| It is well known that the tumor growth and metastasis are closely related to tumor angiogenesis. Fibroblast growth factor receptor 1 (FGFR1) is one of the most important angiogenesis factor, and plays a role in its biological activity primarily through its high-affinity and ligand-bFGF. Therefore, blocking the signal transduction pathway between the bFGF/FGFR1 can inhibit the formation of tumor blood vessels, and sequently suppress tumor growth. Foreign scholars and our preliminary studies confirmed that the active or passive immunotherapy targeting FGFR1 can effectively inhibit tumor angiogenesis to achieve the purpose of tumor treatment. Accordingly, FGFR1 are regarded as the ideal target for anti-tumor angiogenesis and targeted therapy and can be used for further anti-cancer research.Gemcitabine is a new deoxycytidine analogue and has shown cytotoxicity against solid tumors. Studies have shown that traditional low-dose chemotherapy for a long time has the same effect on the treatment of anti-angiogenesis but no toxic side-effects. Thus, it is possible to achieve better treatment of tumor by combining the active immunotherapy with low-dose chemotherapy.Microvessel density (MVD) can better reflect the angiogenesis activity, however, its can not be detected in vivo. Eflow combination with high-frequency ultrasound imaging is more sensitive and has more time and high spatial resolution, which significant manifests the low-speed flow and small blood vessels, making it possible to detect the tumor microvessel. In recent years, the application of ultrasound contrast agents and its related technology provides a new way for ultrasound diagnosis, and greatly improved the diagnostic sensitivity of the blood flow. Harmonic gray-scale contrast-enhanced ultrasound technology is a new method to evaluate the blood flow microcirculation in recent years. Gas microbubble of the contrast agent can increase the acoustic reflex, and significantly enhance the two-dimensional image and Doppler ultrasound signal, improving the display rate of tumor blood flow and manifesting the low blood flow that can not show before ultrasound contrast.Taken together, we investigated the anti-tumor angiogenesis effect of combining the chicken FGFR1 recombinant protein vaccine with low doses of gemcitabine chemotherapy in mouse tumor model, and explored the related mechanism. Meanwhile, we studied the function of the CDFI, high-frequency ultrasound combined with eflow and ultrasound contrast imaging technology in quantitatively detecting tumor microvessel, and investigated the role of ultrasound imaging technology in evaluating the treatment of anti-tumor angiogenesis by chicken FGFR1 recombinant protein vaccine combined with low-dose-gemcitabine.This study is divided into three parts as follows:This part is based on the preliminary studies. we aim the MethA fibrosarcoma tumor model in mice as the object for experimental observation about the tumor volume, microvessel density, apoptosis and proliferation and whether they could produce their own anti-FGFR1 antibody or not after anti-tumor treatment. All the mice were divided into 4 Groups:combined therapy group (C+Ggroup), recombinant protein vaccine of chicken FGFR1 alone treatment group (cFR group), gemcitabine alone treatment group (G group), non-treatment control group (NS group), to know how the chicken FGFR1 recombinant protein active immunization vaccine combined with low-dose gemcitabine chemotherapy can improve the anti-tumor effects and to explore its mechanism, so that we can open up a new way of tumor therapy.Results:①In the 27th, after tumor inoculation, the tumor size (mm3) of the mice were 876±46.78 (C+G group),1123±82.12 (cFR group),1689±160.11 (G group) and 4321±182.80 (NS group). C+G combination therapy group made statistical difference compared with the rest of the groups (P<0.05).②C+G group was significantly prolonged the survival time of mice. After inoculation tumor cells for 46 days the survival rate of C+G group was 90%, C+G group and the control group, the difference between the survival rate of mice with statistical significance (P＜0.05).③Western blot and immunofluorescence assay have found that it have generated anti-FGFR1 antibody in C+G group and cFR groups. Without any anti-FGFR1 antibody has been found in G group and NS group.④ELISPOT detection the number of C+G group, cFR group, G group and the NS group of the B-lymphocyte which can generate anti-mouse-FGFR1 antibody, are (113.75±11.08)/106, (112.26±12.47)/106, (5.62±1.79)/106, (5.51±1.92)/106.⑤The count of tumor microvessel density in high-power microscope field of vision of each group were 9.41±1.77 (C+G group),15.5±3.20 (cFR group),31.9±3.60 (G group) and 39.3±2.87 (NS group). Among C+G group, G group and NS group, the differences were significant (P<0.05).