| Inherited metabolic diseases(IMD) are a group of over 500 inherited conditions caused by defective activity of a single enzyme. It is essential for normal protein, fat or carbohydrate metabolism. Deficiency of an enzyme should lead to accumulation of toxic. These conditions are often life-limiting, multi-systemic and relentlessly progressive. The vast majority of IMD present in childhood, but milder forms present in adulthood. The treatment of severe IMD is still limited. The principle of treatment is to reduce the toxic accumulation, supply the need, and promote e×cretion. Such as diet therapy, enzyme replacement therapy(ERT), substrate reduction therapy(SRT), bone marrow transplantation, liver transplantation, and so on. There are disadvantages with any kind of treatment methods. Diet therapy can lead to malnutrition. ERT is not able to across the blood-brain barrier and the treatment is expensive. SRT is often hindered by the generation of anti-protein antibodies that can lead to hypersensitivity reactions, anaphylactic shock, and so on. Serious brain damage fails to improve by hematopoietic stem cell transplantation(HSCT). Serious IMD patients often present brain damage, the clinical manifestation mainly for the intelligence and mental decline. Serious nervous system damage results in poor prognosis, often leading to the death.Animal studies have shown that transplantation of HSC in the differentiation of microglia, releasing a functional enzyme through the blood-brain barrier, in treatment of brain injury. But a long time was required for microglia differentiation from HSCT, about 6 months in mouse and about 6 to12 months in human. So HSCT can't timely improve the brain damage.Neural stem cells(NSC) have the ability to self-renewal and differentiation function, successfully used in the treatment of a variety of nervous system damage and neurodegenerative diseases model. Such as acute cerebral ischemia injury, neonatal rat brain after hypoxic ischemic brain damage, Alzheimer's disease, multiple sclerosis, Parkinson's disease, etc. There are certain curative effect by NSC treat ting the nervous system disease models, but rejection and the low survival rate limit the best valuable role. In theory, human cells are the most valuable source, because only the human cells may be approved for clinical application test in the future treatment of IMD.Some researches have been reported that human cells can be toleranced by neonatal mice and rats' desensitization. In 2009, Claire M Kelly et al. reported that SD neonatal(P2-P5) rats received a single i.p. injection of 105 human NSC(h NSC). After 10-12 weeks, adult mice were assigned to receive cerebral cortex or striatum injections with(2.5-5) ×105 h NSC. After 12-40 weeks, implantation rate increased. The transplanted cells can survive for a long time. In 2012, researchers reported that neonatal BALB/c mice and Wistar rats received a single i.p. injection of 105 h NSC. After 6 weeks, adult mice were assigned to receive brain parenchyma injections with 3×105 h NSC. 2-3 weeks after the transplantation, h NSC cannot be detected. In 2014, Virginia B.Mattis et al. reported that neonatal(P2-P3) mice received a single i.p. injection of 105 h NSC. After 8 weeks, adult mice were assigned to receive cerebral injections with 2×105 h NSC. 4 weeks after the transplantation, h NSC cannot be detected. In 2013 researchers reported that wistar rats infarction model were received h NSC desensitization or immunosuppressive therapy. 2 weeks after h NSC transplantation, h NSC almost can't be detected. Local brain injury in Wistar rats, various ways to immunosuppression cannot extend NSC survival from cord blood transplantation. In 2016, Andreas heuer et al. reported that SD pups on post-natal days 2 and 4 i.p. were injected with pre-differentiated human embryonic stem cells. Once adult(minimum 15 weeks), all animals received lesions to the ascending dopaminergic fibre bundle. 4-5 weeks after the lesion the rats were engrafted using human embryonic stem cells-derived neural progenitor cells. 