The Research On The Influence And Intervention Mechanism Of17AAG On Diaphragm Vascular Extra-pathway Of The Model Mice With Liver Ascites Tumor

Cancer has now become the first major disease that caused human death, its transfer has been the science research direction, among which, lymphatic metastasis is one of the most common and the most important transfer pathway of the proliferation of malignant tumor. Therefore, the restrain of lymphatic metastasis pathway is a new research direction for treating cancer.Abdominal tumor is vulnerable to celiac transfer, at the same time with a big increase in the number of ascites. In1950s, there were scholars investigating the absorption and circulation of the ascites morphology, and forward the concept that there are cribriporals in the diaphragm. The ascites could go into the inside of the lymph vessels through cribriporals, then enter the lymph circulation. Before entering the lymphatic loop, it neight belonged to the lymph circulation nor the blood circulation. Thus their research put forward the concept of "the body fluids vascular pathway". However the research was only limited to the morphological concept, with little research ultrastructure and ascites cycle of adjustment mechanism, especially on morphological changes and regulating mechanism of ascites cycle in pathological situations. During the transfer of tumor celiac, the ascites fluid which went into the diaphragmatic lymph circulation through the vascular pathway increased a lot, with the lymphatic metastasize of tumor cells at the same time. The pathway of the body fluids vascular morphology was inevitably changed correspondingly and the morphological regulation and functional regulation of the mesothelial cells in diaphragm peritoneal was especially the changed. Thus, we use all sorts of morphological research techniques and molecular biology technology to observe the morphological change of mode of the normal mice liver cancer cell lines (H22cells) in the intra-abdomin, especially the change of the mesothelial cells and the subcutaneous tissue structure. The purpose of this paper is to discuss the pathological change of the diaphragm vascular pathway in the ultra-structure and molecular level, explore the molecular mechanism of change of the vascular pathway of body fluids by detecting the protein and gene expression changes of Hsp90, VEGF-C and LYVE-1, and, with the application of17-AAG and cisplatin combination intervention, discuss the protection mechanism of the vascular pathway of body fluids, providing new direction for the treatments of tumor lymph node metastases.The family of vascular endothelial growth factors (VEGF) includes: VEGF-A, VEGF-B, placenta growth factor (PIGF), VEGF-C, VEGF-D, and their receptors VEGFR-1, VEGFR-2and VEGFR-3. VEGF-C and VEGF-D are the main lymphatic formation factors, which, along with their receptor VEGFR-3, will be an important factor for the tumor lymph node metastases and prognostic evaluation, providing new research direction for the anti-tumor lymphatic metastases treatment. Hyaluronic acid receptor-1(LYVE-1) in lymphatic endothelial cells is a kind of membrane protein, expressed on the micro lymphatic endothelial cells and distributed on the surfaces and basal surfaces of lymphatic tubes. Its main function is to assist hyaluronic acid from the surrounding tissue penetrate lymphatic endothelial and transport to the lymphatic lymph, its increased expression in blood vessels and lymph vessels has a close relationship with the metastasis of tumor cells. Heat shock protein is a group of protein that is produced by the stressors, especially under the high temperature environment, with high conservative property. Hsp has a variety of functions, served as a molecular partner, which can prevent protein aggregation, and therefore can adjust the cell survival and death. Heat shock protein90, as a molecular partner, is engageg in the regulation and maintain the conformation and function of various proteins in cells in order to help the normal growth of cells under the stress environment. In recent years, studies show that many cancer gene proteins are the targets of Hsp90. Therefore, inhibiting the function of the Hsp90will promote the degradation of these cancer gene proteins,which will help cancer treatment.17-allylamio-17-desmethoxygeldanamycin,(17-AAG) is