⑥TUNEL and PCNA antibody staining was detected the tumor cells apoptosis and proliferation. The results showed that:tumor cells apoptotic index of each group were 28.77±3.33% (C+G group),13.75±1.74% (cFR group),14.69±2.30%(G group) and 2.34±0.78% (NS group). C+G group apoptotic index was significantly higher than other groups (P＜0.05). C+G group of tumor cell proliferation index was significantly lower than other groups (P＜0.05), proliferation index in each group were 7.72±1.35% (C+G group), 14.46±3.60% (cFR group),17.87±3.18% (G group) and 28.26±2.33% (NS group).⑦Combination therapy in tumor volume, microvessel density, apoptosis and proliferation are synergies, and the synergy index is 1.63,1.05, 1.17 and 1.20.In this part, we aim the MethA fibrosarcoma tumor model in mice as the object for experimental observation. All the mice are divided into 4 groups: treatment group (C+G group), chicken FGFR1 recombinant protein vaccine alone treatment group (cFR group), gemcitabine alone treatment group (G group), non-treatment control group (NS group), to exploring the evaluation value of color Doppler imaging and high-frequency ultrasound combined with eFlow imaging for detecting the efficacy of anti-tumor angiogenesis by chicken FGFR1 recombinant protein combined gemcitabine.Results:①40 mice were all successed to be tumor-beared (40/40). The tumor formations are obvious subcutaneous nodules.②Most of the nodules are prominent growth out, the early type was spherical, the latter was the disc-shaped or lobulated.③It is visible to see the large distortion trophoblast vascular of the tumor on the surface of the lower skin. High-frequency ultrasound combined with eFlow Technology showed that tumor microvessel traveling distorted, and the vascular diameter of uneven thickness, we can see some of the centers to the edge of a small vascular branches and ansa. eFlow imaging can show more small branches of tumor blood vessels, and has a higher sensitivity than CDFI.④The vessels-tumor-pixel ratio (%) of C+G group, cFR group, G group, NS Group were 3.41±1.51,6.12±1.85, 12.11±1.91,14.13±3.31, and the trend was higher. Compared with the NS group, either C+G group or cFR group are less than it, with the statistically significant differences (P＜0.05). There was no significant difference between G group and NS group (P>0.05); C+G group, cFR group, G group of inter-group differences were statistically significant (P＜0.05). In this part, we aim the MethA fibrosarcoma tumor model in mice as the object for experimental observation. All the mice were divided into 4 groups: treatment group (C+G group), chicken FGFR1 recombinant protein vaccine alone treatment group (cFR group), gemcitabine alone treatment group (G group), non-treatment control group (NS group), to explore the evaluation value of the effect of chicken FGFR1 recombinant protein combined low-dose gemcitabine in anti-tumor angiogenesis by ultrasound contrast imaging.Results:①Before ultrasound contrast, the tumor 2D echo showed less clearance edge, and CDFI showed that the tumor blood flow within and around being the scarce supply. After contrast-enhanced imaging, the host of blood vessels in tumors can be seen rapid flow bypass and into the tumor after injecting the contrast agents about 3-8s, and the tumor echo showed varying degrees, in real terms and in the overall enhanced which can make the tumor border more clear to see.②Before contrast, color Doppler blood vessels-tumor pixels ratio (%) in C+G组each group were C+G group (1.71±1.34), cFR group (3.81±1.99), G group (5.62±2.22), NS group (9.12±2.77). After contrast imaging, the vascular-tumor pixels in each group were C+G group (5.91±1.37), cFR group (9.52±1.35), G group (15.51±2.42) and NS group (20.72±3.23). Before and after contrast, the blood vessels-tumor pixels of C+G group, cFR group, NS group have statistical significance differences (P<0.05); G group and NS group there was no significant difference (P＞0.05); within the same group, also there is a statistically significant difference (P<0.05).ConclusionBased on the present study, our conclusions are as follows:①The angiogenesis effect of Chicken FGFR1 recombinant protein vaccine combined with low-dose gemcitabine antitumor is better than medication alone; the role of combined anti-tumor active immunization mainly through self-induced anti-FGFR1 antibody to inhibite tumor angiogenesis; Combined therapy have a synergistic effect on inhibiting tumor growth and tumor angiogenesis, tumor-induced apoptosis and inhibited cell proliferation. ②High-frequency ultrasound combined with eFlow imaging technology can show tumor microvessel course and the distribution of small branches of tumor blood vessels, which has a higher sensitivity than CDFI. It can provide a real-time and a new non-invasive method for the evaluation of anti-tumor effect of angiogenesis.③Ultrasound contrast may enhance the tumor and the tumor microvessel display contrast with the surrounding tissue, and enhance the tumor and the tumor microvessel detection rate. It can provide an intuitive, sensitive and real-time methods for evaluation the anti-tumor effect of angiogenesis.
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