【作者】 段小辉；

【导师】 孙维佳；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的探讨MICA及NK细胞活化性受体NKG2D在胰腺癌表达和临床意义方法RT-PCR技术检测10例胰腺癌、6例慢性胰腺炎和6例正常胰腺组织中MICA mRNA水平。免疫组化技术检测64例胰腺癌、13例慢性胰腺炎和11例正常胰腺组织中MICA的表达。ELISA和流式细胞术分别检测53例胰腺癌,7例慢性胰腺炎和20例健康志愿者血清中可溶性MICA (sMICA)水平和外周血中NK细胞受体NKG2D的表达。结果1.胰腺癌组织MICA mRNA表达水平显著高于慢性胰腺炎和正常胰腺组织(P<0.01)。2.免疫组化显示胰腺癌组织中MICA阳性表达率为89.1%,显著高于慢性胰腺炎和正常胰腺组织(P<0.01)。MICA表达强度与胰腺癌分化程度、远处转移和临床分期密切相关(P=0.042,0.028,0.003)；MICA高表达的胰腺癌患者预后较好(P=0.019)。3.胰腺癌患者血清sMICA水平显著高于健康志愿者和慢性胰腺炎患者(P<0.01),Ⅲ／Ⅳ期胰腺癌患者血清sMICA水平高于Ⅰ／Ⅱ期患者(P<0.01),有远处转移的胰腺癌患者血清sMICA水平较无转移患者明显增高(P<0.01)。4.sMICA阳性胰腺癌患者外周血中NK细胞受体NKG2D的表达强度明显低于健康志愿者、慢性胰腺炎患者和sMICA阴性胰腺癌患者(P<0.01)。胰腺癌患者外周血中NK细胞受体NKG2D表达强度与血清sMICA水平呈显著负相关(P<0.01)5.胰腺癌根治性切除可以下调患者血清sMICA水平和上调NK细胞受体NKG2D的表达。结论MICA作为免疫监视分子在早期胰腺癌组织中广泛表达,随着胰腺癌进展,MICA从胰腺癌细胞表面脱落形成sMICA,血清中sMICA含量增高可能导致了NK细胞受体NKG2D表达的下调。胰腺癌根治性切除可以下调患者血清sMICA水平和上调NK细胞受体NKG2D的表达。目的探讨MICA在NK细胞杀伤人胰腺癌PANC-1细胞中的作用方法应用NK细胞分选试剂盒从外周血中负向分选高纯度NK细胞用于实验。体外培养人胰腺癌PANC-1细胞和人NK细胞,应用抗体封闭法,观察NKG2D与膜型MICA识别在NK细胞杀伤PANC-1细胞中的作用。将正常NK细胞在含有sMICA阳性胰腺癌患者血清的培基中培养,观察NK细胞受体NKG2D的表达以及NK细胞对PANC-1细胞的杀伤作用。结果1.经MICA或NKG2D抗体封闭后,NK细胞的杀瘤活性较封闭前显著减弱(P<0.01)2.胰腺癌患者血清中的sMICA可以下调NK细胞受体NKG2D的表达,并降低NK细胞对PANC-1细胞的杀伤活性。结论NKG2D与膜型MICA识别在NK细胞杀伤人胰腺癌PANC-1细胞中起重要作用。sMICA是造成胰腺癌免疫逃逸的重要分子机制之一。目的探讨ADAM10在PANC-1细胞MICA蛋白脱落过程中的作用,观察吉西他滨对MICA蛋白脱落的影响并探讨可能的作用机制。方法免疫组化检测64例胰腺癌和11例正常胰腺组织ADAM10的表达情况。通过化学合成的小分子干扰RNA(siRNA)下调PANC-1细胞ADAM10表达后,流式细胞术和ELISA分别检测细胞表面MICA的表达强度和细胞上清中sMICA的含量。采用MTT法检测不同浓度吉西他滨对PANC-1细胞增殖的影响。选择浓度为5ng/ml的吉西他滨作用PANC-1细胞24小时,RT-PCR和Western blot检测细胞中ADAM10 mRNA和蛋白表达水平,同时用RT-PCR、流式细胞术和ELISA分别检测MICA mRNA、细胞表面MICA的表达强度和细胞上清中sMICA的含量。先用ADAM10 siRNA或阴性对照siRNA转染PANC-1细胞,转染24小时后,再加入吉西他滨(对照组加等体积培基)继续培养24小时,流式细胞术和ELISA分别检测细胞表面MICA的表达强度和细胞上清中sMICA的含量。结果1.ADAM10在胰腺癌组织中阳性表达率(87.5%)显著高于正常胰腺组织(P<0.01)。2.下调ADAM10表达后,PANC-1细胞表面MICA表达增强,而细胞上清液中sMICA的含量减少(P<0.01)。3.当培基中浓度小于7ng／ml时,吉西他滨在24小时内对PANC-1细胞增殖无明显影响(P>0.05)。4.吉西他滨显著下调PANC-1细胞ADAM10mRNA和蛋白的表达水平(P<0.01)。5.吉西他滨(5ng／ml)作用PANC-1细胞24小时后,细胞表面MICA表达增强,细胞上清液中sMICA的含量减少(P<0.01)。吉西他滨对PANC-1细胞MICA mRNA表达无明显影响。6.siRNA下调ADAM 10表达后,吉西他滨对PANC-1细胞表面MICA蛋白的表达和上清液中sMICA的含量无明显影响(P>0.05)。结论ADAM 10在PANC-1细胞MICA蛋白脱落过程中具有重要作用。吉西他滨可以抑制PANC-1细胞MICA蛋白的脱落,其机制可能与下调ADAM 10的表达有关。更多还原

【Abstract】 Objective:To investigate the clinical significance of MICA and NKG2D receptor on NK cells in pancreatic cancer.Methods:MICA mRNA in 10 pancreatic cancer tissues,6 chronic pancreatitis tissues and 6 normal tissues of pancreas was determined by RT-PCR analysis.Expression of MICA in 64 pancreatic cancer tissues, 13 chronic pancreatitis samples and 11 normal tissues of pancreas was examined by immunohistochemistry. The serum levels of sMICA and NKG2D expression on NK cells of 53 pancreatic cancer patients,7 