【作者】 郑旭；

【导师】 张杰；

【作者基本信息】 昆明医学院 ， 口腔临床医学， 2007， 硕士

【摘要】 侵袭性牙周炎是发生于全身健康者的进展迅速、有家族聚集性、不同于慢性牙周炎的一类牙周炎，是造成健康人失牙的主要因素之一。侵袭性牙周炎病因尚未完全明了。以往的研究表明，侵袭性牙周炎的发生与白细胞的功能异常有关。为了进一步研究青少年牙周炎患者中导致白细胞功能异常的致病机理，本实验将正常人与侵袭性牙周炎患者的白细胞进行基因差异性表达分析。目的：研究侵袭性牙周炎患者外周血白细胞的基因表达谱变化，从基因水平了解白细胞在侵袭性牙周炎发生过程中的作用，了解其分子作用机制，寻找与侵袭性牙周炎发病相关的基因。方法：抽取侵袭性牙周炎患者外周血10ml，以正常健康者作为对照，分离其白细胞，抽提白细胞的总RNA和总蛋白，按一步法抽提胞总RNA并纯化mRNA，逆转录合成cDNA进行实时荧光定量PCR，根据实时荧光定量PCR的结果，选择HSP40(DNAJC11的蛋白产物)进行Western-blot。结果：从基因芯片结果中挑选出PTHLH、DNAJC11。通过进行荧光定量PCR，DNAJC11在mRNA水平表现出接近4.5倍的上调。选择HSP40进行Western-blot，HSP40(DNAJC11的蛋白产物)蛋白水平表达显著上调。结论：基因芯片技术虽可有效地筛选出侵袭性牙周炎患者外周血白细胞的差异表达基因，但存在假阳性和假阴性，应用荧光定量PCR技术可以验证芯片检测结果，排除假阳性。在侵袭性牙周炎患者的外周血白细胞中，DNAJC11在mRNA和蛋白水平表达明显升高，其意义有待进一步研究，为进一步研究DNAJC11在侵袭性牙周炎发病中意义提供实验依据。更多还原

【Abstract】 It has been demonstrated that aggressive periodontitis (AP) is one of the majorreasons to cause the loss of teeth for healthy people. Several studies showed thataggressive periodontits has strong family linkage and related to genetic disorders.Others showed that the dysfunction of cellular immune system including PMN mayinvolve in the pathogenesis of aggressive periodontitis. Howver, the exact pathogenicmechanism is not clear until today. In order to better understanding the pathogenicbasis of aggressive periodontitis at a molecular level, we examed the gene expressionof normal and aggressive periodontitis subjects by using microarray technology.Objective: To explore differentially expressed genes of Peripheral bloodleucocytes in aggressive periodontitis, and investigate the pathopoiesis mechanism ofdysfunctions in leucocytes which may cause aggressive periodontitis in gene level andthe molecular mechanism of this disease, especially to find some gene which isassociated with Aggressive periodontitis.Methods: 30 ml Peripheral blood sample were taken from three patients(10mlper one) and 10 ml from the health control. The leucocytes were isolated, then we canget total RNA and protein from the leucocytes. Total RNA of leucocytes was drawnby one-step method then the product mRNA was purified, reversely transcribed tocDNA. We use real-time fluorescent quantitative PCR(FQ-PCR) to detect the expresslevel of DNAJCll and PTHLH, then choose HSP40 which is protein production from DNAJC11 for western-blot.Result: We choose DNAJC11 and PTHLH which come from the result of genechip for FQ-PCR. As in Aggressive periodontitis DNAJC11 was up-regulated neal4.5 times in mRNA level than health controls, we choose HSP40 which is proteinproduction from DNAJC11 for Western-blot.HSP40 was up-regulated significantly inprotein level.Conclusion: Gene chip is an effective way to explore differentially expressedgenes of peripheral blood leucocytes in aggressive periodontitis,but there are falsepositive and false negative in the result of gene chip.Real-time fluorescentquantitative PCR is a good way to eliminate false positive. Through FQ-PCR andWestern-blot, we can find DNAJC11 is up-regulated in mRNA and protein level ininperipheral blood leucocytes in aggressive periodontitis. This result can provide thetheory basis for further studies of DNAJC11更多还原