Studying The Role Of Heat Shock Transcription Factor 2 In The Intestinal Mucosal Barrier Function Of DSS Colitis Mice

Background:Ulcerative Colitis(UC)is a chronic and recurrent colonic inflammatory disease that is difficult to cure,and the etiology of which is not clear.The major clinical manifestations of UC including abdominal pain,diarrhea,mucous bloody stool,etc.Due to its long duration,recurrent and cannot be completely cured,it seriously affects the patient's physical and mental health,and brings a lot of burdens to many families and the society.In recent years,the incidence of UC has been increasing rapidly in the world.Our previous study found that Heat Shock transcription factor 2(Hsf2)was highly expressed in UC patients and positively correlated with disease severity.In cell experiments,HSF2 was found to up-regulate cellular occludin,suggesting that Hsf2 may be involved in the regulation of UC mucosal barrier function.At present,the pathogenesis of Hsf2 in UC is not clear,and there is no study on the relationship between Hsf2 and mucosal barrier function in UC.In this study,Hsf2 gene knock out mice were firstly established,and then the effect of Hsf2 gene deletion on intestinal inflammation and mucosal barrier function in mice induced by Dextran sodium sulfate(DSS)was studied,so as to further reveal its role in the development of UC.Objective:Firstly,Hsf2 gene knockout homozygous transgenic mice were established by CRISPR/Cas9 technology.Secondly,DSS was used to induce the colitis of intestinal in mice,and the Disease activity index(DAI),intestinal injury,intestinal shortening degree,and the changes in spleen volume were observed to comprehensively evaluate the effect of Hsf2 on the inflammatory response of DSS mice in colitis model.Finally,the levels of gene transcription and protein expression about occludin and zonalu occluden-1(zo-1)proteins which related to mucosal barrier in the colon tissues of mice were detected to investigate the effect of Hsf2 on intestinal mucosal barrier function at the animal level.MethodsThis study is divided into two partsPart 1:The establishment of Hsf2 gene knock out micePart 2:Researching the mechanism of Hsf2 which regulates the intestinal inflammation and intestinal mucosal barrier function in DSS colitis micePart 1:The establishment of Hsf2 gene knock out mice1.The establishment of the Hsf2 gene knockout mice:Using the CRISPR/Cas9 technique and micro injection method,the steps including:1)artificial design of sgRNA in vitro according to the target gene;2)the sgRNA and Cas9 mRNA were injected into the male prokaryote of the fertilized egg by the technology of micro-injec;3)the embryos were transferred to the fallopian tubes of female mice with false pregnancy for further development and delivery,and the transgenic mice with Hsf2 gene knock out were obtained.PCR and sequencing methods were used to identify and screen the mice,and the heterozygous mice with Hsf2 gene knock out of FO generation were bred.In this experiment,a number of Hsf2+/-mice of FO generation heterozygous mice constructed by this technique were introduced to preserve and breed.2.The breed and establishment of Hsf2 gene knockout homozygous mouse:The gene knocked out(KO)mice and Wild type(WT)mice of C57BL/6 aged 7-8 weeks were selected for mating that were bred in SPF environment,and the mate ratio was one male and two or three female mice in a cage,the male mice were take away from the cage until the vaginal plug was detected in the vagina of female mice,and the female mice were raised alone until they gave birth,the Hsf2-/-mice were identified by PCR technology,and the level of transcription and protein expression of Hsf2 gene were detected by RT-PCR and Western Blot to verify the knockout effect,Finally,the homozygous Hsf2 gene knock out mice were established.Part 2:Researching the mechanism of Hsf2 which regulates the intestinalinflammation and intestinal mucosal barrier function in DSS colitis mice1.Grouping:The healthy C57BL/6 of SPF were selected for the experiment.According to whether the Hsf2 gene knock out,group will be divided into the wild type mice(WT group,16)and knockout(KO group,16),and by RT-PCR and Western Blot technique validation knockout effect.The above two groups were randomly divided into WT control group(8 WT+H20 group),WT modeling group(8 WT+DSS group),KO control group(8 KO+H2O group)and KO modeling group(8 KO+DSS group)after giving the DSS to induce colitis in mice.2.Using the DSS to induce the colitis model:The model group began to drink distilled water that containing 2.5%DSS solution at 8 o 'clock in the morning of the first day of the experiment,while the control group also drank distilled water freely for 7 consecutive days.On the morning of the 8th day,the mice were sacrificed by cervical dislocation under ethyl ether anesthesia,and their colon specimens were obtained.3.The effect of Hsf2 in intestinal inflammation of mice induced by the DSS:During the experiment,observing the eating habits?spirit?activity?hair luster of the mice,and recording the weight?the feces of the mice,then according to the weight change and bloody stool to assess the Disease activity index(diseases activity index,DAI),Next,observing the colon tissue specimens,measuring the length and weight of the colon and spleen,the colon tissue pathology score were calculated after HE staining,Finally,the intestinal inflammation degree was comprehensively evaluated.4.The exploration of the mechanism of Hsf2 regulating the intestinal mucosal barrier in DSS mice:RT-PCR and immunohistochemistry were used to detect the gene transcription and protein expression levels of tight junction related proteins Occludin and Zonalu occluden-1(zo-1)in mucosal barrier in colon tissues.results:Part 1:The establishment of Hsf2 gene knock out mice1.The Hsf2 gene knock out homozygous mice were successfully established.RT-PCR and Western Blot were used to verify the knockout effect that were identified as Hsf2-/-mice.The results indicated that the level of transcription and protein expression of Hsf2 gene in KO group were significantly lower than that in WT group,and Hsf2 gene knockout homozygous mice were successfully established.2.Hsf2-/-mice grew normally,their weight was similar to KO mice in the same age,their survival rate was high,there was no abnormality,and they could conceive and reproduce normally.Part 2:Researching the mechanism of Hsf2 which regulates the intestinal inflammation and intestinal mucosal barrier function in DSS colitis mice1.Compared with each group,the KO+DSS group showed the most obvious blood stool and the most significant weight loss,the highest DAI score,the highest colonic gross score,the most significant colonic shortening,and the most significant splenic enlargement.HE staining indicated that the colon tissues of KO+DSS group had the most severe inflammation,the most significant structural damage and the highest pathological score.The differences between groups were statistically significant.2.After giving DSS to induce colitis model,the level of gene transcription and protein expression of Occludin and ZO-1 gene of tight junction protein were reduced,with the KO+DSS group being the most significant.The differences between groups were statistically significant.Conclusion1.By using CRISPR/Cas9 technology and micro-injection method,the stable Hsf2-/-mice were obtained.After Hsf2 gene knockout,the survival rate of mice was not affected,no malformation occurred,normal growth and reproduction were possible,and the special mice can tolerate the chemical drugs of DSS.2.2.5%DSS can successfully induce experimental colitis model in mice.After giving 2.5%DSS to the mice.the mice showed symptoms such as weight loss and blood stools,and no mice died during the modeling period,and all of them were able to tolerate the stimulation of DSS.The colon tissues of mice were obtained after death,and the inflammation was more obvious in DSS model group.3.After Hsf2 gene knock out,the mice were more sensitive to DSS-induced colitis.After DSS-induced colon inflammation in mice with low level Hsf2,DAI score of the mice was significantly increased and the inflammatory response of the colon tissues was more obvious.4.After Hsf2 gene knockout,the level of gene transcription and protein expression of mouse intestinal epithelial tight junction protein decreased,suggesting that Hsf2 may be involved in regulating the intestinal mucosal barrier function in mice.