| Objective: Using cell culture technique to logarithmic growth phase C26colon carcinoma cells through spleen injection method to establish BALB/cmice liver metastasis model of colon cancer, observing colorectal cancer livermetastasis formation and transfer, and study the expression of CD44protein indifferent period of colon cancer tumor effects on liver metastasis, providingtheoretical basis for the mechanism of colon cancer liver metastasis.Methods:1The cultivation of the colon (C26) cells in mice: cryopreserved C26cells recovery to DMEM containing10%fetal bovine serum-F12medium,plus a concentration of100u/ml of penicillin and streptomycin resistance,culture at37℃and5%CO2incubator, saturated humidity. With0.25%trypsin digestion represented. Observe the cell growth, access to adequatelogarithmic phase of active growth experiment with colon cancer cells.2The way to construct animal models of colon cancer liver metastases:the48mice were divided into control group (8) and experimental group (40).The control group and experimental group reared under the same conditions,the control group did not receive any treatment; the40experimental mice tomodel as described below, After the mice were weighed, with3%sodiumpentobarbital, according50mg/Kg dose intraperitoneal injection of anesthesia,after fixing mouse limbs, skin disinfection, in particular in the left upperquadrant of mice for about2cm incision, and then drill into the abdomen,sterile gauze protection of incision, found in the ventrolateral, willow leafshape the spleen to reveal it cautiously after incision, the1ml syringe withneedle5from the spleen into the pole parallel needles about2mm, payattention not to wear the spleen broken to prevent bleeding, The film is then injected into the spleen at a concentration of1×106cells/ml, single-cell suspe-nsion0.1ml, visible white swelling at the injection site the spleen capsule, pullout the needle and oppression hemostasis with tampon for5minutes, nobleeding after careful observation, spleen back into the abdominal cavity, andshut abdominal one by one3observation of laboratory mice: after injection of tumor cells in thespleen of40mice just randomly divided into4groups, conducted to observethe daily mental condition and growth of the mice in each group.atpostoperative3,6,9,12days survival rates were calculated for each group andthen put to death of animals. Splenic primary tumors and liver for pathologicexamination, calculated for each group of mice tumor formation rate and therate of liver metastases.4different time animal tumor tissue CD44protein target detection: afterkilled, mouse spleen by immunohistochemical method and quantitative imm-unohistochemistry and image analysis software to detect primary tumor tissueCD44protein expression, inference of CD44protein expression differencesaffect liver metastasis.Results:1recovery of C26cells were adherent monolayer growth, no bacterialand fungal contamination, in good condition.2After cultivation of tumor cells in spleen,In the first two days and thefirst11days after surgery,one mice was respectively dead. the mice survivalrates are respectively90%,100%,100%,90%in the third, Sixth, Ninth,Twelfth, postoperative day group. In the third postoperative day group, thedead mice was dissected and not observed tumor formation, obvious abdo-minal bleeding in the naked eye. Consider individual mice were significantlydifferent tolerance for anesthesia-induced immune responses. The remainingmice were killed. Spleen solitary tumor’s slice during0.3-0.5cm, microscopicobservation showed that the distribution of tumor cells into the group, atypic,and spleen clear structural boundaries. Under no visual observation of livertumor formation, but in a mouse liver tissue were observed but found micrometer performance. The rate of spleen tumor is90%,10%of liver tumor.In the first six days after the group, the mice were sacrificed after the nakedeye visible, spleen tumors significantly increased the size of the largest tumordiameter of about0.8cm, was prominent spleen surface, yellowish white, partof the liver of mice can be observed visually and more divergence in grainsamples, needle-like metastases. Microscopic tumor cells found in the spleenwithout ductal, acinar structures were poorly differentiated adenocarcinomaperformance microscope shows a large number of liver tumor cell growth,squeezing the surrounding liver tissue shrinking performance, The rate ofspleen tumor is100%, liver metastasis60%.In the first group after9days, themice were observed under the spleen anatomy visible primary tumorscontinued to increase, the advent of satellite nodules around the lesion, asmall tumor ulcerated surface forming the visible surface of the spleen andperitoneal tumors of other organs, peritoneal adhesions, microscope, diffuselarge number of tumor cell growth, the naked eye observation of varying sizesformed on the surface of liver metastases, close metastases mutual integrationbetween microscopic tumor cells is still a lot of visible part of normal livertissue, showing the distribution of the island, tThe rate of spleen tumor is100%,100%of liver metastases. In the first12days after operation, the first11days of death in mice example, autopsy spleen under visible tumor andsurrounding tissue adhesion closely, tumor compression of the intestine, alarge number of peritoneal ascites, considering the animals died of tumorprogression and caused widespread metastases, tumors in this group of micewas significantly increased compared with the previous planting, withextensive intra-abdominal adhesions to other organs, the liver appeared sizesof diffuse liver metastases, and has extensive disseminated intraperitonealperformance, mirror under most of the visible tumor cells in the spleen, thespleen than the proportion of normal cells, poorly differentiated, and novascular invasion, endoscopic liver tumors seen roughly the same distributionand spleen. The rate of spleen tumor is100%,100%of liver metastases.3Using immunohistochemical method to detect rate of CD44protein expression in experimental mice spleen tumor and then statistical analysis:IOD of CD44protein expression in the third days group after surgery is102.77±5.51; IOD of CD44protein expression in the sixth days after surgerygroup is132.84±5.11;IOD of CD44protein expression in the ninth after daysgroup is164.65±5.50;IOD of CD44protein expression in the twelfth daysgroup after surgery is167.14±6.12;IOD of CD44protein expression werecompared between groups, P <0.05, the difference was statistically significant.Conclusions:1The rat colon cancer cells (C26) through spleen injection method canbe successful to establish animal model of colon cancer liver metastasis, themodel has high rate of tumor and metastatic tumor, experimental time isshort,and can simulate the real actual process, provides a useful experimentalbasis for liver metastasis of colon cancer.2With the proliferation of colon cancer cells in mice, the spleen tumorcells in the expression of CD44protein increased significantly, the degree ofliver metastasis also increased accordingly, speculated that the expression ofCD44protein increases, can promote the formation of liver metastases, closelyrelated to the development of colon cancer liver metastases.3As a carrier for further research on mouse liver metastasis model,expounds the mechanism of the colon cancer liver metastasis and step by stepscreening effective intervention measures, to provide a theoretical basis forpreventing colon cancer liver metastasis. |
PDF Full Text Request