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Objective \The gallbladder as low rate of surgical resection, recurrence transfer high malignancy with poor prognosis whether there VM, have not been reported to date. In this study, paraffin-embedded specimens by human gallbladder cancer, in vitro three-dimensional culture intervention experiments Preliminary gallbladder whether there is a VM, as well as its morphological characteristics, molecular mechanisms, clinical significance and the NCTD gallbladder VM and mechanism. Methods ① collected surgical resection with pathologically confirmed primary gallbladder, gallbladder adenoma and chronic cholecystitis paraffin specimens of 74 cases, 10 cases, 10 cases and clinicopathologic parameters; the HE staining CD 31 and PAS double staining observed gallbladder exists VM, and correlation analysis for clinicopathological parameters, Cox analysis, the Kaplan-Meier Curve. (2) through in vitro cell two-dimensional culture, Transwell chamber invasion assay, collagen contraction experiments, cytotoxicity experiments and CD 31 immunofluorescence detection, clear GBC-SD and SGC-996 cell invasion and migration potential, NCTD IC 50 and HUVEC cells were identified. On this basis, the three-dimensional culture of rat tail collagen type I carried the gallbladder VM vitro simulation and Drug intervention experiments GBC-SD cells were divided into the control group, SGC-996 cells in the control group, TIMP 1 group, TIMP 2 the NCTD group, by HE and PAS staining, optical and electronic microscopy morphology of the three-dimensional cultured tissue slices in vitro simulation VM. To ③ application SABC Envision immunohistochemical and ELISA measured by radioimmunoassay paraffin sections of human gallbladder cancer, three-dimensional culture tissue sections and three-dimensional culture supernatant the the VM related molecule MMP 1 , MMP 2 , MMP 9 , MT 1 -MMP, VEGF, VE-cad and Ⅳ collagen expression, RT-PCR determination of gallbladder cancer cells Cultured cells related genes. Results ① the presence of 13.5% (10/74) VM, gallbladder VM with patient gender, age, tumor location, tumor size, degree of differentiation, Nevin stage, depth of invasion, local lymph node metastasis was found in 74 cases of human gallbladder ; but with the type of organization (the χ 2 = 10.241, P = 0.017), survival time (χ 2 = 5.7221, P = 0.0168) related, may be due to the VM () positive the specimens less, this group gallbladder VM and liver metastases was no significant correlation. ② GBC-SD and SGC-996 cells NCTD IC 50 for 56.18μg · the ml -1 and 22.66μg · ml -1 , GBC -SD cells in vitro through the the Transwell chambers invasion ability (P = 0.0013) and contraction migration (P <0.001) was significantly higher than the SGC-996 cells, so the GBC-SD and SGC-996 cells belong high, low invasive potential of cell lines . By HE and PAS staining, high-powered, inverted optical phase contrast microscope, intensive, such as cancer nest rat tail collagen type I and Matrigel plastic three-dimensional culture GBC-SD cells that the formation of tumor cells lined the matrix monocyclic or polycyclic, 48h The central cavity network-like pipeline structure, 14d lumen mature; the low aggressive SGC-996 cells has not formed pipe-like structure of the simulation VM. ③ molecule expression, gallbladder paraffin sections detection: VM, the VM (-) gallbladder cancer cell MMP 1 , MMP 2 , MMP 9 < / sub>, MT1-MMP, VEGF expression was significantly higher than the gallbladder adenoma, cholecystitis (P <0.0001), VE-Cad, type IV collagen was significantly lower than gallbladder adenoma, cholecystitis (P <0.0001); VM () gallbladder MMP 2 (F = 5.74, P = 0.0187), MT1-MMP (F = 8.78, P = 0.0039) was significantly high, VEGF (F = 5.78, P = 0.0182), IV Collagen type (F = 5.80, P = 0.0181) was significantly lower than the VM (-) gallbladder cancer, MMP 2 , MT1-MMP and gallbladder VM was positively correlated with VEGF, type IV collagen, and gallbladder VM There was a negative correlation; VM () gallbladder MMP 1 (F = 0.27, P = 0.6082), MMP 9 (F = 2.80, P = 0.0976), VE-Cad (F = 1.37, P = 0.2450) expression and VM (-) the gallbladder no difference. Tissue sections detection of three-dimensional culture: GBC-SD MMP 1 , MMP 2 , the expression of MMP 9 , MT1-MMP, VEGF expression was significantly higher at the SGC-996 cells. MMP-1 in the VM (-) group expression and degree of differentiation, Nevin stage, lymph node metastasis, liver metastasis, and with the lower degree of differentiation, Nevin staging later (S3 ~ S5), lymph nodes and liver metastases expression levels increase, there is significant with differences (P <0.05) in the VM () group with clinicopathological parameters unrelated (P> 0.05), the same as the liver metastasis group VM () group expression of MMP-1 expression significantly with higher than the VM (-) group ( P <0.05). MMP-2 in the VM (-) group expression and depth of invasion, degree of differentiation, Nevin stage, lymph node metastasis, liver metastasis is closely related to, and with the invasion of serosa, the lower the degree of differentiation, Nevin staging later (S3 ~ S5 ), lymph nodes and liver metastases expression levels increase, there is a significant difference (P <0.05) in the VM () group clinical pathological type adenocarcinoma (P <0.05), with the lymph nodes, liver metastasis, poorly