| BACKGROUND:Amyotrophic Lateral Sclerosis(ALS)is a fatal neurodegenerative disease that selectively invades upper and lower motor neurons and causes progressive paralysis and muscle atrophy.Its clinical manifestations are mainly muscle atrophy,weakness,and tendon hyperreflexia,pathological reflexes,etc,after the onset of progressive aggravation,mostly due to bulbar paralysis,respiratory muscle weakness combined with pulmonary infection and death within 3 to 5 years.At present,the pathogenesis of ALS is still unclear.Many experiments have shown that the immune inflammatory response plays an important role in the pathological process of ALS disease.In the early stage of ALS disease,mainly protective neuroimmune inflammation is regulated.The body exerts immunomodulatory effects by releasing some anti-inflammatory factors such as IL-4,IL-10 and TGF-β.In the middle and late stages of the disease,neurotoxic inflammatory responses predominate,and neurons are damaged by release of various inflammatory factors such as TNF-a,IL-6,and IL-1β.At present,because the etiology of ALS remains unclear and the pathogenesis is complex,there are still no effective prevention and treatment measures in the clinic.Most researchers explore meaningful treatments from three perspectives:drug therapy,gene therapy,and stem cell therapy.However,due to the lack of effective drugs,gene therapy and other clinical trials are not optimistic.Therefore,stem cell therapy has become the most promising therapeutic strategy for ALS.Compared with neural stem cells and bone marrow mesenchymal stem cells,amniotic mesenchymal stem cells(hAMSCs)have the advantages of abundant sources,convenient materials,no tumorigenicity,low immunogenicity,and immunosuppressive effects,and have stronger proliferation.The ability and stem cell characteristics become ideal immune privileged cells.Therefore,hAMSCs may provide a new treatment for ALS treatment of seed cells,which has potential clinical application value.Previous research in our group showed that in the early stages of disease in transgenic ALS mouse models,multiple hAMSCs intravenous injections have a therapeutic effect,which can delay the disease progression in transgenic ALS mouse models,improve its motor function,and reduce the spinal nerve inflammatory response.Reduce the loss of motor neurons;hAMSCs and microglial cells can significantly inhibit their proliferation and migration when co-cultured in vitro,but the specific mechanism remains unclear.Since the majority of clinical patients have been diagnosed at the middle and late stage of the disease,it is of great significance to provide hAMSCs transplantation therapy in the late and advanced stages of ALS disease and explore its therapeutic effects and mechanisms.Therefore,this study intends to use ALS(hSOD1-G93A)transgenic mouse as a research object,the hAMSCs were intravenously transplanted to the middle and late disease transgenic mice in vivo,from the perspective of stem cell immune immune inflammation,to explore hAMSCs for ALS transgenic mice Therapeutic mechanisms provide a theoretical basis for the clinical treatment of ALS.OBJECT:From the perspective of stem cell immuno-inflammatory regulation,the therapeutic effects and mechanisms of hAMSCs transplantation on the late stage of ALS transgenic mice were explored,thus providing a theoretical basis for the clinical treatment of ALS.METHODS:Fresh amniotic membrane voluntarily donated by healthy cesarean section mothers was selected,and the hAMSCs in the amniotic stroma were gradually separated and purified by a combination of trypsin and collagenase digestion method and tissue block crawling method.The hSODl-G93A hemizygous transgenic male mating mice were mated with female background mice to breed progeny mice,and the progeny mouse genotypes were identified by PCR.The experimental mice were divided into 3 groups:hAMSCs-treated group,PBS-treated group and normal control group,12 mice in each group.At the age of 16 weeks,approximately 1’10 6 cells were injected from the tail vein(resuspended in 300μl of PBS)or 300μl PBS.At the age of 18 weeks,blood,spleen,spinal cord(lumbar enlargement)and other tissues were taken respectively.Corresponding methods were used to detect the corresponding indicators,and the role and mechanism of hAMSCs in the regulation of immune inflammation were analyzed.RESULTS:(1)By ELISA,the levels of anti-inflammatory cytokines such as TGF-p in serum of hAMSCs treatment group were higher than those of PBS treatment group,and the inflammatory factors such as TNF-α,IL-1α,IL-1β,IL-6 were lower than those of PBS treatment.There were differences,but there was no statistical significance(p>0.05).(2)By flow cytometry,compared with the PBS treatment group,the spleen,blood Foxp3,Tregs,tDC,etc.in the hAMSCs treatment group were all increased,with statistically significant differences(p<0.05).(3)Immunohistochemistry showed that the number of ChAT and NeuN-positive motor neurons in the spinal cord tissue of the hAMSCs-treated group and the PBS-treated group was lower than that of the wild-type control group,while the hAMSCs-treated group was higher than the PBS-treated group,but there was no statistical difference(p>0.05);The glial cell markers GFAP and Ibal in the spinal cord were higher in the hAMSCs-treated group and the PBS-treated group than in the wild-type control group,but the PBS group was higher than the hAMSCs-treated group,but there was no statistical difference(p>0.05).CONCLUSION:In the treatment of hAMSCs at the advanced and advanced stages of ALS disease,the anti-inflammatory cytokine TGFβ in the body has a tendency to increase,and the inflammatory factors such as TNF-α,IL-1α,IL-1β,and IL-6 have a decreasing trend.Lymphocyte Treg,tDC,etc.The index has an increasing trend,suggesting that hAMSCs have a therapeutic effect in the late stage of ALS disease,but the disease progresses rapidly,has been irreversible,stem cell treatment is late,and may play a limited role,so it can not achieve an obvious therapeutic effect. |
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