Isolation And Identification Of Clostridium Butyricum And The Function And Mechanism Of Colon Cancer Research

Objective: Intestinal bacteria play an important physiological role inthe human body, proved intestinal flora on the human body has a veryimportant significance. From healthy human feces culture, select1intestinal bacterial strain using TSN medium, the strain was adopted relatedmethods to carry out molecular identified as Clostridium butyricum(CB).The effects on cells were observed by the identified CB on the SW-480colon cancer cells, and the expression of cell apoptosis related proteins Baxand Bcl-2were determined, then to further explore the possible mechanismof CB inhibit the growth of colon cancer cells.Methods:40cases of healthy adult fresh dejection were collected forCB culture. By bacterial culture and the bacteria colony morphology, CBwere detected with Gram staining, separation and purification culture,observed with the identification of biochemical test and combined with themolecular identification of16S rDNA methods. The SW-480colon cancercells and identified CB with different concentrations were1.5×103，1.5×104，1.5×105，1.5×106，1.5×107，1.5×108CFU/ml interaction, the inhibition of cell proliferation was detected by CCK8kit after24h48h,72h, and96h. The concentration of screening effect (1.5×106，1.5×107，1.5×108CFU/ml) and effective duration were more noticeable for follow-upexperiments. The ultra structure of SW-480cells were observed by TEM,meanwhile, cell apoptosis rate were determined by FCM after48h. And theactivity of the SW-480cells of Caspase-3and Caspase-9were surveyed byCaspase kits and the total cell protein were extracted, Western blot methoddetermination of two main proteins associated with mitochondrialapoptosis Bcl-2and Bax. All the data by using Graph Pad Prism5.0software two-factor variance analysis, drawing, P＜0.01,with statisticalsignificance.Results: Develop colonies of CB edge was not neat, milky white,slightly convex surface, the individual was seldom and colony was larger,the whole colony was aggressive, bacteria was cultured under the mediumto growth after36hours and generated a large amount of gas in line withthe literature described characteristics. Then gram stain violet, fusiform,single or paired, times of extreme buds; Only the gelatin liquefaction testand indole test were negative in the biochemical test, other were all positive,these were all accord with the literature description, according to the resultof16s rDNA molecular identification, this plant was a special function ofCB. CCK-8revealed that normal controls and the concentration of1.5×103，1.5×104，1.5×105，1.5×106，1.5×107，1.5×108CFU/ml of CB in colon cancer, the cell inhibitory effect of CB group of24h、48h、72h and96h were higher than the normal control group, P<0.01, there is asignificant difference. The Electron microscopy, according to the results of1.5×107、1.5×108CFU/ml of CB colon cancer cells after48h had cells andorganelles shrivel, rough surface, dense cytoplasm, nuclear chromatin edgeset, part of the cell cytoplasm and nuclei lobulated swelled and formcompounds nuclear fragments and organelles of the multiple apoptoticbody, and separated from the cell body, then presents the typical apoptoticmorphological characteristics, While there was no significant difference inthe normal control group. The apoptosis rate of CB group was higher thannormal control group after48h, P<0.01, the result had significant difference;After48h CB groups: the expression activity of Caspase-3and Caspase-9were higher than in normal controls, statistical differences after1.5×107，1.5×108CFU/ml of CB processing, Caspase-3and Caspase-9activityincreased by68.3%,89.5%and58.8%,89.5%respectively, P<0.01. Proteinexpression results showed that CB effect on cancer cells after48h,theprotein expression of Bax were significantly higher than that of normalcontrol group, P <0.01, statistically significant; Compared with the normalcontrol group, the amount of protein expression of Bcl-2was reduced,t=6.1,P<0.01,the differences were statistically significant.Conclusion: The proliferation inhibition mechanism of colon canceris associated with the effect of CB, the composition of CB or metabolites produced in a certain environment to some extent inhibits the growth ofcolon cancer cells, and then induce the SW-480cell apoptosis, themechanism may be enhanced the activity of Caspase-9and Caspase-3andincreased the expression of Bax, then reduced the Bcl-2, thus suppressingSW-480cell growth and inducing apoptosis of tumor cells.