| 1 Background and objective:Hepatocellular carcinoma (HCC) is one of malignant tumor with incidence in china. Comprehensive therapy including surgical resection has been used to treat this disease, but the result is bad. It threatens people's life severely. HCC is characteristic of rapid development, easily metastasis, and bad prognosis. The metastasis and recurrence of HCC is a complicated process with mangy steps and stages influenced by multiple factors. The mechanism of occurrence, development, invasion and metastasis of HCC is unclear. Thrombospondins (TSP) are a series of glycoprotein with multiple functions. TSP-1 is the most important member. It can be secreted into extracellular matrix by many cells and play many physiological function through interaction with the receptor on cell membrance. Other studies have demonstrated that the synthesis and secretion of TSP-1 was closely related to development of other malignant tumor. There is no report about the influence of TSP-1 on the biological behaviour of liver cancer. So we explored the role of TSP-1 on the biological behaviour of liver carcinoma cell line and the mechanism among it. And the role of TSP-1 is testified in transplantational tumor on nude mice.2 Methods:(1) The expression of TSP-1, CD36, CD47 in human hepatic carcinoma cell lines and selection of cell line.The expression of TSP-1 and its receptors included CD36 and CD47 in human hepatic carcinoma cell lines: HCCLM3, MHCC97, HepG2, SMCC-7721 was examined using western blot. The cell line with no or weak expression of TSP-1 and strong expression of its receptors: CD36 and CD47 was chosed as the research subject.(2) The choice of cell concentration in MTT testThe cell line was divided into 4 groups according to the cell concentration:5 x 103/ml, lxl 04/ml ,2x104/ml ,5x104/ml. MTT test was executed with appropriate cell concentration.(3) The choice of the concentration of TSP-1 and the time point of actionTSP-1 was divided into 6 groups according the concentration: Oug/ml, 10ug/ml,20ng/ml, 30ug/ml, 40ug/ml, 50ug/ml. MTT test was used to detect the role of TSP-1 on cell growth at different concentration and time point.(4) The influence of TSP-1, CD36, CD47 on the biological behavior of human hepatic carcinoma cell line.The cell line was divided into 4 groups: control group, TSP-1 group, CD36-blocked group, CD47-blocked group. The concentration of CD36-blocked group, CD47-blocked group was: lug/105cells, 2ug/105cells, 5ug/105cells. The appropriate concentration of antibody was used to explore the role of CD36, CD47.1) To detect the role of TSP-1, CD36, CD47 on cell growth by MTT test.2) To detect the role of TSP-1, CD36, CD47 on cell adhesion by MTT test and cell counting3) To detect the role of TSP-1, CD36, CD47 on cell cycle and apoptosis by Flow cytometry.4) To detect the role of TSP-1, CD36, CD47 on the change of cell ultractructure by transmission electron microscope.5) To detect the role of TSP-1, CD36, CD47 on cell invasion and metastasis in vitro by Boyden chamber.(5) The primary research on the mechanism of the influence of TSP-1, CD36, CD47 on cell biological behavior of human hepatic carcinoma cell line1) To detect the influence of TSP-1, CD36, CD47 on the secretion of TGF-Betal by ELISA.2) To detect the influence of TSP-1, CD36, CD47 on the expression of Caspase3 and MMP-9.(6) The research of TSP-1 on the transplant tumor in null mice0.2 ml of cell (5xlO6cell) was inoculate into the back of Balb/c nude mice (4-6weeks). The mice was divided into control group and experiment group after thetumor could be touched. The mice of experiment group was injected subcutaneously with TSP-l(40mg/Kg, qod), the mice of control group was injected with aline. The mice were sacrificed three weeks later and the weight and the volume of the transplanted tumour was measured.. Then tumor fixed and embedded by formalin and paraffin were examined for microvessel density with immonohistochemistry method. 3 Results:(1) The expression of TSP-1, CD36, CD47 in human hepatic carcinoma cell lines and selection of cell lineThe expression of TSP-1 was significantly lower in HCCLM3 and HepG2 than in MHCC97 and SMCC-7721. There was no significant difference in HCCLM3 and HepG2. The expression of CD36 was significantly lower in SMCC-7721 than in HCCLM3 and HepG2. There was no differenceabout the expression of CD47 between the SMCC-7721 and the HCCLM3. So HCCLM3 was chosen as research object.(2) The choice of cell concentration in MTT testThe growth curve demonstrated that the time to get to platform period of concentration of 2 x 104/ml, the cells with the concentration of 2 * 104/ml, 5 * 104/ml grew to the platform at 6 days and 4days respectively. 5 x 103/ml, 1 x 104/ml were not get to platform period after 7days. So the concentration (2x104/ml) was chosed as experimental concentration.(3) The choice of TSP-1 concentration and time pointThe inhibitory effect of TSP-1 on HCCLM3 was dose-dependent when the concentration of TSP-1 lower than 40|j,g/ml. There was no difference between 40jig/ml and 50\ig/m\. The inhibitory effect was also dependent on the time point, the inhibitory effect began to occur at 24 hour, but significant at 72 hour. So 40p.g/ml was chosed as experimental concentration and 72 hour as the time pomint.