| Purpose:MCF-7 is from human breast cancer, ER,PR and VEGF are all positive expression。This experimental research was consist of two parts , Accoring to observe the influence to the apoptosis of MCF-7 cell by Ruyanning decoction in vitro ,and According to the experiments in vivo about tumor-inhibitition effect, inhabiting angiogenesis of breast cancer ,the influence to the expression of phosphorylatal Akt protein on MCF-7 tumor bearing nude ,to investigate the mechanisms of Ruyanning decoction were related to signal transduction passageway of PI3K/Akt .Material and method:Partâ… The preparation of mice'blood serum of Ruyanning Decoction. Choosing fifty BALB/C Nude mice of SPF rank, weight between 18 and 22 gram, age between 6 and 8 weeks, rats were randomly assigned into two groups with 25 in each group (the normal blood serum group, the herbal blood serum group), the herbal blood serum group:Taking in Ruyanning Decoction whose dose was converted according to ratio of body surface area with mice , amount to ten times of clinical dose , intragastric administration was forty gram every kilogram everyday, while giving the same dose normal saline to the control group, the frequency of intragastric administration was twice a day at the same time everyday, 12 hours interval,congtinuing 7days. Ruyanning Decoction was boiled in routine, At last time,mice weren't given the food , given the herb two times as original dose,the interval of the two times was one hour ,after 1 hour at the last time, then took blood in a sterile operation manner 1 hour before gave herb.Mice were anesthesed by chloral hydrate.The sample of blood was laid up in a four centigrade degree refrigerator till the next day after placed in normal temperature 4 hours. 2000 rpm in the centrifuge for 15 minutes, then filtrated though 0.22um filter membrane, and packed the blood serum respectively in EP pipe, conserved in negative 80 centigrade degree in refrigerator, inactivated it in 56 centigrade degree water for 30 minutes before blood serum being used.Partâ…¡The influence of blood serum of Ruyanning Decoction to proliferation of human breast cancer cell of MCF-7 were detected using MTT color metric assay. Experimental process:1. Took cell of ogarithm period of growth,then were digested by pancreatic enzymes /EDTA of 0.25 percentage and made into single cell fluid with Dulbecco's modified Eagle's medium(DMEM) composed by 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. 2.Adjusting the density of MCF-7 cell to 2×105/ml ,then inoculated 200ul in the three boards with ninty-six holes each, Then the cells were maintained in 37 centigrade degree ,a humidified incubator with 5% CO2 and grown on 100-mm culture dishes in Dulbecco's modified Eagle's medium(DMEM) with low glucose for 24H.3. After 24 hours exchanged Dulbecco's modified Eagle's medium with 5 percentage fetal bovine serum,continued to cultivate in 37 centigrade, a humidified incubator with 5% CO2 for 24H,in correspondence with the cell stage of G0/G1 .4.There are six groups in all according to different proportion blood serum of Ruyanning Decoction,the concentration of herbal blood serum was five percentage, ten percentage and twenty percentage, corresponding to the normal blood serum.There were ten repeated holes in each group.5. Added different proportion blood serum and culture dishes into each hole ,the volume of each hole was 200ul,the five percentage blood serum and ten percentage blood serum were consisited of twenty percentage blood serum and the normal blood serum,every hole kept concentration as twenty percentage blood serum . Cultivated for 24 hours,48 hours,72 hours respectively.6. Then exchanged Dulbecco's modified Eagle's medium with no fetal bovine serum at different time , then added MTT (5mg/ml ) 20ul,continued cultured for 4 hours,throwed the nutrient solution away ,detected the each time point value of OD by Microplate reader device, Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure,then caculated the inhibition rate of cell proliferation.Partâ…¢To observe the influence of blood serum of Ruyanning Decoction to the apoptosis of MCF-7 cell in the way of AO/EB fluorescent staining. Took cell of ogarithm period of growth,washed by PBS once, then were digested by pancreatic enzymes /EDTA of 0.25 percentage and made into single cell fluid with Dulbecco's modified Eagle's medium(DMEM) composed by 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. Adjusting the density of MCF-7 cell to 1×104/ml ,then inoculated 2ml in the boards with six holes each. After two days ,cell was divided into four groups with normal blood serum and herbal blood serum, acted on the MCF-7 cell for 24 hours repectively, then droped the nutrient solution, fixed by 10 % formaldehyde for 30 minutes ,AO/EB fluorescent staining , carbonate buffer glycerine seal ,cell was observed by fluorescen- ce microscope with 405nm wavelength.Each group was calculated for three times with 300 cell each time ,then calculated the apoptosis rate. Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure by SPSS 10.0,determining the optimal experimental concentration.Partâ…£According to the result of MTT, choosed the optimal concentration of herb serum, cells was divided into normal blood serum and herbal blood serum. The media were then removed and fresh growth media with drugs were added, acted on the cell for 12hours and 24hours respectively. The medium was carefully removed and combined with a 1ml phosphate-buffered saline (PBS) rinse to constitute the"detached sample".The adherent cells were trypsinized and suspended in growth medium. Samples of the adherent and detached suspensions were counted and asses -sed for viability by exclusion of trypan blue. The remaining cell suspensions were washed 1×with PBS and then suspended in PBS before being fixed and processed for flow cytometric analyses of DNA content as described