Preliminary Evaluate The Safety And Investigate The Immune Effect Of Gene Therapy Agents TPKH And TPIN

Objective?1?To preliminary evaluate the safety of KGF and HIF-1?transferred by attenuated salmonella?TPKH?as well as NK4 and IL-2 transferred by attenuated salmonella?TPIN?by acute toxicological test,general pharmacology test,gene protein expression distribution test in BALB/c mice and long-term toxicity test in Wistar rats.?2?To investigate the effects of TPKH and TPIN on cellular immune function and intestinal mucosal immunity in mice.Methods?1?In acute toxicological test,the effects of different doses(1.0×10⁸?1.0×10⁹?1.0×10¹⁰cfu/0.1mL per one)of TPKH or TPIN on mental state and body weight of mice were observed after gavage.In addition,blood routine,liver function and kidney function index and organ index were measured on the 14th day after gavage.Meanwhile,pathological sections were made in the high dose group and control group(1.0×10¹⁰cfu/0.1mL per one)to observe the morphology of each organ.?2?In general pharmacology test,the effects of different doses?1.0×10⁷?1.0×10⁸?1.0×10⁹cfu/0.1mL per one?of TPKH or TPIN on general behavior,spontaneous activity,cooperation movement ability of mice were observed after gavage.?3?In gene expression protein distribution test,the mice of control group were given 0.1mL10%NaHCO₃ by gavage,and the mice of experimental group were given TPKH or TPIN of1.0×10¹⁰cfu/0.1mL per one by gavage.Four mice were killed each time at 0,12,24,48,72,96 hours and homogenate was prepared from heart,liver,spleen,lung,kidney,stomach,small intestine,muscle,brain tissue to obverse the expression levels of KGF,HIF-1?after TPKH was given or HGF?NK4 is the antagonist of HGF?,IL-2 after TPIN was given in different tissues by ELISA,so as to observe the distribution and expression of TPKH or TPIN in mice.?4?In long-term toxicity test,the control group was given 0.1mL 10%NaHCO₃ by gavage,and the experimental group was given different doses(1.0×10⁷?1.0×10¹⁰cfu/0.1mL per one)of TPKH or TPIN by gavage.The rats were given for 7 weeks by gavage,twice a week.The blood routine,serum biochemical index,four items of hemagglutination and organ index were examined at 1st day,4th week,8th week,12th week,16th week after the last gavage,respectively.Meanwhile,pathological sections were made in the high dose group and control group(1.0×10¹⁰cfu/0.5mL per one)to observe the morphology of each organ at 12th week after the last gavage.In addition,the fecal samples of the control group on the 1st day,the high dose group on the 1st day and the high dose group on the 4th week after the last gavage were taken for 16s rRNA sequencing.?5?In cellular immune function test,the control group was given 0.1mL 10%NaHCO₃ by gavage,and the experimental group was given TPKH or TPIN of 1.0×10⁹cfu/0.1mL per one by gavage.The mice were given for 4 weeks by gavage,twice a week.7 days after last gavage,whole blood were collected.The levels of CD3⁺T cells?CD3⁺CD4⁺T cells?CD3⁺CD8⁺T cells were detected by flow cytometry.In intestinal mucosal immunity,the control group was given 0.1mL 10%NaHCO₃ by gavage,and the experimental group was perfused with TPKH or TPIN of 1.0×10⁹cfu/0.1mL per one.The mice were given for twice,7days apart.After the second gavage,twelve mice were killed on the 14th day,and the small intestinal Peyer's patches?PP patches?and mesenteric lymph nodes were taken to detect the expression of B cell activating transcription factors?GATA3,PAX5?by quantitative real-time polymerase chain reaction?qRT-PCR?.In addition,twelve mice were killed on the 28th day,and the expression of IgA in small intestinal mucosa was detected by ELISA.Results?1?During the acute toxicity test,the mental state in each group has no abnormality.The results of body weight,blood routine,liver function and kidney function index and organ index showed that there was no significant difference among different groups?P>0.05?.The results of pathological tissue sections showed that there were no pathological changes in heart,liver,spleen,lung,kidney,stomach and intestine.?2?In general pharmacology test,TPKH or TPIN had no obvious impact on general behavior,activity and cooperation movement ability of mice?P>0.05?.?3?In gene expression protein distribution test,compared with the control group,KGF and HIF-1 protein were significantly expressed in stomach and small intestine at 12,24,48,72and 96 hours after intragastric administration of TPKH?P<0.05 or P<0.01?,and HGF,IL-2protein were significantly expressed in stomach and small intestine at 12,24,48,72 and 96hours after intragastric administration of TPIN?P<0.05 or P<0.01?,but they were not significantly expressed in heart,liver and other tissues?P>0.05?.?4?In long-term toxicity test,the results showed that the rats no death or obvious poisoning symptoms were found.Compared with control group,body weight,routine blood,hemagglutination and organ index of rats were no difference?P>0.05?.In blood biochemistry analysis,compared with control group,alkaline phosphatase?ALP?had significant difference?P<0.05 or P<0.01?at 1st day after the last gavage and the other indexes were not significant difference?P>0.05?.Meanwhile,the results of pathological tissue sections showed that there were no pathological changes in heart,liver,spleen,lung,kidney,stomach and intestine.After gavage of TPKH,it can increase the abundance of intestinal probiotics and have a beneficial effect on intestinal flora,and after gavage of TPIN,it had a certain effect on intestinal flora.the intestinal flora was affected to a certain extent,but with the extension of time,the effect of TPKH and TPIN gradually weakened.?5?In cellular immune function test,by flow cytometry,it was found that CD3⁺T?CD3⁺CD4⁺T cells and CD3⁺CD4⁺/CD3⁺CD8⁺ratio of TPKH or TPIN groups were significantly higher than that of control group?P<0.05 or P<0.01?.In intestinal mucosal immunity,qRT-PCR results indicated that TPKH and TPIN could improve the expression of GATA3 and PAX5?P<0.05 or P<0.01?.The results of ELISA indicated that the level of IgA in intestinal mucosa of TPKH or TPIN groups were significantly higher than that of control group?P<0.01?.Conclusions?1?It is preliminarily observed that TPKH and TPIN are safe in animal experiments,and no obvious toxic action is found.?2?TPKH and TPIN can promote the cellular immune function and enhance the immunity of small intestinal mucosa in mice.