a kind of heat shock protein90(HsP90) inhibitor, which can inhibit tumor-sourced HsP90targetedly, restrain the function of heat shock protein90, and make the key regulator protein and the effect protein degrade through multiple pathways of cells transmission, thus to make the cell growth differentiation inhibited, cause cell death, and suppress tumor growth. The vivo and vitro experiments have confirmed the Hsp90inhibitor has anti-tumor activity, among which17-AAG, which is in the clinical trials. A number of studies have confirmed that17AAG can have very strong inhibition effects on tumor cells, and especially, can exert an anti-tumor function with the combination of traditional chemotherapy drugs.In this study, we used immunohistochemical to detect LYVE-1, VEGF-C and HsP90s expression, used fluorescence real-time PCR to determine the expression of LYVE-1mRNA, VEGF-C mRNA and HsP90mRNA; the protein expression of LYVE-1, VEGF-C and HsP90by Western blot test. By discussing the relationship between the expression of diaphragmatic tissue LYVE-1, VEGF-C and HsP90, this method provide a new direction for the treatment of tumor lymph node metastasis.Based on the above animal experiments, this experiment made an in vitro observation on the changes of expressions of HsP90and LYVE-1, VEGF-C by the application of pure mesothelial cells HMrSV5and training of in vitro and tumor ascites to provide further experimental supports for the animal experiments.Part One The research on the morphology of diaphragm of normal mice and model mice with liver ascites tumorObjective:Due to the rare researches on the circulation of the lymph of diaphragma, and no research on the tissue structure change of diaphragma under the condition of tumor ascites domestically, we therefore set up the cancerous ascites animal model successfully by using mice H22 intraperitoneal injection, and made a further observation on the diaphragmatic structure change, in order to provide morphological basis for the inhibition of tumor lymphatic metastasis.Methods:Randomly, divide the BALB/c30female mice, SPF, weight (20-21) g, into the control group10mice, model group10mice and the ink group10mice.(1) the control group:intraperitoneal injection of saline0.2m l/per mouse, one time on alternate days;(2) the model group with the injection of abdominal tumor cells H22ascites suspension liquid0.2ml.10days later, after the perfusionfixation, to observe the ultrastructural changes by scanning the diaphragma tissue with the electron microscopy (sem) and transmission electron microscope of both two groups.(3) the ink group mice with the injection of10%2-5ml ink solution, then, after0.5-2hours, to inject the fixed liquid10ml to peritoneal cavity, after15minutes to execute the mice, free the diaphragmatic, with buffer rinse, observed directly under optical microscope.Results:1HE dyeing resultsThe mice of control group showed the closely spaced peritoneal diaphragma epithelial cells with the oval nucleus. The mice of model group showed the disorderly arranged diaphragmatic epithelial cells, heighten, cubic, with the enlarged and circular nucleus. The surface has the tumor cells attached, A large number of connective tissue hyperplasia beneath the epithelial cells and there are many irregular lumen. There are attached tumor cells infiltrating through the lumen.2By Intraperitoneal injection by ink, the different distribution of the diaphragma tissue lymphatic vascular and vessels can be observed, position of lymphatic is shallow,scattered in the connective tissue beneath the epithelial cells, whereas, position of vascular is deep, dendrimer-like and intercalated in the muscle fiber.3the results of scanning electron microscope (sem)By the control observation of the scanning electron microscope (sem) and NaOH digestive scanning electron microscopy (sem), there are two kinds of the diaphragma mesothelial cells of the control group:one kind is small cell, which is about10-12microns in diameter,and in which many macula cribriformis can be observed in its connective tissue, with many circle, elliptic cribriporals.By high-magnified scanning electron microscopy (sem), reticular fiber intertwined, with the traverse of macrophages and red blood cells; Anothor kind is flat large cell, with diameter of about20microns, and with the short microvilli on