| With the gradual amelioration of people's living style and living standard ,colorectal carcinoma's incidence and mortality are obviously rising with the ages and situated at the second- fourth rank among the common carcinoma. To date , colorectal carcinoma' prognosis has important effects on the living quality of senile population. Hepatic organ-oriented metastasis of colorectal carcinoma plays an important role among many factors and becomes the research emphasis and focus of colorectal carcinoma. In 1889, Paiget forworded the "seed and soil" theory which mainly emphasized tumor cell 's progressiveness and invasion of itself , passive adaptability to tissue microenvironment, tissue and organ's anatomical factors about tumor metastasis and ignored the active contributions of organism. At present, through more than 100 years' research , plentiful and substantial outcomes have been made in the many levels and fields such as oncogene , biomacromolecule , subcell, cell, mesenchyma microenvironment and so on. On this basis, the modern tumor metastasis theory is formed gradually that tumor metastasis is a multisteps and sequential pathological process and emphasizes the balance between numerous metastasis and antimetastasis factors, active plasticity and specificity of host target organ microenvironment. Publishing of the article "An attractive force in metastasis"in the Nature in 2001 indicated that chemokines' research went into tumor field. In the complicated chemokines networks , CXCL12-CXCR4 biological axis is one of biological axises with point to point signal transduction and paid widely attentions .It is reported that CXCL12-CXCR4 biological axis plays an key role in the organ-oriented metastasis of chromoma ,breast cancer, prostate cancer , synoviosarcoma and the like, but for the details of its concrete mechanism and steps, we are not yet clear .Liver's parenchymal and emsenchymal tissue holds very strong plasticity and adaptability ,at the same time liver is one of main target organ of colorectal carcinoma organ-oriented metastasis. There is a long way to go whether CXCL12-CXCR4 biological axis is involved or not and how to bring action into play. Therefore ,we are determined to unfold the experiment from the CXCL12-CXCR4's point of view.Objective To study the CXCL12-CXCR4's effects on the mechanism of colorectal carcinoma hepatic organ-oriented metastasis from histobiopsy specimen, cell culture ex vivo and animal experiment in vivo levels, especially elucidate the colorectal carcinoma LOVO cell's biological characteristics of migration , invasion, chemiotaxis and mutual relationship of CXCL12-CXCR4 biological axis in the tumor tissue, liver of target organ and blood circulation, enrich the basic theory of tumor organ-oriented metastasis , provide more means to interfere in hepatic organ-oriented metastasis1 prognosis. Methods l.To detect the expressions of CXCR4 receptor and MMP-9 in the LOVO cell ,animal and colorectal carcinoma's histobiopsy specimen with immunohistochemistry. 2.For LOVO cell ex vivo, to observe the CXCR4 receptor's expressions on the LOVO cell, migration, invasion, chemiotaxis by imposing the exogenous CXCL12 or AMD3100 on LOVO cell withcell culture, flow cytometry, immunohistochemistry and Transwell methods. 3.TO establish animal model , to detect the exogenous CXCL12 or AMD3100's impacts on endogenous CXCL12 of target tissue and its ligand CXCR4's expressions and changes from protein and mRNA levels with flow cytometry ,ELISA ,RT-PCR methods. Results 1. The expressions of colorectal carcinoma 's CXCR4 and MMP-9 in the well-differentiated , medium-differentiated, poor-differentiated groups lie in the cytoplasm of tumor cell , are higher than normal control group (p<0.05). With the tissue gradual dedifferentiation , the expressions of CXCR4 and MMP-9 present rising trend, at the same time CXCR4 and MMP-9's expressions have significant difference between the tissue gradings 2.Cell culture ex vivo:(l) General biological characteristic: LOVO cells present epithelial form and have rich pink cytoplasm, prone to adherence growth. (2) Turning to flow cytometry level, the expressions of CXCR4 in the LOVO cell have decreasing tendency with the rising of CXCL12's concentration and extension of time, at the same time has obvious statistical difference among groups beside 25ng group. AMD3100 itself has inhibitory effectives on CXCR4's expressions on.