| OBJECTIVE To observe the relationship between the great omental milky spots and peritoneal metastasis of gastric cancer,and the dynamic changes of the amount of omental milky spots and macrophages'expression after the human gastric cancer cells seeding,so that to explore the role of the omental milky spots in gastric cancer cells cultivation and the mechanisms of that;Study the differences between the shape and number of the omental milky spots and macrophages with or without external effect comparatively.METHODS The human gastric poorly differentiated adenocarcinoma BGC-823 cell lines was subcultured to the logarithmic growth phase using cell culture methods,counting slide counted the cell concentration and adjusted it to 1×107个/ml,then cells were collected for use.Construct animal model of gastric cancer in nude mice.80 nude mice were randomly divided into experimental(56 nude mice)and control groups(24 nude mice),the nude mice of experimental groups were injected cancer cells suspension by the way of 0.2ml/only,small incision of abdomen at the same time point,on the contrary the control group that served as negative control were injected normal saline,then collected specimens at different time points after 1st day,3rd days,7th days,14th days,respectively,14 nude mice of experimental group were collected on each time,the control group were 6 nude mice conversely,half of which were injected by black ink of 0.5ml,after 2h,the omentum were cut off and stretched on the slide glass to count the number of omental milky spots under the microscope,and observe the ultrastructure changes of macrophages in milky spots using electronic microscope,the rest of which were not injected by ink but cut off the greater omentum directly that were made into paraffin to observe morphology and volume changes of the great omental macrophages using HE and immunohistochemistry.RESULTS 1. The comparison of the amount of omental milky spots and macrophages'expression between the experimental and control group the 1st day after implantation:There was no statistical difference of the number of omental milky spots between the experimental group and control group the 1st day after implantation(P>0.05),there was also no statistical difference of the expression of macrophages between the experimental group and control group the 1st day after implantaion(P>0.05).2. The comparison of the amount of omental milky spots and macrophages'expression between the experimental and control group the 3rd days after implantation:There was no statistical difference of the number of omental milky spots between the experimental group and control group the 3rd days after implantation(P>0.05),there was also no statistical difference of the expression of macrophages between the experimental group and control group the 3rd days after implantaion(P>0.05).3. The comparison of the amount of omental milky spots and macrophages'expression between the experimental and control group the 7th days after implantation:There was significant statistical difference of the number of omental milky spots between the experimental group and control group the 7th days after implantation(P<0.001),there was statistical also difference of the expression of macrophages between the experimental group and control group the 7th days after implantaion(P<0.05).4. The comparison of the amount of omental milky spots and macrophages'expression between the experimental and control group the 14th days after implantation:There was significant statistical difference of the number of omental milky spots between the experimental group and control group the 14th day after implantation(P<0.001),there was also statistical significant difference of the expression of macrophages between the experimental group and control group the 14th day after implantaion(P<0.01).5. The amount of omental milky spots and the expression of macrophages would become bigger and changed as time went on:There was no statistical difference of the number of omental milky spots between the 1st day and 3rd days of the experimental group(P>0.05),there was significant statistical difference of the number of omental milky spots between the 1st day and 7th days of the experimental group(P<0.001),there was significant statistical difference of the number of omental milky spots between the 3rd days and 14th days of the experimental group (P<0.001),there was significant statistical difference of the number of omental milky spots between the 7th days and 14th days of the experimental group(P<0.001);the amount of milky spots of the experimental group was correlated with the time of planting,and was a positive correlation(r=0.900,P<0.001);there were no statistical differences of the amount of milky spots among the 1st day,3rd days,7th days,14th days of the control group(P>0.05),the amount of milky spots of the control group was not correlated with the time of planting(r=0.165,P>0.05).There was no statistical difference of the expression of macrophages between the 1st day and 3rd days of the experimental group(P>0.05),there was significant statistical difference of the expression of macrophages between the 1st day and 7th days of the experimental group(P<0.001),there was significant statistical difference of the expression of macrophages between the 1st day and 14th days of the experimental group (P<0.001),there was significant statistical difference of the expression of macrophages between the 3rd days and 7th days of the experimental group(P<0.001),there was significant statistical difference of the expression of macrophages between the 3rd days and 14th days of the experimental group(P<0.001),there was significant statistical difference of the expression of macrophages between the 7th days and 14th days of the experimental group(P<0.001);the expression of macrophages of the experimental group was correlated with the time of planting,and was a positive correlation(r=0.620,P<0.001);there were no statistical differences of the expression of macrophages among the 1st day,3rd days,7th days,14th days of the control group(P>0.05),the expression of macrophages of the control group was not correlated with the time of planting(r=0.200,P>0.05).6. The ultrastructural changes of macrophages:There were all a large number of macrophages on the milky spots the 1st day,3rd,7th and 14th days after planting gastric cells of the experimental group that were observed by transmission electron microscope,with the planting time,the number of the macrophages on milky spots were increasing,macrophage cells were kidney shaped,round,oviod,irregular-shaped,and so on,the size were large,the cytoplasm were riched in mitochondria,Golgi complex,endoplasmic reticulum,and ink particles were phagocytied into cytoplasm,the nucleus were dense,the chromosomein were aggregated,there were many processes around the membrane surface,the processes were long and dense,processes of adjacent cells connected with each other,macrophags were next to cancer cells closely so the gap were very small.The size of macrophages on milky spots in the control groups were smaller than the experimental group the 1st day,3rd,7th and 14th days after planting,the cytoplasmic components were not rich,the processes of membrane surface were few and small,cells were resting on the whole.7. Cultivation of cancer cells at different time points: The 1st day after planting there was no intra-abdominal cancer nodules with naked eyes,but individual cancer cells on the omentum which inflitrated by macrophages and lymphocytes with HE staining;the 3rd days there were several cancer nodules on omentum with nude eyes,and cancer cells on omentum blended into agglomerate and macrophage infiltrated significantly with HE staining.The 7th days the intra-abdominal were filled with cancer nodules,omental nodules which color was gray,texture was tenacious,aspect was swirling,mesenteric nodules were scattered,cancer nodules can be also seen on the liver,spleen,mesenterium,diaphragm,anterior abdominal wall and incision depertment,cancer cells were clearly seen on omentum under microscope with HE staining,macrophages existed among cancer cells which were riched with blood vessels and some were necrosis.The 14th days the intra-abdominal were filled with cancer nodules,gastro-intestinal adhesioned,omentum had been occupied by cancer nodules which color was gray,texture was tough,aspect was swirling,mesenteric nodules were clouded or integrated into agglomerate,the liver,spleen,mesenterium,diaphragm,parietal peritoneum were all cancer nodules,cancer nodules of incision depertment had been prominent upon the skin surface.CONCLUSIONS 1. Gastric cancer cells plant in abdominal cavity is selective,in which the omentum is the easiest and the most suitable part;2. Gastric cancer cells cultivate in the whole abdominal cavity with time prolongs;3. Omental milky spots are specific choice point for implantation metastasis of gastric cancer cells;4. The amount of omental milky spots increases with the time of planting so that can undertake an increasing number of cancer cells;5. Macrophages'mophology changed a certain extent after cancer cells planting. |
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