surface, in which cell borders can be observed clearly, and the adjacent cells are closely connected, without the peritoneal pore amid them. Large cell has no cribriporals and it is the stripe-like collagen fiber with a bunch of high density. Two kinds of cells showed a pattern of stripe-like alternant distribution. The mice of model showed a heighten and circular diaphragmatic mesothelial small cells with irregular arrangement, with increased hyperplasia of connective tissue, and with tumor cells attached on the surface. Two kind of cells showed a disordered distribution of stripe-like fiber. The number of cribriporals increased, the diameter increased, and tumor cells can be observed out of cribriporals. The distribution of Macula cribriformis and collagen fiber is not clear.4The results of transmission electron microscope (tem)By transmission electron microscope (tem), golgi body of the diaphragma epithelial cells, many mitochondria, abundant rough endoplasmic reticulum, ribosomes and many empty bubbles can be observed in the tumor group. These organelles are distributes mainly on the area with thick cytoplasm. Some capillary lymphatic vessels increased and some tumor cells could be seen protrading the tubular-structure branches.Conclusion:we observed the existence of the pathway, and it is composed of macula cribriformis system. In the state of tumor ascites, the diaphragma has ultrastructure change, the lymphatic metastasis of tumor cells exerted through extra-pathway of body fluids out of macula cribriformis, with the corresponding morphological change on vascular extra-pathway of body fluids.Part Two The research on the influence of17AAG on morphological change of the diaphragmatic of the model mice with liver ascites tumor and the lymphatic endothelial-related factorsObjective:By the electron microscope, to observe the morphological change of abdominal planting model diaphragma of H22mice with drug intervention, and by immunohistochemical, Western blo, fluorescence real-time PCR determination, to determine the lymphatic-related factor LYVE-1, VEGF-C of the abdominal planting model diaphragmatic muscle of H22mice and the expression of HSP90protein and genes, to discuss the changes on the related factors of diaphragmatic muscle tissue lymphatic and the intervention mechanism of17-AAG.Methods:Randomly, divide the BALB/c50female mice, SPF, weight (20-21) g, into the control group10mice, model group40mice. The model group with the abdominal injection of H22ascites suspension liquid0.2ml, injected on alternate days. Randomly divide the mice into model group,17-AAG group, cisplatin group,17AAG+cisplatin groups, and10mice for each group and the intervention started after24h.(1) the control group: intraperitoneal injection of saline0.2ml/per mouse, one time on alternate days;(2) the model group:intraperitoneal injection of saline0.2ml/per mouse, one time on alternate days;(3)17-AAG group:intraperitoneal injection of10mg/kg17-AAG0.2ml/per mouse, one time on alternate days;(4) cisplatin group:intraperitoneal injection of4mg/kg cisplatin,0.2ml/per mouse, one time on alternate days;(5)17-AAG and cisplatin/groups: intraperitoneal injection of4mg/kg cisplatin,0.2ml/per mouse, with intraperitoneal injection of17-AAG10mg/kg,0.2ml/per mouse, one time on alternate days;10days later, after the perfusionfixation, to observe the ultrastructural changes by scanning the diaphragma tissue with the electron microscopy (sem) and transmission electron microscope of all groups. To observe the expression of LYVE-1, VEGF-C, HsP90by immunohistochemical method. To determine the expressions of LYVE-1 mRNA, VEGF-C mRNA, HsP90mRNA by fluorescence real-time PCR. To test LYVE-1, VEGF-C, HsP90protein expression by using Western blot.Result:1under electron microscopy, the control group and the model group had the same form according to the Part One. In17-AAG and cisplatin intervention groups, the mice diaphragma mesothelial small cells showed a regular form and were closely arranged. The microvilli of big flat cells reduced or was missing. The boundaries between cells were not clear, which was similar to the control group.2immunohistochemical stains resultsCompared with controls, in the diaphragma tissue of the model group, LYVE-1, VEGF-C, HsP90significantly increased (positive area:0.464±0.011、0.484±0.009,0.494±0.009, p<0.05). Cisplatin,17AAG, cisplatin+17AAG