differentiated adenocarcinoma , depth of invasion, Nevin stage (S3 to S5), VM () group the expression of MMP-2 were significantly higher VM (-) group (P <0.05). MMP-9 in the VM (-) group expression and depth of invasion, Nevin stage, lymph node metastasis, liver metastasis and serosal invasion, with the lower degree of differentiation, Nevin staging the later (S3 to S5), liver transfer of expression increased, there is a significant difference (P <0.05) in the VM () group with depth of invasion, Nevin stage liver metastasis and with serosal invasion, Nevin staging more night (S3 to S5), liver transfer of expression increased, there is a significant difference (P <0.05), the same for invading the serosa, adenocarcinoma, Nevin stage (S3 to S5), liver metastases, VM () group the expression of MMP-9 were significantly higher than the VM (-) group (P <0.05). MMP-14 in the VM (-) group and the VM () group expression and depth of invasion, Nevin stage, lymph node metastasis, liver metastasis is closely related with the invasion and serosal later, Nevin staging (S3 ~ S5) increased expression levels of liver metastases, significant differences (P <0.05), the same as invading serosa, Nevin staging (S3 ~ S5), lymph nodes and liver metastases, VM () group of MMP-14 expression were significantly higher than VM (-) group (P <0.05). (-) Group expression of VEGF in the VM and depth of invasion, Nevin stage, lymph node metastasis, liver metastasis, and with serosal invasion, Nevin staging later (S3 ~ S5), increased expression of liver metastases, significant difference (P <0.05), in the VM () group, only the expression of liver metastasis was significantly higher than that without liver metastases, significant differences (P <0.05). Same pathological indicators, VM (-) group and the VM () group expression of VEGF was no difference (P> 0.05), VE-cadherin VM (-) group expression and depth of invasion, Nevin stage, lymph node metastasis, differentiation degree with serosal invasion, Nevin staging later (S3 to S5), lymph node metastasis, poorly differentiated expression is reduced, there is a significant difference (P <0.05) in the VM () group has nothing to do with clinicopathological parameters (P> 0.05 ), the same pathological indicators, VM (-) group, and VIII () group expression of VE-cad was no difference (P> 0.05) Ⅳ collagen in VM (-) group expression and depth of invasion, Nevin stage, degree of differentiation, liver transfer associated with the invasion and serosal, Nevin staging later (S3 to S5), liver metastases, poorly differentiated expression is reduced, there is a significant difference (P <0.05), group Ⅳ collagen expression and depth of invasion in the VM () , related to the degree of differentiation, and with serosal invasion, poorly differentiated expression is reduced, there is a significant difference (P <0.05), the same as the lymph nodes liver metastasis, VM () group Ⅳ collagen expression were significantly lower than the VM (-) group ( P <0.05). Second, the three-dimensional culture supernatant ELISA test: GBC-SD cells MMP2 protein expression 3D SGC-996 cells, three-dimensional culture dimensional GBC-SD was significantly higher (P <0.05), while three-dimensional culture GBC-SD cells expression of MMP 9 protein expression 3D SGC-996 cells, the two-dimensional GBC-SD increase was not significant (P> 0.05), RT-PCR can display the Ⅷ ability GBC-SD strong Cultured cells related genes, and the SGC the the -996 only weak or no expression (4) Applications NCTD tail type I collagen three-dimensional culture 48h, GBC-SD cells that lose a monocyclic or polycyclic network-like structure capacity, cell sparse, float pyknosis or aggregation karyopyknosis fragmentation, apoptosis and necrosis significantly delayed; expression of TIMP 2 time to play this role, TIMP 1 no effect. , NCTD suppression vitro simulation gallbladder VM capacity, TIMP 2 , TIMP 1 no the gallbladder VM ability to block in vitro. Tissue sections, and the supernatant was detected by three-dimensional culture of 1/2CI 50 The cytotoxicity of NCTD in not only expression of MMP to 2 , MT1-MMP and expression of MMP to 1 , MMP 9 , VEGF-positive GBC-SD cells reduce staining shallow, decreased expression; and over time, the GBC-SD cells MMP 2 , MMP 9 protein expression was significantly inhibited (P <0.05), shows that of NCTD not only effective inhibition of MMP GBC-SD 2 , MT1-MMP, but also inhibit the expression of MMP , MMP 9 , VEGF, RT-PCR NCTD can effectively inhibit the ANG-1 and ANG-2, EphA2, VEGF expression, suppression and destruction in vitro simulation gallbladder VM through the multi-target role the formation and maturation. Conclusion human gallbladder exist VM; the gallbladder cancer VM with histological type, survival is related. High invasive potentials GBC-SD vitro simulated VM ability to form a monocyclic or polycyclic ring network structure of the cancer cells for lining cells in the three-dimensional culture. The molecular mechanisms involved in MMP 2 , MT1-MMP, VEGF, of EPHA2, type IV collagen and other related molecules. NCTD can inhibit effective vitro simulation gallbladder VM; its mechanism may be through with the NCTD multi-target inhibition of the above expression of related molecules, which play a repressor, destruction formation in vitro gallbladder VM role.
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