本文選題：人牙周膜干細(xì)胞 + 慢性牙周炎��； 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：牙周病作為口腔常見慢性疾病會導(dǎo)致牙周支持組織的破壞，包括硬組織如牙槽骨的吸收和軟組織如牙齦的退縮等，如未及時治療，將會導(dǎo)致牙齒的松動甚至缺失，嚴(yán)重影響人們的健康生活。目前牙周支持組織的再生和重建仍然是牙周治療尚未解決的難題，近年來組織工程成為牙周再生研究的熱點(diǎn)方向，其中的種子細(xì)胞來源及其功能表現(xiàn)更是關(guān)鍵性影響因素。牙周膜干細(xì)胞（periodontal ligament stem cells,PDLSCs）由于組織來源相近、高度增殖能力及多向分化能力等特點(diǎn)已成為牙周組織工程的首選種子細(xì)胞。但牙周膜干細(xì)胞通常需要取健康人因正畸或阻生所拔除的牙齒，來源比較局限，因此炎癥組織來源的牙周膜干細(xì)胞（inflammatory derivedperiodontal ligament stem cells,iPDLSCs）也已經(jīng)被分離培養(yǎng)和鑒定，并證明其同樣具有高度增殖能力，多向分化能力等種子細(xì)胞特征。正常和炎癥來源的牙周膜干細(xì)胞均能在成骨、成脂微環(huán)境中正常分化，但兩者的分化能力差異比較目前仍有爭議。組織工程三要素還包括細(xì)胞因子和支架材料，除此之外，血供也是不容忽視的要素。通過組織工程技術(shù)再生的組織只有在血供充足的情況下才能穩(wěn)定生存。而牙周組織工程所涉及的牙根面部位和需要重建的大量牙槽骨缺損處，必須有血管的形成才能為再生組織提供營養(yǎng)，這是保證牙周再生獲得成功不可缺少的一環(huán)。前期骨髓來源、脂肪來源、皮膚來源等的間充質(zhì)干細(xì)胞均被證明在成血管培養(yǎng)基誘導(dǎo)下能向內(nèi)皮細(xì)胞分化且具有成血管能力，并且炎癥環(huán)境可促進(jìn)脂肪干細(xì)胞向內(nèi)皮細(xì)胞分化。因此本實(shí)驗旨在探索牙周膜干細(xì)胞是否具有內(nèi)皮向分化能力，并同時比較正常和炎癥來源的牙周膜干細(xì)胞成血管能力的差異，以期為牙周膜干細(xì)胞成骨及成血管能力的共同實(shí)現(xiàn)提供參考，為牙周組織工程的應(yīng)用提供依據(jù)。 研究目的： 通過成血管培養(yǎng)條件誘導(dǎo)正常和炎癥來源的人牙周膜干細(xì)胞，觀察其內(nèi)皮向分化的能力，并且比較其成血管能力的差異，為牙周組織再生的作用機(jī)制及應(yīng)用提供依據(jù)。 研究方法： 1、牙周膜干細(xì)胞的分離、培養(yǎng)和鑒定：臨床收集正常和炎癥來源的離體牙，通過改良酶組織塊消化法分離培養(yǎng)牙周膜細(xì)胞，經(jīng)有限稀釋法純化得到牙周膜干細(xì)胞。流式細(xì)胞術(shù)檢測干細(xì)胞表面標(biāo)記物；CCK-8法檢測并比較PDLSCs和iPDLSCs的生長曲線及增殖能力；克隆形成實(shí)驗檢測PDLSCs和iPDLSCs自我更新能力并比較克隆形成率的大小。 2、牙周膜干細(xì)胞的內(nèi)皮向誘導(dǎo)：當(dāng)PDLSCs和iPDLSCs生長達(dá)80%-90%匯合時，棄原培養(yǎng)液，加入誘導(dǎo)培養(yǎng)基（含20mL/L胎牛血清，10mL/L青霉素鏈霉素混合液，25ng/mLVEGF，25ng/mLbFGF的-MEM培養(yǎng)液），常規(guī)條件下培養(yǎng)10天。 3、體外檢測并比較PDLSCs和iPDLSCs成血管能力：通過免疫熒光、real-timeRT-PCR、Matrigel assay來檢測PDLSCs和iPDLSCs內(nèi)皮細(xì)胞標(biāo)記物的表達(dá)及體外管腔形成能力。 