diaphragmatic muscle tissues in the intervention group and the treatment group reduced significantly.3the expression results of diaphragmatic muscle tissue geneCompared with control group, in the diaphragma tissue of the model group, LYVE-1, VEGF-C, HsP90mRNA expression raised significantly (p<0.05). Cisplatin+17AAG, compared with model group, the expression of LYVE-1, VEGF-C, VEGF-D, HsP90mRNA reduced significantly (p<0.05).4the expression results of diaphragmatic tissue proteinCompared with controls, in model group, the protein expression of LYVE-1, VEGF-C, HsP90increased significantly (p<0.05). In all treatment groups, the protein expression of LYVE-1, VEGF-C, HsP90reduced significantly (p<0.05) compared with tumor group; Cisplatin and17AAG intervention group reduced most significantly.Conclusion:in the state of tumor ascites, the gene expression and protein expression of diaphragma LYVE-1, VEGF-C, HsP90increased and the expression decreased after the intervention. Deduction:cisplatin and17AAG have inhibiting effect on the diaphragma LYVE-1, VEGF-C, HsP90, and cisplatin+intervention group has joint effect on tumor treatment. Part Three the research on the effect of HepG2supernatant liquid on HMrSV5and the drug interventionObjective:By using the human peritoneal mesothelial cells HMrSV5to culture the mixed culture medium (4ml/25cm2)from liquid supernatant on fresh DMEM and serum-free HepG2simultaneously, with drug intervention on cisplatin and17AAG, to determine the protein and the gene expression of HsP90by the immunohistochemical and Western blot, fluorescence real-time PCR method, thus, to study the influence of combination of drugs in vitro on the cells outside the body.Methods:Divide the human peritoneal mesothelial cells HMrSV5,which is cultured outside the body, into5groups:(1) a normal HMrSV5group;(2) model group (HepG2cultivated supernatant liquid stimulate group);(3) cisplatin intervention group (model group+cisplatin (5μg/ml));(4)17AAG intervention group (model group+17AAG (5μM));(5) joint intervention group (model group+cisplatin (5μg/ml)+17AAG (5μM)). Exert the drug treatment after the adherence of cells in the model group, continue to culture for48h, then abandon the supernatant liquid, use cold PBS to wash once, and add the total RNA extraction TRIzol Reagent (2ml/25cm2), transferred to1.5ml centrifugal tube, freeze-stored at-80℃after the complete cracking of cells. To determine the change of content of LYVE-1, VEGF-C, HsP90by the immunohistochemical, Western blot, real time PCR method.Results:1The morphology of cells and fluorescent resultsDouble dyeing the HSP90, LYVE-1, VEGFC protein antibody marked by CY3with the application of Hoechst33258kit. The protein positive expression inside the cell plasma, represents red fluorescence, and the nuclei represents blue fluorescence. Compared with the normal group, cells are spindle, the red fluorescence inside cytoplasm represents weak, the nuclei are blue, the morphology of model group cells is irregular, fused into round state, and a small amount of apoptosis nuclei are hyper-chromatic densely and represent blue. With the enhancement of protein positive expression in each group, the red fluorescence inside cytoplasm deepened. Compared with the model control group, the protein positive expressions in17AAG,17AAG+cisplatin intervention groups become weak and the number of dead cells is decreased.2gene expression resultsCompared with the control group, the expression of LYVE-1, VEGF-C, HsP90mRNA in model group raised significantly (p<0.05). Compared with the tumor group, the expression of Cisplatin+17AAG intervention group LYVE-1, VEGF-C, HsP90mRNA reduced significantly (p<0.05).3the protein expression resultsCompared with the control group, the expression of LYVE-1, VEGF-C, HsP90protein increased significantly (p<0.05). Compared with the model group, the expression of LYVE-1, VEGF-C, HsP90protein reduced significantly in treatment group,(p<0.05).Conclusion:in vitro, in17AAG,cisplatin group and the combination group, the inhibition of the activity of HsP90in human peritoneal mesothelial cells HMrSV5, which is stimulated by the HMrSV5cells, can reduce the protein expression of LYVE-1, VEGF-C.17AAG, cisplatin may inhibit the protein expression of LYVE-1, VEGF-C by inhibiting the activity of HsP90.