The Study Of Genetic Engineering Lactobacillus Adhering Intestinal Mucosal And Decomposing Small Molecular Urinary Toxins

Objective:1. Explore the capacity of genetic engineering lactobacillus adhering the human colon cancer HT-29lines in vitro and the gastrointestinal mucosa of5/6nephrectomy rat in vivo.2. Investigate the capacity of genetic enginneering lactobacillus decomposing small molecular urinary toxins in vitro and in serum and gastrointestinal trace of5/6nephrectomy rat.Methods:1.Bacteria adhesion experiments:①wild lactobacills and genetic engineering lactobacills were divided into control group and experiental group,adding1ml bacterial concentration of approximate1.5×108cfu/ml into HT-29cells culture medium. At different time (0.5h,1h,2h,4h,8h,24h), compare the adhesion index of two type of bacteria under microscope.②The5/6nephrectomy rats were divided into three groups:1. Experiental group(n=13):genetic engineering lactobacills,1.5×108cfu/ml×2ml/d, i.g, Qd;2. control Group(n=12):wild lactobacill,1.5×108cfu/ml×2ml/d, i.g, Qd;3. Pathological group(n=10):NS,2ml,i.g, Qd.4weeks later, all of rats were executed,and mucosa of stomach,jejunum,colon taken from rats were homogenized,smeared, and compared number of three groups bacteria which adhered mucosa under microscope. 2. Bacteria decomposing urine toxins experiment:①divided into three groups:genetic engineering lactobacills as experiental group, wild lactobacills as control group, serum of patient with renal failure as pathological group. Adding lml serum of patient with renal failure into experiential group and control group culture medium,compared the concentration of creatinine,urea nitrogen,uric acid in two groups with pathological at different time point.②The5/6nephrectomy rat were divided into three groups:1. Experiental group(n=13):genetic engineering lactobacills,1.5×108cfu/ml×2ml/d, i.g, Qd;2. controlGroup(n=12):wild lactobacill,1.5×108cfu/ml×2ml/d, i.g, Qd;3. Pathological group(n=10):NS2ml,i.g, Qd. Before administration,the blood of rats were collected.4weeks later, all of rats were executed, and the contents of stomach,jejunum,colon and blood were collected to detect the concentration of creatinine,nitrogen,uric acid.Results:1. Bacteria adhesion experiments①In vitro experent,there had no significant difference on adhesion index between experiental group and contol group at different time point (0.5h,1h,2h,4h,8h,24h),had no statistical significance(P>0.05).②In vivo experent,,the number of genetic engineering lactobacill adhering mucosa in experiental group were much more than in Pathological group (P<0.05). The number of bacteria adhesing mucosa between experiental group and control group(p>0.05) had no significant difference. In experiental group,the number of genetic engineering lactobacills adhering to colon were much more than to stomach and jejunum.2. Bacteria decomposing urine toxins experiment①In vitro experent,the concentration of urinary toxins in experiental group and control group had no significant difference compared with Pathological group in four(0.5h,1h,2h,4h) time point (P>0.05).The creatinine of experiental group had significantly decreased at8h time point compared with pathological group(P<0.05). At24h time point, the urinary toxins in experiental group and control group had reduced respectly,and had significantly decreased in experiental group compared with pathological group(p<0.05).The creatinine and uric acid had significantly differrence between experiental group and control group at24h time piont(p<0.05).②In vivo experent, there had no significant difference between each group in serum concentration of urinary toxins before administration(P>0.05).4weeks later, creatinine,nitrogen,uric acid had increased in pathological group,but decreased in experiental group and control group. Except urea nitrogen creatinine, nitrogen and uric acid in experiental group much lower than in pathological group (P<0.05). Experiental group compare with control group, serum creatinine, nitrogen, uric acid decline more than36%,7%,30%respectly. The creatinine,urea nitrogen,uric acid concentration of digestive juice in experiental group siginificantly decreased compared with pathological group(P<0.05).The creatinine,uric acid concentration of digestive juice in control group were lower than pathological group but highter than experiental group(p<0.05).Conclution:1. The genetic engineering lactobacillus accepted creatinine hydrolase and uric acid oxidase gene recombination has no difference compared with wild lactobacillus in adhering to HT-29cells.2. There have no difference between genetic engineering lactobacillus and wild lactobacillus in adhering to gastrointestinal mucosa.3. The ability of genetic engineering lactobacillus decompose the small molecular urinary toxins is stonger than wild lactobacillus.4. The genetic engineering lactobacillus can decrease the serum concentration of creatinine,uric acid level by decomposing small molecules in gastrointestinal tract in vivo.