【作者】 许东哲；

【导师】 孙东植；

【作者基本信息】 延边大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的:目前胃癌术后腹膜种植转移的发生率高达50%,是影响胃癌治疗后无瘤生存率的最直接原因之一。早期检测并有效处理腹膜微小种植转移是提高生存率的关键所在。本研究比较逆转录多聚酶链反应（reverse transcription-polymerase chainreaction,RT-PCR）法和转录逆转录协调反应（transcription-reverse transcriptionconcerted reaction,TRC）法检测胃癌腹腔内微量游离癌细胞时间以及对新肿瘤标志物四型再生基因（regenerating gene typeⅣ,RegⅣ）和常用标志物癌胚抗原（carcinoembryonic antigen,CEA）的敏感性比较检测,探讨能提供临床服务的实用方法及RegⅣ可作为胃癌腹膜转移的标志物的可能性。材料与方法:为检测出胃癌细胞,以胃癌腹水中采集的细胞株SNU-719体外培养细胞和10例胃癌患者手术中采集的腹腔冲洗液作为实验组。用急性早幼粒白血病细胞株HL60培养细胞作为对照组。SNU-719和HL60用含有10%小牛血清RPMI-1640培养液,37℃培养箱培养后使用于实验。以2007年在京都府立医科大学附属医院消化外科接受胃癌手术的患者10例做为临床检测标本的对象。开腹后用200ml生理盐水冲洗直肠膀胱陷凹采集后,以此作为腹腔内冲洗液用于实验。对实验组10例患者按常规要求,手术中采集腹腔冲洗液和病理标本,供病理检查。SNU-719和HL60在37℃培养箱培养成10^6-7/ml、腹腔冲洗液,分别离心,取沉淀细胞。加RNase free water提取了SNU-719和腹腔冲洗液中的总RNA,转换成cDNA,根据每次循环结合荧光测定法的Gene Amp 5700序列探测系统,对已合成cDNA进行RT-PCR反应,快速定量检测RegⅣmRNA和CEA mRNA。用TRCR实时监测仪TRCRapid-160和相应试剂盒检测RegⅣmRNA和CEA mRNA,并记录各检查方法的检出时间。结果:（1）RT-PCR法和TRC法检出时间比较。RT-PCR法,从手术室采取腹腔内冲洗液至提取RNA平均时间为40min,转换成cDNA（RT）平均时间为60min,RT-PCR反应需要的平均时间为114min,从采集腹腔冲洗液至检出结果必要的总时间为214±15min。TRC法,采集腹腔冲洗液至提取RNA平均为40min,TRC反应平均33min,从采集腹腔内冲洗液到得出检测结果所必要的总时间为73±7min。TRC法与RT-PCR法比较能在短时间内得到检测结果,统计学分析有显著性差异（P＜0.01）。（2）胃癌腹水细胞株中RegⅣmRNA的检出。RT-PCR法检测结果显示,含有不同比例胃癌腹水细胞株SNU-719的mRNA浓度待测标本中,RegⅣmRNA/β-actin mRNA比值增高,SNU-719的mRNA浓度达100%时比值最高,0%时其比值最小。TRC法中,含有不同比例胃癌腹水细胞株SNU-719的mRNA浓度的待测标本中,RegⅣmRNA/PBGDmRNA比值也增高,RNA浓度达100%时最高。（3）胃癌腹水细胞株中CEA mRNA的检出。RT-PCR法检测结果显示,含有不同比例胃癌腹水细胞株SNU-719的mRNA浓度待测标本中,CEA mRNA/β—actinmRNA比值增高,SNU-719的mRNA浓度达100%时比值最高,0%时其比值最小。TRC法中,含有不同比例胃癌腹水细胞株SNU-719的mRNA浓度的待测标本中,CEA mRNA/PBGD mRNA比值也增高,RNA浓度达100%时最高。（4）腹腔冲洗液中RegⅣmRNA和CEA mRNA检测。10例胃癌患者组织病理学检查,随着胃癌胃壁浸润程度加深以及腹腔冲洗液细胞检查阳性,用RT-PCR和TRC方法检测RegⅣmRNA检出率呈上升趋势,其中检出率最高的是PCY（+）病例。对于腹腔冲洗液的CEA mRNA的检测得到结果与检测RegⅣmRNA结果几乎相同的表现。而且,两种方法之间有高度相关性（Y=0.931X+0.605,γ=0.996,P＜0.00001）。结论:（1）TRC方法与RT-PCR方法比较具有快速简便特点,手术中可以了解腹膜转移信息。（2）新的肿瘤标志物RegⅣVmRNA对胃癌腹水细胞株SUN-719和胃癌病例均有高度敏感性。（3）RegⅣmRNA与CEA mRNA比较同样具有敏感性,在RNA浓度0.01%～100%范围内,用RT-PCR法和TRC法检测得到几乎相同的结果,两种检测方法敏感度比较无显著性差异。（4）随胃癌浸润深度加深,RegⅣmRNA与CEAmRNA检出率增高,对腹膜微量癌细胞检测有一定意义。更多还原

