the number of branch nodes formed by iPDLSCs is higher than that of PDLSCs, and the difference is statistically significant (P < 0.05).
4, after scanning electron microscope, PDLSCs and iPDLSCs can adhere and extend normally on the surface of HA/ beta -TCP. After implantation of nude mice for 3 weeks, HE staining shows the formation of neovascularization, in which the number of blood vessels formed in the induced periodontal ligament stem cells of the inflammation is the most, and the immuno histochemical staining can show the positive expression of CD31. in both PDLSCs and iPDLSCs.
conclusion
PDLSCs and iPDLSCs have high proliferation ability and self-renewal ability. They can differentiate into endothelial cells under the action of vascular induced medium, and the ability of iPDLSCs to form blood vessels is stronger than that of PDLSCs..
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4

【参考文献】