【作者】 李静；

【导师】 宋锦璘；

【作者基本信息】 重庆医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 牙周病、不当口腔临床治疗、外伤等均可造成牙周组织损伤、破坏。随着口腔保健意识及功能、美观等要求的提高,患者希望尽可能保留牙齿。因此,获得牙周组织的再生性修复十分必要,其生物学基础依赖于牙周膜细胞(Periodontal Ligament Cells, PDLCs)的有效增殖[1]。牙周膜(periodontal ligament,PDL)是牙根与牙槽骨之间的一薄层结缔组织,由处于不同增殖阶段或具不定向分化趋势的多细胞亚群构成,在外界刺激下可定向增殖、分化,从而实现牙周组织的再生性修复[2,3,4]。成纤维细胞是人牙周膜细胞(human Periodontal Ligament Cells, HPDLCs)的优势亚群之一,具有较强的增殖能力、多向分化潜能等干细胞的某些特性,在牙周组织的再生性修复中发挥着重要作用[5]。以往研究多探讨细胞因子[6]、中药[7]、物理[8]、化学物质[9]等因素作用下人牙周膜成纤维细胞(human periodontal ligament fibroblasts , HPDLF)增殖、分化的改变及其机理,以期达到对其定向调控并促进牙周组织再生。但尚未发现一种经济、安全、有效的促进HPDLF增殖效应表达的途径。近年来,低强度脉冲超声波(Low Intensity Pulsed Ultrasound,LIPUS)在无创促进骨创伤修复的作用已得到临床认可和应用[10];亦有研究发现其生物、物理学效应可促进某些细胞的增殖、分化[11,12]。因此,LIPUS对HPDLF增殖效应表达的影响具有极高研究价值和潜在应用前景。本研究采用组织块法培养了HPDLCs,并进一步纯化构建了HPDLF的体外纯化培养模型。在该模型基础的细胞组织学来源鉴定证实其为中胚层来源;采用MTT法绘制了不同接种密度(1*104、2*104、1*103)的细胞生长曲线,发现HPDLF的适宜接种密为1*104度;结合细胞形态、生长趋势观察等证实本研究构建的HPDLF体外培养系性状稳定、活性良好。在前述成果基础上,构建了“HPDLF--LIPUS辐照模型”,经MTT法、考马斯亮蓝(coomassie brilliant blue,CBB)染色法等定量、半定量检测方法比较了不同参数(声强:30MW、60MW、90MW,作用时间:10min、20min、30min,重复频率1KHz,脉冲频率1.5MHz)的LIPUS辐照对HPDLF的增殖活性、胞内总蛋白表达量的影响,初步探讨了LIPUS辐照对HPDLF增殖效应表达的影响。遴选适宜本实验条件下促进HPDLF增殖效应表达的LIPUS辐照参数。本研究发现所构建的LIPUS辐照模型具有操作简便,成本低、可有效控制细胞污染等优势,并保证了超声辐照的稳定性、有效性。MTT生长曲线及CBB蛋白测定结果均显示LIPUS辐照对HPDLF的增殖效应表达有促进效应,且90mw,20min/天(重复频率1KHz,脉冲频率1.5MHz)的辐照参数效应最显著。但最长辐照时间(30min)组和最大声强组(90mw)中均未发现抑制效应,并且90mw,20min/天辐照下细胞生长曲线和蛋白浓度变化发现具有近似变化趋势,表明LIPUS对HPDLF的促增殖效应不仅是细胞数量增加的促分裂效应,也同时提高细胞的功能活性。这提示采用LIPUS辐照法有助于获得具有较高功能活性的HPDLF种子细胞。基于前述最适宜参数,本研究进一步结合流式细胞术、RT-PCR法检测了LIPUS辐照下HPDLF的细胞周期及碱性成纤维细胞生长因子(Fibroblast Growth Factor,bFGF)、转化生长因子β1(transforming growth factor-β1, TGF-β1)的表达水平。本研究显示受LIPUS辐照6h、12h时,HPDLF均显著表达bFGF,且12h表达量高于6h,差异有统计学意义(P<0.05),推测HPDLF在受LIPUS辐照后12h内对bFGF的表达有随时间延长而上调的趋势。但是,尚未见TGF -β1的显著表达,可能系HPDLF受LIPUS辐照12h内所表达细胞因子如bFGF等间接抑制了其对TGF -β1的表达,或TGF -β1的表达发生在HPDLF受LIPUS辐照12h后。此外,细胞周期检测结果显示LIPUS辐照后HPDLF的增殖指数(proliferation index,PrI)较对照组显著提高,具有与对照组一致的周期性波动。这可能与HPDLF固有细胞周期循环相关,提示LIPUS对HPDLF促增殖效应表达的机制之一为提高HPDLF的PrI,但不能通过改变HPDLF固有细胞周期时间的方式加快细胞增殖速度。综上所述,LIPUS辐照可促进HPDLF的增殖效应表达,且基于本研究构建“HPDLF--LIPUS辐照模型”的较适宜辐照参数为声强90MW,辐照时间20min/d,重复频率1KHz,脉冲频率1.5MHz。本研究证实LIPUS辐照可提高HPDLF的PrI,从而促进其增殖效应的表达,但不能改变其固有细胞周期时间。同时,发现LIPUS辐照可促进HPDLF对特定细胞因子的表达。上述结果提示90mw,20min/天的LIPUS辐照有助于获得牙周组织工程相关的功能活性HPDLF种子细胞。更多还原

