| Objective: Biotherapy has been the forth tumor treatment methods besides operation, irridation and chemotherapy. Tumor necrosis factor-alpha (TNF- α ) is one of the earliest cytokins used in clinical care, but its use was limited because of its wide and intensive side-effects. Recombinant human tumor necrosis factor-alpha derivative 3a (rhTNF- α D3a) is a novel low-toxicity, high-activity rhTNF- α constructed by our native academicians. Its toxicity is 11 times lower than its proto type. The former has the same direct cytotoxicity as the latter, including many kinds of murine and hominine tumor cells. Hca/16A3-F is a sub-line of Hca, a murine ascites hepatoma, which having high-degree metastatic ability to lymph nodes. This kind of animal tumor is a good model for studying natural lymph nodes metastasis. In order to investigate the effect of rhTNF- a D3a on the growth of transplanted Hca/16A3-F solid tumor, we designed this experiment, and investigated the mechanism of it on local lymph node metastasis.Methods: Hca/16A3-F cells were intraperitoneal injected continuously two times. 615 mice were injected subcutaneously in the right dorsal with 0.2ml the secondary ascitic tumor cells(1 × 106 cells in 0.2ml 0.9% sodium chloride). Tumors were permitted to grow and volume was determined by direct measurement with calipers. When the volume became 0.5cm × 0.6cm, mice were randomly assigned to four different groups, each group had 5 femal mice and 5 male ones. Take 0.9% sodium chloride as the negative-control , etoposide (VP-16) as the positive-control and rhTNF-α D3a as the study object. All drugs were intratumoral and extratumoral injected every other day. Etoposide was given 1 hour's before rhTNF- α D3a . Etoposide was used three times and rhTNF- α D3a ten times. On D28, all the mice were died by disjointed cervical vertebra. All the tumors were taken out, their volume and weight were measured to calculate the tumor inhibition ratio. Take 6 fresh tumor -tissue samples from each groupto analyze cell-cycle with flow cytometry. All the tumors were half departed, one part was stored in 10% formalin, used to pathologic and immunohistochemical analysis of Rb-protein, the other one was stored in -70 ℃ refrigeratory for gelatin zymography analyzing MMP-2 and MMP-9. All the local lymph nodes were take out and stored in 10% formalin for pathologic analyze.Results: There was no intensive side-effect observed. rhTNF-α D3a has definite antitumor activity both alone and combined with VP-16 (P<0.05), the tumor-inhibition-ratio was 38.48% and 75.50% respectively, and combined-drug group got the same antitumor activity (75.41%) with VP-16(P>0.05). There were mass of necrosis focus along the vessel, and accumulation of neutrophils and lymphocytes around the tumor in the pathologic slide of single-druge group through microscope. Analyzed by flow cytometry, the percent of Hca/16A3-F cells stopped in stage G0-G1 was 18.67%,39.17% 27.69% and 29.09% respectively in negative-control , rhTNF-α D3a group, VP-16 group and combined group. Compare with other three groups, proliferation indicator of rhTNF- α D3a group was decreased (60.81%)(P<0.05 ), DNA inhibition ratio (25. 23%) was increased (P<0.05) . Combined-drug group (70.92%, 12.80%) also showed above trend, but has no statistic difference with VP-16 group (P>0.05). Immunohistochemsity for retinoblastoma(Rb) protein revealed that rhTNF- a D3a inhabited the expression of Rb-protein (the expressing ratio is 13.1%, and the negative-control is 23.6%), so did the combined group (the expressing ratio is 18.8%), but combined treatment has no statistic difference with VP-16 alone (22.8%). Lymph node metastatic ratio was decreased in all the three treatment groups: 50% in single group, 12.5% in combined group and 22.22% in VP-16 group. Though the combined group is the lowest and the single group is the highest, there has no statistic difference between them (P>0.05). Gelatin zymography showed that Hca/16A3-F transplanted solid tumor did not express MMP-2 (Matrix metalloprot...
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