MIP-3α联合成骨诱导液诱导大鼠脂肪干细胞向成牙本质样细胞分化的实验研究

发布时间：2019-01-06 13:55

【摘要】：因龋齿或牙周疾病导致的牙齿缺失是人群中的常见病、多发病，世界卫生组织（WHO）将牙齿疾病列为人类发病率最高的三大非传染性疾病之一。临床常用的修复牙齿缺失的方法，如人工材料替代缺失牙齿等，会引起包括免疫排斥在内的诸多不良反应。组织工程技术的出现及发展为牙齿再生提供了新的思路。牙髓干细胞（DPSCs）、牙周膜干细胞（PDLSCs）、牙囊干细胞（DFSCs）、根尖乳头干细胞（SCAP）、人类脱落乳牙干细胞（SHEDs）等牙源性干细胞均表现出了良好的牙向分化潜能，可用作牙齿组织工程的种子细胞，但因其在成人尤其是老年人中来源有限，限制了临床应用；非牙源性干细胞，如骨髓基质干细胞（BMSC），亦能够形成牙齿样及牙周样组织，但其在临床获取相对不便，且BMSC随着供体年龄增加其多项分化能力明显下降，亦存在着诸多不利因素，制约了其在牙齿再生领域的应用。脂肪干细胞（ASC）来源广泛，取材方便，含量丰富，在体内及体外试验中均显示出了良好的自我更新及多向分化潜能，并且其向矿化组织分化的能力不受供体年龄的影响。因此，，本实验首先在体外分离、培养并鉴定了大鼠脂肪干细胞，然后探讨了其向成牙本质样细胞分化的可能性及其条件，以期为牙齿组织工程种子细胞的研究提供新的思路。方法 1.原代分离培养SD大鼠脂肪干细胞，采用倒置显微镜观察细胞的形态和生长状况。将原代细胞培养传代纯化至第三代后采用MTT法测细胞生长曲线，流式细胞仪检测脂肪干细胞表面标志物。 2.取第三至四代生长状态良好的大鼠脂肪干细胞，TNE消化收获细胞，接种于6孔板。分别以成骨及成脂诱导基诱导大鼠脂肪干细胞骨向及脂向分化。而后进行ALP染色、茜素红染色及油红O染色实验。 3.消化收获第三至四代生长状况良好的大鼠脂肪干细胞，接种后用MIP-3α联合成骨诱导基诱导。分别于第1、4、7天行ALP活性检测，并以PR-PCR及Westen Blotting检测成牙本质细胞标志基因及蛋白。以常规培养基、成骨诱导培养基做为对照组。结果 1.大鼠脂肪干细胞生长状态良好，MTT法测细胞生长曲线显示大致“S”型。表面标志物CD29+、CD49d+、CD45-。 2.成骨诱导显示2周时光镜下可见部分细胞向心性聚集生长，胞质内可见高透光结节状颗粒。ALP染色可见胞核周围棕黑色颗粒。3周时茜素红染色可见红色钙化结节。成脂诱导显示1周时细胞周围可见散在脂滴，并不断增大。3周时油红O染色可见胞质分布大量红色大小不一的圆形脂滴，胞核蓝染位于细胞一侧。成骨及成脂诱导表明本实验使用的大鼠脂肪干细胞具有多项分化潜能。 3.成牙本质诱导实验表明，大鼠脂肪干细胞经诱导后ALP活性增高，且在mRNA水平表达成牙本质标志基因及蛋白，高于成骨诱导液组（P0.05）；并且表达量随时间的推移而增加(P0.05)。常规培养基组无成牙本质标志基因及蛋白的表达。结论 1.大鼠脂肪干细胞原代分离培养成功，具有良好的生长趋势及自我更新能力。CD29+、CD49d+、CD45-提示本实验所用细胞为比较单一的未分化的脂肪来源干细胞。 2.大鼠脂肪干细胞在合适的诱导条件下能够向骨及脂肪分化，具有多向分化潜能。 3.成牙本质诱导后大鼠脂肪干细胞矿化能力增加，并能够表达成牙本质特异性基因及蛋白，诱导时间短，诱导效能高，与以往方法相比具有更加切实可行的临床意义。
[Abstract]:Tooth loss due to dental caries or periodontal disease is one of the most common diseases in the population, and the World Health Organization (WHO) is one of the three major non-communicable diseases with the highest incidence of human morbidity. The most commonly used methods of repairing the missing teeth, such as artificial material replacement for missing teeth, can cause a number of adverse reactions, including immune rejection. The emergence and development of tissue engineering technology provides a new way for tooth regeneration. Dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DSCs), apical papilla stem cells (SCAP), and human exfoliated breast stem cells (SHEDs) exhibited good tooth-to-differentiation potential and can be used as seed cells for dental tissue engineering, however, because of its limited source in adult, especially in that elderly, the clinical application is limited; non-dental origin stem cells, such as bone marrow stromal stem cells (BMSC), can also form tooth-like and periodontal-like tissue, but they are relatively inconvenienced in clinical acquisition, in addition, that BMSC, with the increase of the age of the donor, has a significant decrease in its multiple differentiation ability, and there are many unfavorable factors, which restrict the application of the BMSC in the field of tooth regeneration. The origin of the adipose-derived stem cells (ASC) is wide, the materials are convenient, the content is abundant, good self-renewal and multi-directional differentiation potential are shown in the in-vivo and in-vitro test, and the ability to differentiate into the mineralized tissue is not affected by the donor's age In this experiment, the rat adipose-derived stem cells were isolated, cultured and identified in vitro, then the possibility and conditions of the differentiation to the odontoid-like cells were discussed, with a view to providing a new thought for the study of the seed cells of the tooth tissue engineering. Road. Method 1. Primary isolated cultured SD rat adipose-derived stem cells, and the morphology of the cells was observed with an inverted microscope. and the cell growth curve is measured by the MTT method after the primary cell culture is passaged and purified to the third generation, and the fat stem cell is detected by the flow cytometry. Surface marker. 