| Objective: In order to study antitumor and Immuno -regulation mechanism of both different extracts of cortex periplocae in biological and protein level, which are helpful for finding the effective therapeutic measures for curing of human esophageal carcinoma. Meanwhile, which provide us with some experimental bases to develop and exploite Chinese medicine herb of cortex periplocae.Methods: 1.MTT assays was used to detect the suppressive effect of CPP on different human esophageal carcinoma cells such as TE-13, Eca-109, TE-1 and TE-10.2. Affter treatment with CPP for 48h, The cycle of human esophageal carcinma cells TE-13 was studied by flow-cyto -metry (FCM). The apoptosis of TE-13 was measured by Gimsa staining and flow-cytometry (FCM)3. In order to analysize the mechanism of the apoptosis and the cell cycle arresting of TE-13 induced by CPP, expression of CDK2 and CDK4 were detected with western blot.4. The expression of syk mRNA in human esophageal carcinma cells line such as TE-13, Eca-109, TE-1 and TE-10, and the changes of syk mRNA induced by CPP were analyzed by RT-PCR, and the changes of syk protein was measured by FCM.5. Lymphocytes from human peripheral blood were isolataed by density gradient centrifugation with lymphocyte isolation. Effect of CPP on proliferation of lymphocytes and phogocitoxity to macrophages on carcinoma cells were studied wit MTT.6. The influence of CPLA on cytokines production by lymphocytes was analyzed with ELISA and RT-PCR.Results: 1. CPP inhibited the proliferation of human esophageal carcinoma cells such as TE-13, Eca-109, TE-1 and TE-10 in a time and dose-dependent manner(P<0.01). When TE-13, Eca-109, TE-1 cells were treated by CPP for 72h, the inhibitory rate of proliferation rate were(84.03±0.97)%, (80.56±0.65)and (72.49±2.1)%. But the effect of CPP on TE-10 cells was weaker than it on the aboves, the inhibitory rate was only(32.06±1.56)%.2. After treatment with CPP, TE-13 cells shown some typical morphologic features, including cell shrinkage, cytoplasm concentration, nclius forming outwardly acute angle promineney, chromatin concentrating on karyotheca or forming meniscus, nuclear condensation, nuclear fragmentation and formation of apoptotic bodies. The result of FCM showed that CPP can induce apoptosis and cell cycle of TE-13 cells. Affter treated by CPP for 48h, the apoptosis rate of TE-13 increased markely with a dose-dependent manner. Meanwhile, after treatment with CPP, the cell cycles of TE-13 cells were changed, the cells in G0/G1 phase were increased and cells in S phase were decreased.3. TE-13 cells cycle arresting induced by CPP may be related with CDK4 protein decreaing. CDK4 protein expressing can be depressed by CPP (1-5μg/ml), and disparation is meanful (P<0.01); But it have no relation with CDK2 protein expression.4. Expression level of syk mRNA in TE-13 and Eca-109 are very lowly, while the expression level of syk mRNA was high in TE-1 cell and was negative in TE-10 cell. After treatment with CPP for 48h, the expression of syk mRNA(514bp) in TE-13,Eca-109 and TE-1 increase markly, but CPP have no effect on that in TE-10 cells. The result of syk protein measured by FCM are consistent with the FCM result.5. The proliferation of lymphocytes was enhanced by 5μg/ml-40μg/ml CPLA with acting of PHA, and in a dose dependent manner(p<0.01). After treatment with CPLA(20μg /ml) for 72h, proliferation index of lymphocytes was 1.26. CPLA(5-40μg/ml) can strengthen the phagocytosis effect of macrophage on esophageal carcinoma cells significantly(p< 0.01), which may be related to enhanced the production of TNF-αby macrophage.6. The production of IL-2 and TNF-αby lymphocytes could be augmented by treatment with CPLA significantly, which is in a dose-dependment (P<0.01). The expression of IFN-γmRNA in lymphocytes was also enhanced by 20μg/ml and 40μg/ml CPLAConclusions: 1. CPP showed very strong inhibitory effect on the proliferation of esophague carcinoma cells such as TE-13, Eca-109, TE-1 and TE-10 cells.2. Inhabitory effect of CPP on TE-13 cells can be related to induce apoptosis of cells and arrest of cell cycle at G0/G1. It may be related with CDK4 protein expressin and have no related with CDK2 protein expression.3. Syk mRNA and protein expression in human esophageal carcinoma cells are low and it can be enhanced by CPP. The results showed that inhabitory effect of CPP on human esophageal carcinoma cells may be related with syk up -regulation.4. CPLA can regulate the immune response of human peripheral blood cells. The proliferation of lymphocytes was enhanced by CPLA, which also can strengthen macrophage to inhabit the proliferation of esophageal arcinoma cells signi -ficantly. The production of several tumor immunity-related cytokines can be augmented by treatment with CPLA.
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