| Objective: Primary liver cancer (PLC) is a common malignant tumor of digestive system, which is characterized by low rate of early diagnosis, quickly development, easily invasion and metastasis and poor prognosis. Lymph node metastasis and distant metastasis are definitive factors in the prognosis of patient with PLC. Vascular endothelial growth factor family(VEGFs) play an important role in lymph metastasis and distant metastasis. VEGFs include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PLGF (placenta growth factor). Among these, VEGF-D plays an important role in tumor lymph metastasis. It expresses not only in many normal tissues but also in tumor cells. VEGF-D can induce lymphatic vessel generation and promote lymphatic cancer cell proliferation. Expression of VEGFs is closely related to a variety of cytokines. Interleukin-2(IL-2) and Interleukin-6(IL-6), commonly found in human inflammatory mediators, have a complex biological function. They are closely related to VEGFs. We use reverse transcription polymerase chain reaction (RT-PCR) to explore expression of VEGF-D mRNA after stimulating liver cancer cell line by IL-2 and IL-6, further more to define invasion and metastasis mechanisms of PLC. Methods: Liver cancer cells BEL-7402 and SMMC-7721 were cultured in 37℃,5% CO2,saturated humidity sterile incubator in RPMI-1640 culture media (containing 10% FCS,100U/ml penicillin and 100U/ml streptomycin). After the two cell lines were resuscitated, passaged and grew logarithmically, the culture media were changed to media containing 0.1%FCS, and continued to culture for 12 hours to wipe out effect of FCS on cell proliferation. The cells were cultured in the culture media containing 0.1% FCS and IL-2 (0.1μg/L,1μg/L,10μg/L,100μg/L,200μg/L) or IL-6(0.1μg/L,1μg/L,10μg/L,100μg/L,200μg/L) for 3,6,12,24 hours respectively. As control, the cells were cultured in culture media containing isodose phosphate buffered saline. All of the cells were collected in appointment time. Total RNA was extracted using Trizol Reagents, VEGF-D mRNA was measured before and after treatment using RT-PCR,β-actin served as a loading control. Experimental datas were analyzed by SPSS13.0 statistical analysis software.Results: 1. Expression of VEGF-D mRNA in liver cancer cell BEL-7402 after IL-2 treatment: The photometric brightness of electrophoresis strip in experiment groups was significantly darker than those of control group; IL-2 decreased VEGF-D mRNA expressions of experiment groups compared with control group. But there was no statistical difference among every experiment group. In different times interval, relative expression has no difference in the same concentrations. It's clear that IL-2 decreased expression of VEGF-D mRNA of BEL-7402, but it was no connection with concentrations and time.2. Expression of VEGF-D mRNA in liver cancer cell SMMC-7721 after IL-2 treatment: The photometric brightness of electrophoresis strip in experiment groups was significantly darker than those of control group. IL-2 decreased VEGF-D mRNA expression and the expressions were decreased gradually with the increasing concertrations of IL-2 in 0.1μg/L~100μg/L experiment groups. But in different times, the relative expression has no difference in the same concentrations. IL-2 restrained expression of VEGF-D mRNA of SMMC-7721 cell lines and the restraint increased with the concentration increasing in 0.1μg/L~100μg/L experiment groups, but it was no connection with time.3. Expression of VEGF-D of liver cancer cell BEL-7402 after IL-6 treatment: The photometric brightness of electrophoresis strip in experiment groups was significantly brighter than those of control group. There was no statistical difference among every experiment group. In different times interval, the relative expression has no difference in the same concentrations. It's clear that IL-6 increased expression of VEGF-D mRNA of BEL-7402, but it was no connection with time.4. Expression of VEGF-D mRNA in liver cancer cell SMMC-7721 after IL-6 treatment: The photometric brightness of electrophoresis strip in experiment groups was significantly brighter than those of the control group except 0.1μg/L group. The relative expression of electrophoresis strip in experiment groups was more than that of the control group. The inhancements increased with time of 1μg/L and 10μg/L IL-6 experiment groups. The IL-6 increased expression of VEGF-D mRNA of SMMC-7721 and the inhancements increased with time in some groups.Conclusions: 1. IL-2 decreases expression of VEGF-D mRNA in liver cancer cell BEL-7402 and SMMC-7721. It suggests IL-2 inhibits lymph metastasize of liver cancer by inhibiting expression of VEGF-D.2. IL-6 increases expression of VEGF-D mRNA in liver cancer cell BEL-7402 and SMMC-7721. But the effect on biological characters of liver cancer cell by IL-6 is needed to further more study. |
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