| Purpose:PKC is an important enzymes closely related to the function of the spleen, tumor growth and tumor angiogenesis, especially the four isoforms of: PKCδ, PKCε, PKCα, PKCβ, play an important regulatory role in the progression of tumors. Pre-clinical and experimental studies demonstrated that spleen Qi has effects of anti-cancer and improving symptoms;experimental research has begun to show that the effects of improving symptoms and anti-cancer are achieved by improving the function of the spleen,improving PKC expression in liver, spleen kidney tissue is the mechanism of strengthening spleen. To further study the effects in Spleen Qi in tumor and four PKC isoforms(PKCδ, PKCε, PKCα, PKCβ),we conducted a study.In this study, from the intervention to PKC we study the effects of anticancer of Spleen Qi in colorectal carcinoma tumor and microvessel density and apoptosis,and explore effect of Spleen Qi on tissue PKC protein and PKCδ, PKCε protein,and study the effects of Spleen Qi in VEGF, PKCα, PKCβ protein expression in colorectal cancer tissues. This will provide new laboratory laws for the clinical application of strengthening Spleen Qi in the treatment of colorectal cancer. Material and method: 1.Experimental animals and tumor lines: HT-29 human colon cancer cells, the cells were purchased from Chinese Academy of Sciences Library; 30 animal selection nude rats, male and female, weight(20 ± 2 g), aged 4-5 weeks. 2.Preparation of drugs: Strengthening spleen Qi medicine, anti-cancer medicine and strengthening Spleen Qi extract: Spleen Qi medicine: heterophylla, Poria, Atractylodes, licorice, Amomum, woody, Citrus, France Breit composition, the other medicine :Strengthening spleen Qi medicine and indica, Tuckahoe, Fritillaria, diffusa, Yiyiren, Curcuma, banzhilian composition, purchased from Liaoning Provincial Hospital outpatient pharmacy. Respectively, the medicine has 10 times boiling water for two hour, the 8 times boiling water for one hour,and collect two decoctions, 4 ℃ refrigerator spare. 3.Human colon HT-29 cell suspension was prepared and Mice: Select logarithmic growth of tumor cells, the cell concentration was adjusted to 1×107 / ml, nude right armpit iodine disinfection, using the right axillary 1ml syringe in mice Under the inoculation of tumor cells was 0.2ml / mice, the entire procedure require aseptic technique, tumor growth was observed daily. 15 days after inoculation, tumor nodules to appear at the inoculation site in diameter up to 0.8cm, hard texture and other indicators identified as a tumor. 4.Animal grouping and administration: Animal groups: 30 mice numbers randomly divided into model group, Strengthening spleen Qi group, Strengthening spleen Qi and anticancer group. N = 8. Administration: model group were given saline,Strengthening spleen Qi and the other group were given medicine for consecutive 14 days. On day 14, the mice were sacrificed using cervical vertebrae off method, after stripping out the complete tumor placed on ice, after the naked eye tumor tissue, tumor excised full-thickness tissue while avoiding multi-drawn tumor necrosis. 5.Detection targets: tumor and tumor microvascular density(MVD), tumor cell apoptosis detection, tumor cell VEGF expression assay(PCR, Westenblot). Applications of PCR, Westenblot in PKC,PKCδ, PKCε, PKCα, PKCβ expressi in tumor tissue. Results: 1. The cell apoptosis in model group is very little increase,the rate of tumor cell apoptosis of Spleen Qi and combination group are increasing, three apoptotic rates are 3.8 ± 1.2,13.8 ± 2.6, 36.2 ± 6.6, respectively. Strengthening spleen Qi group and combination group comparison with the model group were statistically significant(p <0.01), the apoptosis rate of combination group was significantly increased compared with Strengthening spleen qi(p <0.01). Microvessel density:0.227 ± 0.012,0.218 ± 0.010,0.189±0.011,respectively, which showing a declining trend. Although Strengthening spleen Qi group show the decline, but not statistically significant, while the combination group decreased significantly compared with the model group and Strengthening spleen Qi(p <0.01). 