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Research On Effect Of Melittin Inhibition Of Mouse Colon Cancer

Posted on:2019-08-06Degree:MasterType:Thesis
Country:ChinaCandidate:X Z ZhuFull Text:PDF
GTID:2394330542495776Subject:Traditional Chinese Medicine
Abstract/Summary:PDF Full Text Request
Colon cancer is a common malignant tumor of the digestive tract that occurs in the colon,which accounts for the third place in the gastrointestinal cancer.In recent years,the incidence and mortality of colon cancer have been increasing in China,reaching or exceeding the Western developed countries.The average age of onset is 40 years or older,and the age of high onset is generally 50 to 60 years old,which is 10 years earlier than the average in western countries.At present,clinical treatment of colon cancer is mainly based on surgery,with the purpose of combining radiotherapy and chemotherapy.The 5-year survival rate of colon cancer was 74%,and the radical resection rate was 85%.However,for those patients who have lost their surgical opportunities and cannot tolerate the toxic side effects of chemotherapy,there is an urgent need to find or develop new antitumor drugs.Bee venom is a transparent venom secreted by the bee venom glands and accessory glands,and folk use of bee venom has long history in the treatment of rheumatoid arthritis.The melittin is a small protein molecule extracted from the bee venom,which accounts for about 50%of the dry weight of the bee venom and has a strong biological activity.With the development of modern molecular biology science and technology,the genetic engineering melittin technology has been very mature,and its biological activity has been verified through in vitro and in vivo experiments.The melittin has an anti-tumor effect,can inhibit the growth of digestive tract tumors and promote the apoptosis of tumor cells.Studies on melittin inhibiting proliferation,metastasis and inducing apoptosis of tumor cells at home and abroad have not yet been fully elucidated.In the previous study,the research group found that melittin inhibits the metastasis of gastric cancer cells through the mitochondrial pathway and induces apoptosis of gastric cancer cells.At the molecular and animal level,the optimal drug concentration and therapeutic effect of genetically engineered melittin were explored to investigate the mechanism of melittin in inhibiting colon cancer in vitro and in vivo.This study divided into three parts,as follows:Part I.Study on natural melittin inhibiting colon cancer CT-26 cellsObjective:Different concentrations of melittin inhibited the growth of mouse colon cancer CT-26 cells and explored the effects of melittin on the migration and apoptosis of mouse colon cancer CT-26 cells and its molecular mechanism.Methods:MTT assay was used to detect the proliferation of mouse colon cancer CT-26 cells with different concentrations of melittin(1,2,4,8 and 16 μg/mL)at different time(1,2,4 and 8 h);Transwell chamber test Effects of melittin(1 and 2 μg/mL)on migration of mouse colon cancer CT-26 cells for 1 h;flow cytometry detection of melittin(1 and 2 μg/mL)induced apoptosis of CT-26 cells for 1 h The expression of Caspase-9,Caspase-3 and PARP in the mitochondrial pathway of mouse colon cancer CT-26 cells was detected by Western Blot after 1 h of melittin(1,2 μg/mL).Results:The results of MTT showed that melittin could effectively inhibit the growth of colon cancer CT-26 cells in mice.With the increase of the concentration and the prolongation of action time,the inhibition rate gradually increased and approached 100%,when the concentration of melittin was 1 and 2 μg,respectively.After 1 hour of treatment with/mL,the inhibition rates were 24.83%and 58.16%,respectively,which were significantly different from those in the control group(P<0.01).Transwell chamber experiments showed that after treatment with 1 and 2 μg/mL melittin for 1 h,the number of CT-26 cells permeating membranes was 192 and 53,respectively,which was significantly reduced compared with the control group,with a certain concentration dependence,P<0.01).Apoptosis results showed that the apoptosis rate of CT-26 cells was 8.5%and 11.3%,respectively,after treatment with 1 and 2 μg/mL melittin for 1 h,which was significantly higher than that of the control group and was 5.3%.Concentration-dependent(P<0.01).Western Blot results showed that after 1 and 2 μg/mL melittin treatment of mouse colon cancer CT-26 cells for 1 h,the relative expression levels of Caspase-9 in the cell supernatant were 1.07 and 0.46,respectively,which was significantly lower than the control group 2.25.In a certain concentration-dependent manner(P<0.01),the relative expression levels of Caspase-3 were 3.72 and 2.33,respectively,which was significantly lower than that of the control group(11.03)and showed a concentration-dependent manner(P<0.01).The relative expression levels of PARP were 1.23,respectively.1.10,significantly lower than 1.80 in the control group and showing a concentration-dependent manner(P<0.01).Conclusion:When the concentration of melittin was 1 and 2 μg/mL,respectively,after the mouse colon cancer CT-26 cells were treated for 1 h,the proliferation,migration,and induction of apoptosis were significantly inhibited.The mechanism may be related to the down-regulation of Caspase-9 and Caspase.