| Background and ObjectiveThe cancer terribly endangers people's health, and its morbidity is increasing rapidly in recent 10 years. Using surgical operation is early phase tumor's main treatment tool, but it has stepped into late stage when the tumor is fund, so surgical operation no longer suits, and the chemotherapy becomes the main treatment tool. We often find that the slow living and good pathology differentiation cancer cell has low sensitivity to chemotherapy, but the rapid living and bad pathology differentiation cancer cell has high sensitivity to chemotherapy. It is because that the different pathology type cancer cell stay in different cell division cycle, so cycle-specific chemical drugs fail to completely kill all the tumor cells. In the recent years, multimodality therapy of tumor has been received extensively by clinical doctors, the traditional Chinese medicine is also a very important treatment tool of multimodality therapy on tumor. The mordern pharmacology has already proven that chinese herbs not only can induce the tumor's apoptosis,but aslo regulate immune function,kill the tumor cell directly. Eucommia ulmoides, dodder and herba cistanches are all usual beneficial chinese herbs. It is said that extract of eucommia ulmoides can accelerate the synthesis of rat's collagen protein and the proliferation of epidermic cells; dodder total flavonoids can improve activity of skeletogenous cells and the proliferation of mice's thymus gland and spleen, besides can enhance the engulfing of macrophage, improve the proliferation of lymphocyte; Polysacchrides of cistanche can also enhance the engulfing and secretory function of macrophage,activate the macrophage, and also improve the proliferation of mice's spleen lymphocyte. On these grounds, whether or not can we suppose that the three chinese herbs also improve the tumor cell's proliferation? if can they have the ability to promote the cancer cell's metabolism? And if can the beneficial chinese herbs change the tumor cell's cell cycle, make the cell cycle focus on one cell cycle, such as S phase. G1 phase or M phase, and then we use one cell cycle specific drug to kill them so as to improve the cancer cell's sensitivity to chemotherapy agents?Methods1 Cell culture:esophageal cancer cell Ec9706 were inoculated with culture medium RPMI-1640 containing 10% inactivated fetal calf serum, and conventionally cultured in incubator with 37℃,5%CO2,95% relative humidity.2 Initial screened three benefitial chinese herbs which can make tumor cells proliferate best of all by MTT3 Detected cell proliferation rate:esophageal cancer cell Ec9706 in the logarithmic phase were inoculated in the board with 96 holes, about 1000～3000 cells per hole, and 100μl per hole.24h later, added different concentration Chinese herbs reagent. Setting Chinese herbs groups and control groups. After cultivated in incubator with 37℃5%CO2,95%RH for 48h, cell growth conditions were measured by MTT assay, then recorded the growth rate. And then repeated it for 3 times.4 Draw growth curve:Ec9706 in the logarithmic phase were prepared to single cell suspensions and inoculated into the boards with 96 holes, about 1000～3000 cells per hole, and 100μl per hole.24h later,40mg/ml eucommia ulmoides reagent, 20mg/ml dodder reagent and 30mg/ml herba cistanches reagent were respectively put into boards. Examined one board every day by MTT, and recorded the OD, in total for 6 days, finally drew the growth curve.5 Detected the cell cycle by flow cytometry6 Detected the expression of cyclin D by immunohistochemistry.7 Detected the inhibition to Ec9706 by the combination of Chinese herbs and 5-fu with MTT. Ec9706 in the logarithmic phase were prepared to single cell suspensions and inoculated into the boards with 96 holes, about 1000～3000 cells per hole, and 100μl per hole.12h later, inoculated the chinese herbs reagent and 5-fu into the board holes according to the experimental design, 100μl per hole. Set the only 5-fu group,5-fu unite chinese herbs reagent group and blank controller group respectively. After cultivated in incubator with 37℃5%CO2,95%RH for 24h,48h, 72h respectively, then cell growth inhibition rate were measured by MTT. And then repeated it for 3 times.8 Statistical analysis:using SPSS13.0 statistical software, single factor variance analysis andχ2 test to analyze the results. Size of test wasα=0.05.Results:1. The three benefitial chinese herbs which can make tumor cells proliferate best of all having been detected by MTT were eucommia ulmoides, dodder and herba cistanches.Eucommia ulmoides, dodder and herba cistanches can all make the Ec9706 proliferate in some extents of their concentration. The degree of eucommia ulmoides' concentration to proliferate the Ec9706 was 1-40mg/ml, and the maximum concentration was 40mg/ml. The degree of dodder's concentration to proliferate the Ec9706 was 1-20mg/ml,and the maximum concentration was 20mg/ml. The degree of herba cistanches'concentration to proliferate the Ec9706 was 1-30mg/ml,and the maximum concentration was 30mg/ml. Compared the chinese herbs reagent groups with blank controller group, there was statistical significance(P<0.05).2. Detected the cell cycle by flow cytometry:72h after Chinese herbs'administration, the cells proportion in every stage of cell cycle with experimental group's were significantly different from which was in control group's. Cell proportion on stage S was significantly increased, but significantly decreased on stage G1. Compared the experimental groups'cell proportion on tage S with which was in control group, there was statistical difference(P<0.005).3. Detected the expression of cyclinD in Ec9706 with cell immunohistochemistry:After Chinese herbs'administration, we obtained the expression of cyclinD in both experimental groups'cell and control group's cell, positive position was located in nucleus and endochylema. With eucommia ulmoides, cyclinD expression rate of Ec9706 was 0.642±0.033, with dodder, it was 0.632±0.022, with herba cistanches, it was 0.562±0.023, which in control group was 0.426±0.029.4. The three Chinese herbs reagents in their best concentrations uniting 5-fu improved the inhibition of 5-fu to Ec9706,compared to the only 5-fu group.The difference in two groups have statistical significance(P<0.005).Conclusions:1. Eucommia ulmoides, dodder and herba cistanches can all make the Ec9706 proliferate2. With the effect of eucommia ulmoides, dodder and herba cistanches on Ec9706, cells on stage S of cell cycle accounts for higher rate, but cells in stage G1 accouts for lower rate.3. Eucommia ulmoides, dodder and herba cistanches decreased the number of Ec9706 cells on stage G1, increased the cells on stage S, shorten the cell proliferation cycle, kept more cells in stage S, improved the cell metablism level, which probably was by means of increasing the expression of cyclinD.4. The three Chinese herbs uniting 5-fu group remarkably improved the inhibition of 5-fu to Ec9706 compared with the only 5-fu group,thus the Chinese herbs reagent improved the sensitivity of the Ec9706 cells to 5-fu. |
PDF Full Text Request