(4) The influence of TSP-1, CD36, CD47 on the biological behavior of HCCLM3 1) The choice of antibody concentration and the effect of TSP-1, CD36, CD47,on cell growth and cell cycle:There was no significantly difference of inhibitory effect in different concentration of antibody. It demonstrated that the concentration (>l|j,g/105 cell)could block the interaction of TSP-1 with CD36 and CD47completely. So 5ug/105 cell was chosed as experimental concentration because of cell growth.MTT test demonstrated that the growth of HCCLM3 in each of TSP-1 group, CD36-blocked group and CD47-blocked group were significantly slower than in control group, inhibitory rate was respectively 25.97%, 10.76%, 20.23%. Inhibitory effect of growth in CD36-blocked group was lower than in CD47-blocked group.Flow cytometry for cell cycle indicated that the cell number in GO/Gl phase in TSP-1 group increased thant hat in control group, the cell number in S and G2/M phase in TSP-1 group reduced than in control group. The cell number in GO/Gl phase was not significantly different between the control group and CD36-blocked group. The difference of cell number in GO/Gl and G2/M phase were not significant between TSP-1 group and CD47-blocked group. Cell number in GO/Gl and G2/M phase in CD47-blocked group was lower than in CD36-blocked group. The proliferation index was respectively: 42.70±2.09(control groupX27.85±0.71 (TSP-1 group)>38.04±l.35 (CD36-blocked group), 31.78±0.43 (CD47-blocked group), the difference among groups were significant.2) The effect on cell apoptosisThe apoptosis rate in TSP-1 group (12.44±0.72%) was significantly higher than in control group (4.31±0.29%). There were no significant difference of apoptosis rate between control groupand CD47-blocked group(4.99±0.12%). The apoptosis rate was different among the CD36-blocked group (9.99±0.57%), the control group and the TSP-1 group.The morphological changes were detected by electron microscope. The cell growth in control group and CD47-blocked group were similar: grow vigorously, much module in cytoplasm and many divided nuleus. Apoptosis cell increased in TSP-1 group. There were less cellular module in cytoplasm. Nucleus began to break to pieces and concentrate. There were similar ultrastructure changes in CD36-blocked group and TSP-1 group.3) The effect on cell adhesiveThe cellular adhesive decreased significantly in experimental groups than incontrol group (0.607±0.035;219.83+.14.19). There was no difference on cellular adhesive among TSP-1 group (0.492±0.010;159.17±11.84), CD47-blocked group (0.528±0.016, 177.00±14.39) and CD36-blocked group (0.508±0.014, 161.17±16.19) 4) The effect on invasion in vitroThe cell number penetrating filter membrane was less in TSP-1 group (44.67±3.21) and CD47-blocked group(53.67±3.06) than in control group (77.33±4.51). There was no difference between CD47-blocked group and TSP-1 group. There were significant difference among CD36-blocked group (65.00±3.00), TSP-1 group and control group.(5) The primary study on the mechanism of the influence of TSP-1, CD36 and CD47 on HCCLM31) The effect of TSP-1 on the secretion of TGF-BetalThe average value of TGF-Betal(pg/ml) in each group were: 933.12+33.27, 949.46+42.42, 956.01+45.66, 925.88+29.20. There is no significant difference among all groups.2) The effect of TSP-1 on the expression of Caspase3 and MMP9The expression of Caspase3 are higher in TSP-1 group (0.652+0.024) and CD36-blocked group (0.615+0.020) than in control group (0.398+0.033) and CD47-blocked group (0.432+0.019) (p<0.05) . There is no significant difference between TSP-1 group and CD36-blocked, so is between control group and CD47-blocked group.The expression of MMP9 is lower in TSP-1 group (0.497+0.024)and CD47-blocked group (0.548+0.013) than in CD36-blocked group(0.674+0.019) and control group(0.708+0.021). There is no difference between TSP-1 group and CD47-blocked group, similar result is obtained between CD36-blocked group and control group.(6) The effect of TSP-1 on transplant tumor in nude miceThe volume of transplant tumor decreased significantly in treatment group with TSP-1 than in control group(0.216+0.058 mm3 vs 0.335+0.075 mm3), similar result is obtained from weight of tumor (0.298+0.08 lg vs 0.464+0.092g;p<0.01). TheMVD is lower in treatment group with TSP-1 than in control group(l6.67+4.76 vs 28.17+4.96;p<0.01). 4 Conclusions:(1) TSP-1 is expressioned at lower level in HCCLM3 and HepG2 cell than in MHCC97 and SMCC-7721 cell. The receptor of TSP-1: CD36 and CD47 are expressed in all of these cell lines.(2) TSP-1 can inhibit the growth of HCCLM3 cell through combining with the receptor CD36. The effect depends on the dose and time. The effect do not increase when the concentration of TSP-1 is more than 40^g/ml. The receptor CD47 may play some effect. The inhibit effect may be not related to synthesis of TGF-Betal.(3) TSP-1 can induce apoptosis of HCCLM3 cell line through combined with CD47 which can increase the expression Caspase3. CD36 may play a minor role in inducing apoptosis of HCCLM3 cell line through other than Caspase3.(4) TSP-1 can inhibit the migration and adhesion to extracellular matrix of HCCLM3 cell line. TSP-1 plays the effect through combining with CD36 which can decrease the synthesis of MMP9. CD47 has no effect on these course. The mechanism of inhibiting adhesion is unclear, perhaps CD36 and CD47 do not participate in the course.(5) TSP-1 can inhibit the growth and angiogenesis of transplanted tumor in nude mice of HCCLM3 cell line.
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