18-22 weeks after transplantation, human cells can be found in 89% animals. Human embryonic stem cells-derived neural progenitor cells can prevent xenograft rejection through neonatal desensitization.In addition to neonatal SD rats, others such as BALB/c, C57BL/6, NOD/SCID, B6C3F1/J, FVB(P1-P4) mice and Wistar rats, neonatal h NSC desensitization, adulthood h NSC transplantation cannot tolerate. SD rats can realize human cells immune tolerance, which may be associated with the outbreeding species or the immune of neonatal SD rats is less sound. Establish myeloablative allogeneic hematopoietic stem cell transplantation(allo-HSCT) models to simulate neonatal animals immune unsound, explore weather can realize human cells tolerance. Mice were used to establish HSCT models. Recipient BALB/c and donor C57BL/6 can be successfully establish allo-HSCT models and simulate clinical allo-HSCT unsound. In this study, BALB/c and C57BL/6 were selected to establish allo-HSCT models. The study can provide reference for IMD allo-HSCT patients and there is no report at home and abroad.Objective Based on neonatal h NSC desensitization, BALB/c mice were received lethally total body radiation to destroy immune and hematopoietic function. Mice were received multiple h NSC desensitization during the process of allo-HSCT immune reconstruction. The purpose is to realize h NSC tolerance.Methods Female BALB/c recipients, 9-12 weeks old at time of transplantation were received T-cell-depleted bone marrow cells from female C57BL/6 aged 9-12 weeks. After the success of the allo-HSCT model, 90 model mice were randomly divided into three groups. Group A(NSCT group), group B(desensitization group), and group C(Cs A group). Group B mice were received 3times(1d, 3d and 7d after allo-HSCT) h NSC desensitization. Group A and group C were received 50 ul saline solution. After HSCT stability chimeric and donor T cells implanted into the recipients, the brains were received h NSC injection. One day before h NSC injection, group C mice were daily i.p. injection Cs A(10mg/kg). Group A and group B was received the same volume of saline. 2, 8 and 12 weeks after transplantation, the three groups were received the destruction test in vitro and in vivo survival including h NSC differentiation, rejection and inflammatory reaction test.Results After lethally irradiated, T-cell-deleted bone marrow cells were used to allo-HSCT. 2 weeks after allo-HSCT, all the recipients were totally alive and well condition. 4 weeks after allo-HSCT, the overall survival was 80% and without unhairing.4, 8 weeks after allo-HSCT, the donor chimerism was both76% ±1%. The donor T cells were 37±1% of the implanted cells. 12 weeks after h NSC transplantation, the killing rate of group A, B and C was respectively 28.26%±2.80%?23.94%±2.11%?23.62%±0.15%,P=0.36. There was no significant difference in the three groups. There were no statistically significant difference in the three groups during 2, 8, 12 weeks after h NSC transplantation. 2 weeks after h NSC transplantation, the h NSC survival rate of group A, B and C was 0.42%±0.02%, 0.45%±0.05%, 0.53%±0.01%,P=0.08. 8 weeks after h NSC transplantation, the h NSC survival rate of group A, B and C was 0.21%±0.01%, 0.18%±0.02%, 0.24%±0.02%, P=0.20. 12 weeks after h NSC transplantation, the h NSC survival rate of group A, B and C was 0.16%±0.01%, 0.16%±0.01%, 0.20%±0.01%, P=0.26. 2, 8, 12 weeks after h NSC transplantation, MHC-?, CD68, CD3, Iba–1 were all positive by brain frozen section immunofluorescence staining. 8, 12 weeks after h NSC transplantation, a few positive GFAP cells were detected. Positive ?-tubulin cells cannot be found during 2, 8, 12 weeks after h NSC transplantation.ConclusionsMyeloablative allo-HSCT mice model can be successfully established by T-cell-depleted bone marrow transplantation. Though the recipients were lethally irradiated, allogeneic h NSC transplantation cannot achieve h NSC immune tolerance in allo-HSCT immune reconstruction mice and few h NSC can survive for a long time. Few h NSC can survive for a long time, and differentiated astrocytes can secrete a variety of enzymes at short time. It is timer than donor HSC differentiated into normal white blood cells secreted normal enzymes. The study provides a reference for clinical treatment of IMD brain injury. |
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