本文選題：雌激素　切入點(diǎn)：骨髓間充質(zhì)干細(xì)胞　出處：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：牙周病是炎性骨吸收性疾病，其主要特征是牙齦炎癥、牙槽骨吸收和牙齦萎縮，進(jìn)而導(dǎo)致牙齒松動(dòng)和脫落，直接影響患者口腔牙周組織健康和牙齒使用壽命。在牙周病的組織損傷中主要參與這一過程的是牙周病原體及其代謝產(chǎn)物，尤其是脂多糖（lipopolysaccharide，LPS），它能誘導(dǎo)宿主發(fā)生免疫反應(yīng)，激發(fā)免疫細(xì)胞和局部鄰近組織細(xì)胞增加炎性因子的分泌表達(dá)，改變局部微環(huán)境。以往研究表明，雌激素在骨吸收與骨改建的過程中扮演了十分重要的角色，慢性牙周疾病在發(fā)生和發(fā)展進(jìn)程中就有雌激素參與其中，，例如絕經(jīng)后的婦女骨質(zhì)疏松及口腔牙周病發(fā)展十分迅速，與機(jī)體缺乏雌激素的因素有關(guān)。目前，雌激素影響牙周組織健康的機(jī)制尚不清楚。骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem ce11s,BMSCs)是一種具有多向分化潛能的干細(xì)胞，在體外特定培養(yǎng)條件下能夠向多種細(xì)胞組織等結(jié)構(gòu)分化，常作為組織工程研究的首選種子細(xì)胞。本實(shí)驗(yàn)旨在模擬牙周病局部炎癥微環(huán)境中，若將骨髓間充質(zhì)干細(xì)胞移植至牙周缺損部位，雌激素是否對炎癥微環(huán)境具有抑制作用及其作用機(jī)理，以及雌激素能否調(diào)控骨髓間充質(zhì)干細(xì)胞骨向分化能力，為臨床上應(yīng)用雌激素治療牙周病提供新的理論和思路。 本實(shí)驗(yàn)內(nèi)容如下： 1.大鼠BMSCs的分離、培養(yǎng)和鑒定 選用雌性SD大鼠，鼠齡6-9個(gè)月。用全骨髓貼壁篩選培養(yǎng)法分離培養(yǎng)原代大鼠骨髓間充質(zhì)干細(xì)胞，對BMSCs進(jìn)行成骨誘導(dǎo)培養(yǎng)和成脂誘導(dǎo)培養(yǎng)，應(yīng)用流式細(xì)胞儀對BMSCs表面抗原CD44、CD45、CD29和CD11b進(jìn)行測定。 結(jié)果顯示BMSCs經(jīng)定向成骨誘導(dǎo)后，茜素紅染色為陽性，定向成脂肪誘導(dǎo)后，油紅-0染色為陽性，表面抗原CD29和CD44陽性，CD45和CD11b為陰性，鑒定其為大鼠骨髓間充質(zhì)干細(xì)胞。 2.雌激素對LPS誘導(dǎo)的大鼠BMSCs炎性因子表達(dá)的影響 加入LPS脂多糖、17β-雌二醇和特異性雌激素受體抑制劑ICI182,780。分為對照組，E-782組，LPS組，LPS+E-92組，LPS+E-2組，LPS+E-72組和LPS+E-72+ICI182,780組，刺激6h、12h、24h、48h和72h后，提取細(xì)胞培養(yǎng)上清液和細(xì)胞裂解物，進(jìn)行TNF-α、IL-1β和IL-6的ELISA檢測及Real-time PCR檢測。 結(jié)果顯示，在細(xì)胞培養(yǎng)上清液、細(xì)胞裂解物中和細(xì)胞mRNA水平，LPS刺激后BMSCs中TNF-α、IL-1β和IL-6的表達(dá)量明顯增高，E2可以明顯抑制其表達(dá)，并呈劑量依賴性，ICI182780可以抑制E2的作用。經(jīng)LPS和E2刺激后，三種炎性因子在12h和24h表達(dá)水平達(dá)到峰值，隨時(shí)間延長逐漸減弱。 3.雌激素對LPS誘導(dǎo)的大鼠BMSCs炎性因子表達(dá)的調(diào)控機(jī)制 加入MAPK信號(hào)通路的三種通路抑制劑，即ERK通路抑制劑PD98059，JNK通路抑制劑SP600125，p38α通路抑制劑SB203580。再加入LPS、雌激素，培養(yǎng)12h后，收集細(xì)胞培養(yǎng)上清液，進(jìn)行TNF-α、IL-1β和IL-6的ELISA檢測。 結(jié)果顯示三種炎性因子的表達(dá)量較之單純加入LPS后出現(xiàn)不同程度的降低。