Study On The Effect Of HUC-MSCs-derived Exosomes Transporting Mir-214 On Hepatic Stellate Cells

Objective:To investigate the effect of exosomes derived from human umbilical cord mesenchymal stem cells(hUC-MSCs)transporting miR-214 on hepatic stellate cells(hepatic stellate cells,HSCs),and to observe the proliferation,migration and activation related factors expression of HSCs,so as to explore the effect of exosomes derived from hUC-MSCs transporting miR-214 on hepatic stellate cells,so as to provide new molecular mechanism and new treatment ideas for hepatic fibrosis.Methods:1.Culture and identification of human umbilical cord mesenchymal stem cells:human umbilical cord mesenchymal stem cells were isolated and cultured by tissue adherent method,and the expressions of CD34,CD45,CD90,CD105 and Oct-4 cell surface specific molecules were detected by flow cytometry.Umbilical cord mesenchymal stem cells(hUC-MSCs)were induced to differentiate into osteoblasts and adipocytes to identify their multi-directional differentiation potential.2.Overexpression and inhibition of mir214 expression in human umbilical cord mesenchymal stem cells:according to the instructions of ribofecttmcp transfection reagent,the qualified umbilical cord mesenchymal stem cells were treated,and the overexpression and inhibition of mir214 expression were detected by q-RT-PCR.3.Extraction and identification of exosomes of miR214 transfected umbilical cord mesenchymal stem cells:the supematant of miR214 transfected umbilical cord mesenchymal stem cells was collected and exosomes were isolated The diameter of exosomes was analyzed by particle size analysis.The morphological characteristics of exosomes were observed by transmission electron microscope.4.Coculture experiment of hUC-MSCs derived exosomes and hepatic stellate cells:hepatic stellate cells were divided into five groups:blank control group,mir214 overexpression induction group,mir214 negative control induction group,mir214 inhibition induction group and mir214 negative control induction group.The blank control group was cultured in the exosome free serum medium according to the common method,and the other induction groups were cultured with different groups of exo.After co culture with exosomes for 12,24,48 and 72 hours,CCK-8 was used to detect the proliferation of Ix-2,and scratch test was used to test the migration ability of lx-2 in different groups.Total RNA was extracted from cells of each group,and the mRNA expression of collagen synthesis related proteins was detected by q-RT-PCRResults:1.Culture and identification of hUC-MSCs:umbilical cord mesenchymal stem cells were isolated and cultured to the fourth generation by tissue adherent method.Under the microscope,the fibroblast like cells were observed to grow,arranged closely and showed whirlpool growth.After 80-90%fusion of cells,the flow cytometry results showed that:hUC-MSCs were the surface markers(n=8),hUC-MSCs were the surface markers of hUC-MSCs-The positive expression of CD105(99.12%± 0.64%),CD90(99.25%± 0.34%),CD45(1.60%± 1.04%),CD34(0.84%±0.21%)and Oct-4(68.71%±5.32%)in each group of MSCs,respectively,which indicated that P5 HUC MSCs highly expressed CD105 and CD90,but did not express CD34 and CD45.After 28 days of osteogenic differentiation,the calcium nodules of hUC-MSCs were stained dark red by alizarin red staining.After 28 days of adipogenic induction and differentiation of hUC-MSCs,many tiny fat droplets could be seen under the microscope by oil red O staining.These results indicate that the hUC-MSCs isolated and cultured by tissue block adherent method have the potential of multi-directional differentiation.2.Overexpression and inhibition of mir214 expression in hUC-MSCs:the experiment was divided into blank control group,mir214mimics,mir214mimicsn,mir214inhibitor,mir214inhibitor n transfection group.Mir214 expression was detected by QRT PCR at 24 h,48 h and 72 h after transfection(n=3).The results showed that the expression of mir214 in mir214 mimics group was significantly higher than that in control group(P<0.05).In time,the expression of mir214 in mir214mimics group was significantly higher than that in 24h group(P<0.05).3.Extraction and identification of the exosomes of umbilical cord mesenchymal stem cells transfected with mir214:the exosomes were detected by particle size and observed by electron microscope,and hUC-MSCs were observed by transmission electron microscope The main shape of exosomes is round vesicles,the size is mainly between 30～200nm,most of them are exosomes;using NTA technology to analyze the extracted hUC-MSCs-exo,it is found that the diameter of extracted sample particles is between 50-250NM,and the peak value is about 120nm,which is consistent with the characteristics of exosomes.4.Results:(1)The co-culture experiment of hUC-MSc derived exosomes and LX-2:(1)the morphology ofLX-2 cells was observed under phase contrast inverted microscope.LX-2 cells differentiated in different degrees under the stimulation of different concentrations of TGF-β1.The morphology of LX-2 cells in blank control group showed short fusiform or polygonal shape.Compared with the blank control group,LX-2cells transformed from the original star shaped or short spindle shaped fibroblasts into myofibroblast like cells with spindle shaped and flat fiber structure.The results showed that LX-2 cells were activated significantly by TGF-β1 at the concentration of 10 ng/ml for 48 hours(2)In this experiment,the relative expression level of miR-214 was detected by real-time fluorescent quantitative PCR in control group and TGF-β1 stimulation group.The results showed that the relative expression level of miR-214 in TGF-β1-stimulated group was significantly higher than that in ctrol group(P<0.01)(3)QRT PCR was used to detect HSCs such as collagen type 1(coll)and α-smooth muscle actin(α-SMA)The results showed that the relative expression levels of coll and α-SMA were up-regulated in 10 ng/ml group compared with 5 ng/ml group at 24 h and 48 h after TGF-β1 stimulation(P<0.05),and there was no significant difference between 5 ng/ml group and 10 ng/ml group at 72 h after TGF-β1 stimulation(P>0.05).(4)Methods:LX-2 cells were co cultured with hUC-MSCs-exo-miR214(l0ug/ml)of different transfection groups.After 12h,24h and 48h,CCK-8 assay was used to detect the effect of different groups of exosomes on the proliferation of LX-2 cells Compared with mir214inhibitor N group,the absorbance of LX-2 cells in mir214inhibitor N group was significantly lower(P<0.01).(5)The effect of hUC-msc-exo-mirna214 on the migration of LX-2 cells was detected by scratch test.The results showed that the migration area of lx-2 cells in mir214mimics group was significantly wider than that in mir214mimics N group(P<0.05),Compared with mir214inhibitor N group,the migration area of LX-2 cells in mir214inhibitor group was significantly reduced(P<0.05)(6)Real time quantitative PCR was used to detect the relative expression levels of coll and α-SMA at 48h,and the activation status of LX-2 was detected.The results showed that the relative expression levels of coll and α-SMA in lx-2 cells in mir214mimics group were significantly higher than those in mir214mimics N group(P<0.01),while the relative expression levels of lx-2 in mir214inhibitor N group were significantly higher than those in mir214mimics N group(P<0.01)The relative expression levels of coll and α-SMA were significantly decreased(P<0.01).Conclusion(s):1.Umbilical cord mesenchymal stem cells cultured by tissue adherent method were in accordance with their specific molecular expression characteristics,and had the potential of multi-directional differentiation.2.Overexpression of miR214 hUC-MSC-exo can promote the proliferation,migration and activation of LX-2,and inhibition of miR214 hUC-MSc-exo can inhibit the proliferation,migration and activation of LX-2.