| 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 106-7/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. |
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