| Background: An abnormally microenvironment which is not fit for the living of normal cells is induced and maintenanced by tumor cells through growth rapidly, the abnormal energy metabolism and self-regulation of specific proteins. As the same time, the abnormally microenvironment is the guarantee of the neoplastic transformation ,proliferation,invasion and metastasis of tumor cells.To study the mechanism of acidic microenvironment and changes in biological activity of tumor cells in such an abnormally microenvironment maybe helpful to find ways with high potency and low toxicity to treat tumor, and will further give new insight for integrated traditional and western medicine in cancer research..Through a long time of clinical practice, Professor Wei-pinkang has established the gastric adenocarcinoma phlegm Syndrome theory, and created the Xiaotan Sanje recipe, which is confirmed that can be useful to inhibition the proliferation,invasion and metastasis of gastric adenocarcinoma cells and to prolong survival of patients by experimental studies and experimental research. Pinellia is the monarch drug of Xiaotan Sanje recipe. To study the effect of enthanol extract from Rhizome Pinelliae Preparata on the acidic microenvironment with human gastric adenocarcinoma cells can not only find the possible new mechanism of anti-tumor of Xiaotan Sanjie Recipe, but also discuss the correlations between the gastric adenocarcinoma phlegm Syndrome theory the and acidic microenvironment, which may contribute to clarification the material base of Phlegm. Objective: By studying the acidic microenvironment which is fit for the the living of gastric adenocarcinoma cells, to find the mechanism of enthanol extract from Rhizome Pinelliae Preparata restraining the human gastric carcinoma, and to further enrich the theory of gastric adenocarcinoma phlegm Syndrome theory.Methods: 1,Afetr the drugs has been crushed, 95% ethanol extract, filtration and sterilization, the enthanol extract from Rhizome Pinelliae Preparata can be obtained. The cells were divided into 5 groups: control group and enthanol extract from Rhizome Pinelliae Preparata groups(1mg/ml,0.5mg/ml,0.25mg/ml,0.125mg/ml). All experimental will be repeated 3 times, then combined data for statistical.2,SGC-7901 cells (5×104/m1) were plated in three 96-well plates. There were five groups in each 96-well plates, each group has five wells, and using wells without cells as blanks. The OD of each groups was determined by MTTassay after 24h,48h,72h incubation. We can get the IC50 concentration of 72h enthanol extract from Rhizome Pinelliae Preparata treatment by the OD determined by MTTassay.3,Inverted microscope was utilized to observe morphological changes after 72h drugs with different concentration treatment.4,The cell growth curve was generated by the OD of each groups which was determined by MTT assay after 0,12,24,36,48,60,72h incubation.5,Annexin V-fluorescein isothiocyanate/propidium iodide double label method was used to detect apoptosis rate of SGC7901 cells after 72h enthanol extract from Rhizome Pinelliae Preparata with different concentration treatment.6,The activity of total ATPase of each groups were evaluated with UV spectrophotometric determination, using the following formula (the activity of total ATPase(U/ml)= (OD of drug-treated- OD of untreated sample/ OD of standard pipe- OD of blank pipe)×concentration of standard tube(0.02umol/ml)×6×Sample dilution×7.8) to calculation the activity of total ATPase of each groups with different drug concentration after 72h incubation.7,The pHi value of each groups were measured in the monolayers using the pH-sensitive fluorescent probe 2,7-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) , The pHe values of culture medium were measured by pH211 Calibration Check Microprocessor pH Meter.8,According to the IC50 concentration of 72h enthanol extract from Rhizome Pinelliae Preparata treatment, V-ATP,NHE1 mRNA were examined by the method of RT-PCR after 72h drug treatment.Results: 1,The proliferation of SGC7901 cells can be sharply inhibited by enthanol extract from Rhizome Pinelliae Preparata with different concentration; After treating with enthanol extract from Rhizome Pinelliae Preparata, cellular morphology changed, including brim of cell wall roughening and cell volumes shrinkening, which result in a decline in diopter and the abilty of adhenrence to plate surface; It was observed by flow cytometry that enthanol extract from Rhizome Pinelliae Preparata can induced SGC7901 cells apoptosis. The activity of total ATPase decreased significantly after coculturing SG7901 cells with drugs for 72h. The all effect of enthanol extract from Rhizome Pinelliae Preparata can be enhanced with the increase of its dose.2,Detected by RT-PCR,we can find that the V-ATP,NHE1 mRNA expressed in drug groups are 0.174%±0.079%,0.180%±0.165%,they expressed in controlled groups are 0.318%±0.148%,0.652%±0.323% after 72h incubation. There is obviously different between the controlled group and the drug goup(P=0.020 and P=0.001).3,With the increase of concentration and intervention time of drugs, Enthanol extract from Rhizome Pinelliae Preparata could lower the index of pHi There is statistically different compared with the control group and drug groups ( without 0.125mg/ml group) after 72h drug treatment. At the same time, we also find Enthanol extract from Rhizome Pinelliae Preparata could heighten pHe of each groups, then consequently decrease the difference between pHi and pHe.Conlusion:1,Enthanol extract from Rhizome Pinelliae Preparata can inhibit the SGC7901 cells proliferation and induce cell apoptosis, decrease the activity of total ATPase.2,Enthanol extract from Rhizome Pinelliae Preparata could lower the index of pHi while heighten that of pHe, and consequently decrease the difference between the two.3,Enthanol extract from Rhizome Pinelliae Preparata could inhibit the expression of V-ATP and NHE1 gene.
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