Clinical Study On Blink Reflex Assessment Of Central Nervous System Damage In Type 2 Diabetes Mellitus And Experimental Study On Optimization Of Human Umbilical Cord Mesenchymal Stem Cells To Neural Stem Cells By Astragalus Polysaccharide And Ginsenoside

Clinical study on blink reflex assessment of central nervous system damage in type 2 diabetes mellitusBackground and purpose:Complications of diabetes can involve multiple organs and systems,including the nervous system.Previous researches on electrophysiology mainly focus on the peripheral nervous system,less on the cranial nerve and central nervous system.Therefore,early central nervous system damage in diabetic patients is more likely to be overlooked and misdiagnosed.The blink reflex provides an objective assessment of the cranial and central nervous systems.However,the relationship between body mass index,dizziness,and blink reflex has not been explored in patients with type 2diabetes mellitus(T2DM).In addition,R2 duration,as one of the blink reflection parameters,has not been studied so far.The purpose of this study was to investigate the characteristics and influencing factors of blink reflex in T2DM patients,so as to provide electrophysiological basis for early diagnosis and early treatment of central nervous system damage in T2DM patients and establish a prediction model for central nervous system damage in patients with T2DM.Methods:A cross-sectional observational study was designed to enrol healthy volunteers and hospitalized patients with T2DM.The relationships between these parameters and sex,age,body mass index,duration of T2DM,hemoglobin A1c,distal symmetrical polyneuropathy(DSPN),and dizziness symptoms were analyzed by t-test,one-way ANOVA,logistic regression analysis and ROC curve analysis of two independent samples.Results:The results showed that blink reflex latencies(including R1,ipsilateral R2,and contralateral R2 latency)were negatively associated with body mass index but were positively correlated with the duration of T2DM.There were no correlations between blink reflex parameters and sex,age,and hemoglobin A1c.Patients with DSPN had longer blink reflex latencies and shorter R2 durations than those without DSPN.Patients with dizziness had longer latencies(including R1,ipsilateral R2,and contralateral R2 latency)and shorter R2 durations(including ipsilateral R2 and contralateral R2 duration)than those without dizziness.R2 duration was also a predictive factor for blink reflex abnormality.R2 latency was the most sensitive factor and the optimal predictor of dizziness.Conclusion:Patients with T2DM with low body mass index,long duration of T2DM,DSPN,and dizziness-related symptoms had more abnormal blink reflex parameters,indicating more serious injuries to the cranial nerves or the central nervous system.Experimental study on optimization of human umbilical cord mesenchymal stem cells to neural stem cells by astragalus polysaccharide and ginsenoside Rg1Background and Purpose:Multiple studies have shown that diabetes can cause degenerative changes in the central nervous system,leading to cognitive decline and other symptoms.In addition,with the development of society,the incidence of cerebral apoplexy,neurological degeneration and neurological diseases caused by trauma increases year by year,but the clinical treatment effect of these diseases is not ideal at present.Neural stem cell(NSCs)transplantation has brought new hope for the treatment of these diseases.Human neural stem cells derived from embryonic tissue and the brains of aborted fetuses have been considered the most promising candidates for treating neurodegen-erative diseases and other central nervous system diseases.However,immune rejection,ethical and cost issues limit the use of these allogeneic neural stem cells.Studies have shown that Human umbilical cord mesenchymal stem cells(hUCMSCs)can be transformed into neural stem cells and neuro-like cells and still maintain immunomodulatory and antioxidant activities.Transretinoic acid,Centella asiatica and other inducers can promote the proportion of neural stem cells or neuro-like cells transformed by hUCMSCs.However,these drugs can reduce cell proliferation or promote cell apoptosis while promoting cell transformation.The aim of this study was to investigate the effect of Astragalus polysaccharides(APS)and ginsenoside Rg1 on the transformation of hUCMSCs into neural stem cells.Methods:1.Isolation,culture and identification of hUCMSCs:1)Wharton jelly was