Evaluation Of The Efficacy Of Double Protein In Hematopoiesis And Immune Reconstitution Of Mice After Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation(Allo-HSCT)as a curative therapy for malignant hematological tumors such as leukemia and lymphoma.The most obvious feature after transplantation is the long-term immunodeficiency,which leads to higher risk of mortality caused by opportunistic infections and cancer relapse.Therefore,how to accelerate the immune reconstruction of patients after HSCT has been a hot topic in this field.Studies have shown that protein malnutrition in patients after HSCT is a very common phenomenon,which may cause delay the time for immune reconstruction.Therefore,it is necessary for patients after HSCT to supplement extra protein nutrition in the clinic.There have been reports on the role of clinical nutritional support of a single protein in bone marrow transplantation,but whether double proteins from different animals and plants can accelerate hematopoietic and immune reconstruction after transplantation has not been reported.In this study,a mouse model of allogeneic hematopoietic stem cell transplantation was established,and a double protein consisting of soy protein isolate and whey protein was selected as the research object.Protein nutrition was supplemented after HSCT in mice to make a preliminary study of the effects of double protein on immune reconstitution.Experiment 1:Establish and evaluate a mouse model of allogeneic hematopoietic stem cell transplantationBALB/c(H-2Kd)mice were subjected to the dose of total body irradiation(TBI)(7.5 Gy,8 Gy)and the dose of bone marrow mononuclear cells(BMNCs)(H-2Kb)(5×10~6,2×10~7)from donor.Grouped as follows:RHTH(8Gy irradiation+2×10~7 BMNCs infusion);RHTL(8Gy irradiation+5×10~6 BMNCs infusion);RLTH(7.5Gy irradiation+2×10~7 BMNCs infusion);RLTL(7.5Gy irradiation+5×10~6 BMNCs infusion);RHNT(8Gy irradiation);RLNT(7.5Gy irradiation).The survival status,survival rate,GVHD and donor chimerism of each group were observed after HSCT.The results shown that irradiation doses of 7.5Gy and 8.0Gy can achieve myeloablative purposes,but 8Gy total body irradiation and infusion of 5×10~6 BMNCs means that the mortality of the RHTL group is the lowest and the donor chimerism rate is fully implanted.In addition,there was no obvious GVHD phenomenon after transplantation in mice in each group.Therefore,8Gy total body irradiation and infusion of 5×10~6BMNCs are better model building schemes.Experiment 2:The effect of double protein intervention on hematopoietic and immune reconstitution in mice after HSCT150 BALB/c mice were randomly divided into double protein intervention group(DP),whey protein intervention group(WP),and non-intervention group.(NP),normal control group(CON),and irradiated non-transplanted group(RNT).According to the principles of protein solubility in normal saline and nitrogen balance,mice in DP and WP groups were given nutritional support of 34mg biprotein and 33.3mg whey protein,respectively,after HSCT.Protein nutrition intervention was started on the first day after transplantation for 4 weeks.During the intervention period,evaluating comprehensively hematopoietic and immune reconstitution of mice after HSCT.The results shown that compared with NP group,double protein intervention can significantly accelerate peripheral white blood cells,lymphocytes and NK cells counts,and the recovery of lymphocytes and NK cells was significantly faster than that of WP group in mice after transplantation.The number of c-Kit+cells,short-term hematopoietic stem cells(ST-HSC)and multipotent progenitor cells(MPPs)in the bone marrow of the DP group were dramatically increased than NP group,and the number of short-term hematopoietic stem cells(ST-HSC)was also significantly higher than that of WP group.The visceral index and absolute cell count of thymus and spleen in DP group were significantly higher than those in NP group.The analysis of lymphoid subpopulation in splenocytes illustrated that the number of CD3+and B220+cells in DP group was obviously higher than that in NP group.However,there was no significant difference in organ index and cell number between WP and NP group.Experiment 3:The effect of double protein intervention on the serum biochemical indexes and GVHD of mice after HSCTTo further evaluate the effect of double protein intervention on the body condition and the occurrence of GVHD in mice following HSCT.The body weight was measured regularly,the pathological status of liver,intestine and spleen were analyzed by the HE staining and the blood biochemical indicators of mice after transplantation were also determinated.The results shown that the survival rate of mice in each group after HSCT was higher and there was no significant GVHD occurrence.Blood biochemical indexes shown that the serum total protein content of DP group was significantly higher than that of WP and NP group.Compared with NP group,the contents of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in DP and WP groups were obviously lower.Meanwhile,the levels of serum malondialdehyde(MDA)and 4-hydroxynonenal(4-HNE)in DP and WP groups were also significantly lower than those in NP group.Therefore,Double protein intervention had no significant effect on the survival rate and GVHD of mice after transplantation,but it could significantly increase serum total protein content,improve the body’s antioxidant capacity and protect liver function.