2. The model group, Strengthening spleen group, the combination group were 0.596±0.021, 0.540±0.015,0.412±0.040.Strengthening spleen qi comparison with the model group were significantly different(p <0.05), the combination group with the model group was significantly(p <0.01), there was a significant difference(p <0.05) between groups with Strengthening spleen Qi and the combination group; PKC protein were 0.665±0.016,0.498 ±0.018,0.261±0.019, Strengthening spleen qi comparison with the model group were significantly different(p <0.05), while the combination group with model group, and Strengthening spleen qi groups were more significantly(p <0.01). Three groups PKCδ m RNA were 0.233±0.025,0.261±0.034,0.410±0.030, there were significant differences(p <0.01) between groups. Three groups PKCδ protein were 0.301±0.041,0.361±0.044, 0.516±0.029, Strengthening spleen qi comparison with the model group were significantly different(p <0.05), while differences between the combination group and model group were more significantly(p <0.01). Three groups PKCε m RNA were 0.368±0.044,0.359±0.029,0.224±0.029, there were significant differences(p <0.01) between Strengthening spleen qi group and the model group; three groups PKCε protein were 0.362±0.021,0.314 ±0.031,0.215±0.021, Strengthening spleen qi group compared with the model group were significantly different(p <0.01), while the combination group compared with model group,Strengthening spleen qi group were significantly different(p <0.01). 3. VEGF m RNA of Three groups was 0.447±0.024,0.405±0.033,0.307±0.026, there was a significant difference in comparison(p <0.05) with the model group and strengthening Spleen Qi, the difference of the combination group with model group is more significantly(p <0.01), there is also different between strengthening Spleen Qi and the combination group(p <0.01).VEGF protein of three groups were 0.325±0.037,0.303±0.022,0.230±0.031; strengthening Spleen Qi compared with the model group were significantly different(p <0.05), while the difference between the model group and the combination group was more pronounced differently(p <0.01). PKCαm RNA were 0.590±0.017,0.472±0.025, 0.392±0.025, there are significant differences between(p <0.01)the model group and strengthening Spleen Qi, there were significant differences between the combination group and the others(p <0.01).PKCα protein were 0.566±0.024,0.522±0.021,0.335±0.025, strengthening Spleen qi compared with the model group significantly different ly(p < 0.05), while the difference between the combination group and the model group is more differently(p <0.01). PKCβm RNA were 0.583±0.029,0.471±0.039,0.400±0.024, there is significantly difference between(p <0.01) the model group and strengthening Spleen Qi, the combination group with model group was significant different(p <0.01); PKCβ protein were 0.542±0.061,0.414±0.081,0.292±0.040, there are significant differences between strengthening Spleen Qi and the model group(p <0.01), while the difference between the model group and the combination group was significantly different(p <0.01). Conclusion: 1. Strengthening Spleen Qi and the combination of strengthening Spleen and anti-cancer have a therapeutic effect on colorectal cancer,and promotion of apoptosis of tumor cells is one of its mechanism of action, the combination method is more improved. 2. Strengthening Spleen Qi method can suppress PKC, PKCε m RNA and protein,the combination method promotes the anti-cancer effect significantly. Strengthening Spleen method can promote PKCδm RNA and PKCδprotein, this inhibition of the combination method is more increasely. The inhibition of combination method on PKC, PKC εand PKCδ ultimately inhibit the growth of colon cancer cells, this may be the mechanism of anticancer treatment of Strengthening Spleen Qi and anti-cancer. 3. Strengthening Spleen Qi with anticancer can inhibit tumor angiogenesis and expression of related genes VEGF, and can inhibit tumor angiogenesis PKCα, PKCβm RNA and protein expression, and thus play an inhibition action in tumor vascular.
|
PDF Full Text Request