| Objective: To study the molecular mechanism of the intervention of strengthening spleenand harmonizing stomach method on esophageal cancer from the relationship between tumorcells and endothelial cells, and provide evidence for clinical application as well, there areseveral steps to follow. First, to observe the inhibiting effects of Liujunzi Decoction(representative formula of trengthening spleen and harmonizing stomach method) on theproliferation of esophageal carcinomas cells Eca-9706. Second, to observe the effects ofLiujunzi Decoction on neovascularization of chicken chorioallantoic membrane as well asthe interventions on the mutual relation of tumor cells and human umbilical vein endothelialcells (HUVECS), and the last step is to make comparisons of Liujunzi Decoction and QigePowder (representative formula of regulating qi-flowing for eliminating phlegm method).Method: Experiment objects was selected from esophageal carcinomas cells Eca-9706andHUVECS, we could test the inhibiting effects of Liujunzi Decoction and Qige Powder on thecell lines growth by means of MTT method. After establishing the model of chickenembryos, observing the angiogenesis, and processing the photos by using Image-pro plussoftware, we could make angiogenesis area statistics. Tumor cell and endothelial cellco-culture were cultivated by means of Tanswell, the process of angiogenesis and cellmigration were tested, and the process of tubular generating of vascular endothelial cellswere examined by the method of matrigel plug. Then we observe the effects of LiujunziDecoction and Qige Powder drug supernatant on the index above. Meanwhile, we examinethe intervention effects of Liujunzi Decoction and Qige Powder on IL-6and VEGF secretingmade by tumor cells and endothelial cells.Results:1. The experimental results of MTT method indicated that, compared with thenegative control group, the OD values of cell lines could be reduced after the differentconcentrations of both Liujunzi Decoction and Qige Powder acted on esophageal carcinomascells Eca-9706for48hours. And the inhibiting rate increased with the rising of the concentrations of them. When concentrations and durations changed (600μg/ml,400μg/mland200μg/ml for Liujunzi Decoction,96μg/ml,64μg/ml and32μg/ml for Qige Powder,24h,48h,72h and96h for durations), the varying degrees of OD values reduced, compared withthe negative control group, which showed great concentration and duration dependency.2. we made quantitative analysis of new tubules by establishing a model of chickenembryo and calculating chorioallantoic membrane and vascular area, and found thatcompared with that of the blank control group, the total number of new tubules in tumorsupernatant group increased(n=10, P<0.05), while that of total number of new tubules inLiujunzi Decoction drug supernatant group and Qige Powder drug supernatant groupremarkably decreased(n=10,P<0.05).3. The experimental results of MTT method demonstrated that, in the experiment of theeffects on human umbilical vein endothelial cells growth, the OD values decreased in bothLiujunzi Decoction group and Qige Powder group compared with negative control group,which indicated that the medicinals in both groups could provide relatively weak inhibitingeffects.4. The experimental results of Transwell indicated that, the number of cell immigrationin tumor supernatant group increased apparently when comparing to blank controlgroup(P<0.05), and slight inhibiting effects on cell immigration are made in LiujunziDecoction drug supernatant group comparing to tumor supernatant group.(P<0.05) On thecontrary, slight promoting effects on cell immigration are made in Qige Powder drugsupernatant group comparing to tumor supernatant group.5. The testing results made by means of matrigel plug method indicated that, the ODvalues in both Liujunzi Decoction drug supernatant group and Qige Powder drug supernatantgroup are less than that of tumor supernatant group, which showed that the number of newtubules in both drug supernatant groups was less than that of tumor supernatant group. Theresults indicated that Liujunzi Decoction and Qige Powder could both inhibitneovascularization.6. The testing results made by means of ELISA method revealed that it provided themost IL-6and VEGF factors in tumor supernatant group. The number of these factorsdecreased in all the Liujunzi Decoction drug supernatant groups which are separately at high,middle and low concentrations while mild proliferation effects are taken in Qige Powderdrug supernatant groups at the same concentrations.Conclusion:1. Liujunzi Decoction and Qige Powder could both inhibit the proliferationof esophageal carcinomas cells Eca-9706, the inhibition follows the dose-time-effect relationship.2. Liujunzi Decoction and Qige Powder could inhibit angiogenesis, endothelial cellproliferation and migration, and tubules angiogenesis caused by tumor supernatant. Themolecular mechanism might be that it decreases the secretion of VEGF and IL-6in tumorcells and human esophageal squamous carcinomas cells. |
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