patients with chronic pancreatitis and 20 healthy volunteers was assessed by ELISA and Flow cytometry analysis.Results:There was a significant increase in MICA mRNA levels in the pancreatic cancer samples compared with the chronic pancreatitis and normal tissues of pancreas(P<0.01).The positive rate of MICA immunostaining in pancreatic cancer tissues was 89.1%,whereas fewer was expressed in inflammatory and normal pancreatic tissues(P<0.01). Significant difference was noted between the MICA expression with respect to the histological grade(P=0.042),distant metastasis(P=0.028) and TNM stages (P=0.003).MICA expression was found to be a prognostic factor in resected pancreatic cancer (P=0.019).The serum levels of sMICA were frequently elevated in patients with pancreatic cancer (P<0.01).The level of sMICA was significantly higher inⅢ/Ⅳstage pancreatic cancer patients than that in I/II stage patients (P<0.01).Notably, our data also showed that sMICA levels correlated significantly with the presence of metastasis(P<0.01).NKG2D expression on NK cells from sMICA-positive pancreatic cancer patients was markedly reduced compared to that from sMICA-negative pancreatic cancer patients, chronic pancreatitis patients or healthy volunteers (P<0.01).Significant inverse correlation between NKG2D expression and levels of sMICA was observed in pancreatic cancer patients (r=-0.499,P<0.01).The successful radical resection of tumor significantly decreased the serum levels of sMICA and increased the NKG2D expression on NK cells.Conclusions:MICA is induced and expressed widely in the early stage of pancreatic cancer.With the progression of pancreatic cancer, MICA can be released into the bloodstream or tissue culture medium as sMICA.The elevation of sMICA may be associated with down-regulated NKG2D expression on NK cells.The successful radical resection of tumor significantly decreased the serum levels of sMICA and increased the NKG2D expression on NK cells. Objective:To investigate the role of MICA on anti-human pancreatic cancer cell line PANC-1 mediated by NK cells.Methods:NK cells were purified by negative depletion using NK cell isolation kit by Vario MACS system.Studyed the reaction between MICA and NKG2D by purified-antibody blockade.Cultured NK cells from healthy donors with sMICA-positive serum for 24 h and assessed the NKG2D expression and Cytotoxicity of NK cells to PANC-1 cells.Results:1.Cytotoxicity of NK cells to PANC-1 cells was decreased because of NKG2D or MICA antibody blockade(P<0.01)2.The expression of NKG2D was decreased after NK cells cultured with sMICA-positive serum.NK cells pre-treated with sMICA-positive serum were not capable