| Objective To research whether does the endoplasmic reticulum stress(ERS) exist andthe effect of ERS in the process of adipose stromal cells(ADSCs) differentiation intoneurons, study the relationship between ERS and caspase-dependent apoptosis during theinduced research. So as to improve the efficiency of the induction and increase thenumber of ADSCs derived-neurons, prolong their survival time and provide a theoreticalbasis for further clinical drug screening and cell transplantation.Methods1) ADSCs were isolated and cultured. We observed the morphology ofADSCs under inverted phase contrast microscope and calculated the differentiation rateof ADSCs from passages2.2) ADSCs from passages2were inducted by β-mercaptoethanol, and as the extension of induction time, the cells were assigned touninduction, pre-induction, induction for1,3,5, and8h group.3)The expressions ofneuron specific enolase (NSE), glucose-regulated protein94(GRP94), transcriptionfactor C/EBP homologous protein (CHOP) and Caspase-3were detected byimmunocytochemistry in ADSCs and pre-induction, induction for1,3,5and8hrespectively.4) The expressions of NSE, GRP94, CHOP, Caspase-3were detected byWestern-blotting in ADSCs and pre-induction, induction for1,3,5and8h respectively.5)Endoplasmic reticulum ultrastructure characteristics of induced ADSCs were observed bytransmission electron microscope during the process of ADSCs differentiation intoneuron.6) Using MTT assay to detect the OD values of the living cells of each groupduring the process of ADSCs differentiation into neurons.7) Statistical analysis: Theexperimental data was managed with WPS2013database. We used statistical softwarepackage SPSS17.0to analysis the data, and measurement of data was expressed asmean±SD. The differences in the different time points in the same group were comparedusing the one-way analysis of variance, and P<0.05represented that the difference wasstatistically significant.Results1) The suspension of ADSCs adhered at about the48hour, and the cellsmorphological were diversity, such as long spindle, spindle, round and irregular form andso on. Adherent cells began to proliferate and form converge gradually after the originalgeneration3days. Cells were70%~80%confluent at10~14days. Cells were for passageat this time.2) In the7th day of ADSCs2th generation,3.00±0.70%of cellsdifferentiated; The pre-induction cell differentiation rate was4.80±0.84%. The pre-induction cells protruded short processes and part of the cells floated to death. Wheninduced on1h,63.40±1.14%of the cells differentiated. The nucleus changed bigger androunder than that of the pre-induction cells. The cytoplasms retracted and the cells bodystretched out long protrusions as neuron axons. When induced on3h,72.60±0.89%ofthe cells differentiated. The cytoplasmic refractivity increased, and extracellular halationwas clearly observed. The cells’protrusions became longer. When nduced on5h,88.20±0.83%of the cells differentiated. The cytoplasmic refractivity was further increaseand the cells connected to each other as a mesh. The end of processes appeared branchesand the outcome was similar to neuronal dendrites. At induction for8h,86.4±1.82%of the cells differentiated. There was no significant difference between5and8h (P>0.05).Morphology of the differentiated cells at induction for8h was similar to the cells at5h.3)The expression of NSE in uninduced ADSCs was positive. The positive expression partswere mainly located in cell bodies and protrusions. NSE positive expression rate on5hwas significantly higher than that on1h and3h (P<0.05), but there was no significantdifference between5and8h (P>0.05); The expressions of GRP94in uninducted ADSCspre-induction, induction for1,3,5, and8h were positive. The positive expression partswere mainly located in cell bodies and protrusions. Cell positive expression ratedecreased gradually (P<0.05), but there was no significant difference between5and8h(P>0.05); The expressions of CHOP in uninduction, pre-induction, induction1,3,5,8hgroups were positive, and the positive expression rate increased gradually (P<0.05). Thepositive expression parts were mainly located in nucleus, perinuclear and projections ofthe cells in each time point, and the positive expression level in the nucleus wassignificantly higher than that in the perinuclear and projections; The expressions ofCaspase-3in uninduction, pre-induction, induction1,3,5,8h groups were positive, andpositive expression rate increased gradually (P<0.05). The positive expression parts weremainly located in cell bodies and protrusions. The positive expression level in the cellbodies was significantly higher than that in the protrusions.4) NSE levels in uninduction,pre-induction, induction for1,3,5,8h group increased gradually (P<0.05). However, ithad no significant difference between5and8h (P>0.05); GRP94levels in uninduction,pre-induction, induction1,3,5,8h decreased gradually (P<0.05). It had no significantdifference between uninduction and pre-induction (P>0.05); CHOP levels in uninduction,pre-induction, induction1,3,5,8h increased gradually, and reached the peak on8h(P<0.05); Caspase-3levels in uninduction, pre-induction, induction1,3,5,8h increasedgradually, and reached the peak on8h (P<0.05). It had no significant difference between5and8h (P>0.05).5) At induction for5h, parallel arrangement and non-aligned parallelof rough endoplasmic reticulum and nissl bodies were observed by TEM indifferentiation cells; In part of the induced cells, mild to moderate expansion, even severeswelling expansion and vesicular changes and degranulation of the endoplasmic reticulumscould be observed.6) The absorbance of the living cells decreased gradually inuninduction, pre-induction,induction for1,3,5and8h, and it reached the peak on8hgroup (P<0.05).Conclusions In the process of ADSCs differentiation into neurons, UPR was depressedsignificantly, and the activity of apoptosis caused by ERS was significantly enhanced,and apoptosis caused by ERS signaling pathway was one of the important reasons of cellsdeath in the process of the induced reaction. |
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