| With an increasing trend of primary lung cancer incidence and mortality, to delve into relevant factors on the incidence of lung cancer and improve treatment to early detection, accurate diagnosis and good treatment, has been the pursuit of the goal of the researchers. Many recent-year studies have shown that malignant tumor growth, metastasis depend on angiogenesis. Neovascularization provide plenty of nutrients for the growth of malignant tumors, excreted metabolic waste, and provided a good channel for the transfer of the tumor cells. Tumor angiogenesis plays an important role in the incidence, development, invasion, metastasis and recurrence of malignant tumors. So anti-angiogenic therapy combined with cytotoxic drugs is expected to improve the effect of cancer treatment. Recombinant human endostatin (Endostar) has been developed and produced by our Chinese researchers. Vascular endothelial growth factor (VEGF)、hypoxia inducible factor1α (HIF-1α)、Semaphorin3A (Sema3A)、CD105involve in tumor angiogenesis and development process, and they play an important role in the biological behavior of malignant tumors. The experiment through dynamic observation of weight and tumor growth in different groups of Lewis lung cancer C57BL/6mice model, changes in the expression of VEGF、HIF-1α、Sema3A、CD105, and the change of the tumor cells and tumor vascular morphology can explain that Recombinant human endostatin combined with DDP may improve the therapeutic effect, and can describe the possible mechanism, so as to provide an experimental basis for endostar widely used in clinical and research lung vascular normalization mechanism. The study consisted of a few parts as follows:1The way of making model rats, feeding dosage and their group divisionThe cultured murine Lewis lung carcinoma cells were inoculated in C57BL/6mice right armpit to establish xenograft model. At8-10days after the inoculation, could touch the the size of soybean mass at subcutaneous inoculation, and gradually increased with the time. After two weeks,56tumor diameter about8mm tumor-bearing mice were randomly divided into four groups:group A:saline group; group B:Recombinant human endostatin group; group C:DDP group; D: Recombinant human endostatin+DDP group. Before dosing every time, adjusted the dosage leading to weight. After dosing, at the2nd、4th、6th、8th、10th、12tth、14th day, two mice were sacrificed each group. The tumor blocks were taken out and fixed.2The general observation of mice after administrationSelect the general condition of group A mice as standard, compare with group A, with the administration time to extend, in group B, the mice eating、activities were basically normal, the growth of weight and tumor volume slowed down. Group C mice gradually performanced the poor spirit、diet and activities significantly reduced、gray color without burnish、 huddled、got together, and the most obvious phenomenon were weight-loss and tumor volume smaller than that in group B.Group D mice’s general condition was better than group C, but worse than group B, the weight heavier than group C, and the tumor volume less than group C.3Tumor tissue pathological observationGeneral observation:the characteristic of tumor blood vessel is rich, quality crisp, bleeding easily, tumor cells infiltrating surrounding tissue and skin, boundaries not clear, split difficultly, bleeding obvious, cut fish-shaped, necrotic area in the middle. HE:a large number of cancer cells, the cells arrange closely, cell volume and nuclear big, stromal vascular rich.4Observing the expression of tumor angiogenesis factors After removed all the tumor organization, observed the expression of the pro-angiogenic factors VEGF、HIF-1a, and inhibiting angiogenesis factor Sema3A with immunohistochemical methods. The results showed: the four groups were visible the positive expression of VEGF, group A expressed the highest (153.67±5.40), and group D the lowest (78.79±7.83). So as HIF-la positive expression in the four groups, group A expressed the highest (142.10±4.18), and group D the lowest (60.05±9.70). Both in group B and D after chemotherapy began, VEGF and HIF-1a expressed supreme at the2nd day, minimum at the4th-8th days. At the9th-14th days, the expression rised dayly, but lower than them at the2nd day. Sema3A did not express in group A and C. In group D, it expressed the highest (75.48±15.77), in group B (66.65±10.64). After chemotherapy began, at the2nd day, it did not expressed. At the4th-8th days, it showed high expression. At the9th-14th days, its expression decreased gradually.5Detection of MVD in tumor origantionDetected CD105with immunohistochemistry. Obseved the blood vessels within the tumor tissue with light microscope, and the last counted microvascular density (MVD) with the Weidner method. Having taken the average of the five high power fields, can display:group A20.00±3.45, group D16.49±7.26. And its expression differences are related to the different time points after administration. It expressed supreme on the2nd day, minimum on the4th-8th days. On the9th-14th days, the expression rose daily, but lower than them on the2nd day.6The observation of the tumor cells and vascular structures with electron microscopeObserved with electron microscope, the tumor cells in the tumor tissue were similar to each group. Performanced large nucleolus, developed organelle, such as mitochondria, endoplasmic reticulum, Golgi. Vascular structures have no significant difference. Performanced:vascular endothelial cell swelling, developed organelles. As the above shows, about two weeks after the Lewis lung carcinoma cells were injected into C57BL/6mice, all could develop tumor.It provided an available way for the mouse lung carcinoma experiment. By observing the mice general condition、weigh、volume changes、angiogenesis-related factor expression and microvascular density, it is proved that Recombinant human endostatin can not only anti-angiogenesis, but also enhance the effect of DDP in the treatment of tumors, the mechanism may recombinant human vascular endostatin improve blood infusion through normalized tumor vascular, so as to enable cytotoxic drugs better role in tumor tissue. But electron microscopy shows that tumor vascular morphology has no significant change in each group at different time points. The reason may be due to the vascular normalization, it’s only the researchers’duction, or the administration time short, yet not cause morphological changes. Sema3A is an inhibited factor of tumor angiogenesis, developing into formulations may offer new avenues for the clinical treatment of tumor angiogenesis. CD105expression in the value-added vascular endothelial cells, the development of anti-CD105preparations may also provide new ideas for treatment of lung cancer. |
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