| PART 1 STUDY ON CELLULAR AND HISTOMORPHOLOGICAL FEATURES IN LARGE PATHOLOGICAL SLICES OF COLORECTAL CANCERObjective: To explore the histomophological features of primary colorectal cancer,liver metastasis,ovarian metastasis and lymph node metastasis,and to analyze their relationship with the biological characters and behavior characteristics of tumor by using large pathological sections.To further explore the biological features and cell behavior of tumor cells by observing the tissue and cell morphology,and to reveal the mechanism of infiltration and metastasis of colorectal cancer.Methods: A total of 52 patients with stage III-IV colorectal cancer were selected,including 52 cases with lymph node metastasis,11 cases with liver metastases and 6 cases with ovarian metastases.The primary lesions,liver metastases,lymph node metastases and ovarian metastases were collected and made into large pathological sections and routine HE staining was performed.With the help of the EVOS auto intelligent full automatic fluorescence microscopic imaging system,full-scale scanning of the large pathological section was carried out to obtain the full-scale and detailed images of the pathological sections.For comparison,the primary lesions were divided into four sections based on tumor cell distribution under low magnification: a)main lesion area(the area where most tumor cells concentrated);b)periphery area(the area where tumorous tissue and structures extended from the main lesion under low magnification.);c)tumor spreading area(extending from the periphery section,where tumor cells were sporadically,spared,discontinuously distributed.)and normal tissue(the regions where no tumor cells existed).The characteristics and patterns of cell distribution and abnormal cell structure were observed and compared among different regions of primary lesions,liver metastases and ovarian metastases,and the lengths of the relevant radial lines was also measured.In this study,IBM SPSS Statistics 16 software was used for data analysis.The measurement data that obeyed the normal distribution or the approximate normal distribution were presented as mean ± standard deviation((?)±s),and the students' t-test for independent samples was used between two groups;the skew distributed data were presented as median with interquartile range(Q1–Q3)(median [IQR]),and the Mann-Whitney U test was used for the comparison between two indexes.The Kruskal-Wallis H test was used to compare multiple indicators.If the difference was statistically significant,Bonferroni test was conducted to confirm it.The difference was considered significant when P<0.05.Results:(1)Primary lesion: tumor cells distribution showed similar pattern in the four zones of most specimens.The structure of basal layer in main tumor area was obviously destroyed.The tumor extended to both the surface of the mucous membrane and the deep muscle layer,even completely destroyed the surface of the mucous membrane to form the ulcer.The structure of basal layer distant from the border of main lesion area was gradually become normal.Malignant morphological changes in the basal layer cells were discovered and with a large number of continuous distributed tumor cells,and those cells often invaded into the mucosal layer.In the extention area,the more closer to the outer edge of the main lesion area,the thicker the normal mucosa of the surface,the thicker the normal mucous membrane on the surface,and the cells of the lesion gradually shrink to the basal layer and less cells infiltrated into the myometrium.The tumor cells were obviously dispersedly distributed.The cells or the small lesions were disconnected with each other,but almost all were distributed close to the basal layer.It seemed that tumor cells extended from the main area to the periphery along the basal cell layer.The measurement along the basal layer showed that the length of the lesion extending along the basal cell layer was much longer than the length of the main lesion area(P<0.05).As for the extension in the basal region of the junction with the normal tissue,it was easy to see atypical cells and morphological characteristics of the gradation,the number of abnormal cells which is significantly more than other regions(P<0.05),and the difference was statistically significant.However,there was no morphological changes indicated that tumor cells produced a holistic extrusion or shift on the normal structure.(2)Liver metastases: the adjacent area between tumor mass and normal tissues was different from that of the primary tumor.Fibrous structure surrounded the metastases,forming a membrane-like structure.Scattered tumor cells or abnormal cells could be seen in the adjacent tissue closed to the fibrous tissue.The main lesion cells were nested without regular distribution or direction,and the morphology was closely to the coresponding primary lesion.The consistency of cell size and morphology were of less hetergeneity than those of primary tumor.The difference between the primary lesion and the lymph node metastases in morphology was not significant.(3)Ovarian metastases: tumor cells were distributed in the parenchyma of the ovary,but rarely reached the outburst of the membrane.The tumor cells were nested in the ovarian parenchyma,close to the primary tumor,and were scattered around the neoplastic foci.There was no fibrous tissue around the neoplastic foci and spared tumor cells distributed randomly.The morphology of the ovarian metastases was undistinguishable from the liver and lymph node metastases.But the tissue and cellular morphology were diversed,the small round cells were commonly be seen but it was difficult to identify the source of cells.There were obvious morphological differences compared to the primary and other metastatic foci.(4)Lymph node metastases: the tumor cells were nest-like distribted,similar to the primary lesion,and with no fibrous tissue around.The scattered tumor cells or heteromorphic cells were found around the neoplatic foci.Tumor cells or heteromorphic cells were spared among normal lymphocytes.The tumors cells of lymph node metastases could not be distinguished from those of primary lesions and liver metastases.Conclusion: The results of morphological observation by large pathological sections suggested that:(1)There was a certain regularity in the changes of tissue and cell morphology of different lesions of colorectal cancer.It can reflect some biological characters and behavior characteristics of tumor cells of different foci.