炎症微环境下雌激素对大鼠BMSCs炎性因子表达及骨向分化的调控作用研究

发布时间：2018-04-01 08:14

  本文选题：雌激素　切入点：骨髓间充质干细胞　出处：《第四军医大学》2014年博士论文

【摘要】：牙周病是炎性骨吸收性疾病，其主要特征是牙龈炎症、牙槽骨吸收和牙龈萎缩，进而导致牙齿松动和脱落，直接影响患者口腔牙周组织健康和牙齿使用寿命。在牙周病的组织损伤中主要参与这一过程的是牙周病原体及其代谢产物，尤其是脂多糖（lipopolysaccharide，LPS），它能诱导宿主发生免疫反应，激发免疫细胞和局部邻近组织细胞增加炎性因子的分泌表达，改变局部微环境。以往研究表明，雌激素在骨吸收与骨改建的过程中扮演了十分重要的角色，慢性牙周疾病在发生和发展进程中就有雌激素参与其中，，例如绝经后的妇女骨质疏松及口腔牙周病发展十分迅速，与机体缺乏雌激素的因素有关。目前，雌激素影响牙周组织健康的机制尚不清楚。骨髓间充质干细胞(Bone marrow mesenchymal stem ce11s,BMSCs)是一种具有多向分化潜能的干细胞，在体外特定培养条件下能够向多种细胞组织等结构分化，常作为组织工程研究的首选种子细胞。本实验旨在模拟牙周病局部炎症微环境中，若将骨髓间充质干细胞移植至牙周缺损部位，雌激素是否对炎症微环境具有抑制作用及其作用机理，以及雌激素能否调控骨髓间充质干细胞骨向分化能力，为临床上应用雌激素治疗牙周病提供新的理论和思路。 本实验内容如下： 1.大鼠BMSCs的分离、培养和鉴定 选用雌性SD大鼠，鼠龄6-9个月。用全骨髓贴壁筛选培养法分离培养原代大鼠骨髓间充质干细胞，对BMSCs进行成骨诱导培养和成脂诱导培养，应用流式细胞仪对BMSCs表面抗原CD44、CD45、CD29和CD11b进行测定。 结果显示BMSCs经定向成骨诱导后，茜素红染色为阳性，定向成脂肪诱导后，油红-0染色为阳性，表面抗原CD29和CD44阳性，CD45和CD11b为阴性，鉴定其为大鼠骨髓间充质干细胞。 2.雌激素对LPS诱导的大鼠BMSCs炎性因子表达的影响 加入LPS脂多糖、17β-雌二醇和特异性雌激素受体抑制剂ICI182,780。分为对照组，E-782组，LPS组，LPS+E-92组，LPS+E-2组，LPS+E-72组和LPS+E-72+ICI182,780组，刺激6h、12h、24h、48h和72h后，提取细胞培养上清液和细胞裂解物，进行TNF-α、IL-1β和IL-6的ELISA检测及Real-time PCR检测。 结果显示，在细胞培养上清液、细胞裂解物中和细胞mRNA水平，LPS刺激后BMSCs中TNF-α、IL-1β和IL-6的表达量明显增高，E2可以明显抑制其表达，并呈剂量依赖性，ICI182780可以抑制E2的作用。经LPS和E2刺激后，三种炎性因子在12h和24h表达水平达到峰值，随时间延长逐渐减弱。 3.雌激素对LPS诱导的大鼠BMSCs炎性因子表达的调控机制 加入MAPK信号通路的三种通路抑制剂，即ERK通路抑制剂PD98059，JNK通路抑制剂SP600125，p38α通路抑制剂SB203580。再加入LPS、雌激素，培养12h后，收集细胞培养上清液，进行TNF-α、IL-1β和IL-6的ELISA检测。 结果显示三种炎性因子的表达量较之单纯加入LPS后出现不同程度的降低。尤以JNK通路抑制剂SP600125作用最为明显。 4.雌激素对LPS诱导的大鼠BMSCs骨向分化能力的影响 分为对照组，E-72组，LPS组，LPS+E-7-72组和LPS+E2+ICI182,780组，加样刺激6h、12h、24h、48h和72h后，提取细胞培养上清液和细胞裂解物进行OPG和RANKL的ELISA检测，以及进行OPG和RANKL的mRNA检测。 结果显示，在细胞培养上清液、细胞裂解物中和细胞mRNA水平，加入E2后，OPG表达量明显增高；加入LPS后，RANKL的表达量明显增高；加入LPS和E2后，OPG表达量明显增高，而RANKL的表达量明显下降。经LPS和E2刺激后，OPG在12h和24h达到并维持最高峰值，RANKL在24h表达水平达到最高峰。OPG/RANKL比值在E2组最高，LPS组最低，同时加入LPS和E2后的比值较之单纯加入LPS组明显升高。 5.雌激素影响LPS诱导的大鼠BMSCs表达OPG/RANKL的炎性因子调控途径 加入LPS、E2，并分别加入TNF中和性抗体anti-TNF-α，antiIL-1β，以及IL-6可溶性受体sIL-6R。分别培养6h、12h、24h、48h和72h后，收集细胞培养上清液进行OPG和RANKL的ELISA检测。 结果显示，加入antiTNF-α、antiIL-1β和sIL-6R后，OPG表达与单纯加入E2无明显差别，与加入LPS和E2比较略有下降，至24h达高峰，随后逐渐下降；RANKL的表达与单纯加入LPS相比，以及与加入LPS和E2相比，均有不同程度的下降，其中以加入sIL-6R后最为明显，至24h表达水平达到最高峰。 结论： 1.通过大鼠全骨髓贴壁筛选法可以对BMSCs进行分离、纯化、培养和扩增，将其生物学特性和流式细胞术检测表型特征相结合，可以鉴定为大鼠BMSCs。 2.LPS可以诱导BMSCs分泌表达炎性因子TNF-α、IL-1β和IL-6，雌激素可以显著抑制炎性因子的高表达，并且呈浓度依赖性，表明雌激素具有明显的抗炎作用。 3.LPS和雌激素共同刺激下的BMSCs炎性因子表达量6h时处于上升期，12h和24h处于高峰，48h和72h时表达下降，表现为明显的时间依赖性。 4.MAPK信号通路可能是雌激素调控BMSCs炎性因子表达的途径之一。 5.雌激素可以明显促进BMSCs中OPG的表达，LPS可以明显促进BMSCs中RANKL的表达，雌激素可以通过调控OPG/RANKL的比值提高LPS作用后BMSCs的骨向分化能力，促进炎症微环境下BMSCs的骨向分化。 6.经LPS与E2作用后，大鼠BMSCs的OPG表达在6h开始上升，12h到24小时到达高峰，随后下降，而RANKL的表达在6h上升，12h以后一直保持较高水平。 7.雌激素可以通过调控炎性因子TNF-α、IL-1β和IL-6表达的途径对LPS诱导的BMSCs表达OPG/RANKL进行调控，雌激素调控炎性因子的表达是其发挥生物学功能的途径之一。
[Abstract]:Periodontal disease is an inflammatory disease of bone absorption, its main feature is the absorption of alveolar bone and gingival inflammation, gingival atrophy, and cause the teeth loose and fall off, directly affect the periodontal tissue of patients with oral health and dental service life. The main part of this process in the periodontal tissue damage in periodontal pathogens and metabolism the product, especially lipopolysaccharide (lipopolysaccharide, LPS), it can induce host immune response, stimulate immune cells and local adjacent tissue cells increased expression and secretion of inflammatory factors, change the local micro environment. Previous studies have shown that estrogen absorption plays an important role in the process of bone remodeling and bone, chronic