| Objective:According to the important role in the signal transduction in the metastasisingof tumor of GHR,through setting up a growth hormone receptor plasmi and establishing the liver metastasis model in BALB/c mice of human colon cancer cells SW480, and combining 5-fluorouracil to research of liver metastasis of colon carcinoma on /BALB/c mice by small interfering RNA technique to target inhibition of human growth hormone receptor.And then provide a new strategy for the liver of clinical prevention and cure of colon cancer thus to the influence that the cell liver of person's colon cancer metastasis.Methods:1. Constructing The GHR plasmi:According to the gene sequences of Homo Sapiens growth hormone receptor, design 4 pairs of miRNA oligos according to Homo Sapiens receptor gene sequence design of the growth hormone, and then annealing the 4 pairs of oligos to double-stranded DNA, and select 3 clones to sequenced, in order to verify whether the inserting gene fragments of restructuring clones is in accordance with the oligo sequence of design.2. Establishing the liver metastasis model in BALB/c mice of human colon cancer cells SW480:10 female BALB/c mice aged 8 weeks, weighed 20-22 grams before surgery, were feed in SPF environment.After the mice were anaesthetized successfully, takes left back inclined slice of 0.5～1.0 cms, enter into celiac and expose spleen fully, put spleen out of celiac very softly, inclined with 5# needles follow spleen along the length-diameter about 0.5 cm, each mice injects human colon cancer SW480 cells levitation liquid 0.1 ml(1×107cells/ml)slowly, after completed. Put back spleen to in-situ, close celiac.Breed those mice in II laboratory normally.Killed those mice after operation and checked organization,it was be tested that those models were established successfully.3. Combined 5-fluorouracil to research of liver metastasis of colon carcinoma on BALB/c mice by small interfering RNA technique to target inhibition of human growth hormone receptor:36 mice which had been established the liver metastasis model of human colon cancer SW480 cells successfully were divided randomly into 6 groups:physiology brine group(NS group), plasmi group(G4 group), growth hormone group(GH group),5-fluorouracil(5-FU group),5-fluorouracil and plasmi group(5-FU+G group),5-fluorouracil and plasmi and growth hormone group(5-FU+G+GH group)(n=6).The mice were given medicine on the first day after surgery,and the amount of giving medicine is 10.the methods and dose of giving medicine is that:①NS group:NS10μl/each/3 days;②G group:Subcutaneous injected Eukaryotic expressing plasmid, G4 10μg/each/3 days;③GH group:The growth hormone with Physiological dose was Subcutaneous injected, rhGH2IU/kg/3 days;④5-FUgroup:Intraperitoneal injection,5-FU,20mg/kg/3 days;⑤G4+5-FU group: Intraperitoneal injection, rhGH 2IU/kg/3 days+5-FU,20mg/kg/3 days.⑥G4+GH +5-FUgroup:Intraperitoneal injection, rhGH 2IU/kg/3 days+rhGH 2IU/kg/3days+5-FU 20mg/kg/3days. Between the period of experiment. Observe the case of drinking water, feeding, mental state and Active state regularly,weight the mice every 5 days. Then the mice were executed at the 30th days after surgery, put out the liver and the spleen, embedding section with paraffins(slice thick as 4μm), dyeing wih HE, observe the performance of pathology tissue, count the rate of metastasis liver and the number of metastasis.Results:1, The change of weight:All of the BALB/c mice which had been Inoculated the cells SW480 of person's colon cancer survived, and we found the weight of all of the mice had not difference before operation. In the 1st day after operation, those mice of all groups had less dynamic, Spiritual statuses, the quantity of drinking water and ingestion were reduced and the weights of them declined and there had not difference between those groups. In the 1st day after operation, the weight of the mice of GH were recovered to the weight of preoperative, and the weight of others were lessen than the weight before operation(P<0.05), and the weight of G4 group were lessen than the weight of NS group. After 15 days of operation,the weight of the mice of G4+GH+FU were recovered to the weight of preoperative, and the weight of NS group, G4 group,5-FU group and G4+FU group were lessen than the weight obfore operation(P<0.05). Compare to NS group and G4 group and G4+FU group,the weight of G4+GH+FU group were bigger after operation 10 days (P<0.05). Compare to NS group and G4 group and G4+FU group,the weight of G4+GH+FU group were bigger after operation 10 days (P<0.05). Compare to NS group,the weight of G4+GH +FU group were lessen in the 10st and 30st days after operation (P<0.05). Compare to NS group,the weight of FU group and G4+FU group were lessen in the 20st days after operation (P<0.05).2, The change of morphology:The size of liver and spleen were lessen and the texture became more brittle and hard, and there were some big and little tumour knot on spleen and liver,and the metastasis knot located in the surface of the liver were gathered at the ledge of the liver leafs and the surface next to viscera, the change of on right liver leafs was more obviously. The metastasis knot were sharp size and scattered uniformityly in some mice, and the metastasis knot is grey. Some metastasis knots had cankered in individual 1 mice in NS group and 2 mice in GH group,and the shape of liver had been destroyed seriously. There also were hemorrhagic ascites in some mice, eg:5 in NS group,4 in GH group,3 in FU group,3 in G4 group,2 in G4+FU groupand 1 in G4+GH+FU group.3, Microscopic performance:①The growth rate of tumour in spleen and the rate of metastasis in liver There were tumors on spleen and liver in 36 BALB/c mice, the growth rate of tumour in spleen was 100%, the rate of metastasis was 100%,too.②Spleen:Observing with microscope, we found the cancer cells accumulate to clouds, most of the normal structure of spleenlobule had been destroyed, the size of cells was lessen, and the cancer cells differentiated poorly, and atypia obviously, cytoplasm and nuclei became pyknotic, chipped, dissolved, fission phase increased.The case was accord with Colon low differentiated adenocarcinoma. The change in NS group and GH group are seriously, Compare to NS group and GH group, the change in G4 group,FU group,G4+FU group and G4+GH+FU group are slightly and the comparison were significanced in principle of Statistics(P<0.05).③Liver:The cancer cells accumulate to clouds, most of the normal structure of hepatic lobule had been destroyed, the size of cells was lessen, and the cancer cells differentiatedpoorly, and atypia obviously, cytoplasm and nuclei became pyknotic, chipped, dissolved, fission phase increased, and were similar to the performance of the cancer knot on spleen. The case was accord with Colon low differentiated adenocarcinoma, and the tumour cells were be sured metastasis form the tumour growthed in spleen. The change in NS group and GH group are seriously, Compare to NS group and GH group, the change in G4 group,FU group,G4+FU group and G4+GH+FU group are slightly and the comparison were significanced in principle of Statistics(P<0.05).Conclusion:1, Maybe GHR play an important role in the liver metastasis of human colon cancer;2, Through targeted inhibiting GHR with siRNA, the ratio of hepatic metastases of human colon cancer SW480 cells;3, Combined with 5-fluorouracil, small interfering RNA,using GH will improve the nomal case of mice,but not increased the rate of the liver metastasis of human colon cancer SW480 cell;4, Combined with 5-fluorouracil, small interfering RNA technique to target inhibition of human growth hormone receptor can not reduce the ratio of the liver metastasis of human colon cancer SW480 cell.
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