-3 is directly related to PARP protein levels.Part II.Inhibitory effect of attenuated Salmonella carrying melittin gene on mouse colon cancer CT-26 cells in vitroObjective:The growth of colon cancer CT-26 cells was inhibited by different concentrations of each group of bacteria,and the effects of attenuated Salmonella(Mel)with melittin gene on migration and apoptosis of colon cancer CT-26 cells were investigated and its molecular mechanisms.Methods:MTT assay for different concentrations of Mel and attenuated Salmonella(X4550)and attenuated Salmonella(GFP)carrying fluorescent genes(15,20,25,30,35,40,45,and 50 CFU/cell)Effect of mouse colon cancer CT-26 cells on proliferation of cells after 24 and 48 hours;Transwell chamber assay of Mel bacteria 25 CFU/cell,control group X4550 bacteria 45 CFU/cell and control group GFP bacteria 30 CFU/cell effect was small Mouse colon cancer CT-26 cells 24 h after migration ability;Flow cytometry detection of Mel bacteria 25 CFU/cell,control group X4550 bacteria 45 CFU/cell and control group GFP bacteria 30 CFU/cell role CT-26 24 hours later,the cells were induced to undergo apoptosis;Western Blot was used to detect colon cancer in mice after 24 hours of 25 CFU/cell treated with Mel bacteria,45 CFU/cell of control group X4550 and 30 CFU/cell of GFP strains of control group.The protein levels of Caspase-9,Caspase-3,and PARP in the mitochondrial pathway of CT-26 cells.Results:The results of MTT showed that each group of bacteria could effectively inhibit the growth of mouse colon cancer CT-26 cells.With the increase of the concentration and the prolongation of the action time,the inhibition rate increased gradually,approaching 100%,when the concentration of Mel bacteria was 25 CFU/cell.The inhibition rate was 50.26%for CFU/cell,50.12%for X4550 bacteria,45 CFU/cell,and 50.02%for GFP cells when the concentration was 30 CFU/cell.At 24 h,there was a significant difference from the control group(P<0.01).Transwell chamber experiments showed that the number of CT-26 cell membranes after treatment with 25 CFU/cell Mel 24 h was 41.8,which was significantly reduced and was in a concentration-dependent manner(P<0.01)compared with the control group of 161.8.Apoptosis results showed that the apoptosis rate of colon cancer CT-26 cells after treatment with 25 CFU/cell Mel 24h was 13.98%,which was significantly higher than the control group 5.73%and showed a certain concentration dependence(P<0.01).Western Blot showed that the relative expression levels of Caspase-9,Caspase-3,and PARP were 0.43,0.37,and 0.44,respectively,in the cell supernatant after 24 hours of colon cancer CT-26 cells treated with 25 CFU/cell Mel bacteria.Compared with the control group,the concentrations of 1.91,1.3,and 2.2 were significantly lower,showing a certain concentration-dependent(P<0.01).Conclusions:The attenuated Salmonella(Mel)strain carrying the melittin gene has the activity of natural melittin.When the concentration is 25 CFU/cell,the mouse colon cancer CT-26 cells can inhibit cell proliferation and migration significantly after being used for 24 h.,And induce apoptosis,its mechanism may be directly related to down-regulation of Caspase-9,Caspase-3 and PARP protein levels.Part III.Study on treatment of urine CT-26 Cell tumor of mouse with attenuated salmonella carrying melittin geneObjective:To observe the effect of attenuated Salmonella with melittin gene on mouse colon cancer CT-26 cell xenograft model.Methods:60 BALB/c mice were adapted for feeding for one week after the purchase.Except for the normal group,all mice were injected subcutaneously into the right armpits with the mouse colon cancer CT-26 cell suspension concentration of 3×106/0.1 ml/rat.The method induces the mouse model of mouse colon cancer CT-26 cell xenograft.After the injection of the cell suspension,the mice were examined every day for the right infraorbital skin mound,palpable masses,and enlarged masses to determine the success of the model.They were divided into a model group,a control group X4550 group,a control group GFP group,a Mel group,and a positive control ginsenoside control group(Rg3)according to a random number method.Three days after tumor formation,X4550 bacteria group,GFP bacteria group,and Mel bacteria group were intragastrically administered with 1 ×106CFU/0.2 ml/mouse corresponding bacteria,and each day of Rg 3 group was given 0.1 mg/kg/0.2 ml/rat Rg 3 by gavage.The normal group and the model group were intragastrically administered with 0.2 ml/mouse of normal saline.After 3 days of gavage,the body weight of the mice was measured,the length and width of the tumor were measured,and the general condition of the mice was recorded.After the administration for 4 weeks,the mice were given last time.After 12 hours of drug administration,blood samples were collected by eyeball sampling.The relevant factors(IL-2,IL-6,IL-10,VEGF,IFN-γ)were detected by ELISA.The tumor tissues of mice were used for RT-PCR detection of tumors.VEGF,KDR and p53 mRNA expression.Results:Visual observation showed that except for the normal group,all the other groups of mice were successfully tumor-bearing.Through comparison of mouse tumor measurement volume,it was found that the tumor volume of mice in the administration group was relieved,among which the tumor volume of Mel bacteria group mice.The degree of growth was slower than that of other groups(P<0.01);ELISA was used to detect IL-2,IL-6,IL-10,VEGF,IFN-γ and other inflammatory factors in mice serum,and the immunological indicators of mice in the Mel group were detected.The changes were better than those in other groups(P<0.01).Compared with the model group,the relative expression levels of VEGF and KDR mRNA genes were down-regulated(P<0.01),and the relative expression of p53 mRNA genes was increased in the treatment groups.The difference was statistically significant(P<0.01).Conclusions:The attenuated Salmonella carrying the melittin gene has the activity of natural melittin and can effectively relieve the tumor growth of tumor-bearing mice by regulating the expression of IL-2,IL-6,IL-10,VEGF,IFN-γ and other inflammatory factors.And inhibit the activation of key signaling molecules in the VEGF/KDR pathway and activate the expression of the tumor suppressor gene p53 to inhibit tumor growth.
Keywords/Search Tags:melittin, attenuated salmonella, colon cancer, apoptosis, VEGF/KDR, animal model
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