| With the rapid development of industry, environmental pollution and food safety problems have become increasingly prominent, the trend of population aging is obvious, with the existence of smoking, drinking and other unhealthy living habits, the incidence and mortality of malignant tumors increased year by year. The incidence and mortality of primary bronchial lung cancer is in the first place among malignant tumors, but the prognosis of current clinical applied treatment on patients is still of poor effect. It can be found from the study of malignant tumors that the formation of new vessels plays a crucial role in tumor growth, infiltration and metastasis, and that sustained angiogenesis is the essential condition for the rapid growth of tumor. The formation of tumor neovascularization is in a certain period of time, under certain circumstances, by a number of factors, multiple steps caused by a series of cytokines mediated cascade biochemical reaction process. Angiogenesis can affect tumor invasion and metastasis, and reduce the survival rate of patients. It has been proposed that anti-angiogenesis can cure cancers as early as 20 years ago. Vascular endothelial growth factor(VEGF) is the most representative angiogenic factors so far, and has become the main drug target for anti-angiogenesis.90 K, also called Gal-3BP ? Mac2-BP or LGALS3 BP, is a highly glycosylated secretory protein, exerting its biological characteristics as a ligand combined with galectin family and E-selectin. By mediating cell proliferation, cell apoptosis, cell cell and cell matrix interactions, 90 K can lead to the tumor angiogenesis, and is closely related with the prognosis of patients with malignant tumor. Arresten is a type of endogenous angiogenesis inhibitor derived from noncollagenous domain of chain ?1 ofcollagen type IV on the basement membrane, composed of 229 amino-acid residues. The effects of this structure fragment on inhibition of vascular endothelial cell proliferation and angiogenesis in vitro are 3 times and 10 times that of endostatin. Tetramethylpyrazine(TMP) is an anti-angiogenesis alkaloid extracted from traditional Chinese medicine Chuanxiong, and is of strong anti-tumor effect. Cisplatin(DDP) is a common first-line chemotherapy drugs for malignant tumor. By observing survival state, tumor size, tumor weight, tumor inhibition rate of 4 groups of mice and detecting the expression of angiogenesis-related factors(90K?Arresten ? VEGF), this experiment explored the possible mechanism of TMP for inhibiting tumor angiogenesis, and found that TMP combined with DDP can improve the effect of chemotherapy.In this thesis, Lewis lung carcinoma cells were inoculated into the subcutis of C57BL/6 mice. The tumor were formed about ten days later after the subcutaneous injection. Forty mice were randomly divided into four groups: normal saline group(NS group), tetramethylpyrazine group(TMP group), cisplatin group(DDP group), tetramethylpyrazine plus cisplatin group(TMP+DDP group). The mice were treated continuously for14 days. The survival status of mice was observed. Tumor size was measured every day to calculate the tumor volume. The tumor was completely stripped and weighed to calculate the anti-tumor rate after the mice sacrificed. Transmission electron microscopy were used to observe the tumor cell structure. The expressions of 90 K, Arresten and VEGF were determinated by immunhistochemistry and Western blot. The results shows that the survival quality of TMP group is better than NS group, while the changes in the DDP group is reducing diet and activity, poor mental condition, dull hair, poor response to external stimuli, significant weight loss, slow tumor growth. The survival quality of TMP+DDP group is between TMP and DDP groups. The results show that TMP combination with DDP can enhance the effect of chemotherapy and TMP can reduce thetoxicity of cisplatin. Tumor volume size of each group is as follows: NS group > TMP group > DDP group > TMP+DDP group. Compared with NS group, the tumor weight of TMP, DDP and TMP+DDP groups is significantly lower. The tumor inhibition rates are 25.57%, 45.24%, 66.5%,respectively. TEM observation of NS group shows that tumor cells intensively distributed, intact membranes, rich organelles, large nuclei,prominent nucleoli. While the TEM observation of the other three groups shows that tumor cell volume decreases, incomplete membrane, lipid droplets in the cytoplasm increases, organelles decline, partly nuclear membrane unclear, visible chromatin margination. The Immunohistochemical results show that compared with NS group, the expression of 90 K in the other three groups decreased, and the TMP+DDP group exhibited the lowest expression. While the expression of Arresten in the other three groups was higher than NS group, and the TMP+DDP group exhibited the highest expression. The results deduced by immunhistochemistry and Western blot were consistent with each other.Kim's formula was used to evaluate the synergy of combined treatment:the q values for TMP+DDP group was 0.85 < q < 1.15, that showed the additive inhibition of combined TMP and cisplatin in mice lewis lung cancer.In conclusion, TMP can inhibited the growth of Lewis lung carcinoma.Moreover, in combination with cisplatin, TMP can also improve the effect of chemotherapy and reduce the side effects of cisplatin chemotherapy. The anti-tumor mechanism of TMP combined with cisplatin might be: 1.Suppress tumor angiogenesis by down-regulating expression of 90 K and VEGF and up-regulating expression of Arresten. 2. Improve tumor microcirculation to make the cisplatin reach the site of action successfully and suppress tumor growth. |
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