(2)The heterogeneity of biological characters and behavior exists in tumor cells in different lesions of large intestine cancer.This heterogeneity may be related to the multidirectional differentiation and generation of tumor cells.(3)The invasion,metastasis and development of tumor cells is related with the biological characters of tumor cells.The cell proliferation may be the main mode of growth of the metastasis of colorectal cancer and the main lesion area of the primary cancer.(4)The obvious morphological differences in the ovarian metastasis suggest that tumor cells is not the only reason responsible for the ovarian metastasis of colorectal cancer,The special biological structure and environment of the ovaries and the whole body may also contribute.PART 2 STEM CELL INDUCTION EXPERIMENT AND DETECTION OF THREE MOLECULE PROTEINS IN PRIMARY AND METASTATIC TUMORSObjective: On the basis of histopathological study of large pathological sections,through the comparison of protein expression in different regions of primary lesions and different metastatic foci and investigation of effect of ascites on umbilical cord mesenchymal stem cell,we further explored the differences in primary lesions and metastases.the possibility of multi-potent stems cells being induced into malignant cells under specific tumor environment.Methods:(1)Immunohistologic analysis of specific tumor related genes:immunohistochemical staining of P53,VEGF,and ?-catenin was performed on primary tumors and metastatic tissues of different sites,respectively,and the slices were fully processed by the EVOS auto intelligent automatic fluorescence microscopy imaging system.Multi-slice scans was performed to obtain full-scale views of the sections.The primary lesions were divided into four sections according to HE staining.The expression of protens were observed and analyzed by two senior pathologists.In case of contradictions,the results was determined after discusion.(2)Induction culture of umbilical cord mesenchymal stem cells: qualified umbilical cord mesenchymal stem cells were divided into control group and experimental group.the experimental group was culture in medium with different concentration of ascite,while the cells in control group cultured in medium with the same amount of PBS.Cell growth and cell morphology were observed at 24 and 48 hours after culture.The cell proliferation was analyzed by CCK-8 viability assay.In this study,statistics were analyzed using IBM SPSS Statistics 16.0 software.Measured data that follow a normal distribution or a nearly normal distribution are described in the form of mean±standard deviation((?)±s).Students t-test was used for comparison between groups;median is used for severely skewed data.In the form of "median [IQR]",the Mann-Whitney U test was used for the comparison between the two indicators,and the Kruskal-Wallis H test was used for the comparison among multiple indicators.If the overall difference was statistically significant,Bonferroni test was used to confirm the result.When P<0.05,the difference was considered statistically significant.Results: 1.Protein expression in different regions of primary lesions and different metastatic foci:(1)Primary lesion: P53 was expressed in all regions of primary lesion,but were most prominent in the main lesion regions;VEGF was also expressed in all regions of the tumour,but the expression in the peripheral and extended regions of the tumour were significantly higher than that in the main lesion area.The expression of ?-catenin was most obvious in the peripheral area of the tumor,and was also expressed in the main lesion area,extended area and normal tissue area.The expression of P53,VEGF and?-catenin P53,VEGF were all statistically significant(P<0.001)among primary tumor tumor area,tumor peripheral area,extension area and normal tissue area.The comparison between the two groups also showed that there was a statistically significant difference in the expression of P53 and VEGF in any two regions(P<0.05).(2)Liver metastases: The expression of three protein was also different from other lesions.VEGF was mainly expressed in the peripheral area of liver metastases(8/11,73%),but P53 was expressed in the whole tumor gegions.(3)Ovarian metastases: P53,VEGF,and ?-catenin proteins were highly expressed in ovarian metastases,but their expression intensity was still different.(4)Lymph node metastases: The expression of three protein in lymph node metastases was similar to that in ovarian metastases,but there was significant difference in the expression of three protein expressions in different regions of the primary tumor.2.Effct of ascited on umbilical cord mesenchymal stem cells: the morphology of mesenchymal stem cells changeed significantly after induction culture,the morphology of the cells before induction was uniform,flat,and similar to fibroblasts.After 24 and 48 h of exposure to ascite,compared with control cells,the morphology of MSCs changed significantly.The cells became rounder and shorter,and the nucleus increased.Compared with control group,the proliferation rate of the cells was significantly increased,Conclusion: According to the results of immunohistochemistry,mesenchymal stem cell culture,and results of conventional large-scale pathological biopsy:(1)There are differences in the biological expression of cells in different regions of the primary tumor of colorectal cancer,indicating that there are differences in biological characteristics,and it is also possible to initially explain the possible causes of ethology and the corresponding histomorphological differences.(2)There are differences in the biological expression of different regions of the primary tumor and the metastases of colorectal cancer,suggesting that different metastatic cells may originate from different regions or cells of the primary tumor.(3)The heterogeneity of the biological characteristics and behaviors of the primary tumor and different metastases of colorectal cancer may be related to the multi-directional differentiation of the primary tumor of colorectal cancer.(4)The results of induction culture of umbilical cord mesenchymal stem cells suggest that tumor-like biological environment is likely to have biological induction effect on cells with differentiation potential.Whether it has biological significance for tumor metastasis and infiltration mechanism should be further studied.the study. |
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