The Application Of A Novel Isolation Technique For The Experimental Study In Xenoplanted Gastric Carcinoma

BackgroundMorbidity and mortality is high in gastric cancer.Gastrectomy is the most important way to treat gastric cancer. Despite the development of surgical oncologic procedures,distant metastases and local recurrences are still major problems after radical gastrectomy.The manipulation of tumor during the surgery may result in metastases and relapse of tumor.Animal studies has shown that malignant cells are shed into the blood stream during surgical resection of a primary tumor and that this may enhance the development of metastases.The no-touch isolation technique was proposed to reduce intraoperative shedding of tumor cells into the portal vein during resection of colorectal cancer years ago.ObjectiveThis experiment will study the use of a novel blockade technique in rabbit VX₂ gastric carcinoma.We will study whether the novel blockade technique decreases metastatic tumour cells from spreading not only into bloodstream effectively but also lymph flow.MethodsWe established twenty rabbit xenoplanted gastric carcinomas in two ways.We established ten xenoplanted gastric carcinomas by injecting the VX₂ carcinoma cells suspension into the body of stomach.Another ten were underwent surgery by implantation of fragments of VX₂ carcinoma into the body of stomach.Then 2, 4 and 2 rabbits were sacrificed 1, 2 and 3 weeks after xenoplanting in each group.And the remaining 2 rabbits underwent autopsy when they died more than 4 weeks after xenoplanting in each group.So,we can eveluate the progression of the growth and metastases of the xenoplanted gastric carcinomas.Then another eighteen rabbits with xenoplanted VX₂ gastric carcinomas,established by implanting the fragments of VX₂ carcinoma into the body of stomach,were diveded into three groups.Six were under operation with conventional partial gastrectomy without using the novel isolation technique.Six were done with the novel isolation technique first,and then under partial gastrectomy. The vessels around the gastric cancer were ligated first,then silk sutures were placed on the gastric wall near the tumor to block the lymph-vascular,the vessels and the gastral cavity.We examined blood samples of the portal vein collected before and during manipulation of tumors and the gastric lymph nodes after surgery in both group. The remaining six were only under surgery of dissection of the gastric lymph nodes lonely with gastric tumors intact. The blood was prepared into cell suspension and the lymph nodes were prepared into homogenate at the concentration of 1×10⁶/ml.When clone AE1/AE3 of rabbit anti human CK antibody,goat anti rabbit IFTC coagulated IgG and PI were added,at least 105 cells per time were evaluated using FCM assay.Adjusting the forward scatter settings (FSC) and side scatter settings (SSC) is used to exclude cell debris,cell doublets and clumps. FITC signals and the PI signals were recorded as dot plot amplified data.The CK- PI double positive cells were the VX₂ carcinoma cells.The percentage of CK-PI double positive cells was the the percentage of VX₂ carcinoma in samples.Statistical analysis was performed with Stata 7.0.Differences between means were determined using the Student's t test when groups passed both a normality test and an equal variance test.In all cases,P values of <0.05 were regarded as being statistically significant.ResultsWe can established xenoplanted gastric carcinoma using suspension and fragment of VX₂ carcinoma cells successfully.The tumors began to grow 1 week after xenoplanted.It continued to grow in the next week.And it growed at the highest speed 3 weeks later.Extensive metastases,including the gastric lymph nodes melted together,metastases in diaphragm,metastases in liver.There were metastases in the abdominal cavity and abdominal wall in the suspension group but not present in the fragment group.The diameter of tumors is 0.15±0.07 cm,0.70±0.42 cm,1.65±0.21 cm and 2.25±1.06 cm at the first week,the second week,the third week and the fourth week and later after implanting respectively in the suspension group.The results were 0.40±0.10 cm,1.18±0.21 cm,1.78±0.39 cm and 3.50±1.08 cm respectively in the fragment group.The mean positive rate of VX₂ tumor cells in porta vein collceted during manipulation of tumor was 60.10% and it was 20.59% before manipulation in the conventional cases.There is significant difference(P<0.05). The mean positive rate of VX₂ tumor cells in gastric lymph nodes collceted after surgery was 51.59% and it was 22.08% before manipulation in the conventional cases.There is significant difference(P<0.05) too.The mean positive rate of VX₂ tumor cells in porta vein collceted during manipulation of tumor was 43.30% and it was 32.62% before manipulation in the novel isolation group.There is not significant difference(P>0.05). The mean positive rate of VX₂ tumor cells in gastric lymph nodes collceted after surgery was 35.86% and it was 22.08% before manipulation in the novel isolation group.There is not significant difference(P>0.05) either.The mean positive rate VX₂ carcinoma cells spreaded into bloodstream in the conventional group was 39.51% and it was 10.67% in the novel isolation cases. There is significant difference(P<0.05).The mean positive rate of VX2 tumor cells spreaded into lymph flow in the conventional group was 27.84% and it was 9.42% in the novel isolation cases. There is significant difference(P<0.05).ConclusionsWe can establish rabbit model of xenoplanted gastric carcinoma using suspension and fragment of VX₂ carcinoma cells successfully.And the ways of growth and metastasis of xenoplanted rabbit gastric carcinomas are the same as human gastric cancer.We can establish ideal xenoplanted gastric carcinoma using fragment of VX₂ carcinoma more conveniently and efficiently.VX₂ carcinoma cells are shed into the bloodstream and lymph flow during surgical resection of tumors.The novel isolation technique may be useful to prevent VX₂ carcinoma cells from spreading into not only bloodstream but also lymph flow effectively.