| Objective: The chemotherapy holds the very important status in the tumor treatment. Although the recent years new chemotherapy medicine appears and the chemotherapeutical plans are improved unceasingly, the drug resistance of the malignant tumor displaying is always the primary cause that tumor could not be cured successfully. Some researches demonstrated that, the refractory and recrudescent patient could achieve remission again after being treated with a combination of the cytokine and the anticancer drug, then the cytokine,such as TNF,IL-2,IFN-a,GM-CSF,G-CSF, had possibly some function to reverse drug resistance. Many literatures reported cytokine including TNF had changed the drug resistance of the tumor cell by downregulating MDR mRNA expression, reducing P-gp synthesis, suppressing P-gp the function and so on, but it was infrequent to reverse drug resistance by inducing the tumor cell apoptosis. The cell apoptosis has close relations with the tumor occurrence and development, and induction tumor cell apoptosis may become a new strategy of treating malignant tumor. In recent years, the domestic and foreign researches had proven many factors could regulate tumor cell apoptosis , such as cytokine,oncogene,some traditional Chinese drugs. The tumor necrosis factor originates from the macrophage and the monocyte and has anti-tumor activity in vivo, not only can activate the tumor blood vessel to cause the organization hemorrhage and necrosis, but also induce the tumor cell apoptosis. This experiment took Hca/16A3-F as a cell line and established mice hypodermic implanted tumor model. We treated them with Vp-16 and induced tumor cells drug-resistance. After a week we built drug-resistance model, again used Rh-TNFα-Dk2 to treat them, through morphologyobservation under the electron microscope and proapoptosis bax and antiapoptosis bcl-2 protein expression situation we investigated if TNF-αcould induce tumor cell insensitive to Vp-16 apoptosis in order to seek a new way to overcome drug resistance.Methods:Hca/16A3-F cells were intraperitoneal injected continuously two times.615 mice were injected subcutaneously in the right anterior limb with 0.2ml the secondary ascitic tumor cells(1×106cells in 0.2ml 0.9% sodium chloride) .Tumors were permitted to grow and volume was determined by direct measurement with vernier caliper. When the diameter became 6-10mm, mice were randomly assigned to four different groups. Take physio-growth tumors as the negative-control group, etoposide(VP-16) as the positive-control group, TNF-αas single-drug group and TNF-α+ VP-16 as combined-drug group. Each group had 10 femal mice and 10 male ones.Each group mice started to give medicine from the 8th day. All drugs were intratumoral injected. Etoposide was used three times and TNF-αeight times. Two kinds of medicine should be separated a week. From the day before treatment, used vernier caliper to measure the longest diameters and its the widest vertical diameters of implanted tumor every one day, recorded and calculated mean tumor diameters. On D28, half mice of each group were died by disjointed cervical vertebra. All the tumors were taken out, their weight was measured to calculate the tumor inhibition ratio. Taken fresh tumor-tissue from each group, one part was stored in 10% formalin, used to pathoiogic and immunohistochemical analysis of bcl-2 and bax protein;the other one was stored in Glutaric dialdehyde, used to make the electron microscope specimen. The apoptotic difference of each group was observed under transmission electron microscope. Each group of another half mice were fed under the same condition, and observed survival time of each group mice.Results: 1.Vp-16 and TNF-αhad toxicity to the animals. 2. The combined-drug-treated tumors were significantly smaller in volume and lower in weight than those in other group (P<0.05 or P<0.01). On D28, TNF-αhad definite antitumor activity both alone and combined with Vp-16 (P<0.01), the tumor-inhibition-ratio was 24.20% and 72.49% respectively, and combined-drug group got the higher inhibition ratio than Vp-16(48.97%,P<0.01). 3. Mice treated by combined drug had longer survival time than the others (P<0.01). 4. Hematoxylin-eosine(HE) stain could been seen there were mass of necrosis focus on combined-drug group compared with the other three groups(P<0.05). 5. Under the electronic microscope, there were a lot of apoptotic cells at more fields of visions in combined-drug group. These apoptotic cells were small, and cytoplasm was thick. There were typical apoptotic cells, and had demonstrated the process of apoptosis in different stages. 6. The expression of bcl-2 protein reduced in VP-16 group,TNF-αgroup and combined-drug group, and had significantly statistic difference compared with negative-control group(P<0.01), but combined-drug group had no statistic difference with Vp-16 alone(P>0.05). There had significantly statistic difference between Vp-16 group and TNF-αgroup (P<0.01), so did Vp-16 group and combined-drug group (P<0.05). The expression of bax protein in combined-drug group was the highest compared with the other three groups, but no obvious statistic difference compared with TNF-αgroup (P>0.05). There had no significantly statistic difference between Vp-16 group and TNF-αgroup (P>0.05). The expression of bax protein in negative-control group was the lowest compared with the other three groups, and had obvious statistic difference compared with them( P<0.01).Conclusions: 1. Combined-drug group had the obvious superiority in inhibiting tumor proliferation. 2. The survival period of mice bearing tumor was significantly prolonged in Combined-drug group. 3. The mechanism of the inhibition of the implanted tumor growth and induction cell apoptosis with combined-drug treatment may be associated with the downregulation of bcl-2 and upregulation of bax expression, which the implanted tumor of liver cancer is insensitive to chemotheraphy.
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