(3) When it comes to migration and invasion ,results showed that LOVO cell 's chemiotaxis and invasion were remarkably enhanced with the concentration ascending of exogenous CXCL12 .when we administer O.lmmol/L AMD3100 to LOVO cell ex vivo ,its chemiotaxis and invasion were markedly inhibited .3.Animal experiment in vivo: (1) exogenous CXCL12 and AMD 3100 haven't marked impacts on the CXCL12-CXCR4 biological axis existed in the liver, kidney and blood circulation of normal experiment SCID mice. (2) Hepatic metastasis rate of colorectal carcinoma is 33%,62.5%,0% respectively in the tumor-beared group, CXCL12, AMD3100group. (3) For immunohistochemistry, in the normal group, normal CXCL12 and normal AMD3100 group ,the CXCR4 and MMP-9 's expressions in the cytoplasm are negative while in the tumor-beared group, tumor-beared CXCL12 and tumor-beared AMD3100 group , CXCR4 and MMP-9's expressions are upregulated greatly with statistical difference (p<0.05) for normal group and between groups .expressions of CXCR4 and MMP-9 have obvious changes. (4)From the cytometry point of view, These are low or no expressions of CXCR4 in the normal group, normal CXCL12 group,,normal AMD3100 group. CXCR4 receptor's expressions on the surface of tumor cell and hepatic cell and tumor cell in the tumor-beared group, tumor-beared CXCL12 and tumor-beared ADM3100 group upregulated dramasticly . Slight changes of CXCR4's expressions are found in the kidney tissue and poorer obviously than hepatic and tumor's. Exogenous CXCL12 can markedly upregulated the CXCR4's expressions(p<0.05) in the liver and tumor but can't in the kidney. In addition , exogenous AMD3100 makes the expressions of CXCR4's in the liver and tumor drop obviously but those of kidney tissue. (5) protein expression level: exogenous CXCL12 and AMD 3100 but for tumor-beared facor haven't marked actions on the CXCL12' contents existed in the blood circulation of normal experiment SCID mice. CXCL12 's expressions in the liver of tumor-beared SCID mice are greater than normal group and tumor tissue of tumor-beared groups .exogenous CXCL12 has a positive feedback action on CXCL12's contents in the liver and tumor tissue but not in the kidney and at the same time , the CXCL12 's expressions in the liver and tumor tissue are subjected to the exogenous AMD3100. (6) RT-PCR detecting: In the normal liver and kidney tissue lies inherent expressions of mRNA of CXCL12 and CXCR4 and exogenousCXCL12 and AMD3100 have no obvious effects on them. mRNA's abundence of CXCL12 and CXCR4 in the liver and tumor tissue of tumor-beared mice is 1.25±0.16. 0.82±0.07;1.64±0.1K 1.73±0.05 respectively which are upregulated to 2.13 + 0.24. 1.14±0.09;1.67±0.1K 1.97 + 0.11 and subjected to AMD3100. Exogenous CXCL12 and AMD3100 have no obvious effects on the expressions of the adundence of CXCL12 and CXCR4 in the kidney. Conclusion (1) CXCL12-CXCR4 biological axis' system plays an important role on liver-specific metastasis of human colorectal carcinoma .(2) The expressions have manifest organ and tissue specificity that in the blood circulation has the greater contents of CXCL12 than the others. CXCL12-CXCR4 biological axis in the kidney and blood circulation are insensitive to exogenous CXCL12 and AMD3100 In the liver, CXCL12 and CXCR4 both are enhanced obviously while in the tumor tissue only CXCR4's expression is upregulated, at the same time , expressions of different tissues are not only independent but also correlative and play the different action on liver-oriented metastasis of colorectal carcinoma . (3).Exogenous CXCL12 play agitating effects on the invasion and chemiotaxis of LOVO cells ex vivo which are in conformity with those of animal experiment.(4) CXCL12-CXCR4 biological axis and MMP-9 have mutual synergism and play an important role on tumor cell's invasion and chemiotaxis.
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