| Background and Objective:Short bowel syndrome(SBS)is a malnutrition syndrome in which most of the small intestine is caused by physical or functional loss,resulting in an effective absorption of the small intestine.Clinically,diarrhea,water and electrolyte disorders,malabsorption and progressive malnutrition are the main features.Generally,when the residual intestinal length is ≤ 35 cm,it can be defined as ultra short bowel syndrome(USBS).At present,the clinical treatment of SBS is mainly non-surgical treatment,and mainly rely on parenteral nutrition(PN)to provide additional nutritional supplements for SBS patients to maintain physiological metabolic needs.But PN is also a high-risk,expensive,and potentially inadequate treatment.For example,PN-related ductal infections,intestinal mucosal atrophy,liver failure,etc.,which seriously threaten the lives of SBS patients.Especially for USBS patients,because the remaining small intestine is extremely short or completely lost to the small intestine,it is necessary to rely on PN for life and suffer from the above risks.Therefore,finding a safe and effective method to promote the compensation of residual colonic function in such patients,reducing their use of PN or getting rid of dependence on PN is a new idea for the treatment of patients with USBS.The previous study of our group has confirmed that the residual colon can undergo compensatory changes both in morphology and function by constructing a USBS rat model,and found that protein disulfide isomerase A3(PDIA3)is significantly higher.Expressed in the compensated colon tissue.As a resident oxidoreductor of ER,PDIA3 has been shown to be involved in many signal transduction and regulation processes related to cell survival,and has potential proliferation-promoting effects on cells.So,does PDIA3 also play a role in promoting colon compensation as a key regulatory protein in USBS colon compensation? Our previous studies have shown that PDIA3 promotes proliferation and inhibits apoptosis in SW480 cells by up-regulating and down-regulating the expression of PDIA3 in colon cancer cells(SW480).So,does PDIA3 have the same effect in human normalcolonic mucosal epithelial cells? In this experiment,the expression level of PDIA3 in human normal colonic mucosal epithelial cells(NCM460)was bidirectionally regulated by constructing an overexpression and silencing plasmid,and whether it could promote proliferation and inhibit apoptosis in NCM460 cells.To provide an experimental basis for further study of whether PDIA3 functions as a key protein in colonic compensation.Methods:1.Construction of eukaryotic cell overexpression plasmid(PDIA3-GV141)and silencing plasmid(PDIA3-shRNA),using DNA sequencing to verify the success of the construction;2.Using Lipofection technique to transfect overexpression plasmid and the silencing plasmid into the NCM460 normal human colonic epithelial cells.3.Western blotting method was used to detect the changes of PDIA3 protein expression in transfected different doses of over-expression and silencing plasmid of NCM460 cells;4.CCK-8 method was used to detect the cell proliferation rate at 24 hours,48??hours and 72 hours after transfection of different doses of overexpression and silencing plasmids;.5.Flow cytometry was used to detect the apoptosis rate of cells transfected with different doses of overexpression and silencing plasmids.Results:1.Eukaryotic cell overexpression plasmid(PDIA3-GV141)and silencing plasmid(PDIA3-shRNA)was confirmed to be successfully constructed through DNA sequencing.2.Western blotting tips: In the overexpression experiment,the expression level of PDIA3 in the experimental group NCM460 cells was higher than that in the empty vector group and positively correlated with the dose of the transfection plasmid;In the silencing experiment,the expression level of PDIA3 in the experimental group NCM460 cells was lower than that in the empty vector group and negatively correlated with the dose of the transfection plasmid.3.CCK-8 shows: In the overexpression experiment,the proliferation rate of the experimental group at 24 h,48h,72 h was lower than that of the empty vector group(P<0.05,the difference was statistically significant);In the silent experiment,the proliferation rate of the experimental group at 24 h,48h,72 h was higher than that of the empty vector group(P<0.05,the difference was statistically significant).4.Flow cytometry results shows: In the overexpression experiment,the apoptosis rate of the experimental group was higher than that of the empty vector group(P<0.05,the difference was statistically significant);In the silencing experiment,the apoptosis rate of the experimental group was lower than that of the empty vector group(P<0.05,the difference was statistically significant).Conclusion:PDIA3 inhibits the proliferation of human colonic epithelial cells(NCM460)and promotes its apoptosis. |
