| Parabiosis is an experimental method of building a medical model, which can well reflect the interaction of blood and body fluids between the two conjoined animals after surgery that is aiming to achieve mutualism and observe all the effects between conjoined bodies ranging from blood to immunity. Mice is usually used to build parabiosis models which method has been applied more than 150 years. While there were more relevant parabiosis models researches carried out in America, Britain Switzerland and Japan, only a few were implemented in China. After Francesco S.L published an article, which immediately attracted extensive attention, about the process of GFD11 (Growth Differentiation Factor-11) reversing related myocardial hypertrophy in Cell Journal in 2013, Science Magazine released two articles subsequently which supported this view in 2014, confirming that GDF11 had improved the function of brain and skeletal muscle in the aging mice and causing more public attraction on this experimental model after.Tumor is composed of malignant cells which have escaped from the body’s immune surveillance. The theory of tumor immune editing invented by R.D Schreiber argues that the immune system not only has the ability to eliminate tumor cells, also have the effect of promoting tumor’s growth. The occurrence and development of cancer cells in the body is a dynamic process of interaction between the immune system and tumor cells. Hence, whether parabiosis model promotes or suppresses the growth of tumor is still unclear. If parabiosis model can provide new direction of immunotherapy of tumor, and whether the healthy mice can give immunology support and thus play a role in combating cancer to decrease the size of tumor of the other mouse after successfully established interaction? If this effect is existing, whether is the result from the influence of both cellular and humoral factors of the healthy mouse on the ill one or is the result from the activation of humoral factors of healthy mouse on the diseased one and the effect of vaccination, secreting a large number of antitumor factors thus resist or against tumor cells. Moreover, how the two parabiosis mice did accomplish symbiotic transplantation tolerance in mice and what the relationship between chimera and the state of the tolerance is, continue to be a meaningful question in future.Therefore, this study focuses on further discussion about the application of parabiosis model in oncology and searching for factors of cells and body fluid that can kill and inhibit tumor cells. This experiment aims to research the following several aspects:frstly, establishing a parabiosis model and observing the physiological changes of this mice model; secondly, after building the above model successfully, testing these mice spleen T cells through flow cytometry technology and interactions of the two T cells in mice, as well as the effect of killing tumors; thirdly, analyzing the state of tumor destruction in parabiosis mice from the perspective of morphological comparison under immunohistochemical technology.This research paper contains four parts as follows:Part I The establishment of parabiosis mice modelObjective:establish parabiosis mice model and observe the changes of tumor size after the conjoined surgery of the two tumor-bearinging mice, as well as demonstrate the rationality of the model.Methods:select allogeneic mices; connect the skin and subcutaneous tissue along the humerus and frame side until the end of the waist line at the point of 4-5 mm away from elbow distal; thus set up parabiosis model mice; observe modeling rate of parabiosis mice, time length of mutual interaction of the two mice in blood, and the changes of tumor volume.Results:the successful rate of surgeries is much higher in mature stage than that in analog phase the rate of surgical success rate of mature stage was obviously higher than that of analog phase (PO.05). The interaction of blood was established on the third day with modeling rate at a percentage of 85.7%. After the surgery, the tumor sizes of parabiosis pairs on d2, d4,d6 and d12 after conjoined surgery are smaller than those of positive control group with a significant difference(P<0.05).Conclusion:successfully established allogeneic parabiosis mice model can well adapt to the conjoined state of mice, also they can be applied in wide medical experiments.Part II Analysis of influence of parabiosis model on T cells of the spleenObjective:Analyze the influence of parabiosis model on B16 tumor cells.Method:examine expressions of CD4+T cells, CD8+T cells and IL 2, IL-4, IL-10, and INF-yin spleen cells ofparabiosis model mice respectively through flow Cytometer technology.Results:after blood interaction, the healthy cells of GFP+ C57 mice enter into the blood circulation of tumor-bearing mice via the connecting capillary network, playing a role of stimulating CD4+ and CD8+ cells in tumor-bearing ones and causing a result that CD4+ cells increase higher in tumor-bearing mice than those in positive control group with a significant difference (P<0.05). The number of GFP+ cells that can be detected in a tumor-bearing mouse is small, but GFP+ cells can stimulate the mouse itself to generate more CD4+IL-4, CD4+ IL-10 (P<0.05).The number CD4+IL-2, CD4+IL-4 and CD4+ IL-10 in GFP+ mice are higher than those in negative control group(P<0.05).The content of IFN-yin both mice of parabiosis model are decreased (P<0.05). The figure of CD4/CD8 in parabiosis GFP+ mice is higher than those in negative control group with a significant difference (P< 0.05).Conclusion:parabiosis model has played an important role in the immune regulation, which provides a basis for the future tumor immunotherapy.Part Ⅲ The effect of T cells in tumor cells by parabiosis modelObjective:Analyze the influence of parabiosis model on B16 tumor cells.Method:choose tumor tissue of parabiosis mice after conjoined surgery at d8 and d14, and observe the changes of CD3, CD4, CD8, CD31, IFN-γ and VEGF through Immunohistochemical analysis.Results:(1) incomplete capsule formation of tumor, tumor cells accumulation in the form of nests, hyperchromatic and big nuclei, visible fission of multi-nuclei, and kinds of different focal necrosis were detected by HE staining microscope. Tan precipitation found in cytoplasm and nuclei is defined as positive ones. (2) The positive expression rates of CD3, CD4, CD8 and IFN-ycells are higher than positive control group and their Optical Density are(0.32±0.63), (0.33±0.00), (0.31±0.91), and(0.28±0.14)respectively, with a significant difference (P<0.05). The positive expression rates of CD31 (0.19±0.50) and VEFG cells (0.19±0.21) are lower when compared with positive control group with no significant difference (P>0.05). CD31 and VEGF positive cells expression is low, respectively (0.19±0.50), (0.19±0.21), compared with positive control group, there is no statistical significance (P-0.05). (3) The positive rates of CD31 and VEGF cells in the positive control group are higher, with Optical Density of (0.32±0.35) and (0.29±0.35) respectively, but when Compared with parabiosis bearing-tumor group, there is no significant difference (P>0.05).The positive expression rates of CD3, CD4, CD8 and IFN-γ cells at d8 are low which are 0.22,0.17,0.15 and 0.16 respectively. When compared with parabiosis tumor-bearing group, there is no significant difference (P>0.05).Conclusion:Parabiosis models can stimulate parabiosis tumor-bearing mice to generate CD3, CD4, CD8 and IFN-y, and play a remarkable role in immune regulation and tumor destruction (P<0.05). The positive expression rates of CD31 and VEFG cells in parabiosis tumor-bearing group are lower; however, when compared with positive control group, there is no significant difference (P>0.05). |
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