previously. Percentages of proliferation cells and cells in the G1, S, and G2/M stages of the cell cycle were determined with a DNA histogram-fitting program. Attempts were made to collect a minimum of 104events/sample for subse quent analyses.Choosed the optimal concentration of herb serum, cell was divided to normal blood serum and herbal blood serum, collected cell after blood serum acted on the cell for 6 hours,12 hours,18 hours,24 hours respectively .Then MCF-7 cell were starved as described above for 24 hours and then treated with the various agents indicated in the text. They were then washed twice with phosphate-buffered saline (PBS) and lysed in ice cold lysis buffer. Cells were detected by FCM. Then carried out statistical analysis on the result. Differences from control were assessed by analysis of variance with ANOVA tests when multiple comparisons were required.Partâ…¤To observe the influence of blood serum of Ruyanning Decoction to the expression of BAX/BCL2, Caspase3 , P-Akt protein of MCF-7 cell. Took cell of ogarithm period of growth,then were digested by pancreatic enzymes /EDTA of 0.25 percentage and made into single cell fluid with Dulbecco's modified Eagle's medium(DMEM) composed by 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. Adjusting the density of MCF-7 cell to 1×104/ml ,then inoculated 5ml in the culture bottle, Then the cells were maintained in 37 centigrade degree ,a humidified incubator with 5% CO2 and grown on 100mm culture dishes in Dulbecco's modified Eagle's medium(DMEM) with low glucose for 24H. After 24 hours exchanged Dulbecco's modified Eagle's medium with 5 percentage fetal bovine serum,continued to cultivate in 37 centigrade, a humidified incubator with 5% CO2 for 24H,in correspondence with the cell stage of G0/G1.Choosing the optimal experimental concentration, divided into normal blood serum and herbal blood serum, acted on the MCF-7 cell for 24 hours repectively, then collected cell.MCF-7 were starved as described above for 24 hours and then treated with the various agents indicated in the text. They were then washed twice with phosphate-buffered saline(PBS) and lysed in ice-cold lysis buffer.To detected the expression of BAX/BCL2, Caspase3 , P-Akt protein of MCF-7 cell in the way of Western blot.Partâ…¥To observe the morphological change of MCF-7 cell by transmission electron microscope,to detect the apoptosis by TUNEL in the way of immunohistochemistry , the expression of MMP-2/MMP-9,MVD was detected by immunohistochemistry, the expression of EGF/VEGF was detected by ELISA.Results:1.The result of MTT color metric assay revealed that there was no significant difference between the five percentage herbal blood serum at different time with 24 hours, 48 hours, 72 hours and the normal blood serum. AS 5 percentage of blood serum was concerned, there was no significant difference between normal blood serum and herbal, while on the percentage of 20 herbal blood serum, there was significant difference about the value of OD comparing to normal blood serum, illuminated that 20 herbal blood serum had obviously effect on the proliferation of MCF-7 cell. The herbal blood serum inhibited the proliferation of rat aortic vascular smooth muscle cell in a concentration-dependent and time-dependent manner.2.The analytical result of Annexin V-FITC/PI double staining detected via Flow cytometry (FCM) showed that:Herbal blood serum of Ruyanning Decoction inhibited the transformation from G0/G1 phase to S phase in a definite range. Comparing to the control group , herbal blood serum delayed the process of cell cycle obviously,the proportion that on G0/G1 phase was more than normal blood serum, while on the S phase the proportion went down sharply, cell blocked on the G1 phase. The herbal blood serum inhibited the proliferation of MCF-7 cell in a concentration-dependent and time-dependent manner.3. The result of Western blot revealed that Ruyanning Decoction serum could down-regulate the expression of Bcl-2 protein and phosphorylatal Akt protein, up-regulate Bax and Caspase3,decreased the ratio of Bcl-2/Bax,in the herbal blood serum, and there was significant difference comparing to normal blood serum.The result show that blood serum of Ruyanning Decoction could induce apoptosis of MCF-7 cell,one of possible mechanism of which effected the expression of phosphorylatal Akt protein in the passageway of PI3K/Akt,then meddled related regulated protein of cell cycle.4.The result of transmission electron microscope revealed that: the morphology of cell in normal blood serum was in a average state, the nucleolus was clear; while in the herbal blood serum the morphology of cell manifested typical apoptosis, for example, cell nucleus was irregular, concentrated cytoplasm, the nucleolus of the cell was disappear, the pyknosis of chromatin, gathered under the caryotheca aside, the swelling of endoplasmic reticulum etc. The result of TUNEL in the way of immunohistochemistry also revealed that Ruyanning Decoction could induce apoptosis of MCF-7 cell. 5.The result of the expression of MMP-2/MMP-9,MVD detected by immunohistochemistry revealed that MVD of MCF-7 mammary cancer loaded on Nude mice falled ,improved the inhibition rate of tumor ,down-regulated the expression of EGF/VEGF of blood serum of micedetected by ELISA ,the expression of MMP-2/MMP-9 reduced ,illuminated Ruyanning Decoction had the effect to inhibite angiogenesis of mammary cancer,had multi-target curable effect.Conclusion:1. The prescription of Ruyanning has the effect of inducing the apoptosis of the cell of MCF-7 breast cancer and MCF-7 tumor bearing nude . and MCF-7 tumor bearing nude by the prescription of Ruyanning are related to signal transduction passageway of PI3K/Akt .2. Angiogenesis of MCF-7 breast cancer on tumor bearing nude can be inhabited by the prescription of Ruyanning.
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