of efficiently killing PANC-1 cells.Conclusions:MICA-NKG2D play a critical role in cytotoxicity of NK cells to PANC-1 cells.sMICA reduce the expression of NKG2D,and impaire NK cell-mediated imnune surveillance in pancreatic cancer.Part 3 ADAM10 is involved in MICA shedding and effects of gemcitabine on MICA protein ectodomain shedding in PANC-1 cellsObjective:To investigate the role of ADAM 10 on MICA protein ectodomain shedding and observe whether gemcitabine affect MICA protein shedding from PANC-1 cells.Methods:1.Expression of ADAM 10 in 64 pancreatic cancer tissues and 11 normal pancreas tissues was examined by immunohistochemistry.2. PANC-1 cells were transfected with ADAM10 siRNA or control siRNA for 48 h. The expression of membrane-bound MICA was evaluated by flow cytometry,and sMICA production in the culture supernatant was evaluated by ELISA.3.The cytotoxicity of gemcitabine to PANC-1 cells was evaluated by MTT.4.PANC-1 cells were treated with a nontoxic dose of 5ng/ml gemcitabine for 24 h,and their expression of ADAM 10 were evaluated by RT-PCR and Western blot.The expression of membrane-bound MICA and MICA mRNA were evaluated by flow cytometry and RT-PCR, respectively. At the same time,24h culture supernatants were subjected to the analysis of sMICA levels by ELISA.5.PANC-1 cells were transfected with ADAM10 siRNA or control siRNA for 24 h and further cultured with 5ng/ml gemcitabine (equal culture medium for control)for 24 h.The expression of membrane-bound MICA was evaluated by flow cytometry,and sMICA production in the culture supernatants was evaluated by ELISA。 Results:1.The positive rate of ADAM 10 immunostaining in pancreatic cancer tissues(87.5%)was significant higher than in normal tissues of pancreas(P<0.01)2.Knockdown of ADAM10 for PANC-1 cells clearly upregulated MICA expression on their cellular surface and downregulated sMICA levels in their culture supernatants(P<0.01)3.When the concentration of gemcitabine was lower than 7ng/ml, gemcitabine maked little effect on the growth of PANC-1 cells in 24h.4.Gemcitabine suppresses ADAM10 expression in PANC-1 cells (P<0.01)5.Gemcitabine(5ng/ml)treatment led to an increase in membrane-bound MICA expression and a decrease in sMICA production in PANC-1 cells(P<0.01).The mRNA levels of MICA did not change after PANC-1 cells exposure to gemcitabine(P>0.05)6.PANC-1 cells were transfected with ADAM10 siRNA and then treated with gemcitabine.Neither upregulation of surface MICA nor downregulation of soluble MICA levels was observed.Conclusions:ADAM 10 is involved in MICA shedding of PANC-1 cells.Gemcitabine inhibits MICA ectodomain shedding through suppression of ADAM10.更多还原