Construction Of Huamn Gastric Cancer Cell Lymph Node Metastasis Model And Screening Of Highly Metastatic Cell Lines And Explore Its Mechanism

Objective: To construct a model of human gastric cancer cell line MGC-803 implanted in a footpad lymph node in nude mice,and to establish a highly metastatic gastric cancer cell line MGC-803-L,preliminary study of its biological behavior and its invasion,metastasis mechanism.Methods: 1.The logarithmic phase of human gastric cancer cell line MGC-803 was routinely digested and made into a single cell suspension.Twelve nude mice were randomly selected for subcutaneous injection of cell suspension into the footpad to observe the tumorigenicity of the footpad in nude mice.2.After being raised at least six weeks,put to death at least,ipsilateral popliteal fossa or pathological and immunohistochemical detection of inguinal lymph node,to find cancer metastasis of lymph nodes,if not found cancer metastasis of lymph nodes,repeat the experiment,to extend the time of feeding,confirmed that gastric cancer lymph node metastasis rate of 70% or higher.3.It will be proved that the lymph nodes with metastatic carcinoma are cut into small tissue blocks,and the primary cells are extracted for primary cell culture,and the cell line MGC-803-L of stable genetic high metastatic gastric cancer isobtained 4.Use short tandem repeat(STR)cell typing technology to detect MGC-803-L.5.The biological behavior differences between MGC-803-L and MGC-803 were compared by using CCK-8 kit,flow cytometry,Transwell chamber and Transwell cubicle with Matrigel glue.6.Real-time fluorescence quantitative PCR was used to detect the expression of related genes such as Chloride intracellular channel 1(CLIC1)and Intergrin(ITG)(?1,?3,?v,?1);The expression of CLIC1 protein between MGC-803 and MGC-803-L and the expression of CLIC1 protein between transplanted tumors and metastatic lymph nodes were analyzed by immunohistochemistry.Results: 1.Human gastric cancer cell line MGC-803 after injection into the foot pad of nude mice,most of the nude mice's footpad implanted tumors grew after 1 week.The growth of the transplanted tumors was observed in the foot pad of the nude mice at 2 weeks and the tumor formation rate was 100%.2.HE staining pathologically confirmed that the axillary and inguinal lymph nodes had metastases of different degrees;immunohistochemical staining suggested strong staining of CK-pan,further confirming lymph node metastasis of cancer cells.Combining the two results,the metastatic rates of axillary and inguinal lymph nodes in nude mice were 75.00%(9/12)and 41.67%(5/12),respectively.3.Take metastatic lymph nodes to extract primary cell culture and successfully establish human high metastatic cell line MGC-803(MGC-803-L).4.The STR data showed that the cell line MGC-803-L was identical to the parental cell line MGC-803(90.7% match)and could be used for subsequent experiments.5.There was no significant difference in proliferation rate between the two groups except 0h(P>0.05).The relative proliferation rates of the two groups of cells MGC-803 and MGC-803-L at 24 h,48h,72 h were(42.62% ± 0.03)(60.18% ±0.07),(75.80% ± 0.08)(107.12% ± 0.09),(136.26% ± 0.03)(164.18% ± 0.11),and the proliferation rate of MGC-803-L group at 24 h,48h,and 72 h was higher than that of MGC-803 group(P<0.05).The apoptosis rates of MGC-803 group and MGC-803-L group were(12.27%±4.84)(12.45%±2.36),and the difference was not statistically significant(P>0.05);The apoptosis rates of MGC-803 and MGC-803-L cells were(12.27%±4.84)(12.45%±2.36),with no significant difference(P >0.05);The number of Transwell cells passing through Matrigel gel in the MGC-803 and MGC-803-L groups was(108.00±11.87)(214.20±13.75).The invasiveness of the MGC-803-L group was significantly higher than that of the MGC-803 group(P <0.01);the number of Transwell cells in the MGC-803 group and MGC-803-L group were(232.40±22.36)(437.00±31.25),and the migration ability of the GC-803-L group was significantly higher than that of the MGC-803 group(P<0.01).6.In group MGC-803-L,the relative expression of CLIC1 mRNA in group MGC-803 was(1.66 ± 0.19),and the relative expression level of the MGC-803-L group was higher than that of the MGC-803 group(P<0.05).The relative expression levels of ITG(?1,?3,?v,?1)mRNA in the MGC-803-L group were(2.51±0.86)(1.47±0.19)(1.50±0.23)(1.19±0.08),and the mRNA expression of ITG(?1,?3,?v,?1)in MGC-803-L group was higher than that in MGC-803 group(P<0.05).The relative expression of CLIC1 protein in the metastatic lymph node group and the transplanted tumor group were(0.499±0.046)(0.226±0.091)and the relative expression of CLIC1 protein in the metastatic lymph node group was higher than that in the transplanted tumor group(P<0.05).The relative expression levels of CLIC1 protein in the MGC-803-L and MGC-803 groups were(0.647±0.138)(0.907±0.078),and the relative expression of CLIC1 protein in MGC-803 group was higher than that in MGC-803-L group(P<0.05).Conclusion: Successfully constructed a human gastric cancer cell lineMGC-803 in a nude mouse footpad xenograft model.The tumorigenesis rate and metastasis rate of this model were high,and a stable and highly metastatic gastric cancer cell line MGC-803-L was successfully established.The acquisition of this cell line lays a good foundation for further exploration of the invasion and metastasis mechanism of gastric cancer.In addition,based on this,the present study preliminarily confirmed that elevated CLIC1 is associated with enhanced metastasis of gastric cancer,and may be related to differential expression of ITG?1,ITG?3,ITG?v,and ITG?1 genes.