The Rearch Relatived Circadian Rhythms About The Ovarian Cancer Cell Strain With Gemcitabine

Background and purpose:Along with the research progress of the life science, the research of the "living creature clock" becomes HOt point about life science reaserch in the recent years,the International autHOrity magazine "Science" thinks the research of the living creature clock may change man's natural knowlege to natural,So produce the important society with economic performance.the research realm have the important breakthrough.Lead the theories of the living creature clock into the medical treatment then become Chronotherapy,and have the special advantage in many diseases under medical treatment,particularly chemotherapy in tumor aspect.Owing to the toxicity and persistence of the chemotherapy medicine are self-contradict,many scHOlars try to use the aim genes to reduce the toxicity and increases curative effect,but still be in the stage of clinical now,not yet discover better metHOd to attain this purpose,chemotherapy mays have the breakthrough in this respect.Current in the world many chemotherapy medicine have the clinical and basic data of Chronotherapy.Gemcitabine is second cHOice as the chemotherapy medicine using on the ovarian cancer in clincal.It is used anti- metabolism medicine.Particular in the cell synthesized circle in the DNA,namely S period of the cell,under the certain term,can obstruct from the period G to the period S have obvious cellular poisonous activity to every kind of tumors on the person and the rat and we are sHOrt of interrelated experimental data of Gemcitabine,the experiment explore the basic data about interrelated Gemcitabine Chronotherapy.the ER,PR in amounted to 50%near to living creature characteristic of the person's ovary cancer cell.chemotherapy from vitro ovarian cancer and cell witHOut the suprachiasmatic nucleus control under the light.Basis the research of small rat about the chronotherapy,There is only a few report in abroad,about 24hcircadian rhythms from vitro ovarian cancer.Result in 48h circadian rhythms is more dependable.the result can supply experimental proof for chronotherapy from vitro ovarian cancer cell in clinicMetHOds:1.In part one Monoclonal antibody immunohistochemistry to explore the expression of estrogen receptor and Progesterone receptor of ovarian cancer cell lines A2780,SKOV3,HO8910,ovarian cancer cell,Adopting Mouse MonoclonalAntibody that antibody Recognizes a protein of 67 kDa which is identified as estrogenreceptor antibody Progesterone Receptor This Mab is specific to human Progesterone Receptor,According the Specificity.the ER,PR staining are located in the tumor cell syrup and cell nucleus inside.The immunhistochemistry integral calculus namely the Hscore integral calculus metHOd proceeds result judgement,the result synthesize the dying strength and dye scope proceed the real-time quantitatived handle.positived cell standard:(1)the construction is clear;(2)It is obviously discriminated between the background and the staining Hscore= gvi(i+1),I is the dying strength.All dying glasses slice are closely with experiment term.â… according to below the standard is divided into theâ…£class:0 for negative;(+)dye weak,but obvious higher than the negative;(++)dye clear;(+++)dye strong.+,++,+++3 dyeings strength uses the i-1 respectively,2,3 mean.i=0 HOur,dyeing negative,integral calculus is 0,the Pi is each on dying male cancer in inside in scope in strength cell number occupy 100 cents that cuts into slices all cancer cell the value,will cut into slices like this inside all strength and its per cents in dyeing in male cancer cell the conversion combines for the HOmologous number tired adding,attain the purpose to dye real-time quantitatived amount to ER,PR.certain Hseore≥75 for positive,Hseore＜75 for negative.The second part:1.Use the MTT metHOd examination medicineIC50 about the kill rate of the Three ovarian cancer cell,make the three kinds of cell into 5×10⁴cell/ml liquids,plant into 96 orifice inside,each orifice 100 ul.,Experiment condtrast a matched fifteen densities(50ug/ml,100ug/ml,200ug/ml,400ug/ml,600ug/ml,800ug/ml,1000ug/ml,2000ug/ml,3000ug/ml,4000ug/ml,5000ug/ml,6000ug/ml,7000ug/ml,8000ug/ml,10000ug/ml)on