separated from the umbilical cord,cut into fragments of about 1mm~2,and evenly spread in a 175cm~2 sterile plastic culture bottle for culture;2)Morphological observation and flow cytometry were used to detect the expression of cell-related molecular markers;3)Osteogenic,adipogenic and chondrogenic differentiation of hUCMSCs was induced in vitro to detect its multidirectional differentiation potential.2.Production and identification of NSCs:1)hUCMSCs were added into the induction medium of neural stem cells for routine culture;2)The self-renewal ability of the induced nerve globules was detected by continuous culture and secondary pellet-forming experiment;3)The ability of these nerve spheres to redifferentiate into neurons and glial cells was detected by immunofluorescence staining;4)Flow cytometry and cellular immunofluorescence staining were used to detect the expression of Nestin and SOX2,markers of neural stem cells in the induced nerve bulb;5)Flow cytometry was used to detect the expression of surface markers CD90and human leukocyte associated antigen D(HLA-DR)of induced NSCs mesenchymal stem cells.3.Determination of the optimal dosage of APS and ginsenoside Rg1:1)Preparation of mesenchymal stem cell media containing different concentrations of APS and ginsenoside Rg1.CCK-8 assay was used to detect the effects of different concentrations of APS and ginsenoside Rg1 on the proliferation of hUCMSCs;2)To prepare neural stem cell induction media with different concentrations of APS and ginsenoside Rg1.CCK-8 assay was used to detect the effects of different concentrations of APS and ginsenoside Rg1 on cell viability during the transform-ation of hUCMSCs to NSCs.4.APS and ginsenoside Rg1 inhibited cell apoptosis during hUCMSCs to NSCs.Cell apoptosis in negative control group,APS group and ginsenoside Rg1 group was detected by Annexin V-PI assay.5.APS and ginsenoside Rg1 improved the conversion efficiency of hUCMSCs to NSCs:CD90,HLA-DR,Nestin and SOX2 proteins in negative control group,APS group and ginsenoside Rg1 group were quantified by flow cytometry and cellular immunofluorescence staining,respectively.6.Preliminary study on the relevant mechanisms by which APS and ginsenoside Rg1 improve the conversion efficiency of hUCMSCs to NSCs:Q-PCR was used to detect the changes in m RNA expression levels of related signaling pathways.Results:1.HUCMSCs can be obtained in large quantities by tissue mass adherent culture method.The cells grew in a uniform spindle-like swirl and had the ability to grow adherently inside a plastic culture bottle.The expressions of CD44,CD73,CD90,and CD105 were all greater than 95%,and the expressions of CD34,CD45,and HLA-DR were all less than 2%.In vitro induction experiments showed that hUCMSCs could successfully differentiate into osteocytes,adipocytes and chondrocytes,with multiple differentiation potential.2.The hUCMSCS-derived nerve globules have the ability of self-renewal and the potential of redifferentiation to neurons and glial cells.The expression of neural stem cell markers Nestin and SOX2 in these nerve globules was positive.3.The optimal dosage of APS and ginsenoside Rg1 to promote the proliferation and transformation of hUCMSCs to NSCs were 1.6mmol/L and 10?mol/L,respect-ively.4.APS and ginsenoside Rg1 can effectively inhibit cell apoptosis during the transformation of hUCMSCs to NSCs.5.The expression of CD90 protein decreased with the extension of induction time,and the decrease of CD90 expression in APS group and Rg1 group was more obvious than that in negative control group.6.NSCs induced by APS and ginsenoside Rg1 still did not express HLA-DR.7.The protein expression levels of Nestin and SOX2 in APS group and ginsenoside Rg1 group were higher than those in negative control group.8.APS and ginsenoside Rg1 can down-regulate the m RNA expressions of Wnt/?-catenin and Notch signaling pathway related genes GSK3?,?-catenin,Notch and Hes1 during the transformation of hUCMSCs to NSCs.Conclusion:1.APS and ginsenoside Rg1 can significantly optimize the transformation of human umbilical cord mesenchymal stem cells to neural stem cells.2.The improvement of conversion efficiency of APS and ginsenoside Rg1 may be related to the inhibition of Wnt/?-catenin and Notch signaling pathways.3.Transplantation of NSCs induced by APS or ginsenoside Rg1 is expected to bring more effective efficacy for the treatment of diabetic central nervous system damage,stroke,neurodegeneration and neurological diseases caused by trauma.