periodontal in the process of disease occurrence and development have estrogen involved, such as postmenopausal osteoporosis and periodontal disease is developing very rapidly, and the lack of female hormones are factors . at present, the mechanism of estrogen affects the health of periodontal tissue is unclear. Bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem ce11s, BMSCs) is a kind of pluripotent stem cells, in vitro specific culture conditions to a variety of cell tissue differentiation, is often used as a tissue engineering research preferred seed cells. The aim of this study is to simulate periodontal local inflammation microenvironment, the transplantation of bone marrow mesenchymal stem cells to periodontal defect, whether estrogen can inhibit the inflammatory microenvironment and its mechanism, and estrogen can regulate bone marrow mesenchymal stem cells to osteoblast differentiation, provide theory and new ideas the clinical application of estrogen for the treatment of periodontal disease.
The contents of this experiment are as follows:
Isolation, culture and identification of BMSCs in 1. rats
Female SD rats were selected for 6-9 months. Bone marrow mesenchymal stem cells were isolated and cultured from whole bone marrow adherent culture. Osteogenic induction and adipogenic induction of BMSCs were carried out. The surface antigen CD44, CD45, CD29 and CD11b of BMSCs were determined by flow cytometry.
The results showed that BMSCs was positive after alizarin red staining after directional osteogenesis induction. After directional fat induction, oil red -0 staining was positive, surface antigen CD29 and CD44 were positive, CD45 and CD11b were negative, and it was identified as rat bone marrow mesenchymal stem cells.
2. effect of estrogen on the expression of BMSCs inflammatory factors induced by LPS in rats
Adding LPS LPS, 17 beta estradiol and estrogen receptor specific inhibitor ICI182780. were divided into control group, E-782 group, LPS group, LPS+E-92 group, LPS+E-2 group, LPS+E-72 group and LPS+E-72+ICI182780 group, 12h, 24h, stimulation of 6h, 48h and 72h after extraction, cell culture supernatant and cell lysate, TNF- alpha ELISA, PCR detection and Real-time detection of IL-1 beta and IL-6.
The results showed that the culture supernatant in cells, cell lysates and cell levels of mRNA, TNF- alpha LPS BMSCs after stimulation, the expression of IL-1 beta and IL-6 were increased, E2 can significantly inhibit its expression in a dose-dependent manner, ICI182780 can inhibit the function of E2. After LPS and after E2 stimulation, three the inflammatory factor expression level reached the peak value at 12h and 24h, decreased with time.
3. estrogen regulates the expression of BMSCs inflammatory factors in rats induced by LPS
The three pathway inhibitors of MAPK signaling pathway, namely ERK pathway inhibitor PD98059, JNK pathway inhibitor SP600125, p38 alpha pathway inhibitor SB203580., were added to LPS, estrogen, and 12h after culture.
The results showed that the expression of three inflammatory factors decreased in varying degrees compared with that of LPS, especially the JNK pathway inhibitor SP600125.
Effect of 4. estrogen on LPS induced BMSCs bone differentiation in rats
They were divided into control group, E-72 group, LPS group, LPS+E-7-72 group and LPS+E2+ICI182780 group. After stimulating 6h, 12h, 24h, 48h and 72h, the cell culture supernatant and cell lysate were extracted for OPG and RANKL detection, and the detection of "Er" and "Yu" was carried out.