4、體內(nèi)實(shí)驗檢測PDLSCs和iPDLSCs成血管能力：掃描電鏡觀察PDLSCs和iPDLSCs在HA/β-TCP材料表面的黏附伸展情況，并植入裸鼠皮下，3周后取材，組織學(xué)觀察其體內(nèi)成血管能力。 5、統(tǒng)計學(xué)分析：用SPSS19.0統(tǒng)計軟件對數(shù)據(jù)（x±s）進(jìn)行方差分析，兩兩比較用t檢驗，檢驗水準(zhǔn)=0.05。 研究結(jié)果： 1、經(jīng)培養(yǎng)純化成功獲取PDLSCs和iPDLSCs，顯微鏡下觀察細(xì)胞均呈長梭形紡錘狀；流式細(xì)胞術(shù)檢測細(xì)胞均陽性表達(dá)間充質(zhì)干細(xì)胞標(biāo)記物：CD29、CD90、CD105、CD146，兩者表達(dá)率無統(tǒng)計學(xué)差異，陰性表達(dá)造血干細(xì)胞標(biāo)記物：CD34、CD45。CCK-8檢測結(jié)果顯示PDLSCs和iPDLSCs生長曲線均呈“S”型，6天前各時間點(diǎn)，iPDLSCs的數(shù)量高于正常PDLSCs，尤其是第3和第4天，具有統(tǒng)計學(xué)意義（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，差異具有統(tǒng)計學(xué)意義。 2、PDLSCs和iPDLSCs經(jīng)成血管誘導(dǎo)10天后，顯微鏡下觀察細(xì)胞呈“鵝卵石”樣，與內(nèi)皮細(xì)胞形態(tài)基本相同。 3、PDLSCs和iPDLSCs內(nèi)皮向誘導(dǎo)10天后，免疫熒光檢測顯示兩者均陽性表達(dá)內(nèi)皮細(xì)胞標(biāo)記物VEGF、vWF，iPDLSCs表達(dá)的陽性率高于PDLSCs；real-timeRT-PCR檢測兩者誘導(dǎo)后均表達(dá)CD31、vWF、VE-cadherin，其中iPDLSCs的CD31、VE-cadherin mRNA表達(dá)水平均明顯高于PDLSCs，差異有統(tǒng)計學(xué)意義（P＜0.05）；誘導(dǎo)后的PDLSCs和iPDLSCs均能在Matrigel基質(zhì)膠上形成管腔樣結(jié)構(gòu)，并相連成網(wǎng)狀，通過軟件半定量分析，iPDLSCs形成的分支節(jié)點(diǎn)數(shù)、管腔長度均高于PDLSCs，差異有統(tǒng)計學(xué)意義（P＜0.05）。 4、經(jīng)掃描電鏡觀察，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染色可見有新生血管生成，其中炎癥來源的牙周膜干細(xì)胞誘導(dǎo)組形成的血管數(shù)量最多，免疫組化染色可見誘導(dǎo)后的PDLSCs和iPDLSCs均陽性表達(dá)CD31。 結(jié)論 PDLSCs和iPDLSCs具有高度增殖能力和自我更新能力，，在成血管誘導(dǎo)培養(yǎng)基作用下均能向內(nèi)皮細(xì)胞分化，而iPDLSCs成血管能力強(qiáng)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..
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 潘 勇,艾玉峰,熊 猛,張琳西,夏文森,彭 湃,黃 蔚,趙玉峰;體外組織工程血管支架內(nèi)皮化的實(shí)驗研究[J];中國美容醫(yī)學(xué);2002年04期

2 談s

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