2. Rat fat stem cells with good growth status from third to fourth generation, and TNE digested and harvested cells. and inoculating to a 6-hole plate to induce the adipose-derived stem cells of the rat in the form of a bone and a fat-forming induction group, respectively. the bone is differentiated to the fat, then the ALP is stained, the fluorescein red is dyed and Oil red O staining test. 3. The third to fourth generation of fat stem cells from the third to fourth generation were digested and harvested, and the cells were inoculated with MIP-3. The ALP activity was detected in the 1st, 4th and 7th day, respectively, and the dentin was detected by PR-PCR and Westen Blotting. cell marker gene and protein. Pei Pei The results showed that the growth status of the adipose-derived stem cells in the rat was good, and the growth curve of the cell growth curve was generally "S" type. Surface marker CD29 + CD49d +, CD45-. long, high light-transmitting nodular particles in the cytoplasm. The staining of ALP can be seen in the dark brown granules around the nucleus. In the week, red and calcified nodules were seen in the red staining of the red blood. In the case of lipid-induced display, the peripheral blood of the cells was scattered on the surface of the lipid droplets, and increased. At the end of the third week, the red O-staining of the oil showed a large number of red and different levels of red blood. A drop of circular fat, and the nucleus of the nucleus is located on one side of the cell. The bone and the fat-forming induce the use of this experiment. The rat adipose-derived stem cells had a number of differentiation potential. 3. The induction of the dentine in the rat indicated that the ALP activity of the rat adipose-derived stem cells increased after induction, and the mRNA level was expressed as the dentin marker gene and the protein, which was higher than that of the bone-inducing liquid group (P0.05); and the expression of the protein was higher than that of the bone-inducing liquid group (P0.05). The increase of the amount over time (P0.05). base group Conclusion: The primary separation of the rat adipose-derived stem cells The culture was successful, with good growth tendency and self-renewal ability. CD29 +, CD49d +, CD45-prompting the experiment The cells used are relatively single undifferentiated fat-derived stem cells. in that induction condition, the bone and the fat can be differentiated and have the multi-directional differentiation potential. 3. the mineralization ability of the adipose stem cell of the rat is increased after the dentin induction, and the dentin-specific gene and the protein can be express, the induction time is short, the induction time is short,
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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