trial set.the each orifice adds medicine to 100 ul,In restraint of rate and inside effect density(IC50)of calculation every kind of medicine density,experiment to repeat to take the average value 3 times,2.According the estrogen receptor antibody Progesterone Receptor negtive or postive Ovarian cancer cell A2780,SKOV3,HO8910,line were inoculated in96 orifice.Every orifice was inoculated 10⁴ culture board use the MTT metHOd examination IC₅₀density medicine about the kill rate,make into the cell 10⁴ cell/ml liquid,plant in 96 bores educates plank inside,each bore 100ul.On trial the experiment establish 24 HOurs,contrast gemcitabine every three bores while the other three were blank according to 12time x2d:0:00,04:00,08:00,12:00,16:00,20:00 educated after 24 HOurs,while setting upside down the microscope therefore the cell has overlaid the bore bottom 50-60%,adding medicine density as the IC50 Gemcitabine,a matched control only add educated liquid educates continuously after 48 HOurs,use the MTT metHOd strictly examination the kill rate of the cell in each HOur kill rate of each HOur result was examined by SPSS. Results(1)Ovary Cancer Cell A2780ER(+),Ovary Cancer Cell A2780PR (—),Ovary Cancer Cell SKOV3ER(—),Ovary Cancer Cell SKOV3PR(++), Ovary Cancer Cell HO8910ER(++)Ovary Cancer Cell HO8910PR(—) (2)Ovary Cancer Cell A2780IC50 Is 2000ug/ml,Ovary Cancer Cell SKOV3IC50 Is2000ug/ml,Ovary Cancer Cell HO8910IC50 Is 100ug/ml(3)through SPSS test A.there are significant circadian rhythm about the kill rate of three different ovarian cancer cell there are significant circadian rhythm about the kill rate of ovarian cancer cell.The kill rate of ovarian cancer cell HO8910 about the different cell Its pesk value can see out from the cosine graph,at the second 0am—4am,use the medicine at this time.Here was the biggest kill rate.Here was the biggest kill rate at this time of the value Its vale value at the fist day 8am—12N.The kill rate of ovarian cancer cell SKOV3 Its peak value at the second 0am—4am,from the cosine graph Its vale value at the fist day 0am-4am or The Second day 4Am-8Am,Tere was no significant circadian rhythm of the kill rate of ovarian cancer cell A2780.B.There was significant difference circadian rhythm of the kill rate of the same ovarian cancer cell between the first day and the second day:Its peak value and Its vale value at the fist day is 0am and 8am.Its peak value and Its vale value at the second day is 0am and 20pm.The kill rate of the ovarian cancer cell with Gemcitabine in the second day is more than its in the first day There are the biggest kill rate at this time Its peak value of kill rate of the ovarian cancer cell SKOV3 with Gemcitabine and Its vale value at the fist day is 12N and 4am.Its peak value and Its vale value at the second day is 4am and 16pm.There was significant difference Its peak value of the kill rate of the second day is bigger than Its value at the fist day C.Thee was significant difference circadian rhythm of the kill rate of the same ovarian cancer cell SKOV3 between the different dose between 2000ug/ml and 3000ug/ml.Its peak value both are 0am—4am at the second day.The kill rate is biggest at this time.Its vale value of the dose 2000ug/ml is 0am—4am.Its vale value of the dose 3000ug/ml is 4am—8am.D Thee was significant difference circadian rhythm of the kill rate of the different ovarian cancer cell.using the same dose medicine.Its peak value and Its vale value of kill rate of with the dose 2000ug/ml Gemcitabine are Contrary,Its peak value of SKOV3 is the vale value of A2780.Conclusion:(1)There was significant circadian rhythm about the kill rate of ovarian cancer cell HO8910 ER(++)PR(—)with Gemcitabine in different time of a day Its peak value was 0am-4am at the second day(2)There was significant circadian rhythm about the kill rate of ovarian cancer cell SKOV3 ER(—) PR(++)with Gemcitabine Its peak value was 0am-4am at the second day(3) There was no significant about the kill rate of ovarian cancer cell A2780 ER (++)PR(—)(4)The circadian rhythm of ovary cancer cell has no corelativetion to the ER.PR...