【Abstract】 Objective:Incidence of peritoneal seeding metastasis after operation of gastric carcinoma is as high as 50%,and the most direct one of the reasons for affectting the survival rates of tumor-free after treatment of gastric carcinoma.Early detection and effective treatment for the peritoneal micro-seeding metastasis is the key events of improving survival rate.This study was that the times of detecting free micro-gastric cancer cells in abdominal cavity were measured by the real-time reverse transcription polymerase chain reaction（RT-PCR） as compared with the transcription reverse transcription concerted reaction（TRC）,and the sensitivity of new regenerating gene typeⅣ（RegⅣ） tumor markers as compared with commonly used carcinoembryonic antigen（CEA） to study the practical method for providing clinical application and explore the possibilities of RegⅣas a marker of peritoneal metastasis of gastric carcinoma.Materials and Methods:In this study,culture cell line of gastric cancer SUN-719 and the intra-abdominal rinse fluid in 10 cases of gastric cancer during operation were used as a experimental group,and acute promyelocytic leukemia cell line HL-60 as a control group. SNU-719 and HL60 were cultured with RPMI-1640 containg 10%calf serum at 37℃.10 cases of gastric cancer operated at the Affiliated Hospital of Medical School,Kyoto,were used as objects of detecting samples in clinic.After laparotomy,the abdominal cavity was rinsed with 200 ml saline and collected at the excavatio rectovesicalis to be used for the examination.10 patients in the experimental group were operated with routine requests and the intra-abdominal rinse fluid during operation and the pathological specimens were used for pathological examination.SNU-719 and HL60 were cultrued at 37℃to 10^6-7/ml and intra-abdominal fluid were centrifuged respectively,and from that were taken out the precipitated cells.And total RNA was extracted with adding RNase free water from the SNU-719 and intra-abdominal fluid,and converted into cDNA to detect the quantity of RegⅣmRNA and CEA mRNA with synthesized cDNA by RT-PCR in accordance with each cycle with fluorescence method of Gene Amp 5700 Sequence Detection System.After detecting the RegⅣmRNA and CEA mRNA by the TRCRapid-160 and the corresponding detection kit,the detecting time of each inspection methods was recorded.Results:（1） The time detected by RT-PCR and TCR:Using RT-PCR,the average time from collecting the intra-abdonima rinse fluid in operation room to extracting RNA was 40min,converting to cDNA was 60min,and detected by real time RT-PCR was 114min, and total time from collecting the intra-abdonima rinse fluid to detecting out the results was 214±15min.Using TRC,the average time from collecting the intra-abdonima rinse fluid to extracting RNA was 40min,detected by real time TRC was 33min,and total time from collecting the intra-abdonima rinse fluid to detecting out the results was 73±7min.The detecting time was more short by TRC than by real time RT-PCR,and the difference was statistically significant（P＜0.01）.（2） The detection of RegⅣmRNA in SNU-719 cell line: the results detected by RT-PCR showed in different proportion of SNU-719 mRNA concentrations of measured samples that RegⅣmRNA/β-actin mRNA ratio increased,and was the highest at the SNU-719 mRNA concentration up to 100%and the lowest up to 0%. The results detected by TRC showed in different proportion of SNU-719 mRNA concentrations of measured samples that RogⅣmRNA/PBGD mRNA ratio also increased and was the highest at the RNA concentration up to 100%.（3） The detection of CEA mRNA in SNU-719 cell line:the results detected by RT-PCR showed in different proportion of SNU-719 mRNA concentrations of measured samples that CEA mRNA/β-actin mRNA ratio increased,and was the highest at the SNU-719 mRNA concentration up to 100%and the lowest up to 0%.The results detected by TRC showed in different proportion of SNU-719 mRNA concentrations of measured samples that CEA mRNA/PBGD mRNA ratio also increased and was the highest at the RNA concentration up to 100%.（4） The detection of RegⅣmRNA and CEA mRNA in the intra-abdominal rinse fluid:In histopathological examination of 10 cases of gastric cancer,along with the increased invasive degree of gastric cancer to gastric wall and positive cells inspected in the intra-abdominal rinse fluid,the detection rate of RegⅣmRNA had upward trend by RT-PCR and TRC,in which the PCY（+） cases had the highest detection rate.The results between CEA mRNA and RegⅣmRNA in the intra-abdominal rinse fluid were almost the same,and there was a significant correlation between two methods（Y=0.931X+ 0.605,γ= 0.996,p＜＜0.00001）.For the peritoneal fluid detection and the outcome of results almost identical performance.Conclusion:（1） The TRC method is quick and easy features and comprehends the information for peritoneal metastasis in operation as compared with real time RT-PCR.（2） New tumor markers RegⅣmRNA has a high degree of sensitivity regardless of gastric carcinoma ascites cells SUN-719 and the cases with gastric cancer.（3） RegⅣmRNA has the same sensitivity as compared with the CEA mRNA,and in 0.01%to 100%range of RNA concentration,the results detected by the RT-PCR and TRC are almost the same and the sensitivity between two methods has no significant difference.（4） The detection rate of RegⅣmRNA and CEA mRNA increases along with the increased invasive degree of gastric cancer that has a certain significance for detecting peritoneal micro-cancer cells.更多还原