尤以JNK通路抑制劑SP600125作用最為明顯。 4.雌激素對LPS誘導(dǎo)的大鼠BMSCs骨向分化能力的影響 分為對照組，E-72組，LPS組，LPS+E-7-72組和LPS+E2+ICI182,780組，加樣刺激6h、12h、24h、48h和72h后，提取細(xì)胞培養(yǎng)上清液和細(xì)胞裂解物進(jìn)行OPG和RANKL的ELISA檢測，以及進(jìn)行OPG和RANKL的mRNA檢測。 結(jié)果顯示，在細(xì)胞培養(yǎng)上清液、細(xì)胞裂解物中和細(xì)胞mRNA水平，加入E2后，OPG表達(dá)量明顯增高；加入LPS后，RANKL的表達(dá)量明顯增高；加入LPS和E2后，OPG表達(dá)量明顯增高，而RANKL的表達(dá)量明顯下降。經(jīng)LPS和E2刺激后，OPG在12h和24h達(dá)到并維持最高峰值，RANKL在24h表達(dá)水平達(dá)到最高峰。OPG/RANKL比值在E2組最高，LPS組最低，同時(shí)加入LPS和E2后的比值較之單純加入LPS組明顯升高。 5.雌激素影響LPS誘導(dǎo)的大鼠BMSCs表達(dá)OPG/RANKL的炎性因子調(diào)控途徑 加入LPS、E2，并分別加入TNF中和性抗體anti-TNF-α，antiIL-1β，以及IL-6可溶性受體sIL-6R。分別培養(yǎng)6h、12h、24h、48h和72h后，收集細(xì)胞培養(yǎng)上清液進(jìn)行OPG和RANKL的ELISA檢測。 結(jié)果顯示，加入antiTNF-α、antiIL-1β和sIL-6R后，OPG表達(dá)與單純加入E2無明顯差別，與加入LPS和E2比較略有下降，至24h達(dá)高峰，隨后逐漸下降；RANKL的表達(dá)與單純加入LPS相比，以及與加入LPS和E2相比，均有不同程度的下降，其中以加入sIL-6R后最為明顯，至24h表達(dá)水平達(dá)到最高峰。 結(jié)論： 1.通過大鼠全骨髓貼壁篩選法可以對BMSCs進(jìn)行分離、純化、培養(yǎng)和擴(kuò)增，將其生物學(xué)特性和流式細(xì)胞術(shù)檢測表型特征相結(jié)合，可以鑒定為大鼠BMSCs。 2.LPS可以誘導(dǎo)BMSCs分泌表達(dá)炎性因子TNF-α、IL-1β和IL-6，雌激素可以顯著抑制炎性因子的高表達(dá)，并且呈濃度依賴性，表明雌激素具有明顯的抗炎作用。 3.LPS和雌激素共同刺激下的BMSCs炎性因子表達(dá)量6h時(shí)處于上升期，12h和24h處于高峰，48h和72h時(shí)表達(dá)下降，表現(xiàn)為明顯的時(shí)間依賴性。 4.MAPK信號(hào)通路可能是雌激素調(diào)控BMSCs炎性因子表達(dá)的途徑之一。 5.雌激素可以明顯促進(jìn)BMSCs中OPG的表達(dá)，LPS可以明顯促進(jìn)BMSCs中RANKL的表達(dá)，雌激素可以通過調(diào)控OPG/RANKL的比值提高LPS作用后BMSCs的骨向分化能力，促進(jìn)炎癥微環(huán)境下BMSCs的骨向分化。 6.經(jīng)LPS與E2作用后，大鼠BMSCs的OPG表達(dá)在6h開始上升，12h到24小時(shí)到達(dá)高峰，隨后下降，而RANKL的表達(dá)在6h上升，12h以后一直保持較高水平。 7.雌激素可以通過調(diào)控炎性因子TNF-α、IL-1β和IL-6表達(dá)的途徑對LPS誘導(dǎo)的BMSCs表達(dá)OPG/RANKL進(jìn)行調(diào)控，雌激素調(diào)控炎性因子的表達(dá)是其發(fā)揮生物學(xué)功能的途徑之一。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781.4

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 路曉淼;王恩群;;神經(jīng)生長因子對骨髓基質(zhì)細(xì)胞成骨分化影響的研究進(jìn)展[J];安徽醫(yī)藥;2009年02期

2 張文鵬;葉發(fā)剛;y囇澡

本文編號(hào)：1694696