【Abstract】 Periodontal disease, improper dental out-patients, physical injury and so on can lead to periodontium injury. With the awareness of oral health, and the requirement of better capability and dental aesthetics, more and more patients would like to keep their teeth. As a result, to acquire periodontal regeneration, which is based on the proliferation of periodontal ligament cells, becomes very necessary.The periodontal ligament is a thin layer of connective tissue locating between tooth and the alveolar bone, and it is composed of multi-cell subsets, which may be at different proliferation stages and have the potential for non-directed differentiation, special external treatment can stimulate the proliferation and committed differentiation of those multi-cell subsets, and this can finally lead to the periodontal regeneration. Especially, the fibroblasts, as one of the dominant subsets of the periodontal ligament cells, have very high capability of proliferation and committed differentiation, and are very important for the periodontal regeneration. Previous related research mainly focuses on how cytokine, medicine, physical and chemical factors and so forth affect the proliferation, differentiation of the periodontal ligament fibroblasts, as well as focuses on the underlying mechanism, to directly control the periodontal regeneration. However, one economic, safe, effective method to promote the proliferation of the periodontal ligament fibroblasts hasn’t been found yet.Recently, low intensity pulsed ultrasound, LIPUS, has already been clinically used for no-invasive bone wound healing. Some previous studies show that LIPUS can promote the proliferation and differentiation of some cells, indicating that the effect of LIPUS on the periodontal ligament fibroblasts may be of great significance.In this study, explant culture was employed to work on human periodontal ligament cells, and an in vitro culture model of human periodontal ligament fibroblast cells was developed here. With the developed model, the cytohistology origin of the cultured cells was tested, and the result showed a mesoderm origin of these cells. With MTT method, the cell growth curves under different inoculation densities (1*104、2*104、1*103) were measured, and the inoculation density of 1*104 was found to fit HPDLF well. These results, together with some further observation of the morphology and growth trend of the cultured cells, showed that the in vitro HPDLF culture system are constant and in good quality. Based on the results above, HPDLF-LIPUS radiation model was constructed, in which, with some quantitative and semi-quantitative methods such as MTT and coomassie brilliant blue staining, the influence of different LIPUS radiation (sound intensity:30MW, 60MW, 90MW; time:10min, 20min, 30min; repeat frequency: 1KHz; pulse frequency: 1.5MHz) on the proliferation and intracellular protein expression of the HPDLF cells was compared, and proper LIPUS radiation parameters for our experimental conditions were acquired. The present study indicates that the constructed ultrasound radiation model, which can guarantee a stable and effective ultrasound radiation, is easy to handle and with low cost, and can make the cell contamination under control. The result from MTT and coomassie brilliant blue methods shows that LIPUS radiation can effectively increase the HPDLF proliferation, especially the radiation with a sound density of 90 mw, and a treatment of 20 minutes per day (repeat frequency of 1KHz and pulse frequency of 1.5 MHz) has the most significant effect. Moreover, the longest daily radiation time of 30 minutes and the largest sound density of 90 mw weren’t found to inhibit the cell proliferation. In addition, under a radiation, which has a sound density of 90mw and a treatment of 20 minutes per day, the cell growth curve and protein concentration changed slightly, indicating that LIPUS promote the cell proliferation not only by increasing the cell number, but also by increasing the cell activity, this means LIPUS radiation may help acquire HPDLF high activity seed cells.Under the LIPUS radiation with the best parameters, flow cytometry and RT-PCR were furtherly used to study the cell cycle and the expression of basic fibroblast growth factor (bFGF) and Transforming growth factorβ1 (TGF-β1). The result showed that, after 6 hours and 12 hours of LIPUS radiation, there was significant expression of bFGF in the HPDLF cells, and the expression at the 12th hour was significantly higher than that at the 6th hour (p<0.05). But no expression of TGF-β1 in HPDLF cells was found within 12 hours of LIPUS radiation, this is probably because its expression has been inhibited by other cytokines like bFGF, or may be because the TGF-β1 won’t express until after 12 hours of HPDLF radiation. Moreover, the cell cycle test showed that, the HPDLF cells under LIPUS radiation have higher proliferation index than, but with the same cyclical fluctuations as, those in the control group, this may be due to the inherent cell cycle of HPDLF cells, indicating that LIPUS may promote the cell proliferation of HPDLF cells by increasing proliferation index rather than changing cell cycle time.In summary, LIPUS radiation can promote the proliferation of the periodontal ligament fibroblast cells. In the present study, the best parameters for the constructed HPDLF-LIPUS radiation model are: sound intensity, 90 MW, treatment time, 20 min/day; repeat frequency, 1KHz; pulse frequency, 1.5MHz. With the HPDLF-LIPUS radiation model with the best parameters, this study showed that LIPUS radiation could promote the proliferation of HPDLF cells by increasing the cell proliferation index, instead of by changing cell cycle time. Moreover, this study also showed that LIPUS radiation could promote the expression of some special cytokine in HPDLF cells. All these results indicate that LIPUS radiation of 90 MW, 20 min/d can help to get periodontal ligament related HPDLF seed cells.更多还原