The results showed that the culture supernatant in cells, cell lysates and cell mRNA levels, after joining E2, the expression of OPG was significantly increased; after joining LPS, the expression of RANKL was significantly increased; adding LPS and E2, the expression of OPG was significantly increased, while the expression of RANKL was decreased significantly. After LPS and E2 stimulation. OPG in 12h and 24h to reach and maintain the highest peak, the expression level of RANKL reached the peak of.OPG/RANKL was the highest in E2 group in 24h, LPS group is the lowest, while adding ratio of LPS and E2 after compared with adding LPS group increased significantly.
5. estrogen affects the regulation pathway of LPS induced BMSCs expression of OPG/RANKL in rats
After adding LPS, E2 and TNF neutralizing antibody anti-TNF- alpha, antiIL-1 beta and IL-6 soluble receptor sIL-6R. respectively, 6h, 12h, 24h, 48h and 24h were cultured respectively, and then the supernatant of cell culture was collected to detect the "Qi" and "Yu".
The results showed that adding antiTNF- alpha, antiIL-1 beta and sIL-6R, OPG expression had no significant difference with the simple entry into the E2, compared with the accession to the LPS and E2 decreased slightly, reaching the peak at 24h, then decreased gradually; the expression of RANKL compared with the accession to the LPS, and compared with the addition of LPS and E2 were decreased in different degree. The most obvious after adding sIL-6R to 24h, the expression level reached a peak.
Conclusion:
1., we can isolate, purify, culture and amplify BMSCs by combining the whole bone marrow adherence screening method, and combine its biological characteristics and phenotypic characteristics with flow cytometry. It can be identified as BMSCs. in rats.
2.LPS can induce BMSCs to secrete inflammatory cytokines TNF- alpha, IL-1 beta and IL-6. Estrogen can significantly inhibit the high expression of inflammatory factors, and in a concentration dependent manner, it indicates that estrogen has an obvious anti-inflammatory effect.
The expression of BMSCs inflammatory factors stimulated by 3.LPS and estrogen increased at 6h, and 12h and 24h were at the peak. The expression of 48h and 72h decreased significantly, showing a significant time dependence.
4.MAPK signaling pathway may be one of the ways that estrogen regulates the expression of BMSCs inflammatory factors.
5. estrogen can significantly promote the expression of OPG in BMSCs. LPS can significantly promote the expression of RANKL in BMSCs. Estrogen can enhance BMSCs differentiation ability through regulating the ratio of OPG/RANKL, and promote the osteogenic differentiation of BMSCs in inflammatory microenvironment.
6. after the action of LPS and E2, the expression of OPG in BMSCs began to rise at 6h, reached its peak at 12h to 24 hours, then decreased, while the expression of RANKL increased in 6h, and remained at a high level after 12h.
7., estrogen can regulate the expression of LPS induced BMSCs expression by regulating the expression of inflammatory factors TNF-, IL-1 and IL-6. Estrogen regulates the expression of inflammatory factors, and it is one of the ways to exert its biological function.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R781.4

【共引文献】

相关期刊论文 前10条

1 路晓淼;王恩群;;神经生长因子对骨髓基质细胞成骨分化影响的研究进展[J];安徽医药;2009年02期

2 张文鹏;叶发刚;y囇澡

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