| Objectives:1.The BALB/c mouse allergic rhinitis(AR)model was established and evaluated to provide experimental subjects for further research.2.Preparation of human umbilical cord mesenchymal stem cells(hUCMSC),including isolation,expansion culture,passage,cryopreservation and preparation of related drops,providing materials for experimental research and clinical use.3.The mouse AR model was used to evaluate the efficacy of transplantation of hUCMSC in the treatment of AR.The distribution of green fluorescent protein(GFP)-labeled hUCMSC in vivo and the therapeutic mechanism of hUCMSC were elucidated,which provided technical methods and theoretical basis for the application of hUCMSC in clinical treatment.Methods:1.Establishment and evaluation of mouse AR model:15 healthy 4-6 week old BALB/c female mice were randomly divided into groups A,B,and N,with 5 mice in each group,which were normally raised in SPF environment.In group A and B,200 ?l of sensitizer with concentration of 25 ?g OVA/2 mg Al(OH)3 and 50 ?g OVA/2 mg Al(OH)3 was intraperitoneally injected once every week for 3 times.The droplets were stimulated with 20 ?l of OVA(10 ?l each of the left and right nostrils)by 7 consecutive times;the N group was treated with 200?l of normal saline instead of OVA,the frequency of intraperitoneal injection and nasal drops was the same as that of groups A and B.The mice were observed daily for sneezing and runny nose after the operation of the nasal drops,and the scores were calculated.HE staining was performed on the nasal mucosa of the mice to observe the changes of histological structure.The IL-4 and IFN-y in the serum of each group was detected by ELISA;the total protein content in the serum of each group was determined by BCA method;the transcription levels of IL-4,6,10 and IFN-y mRNA in the spleen tissues of each group were determined by PCR.The established mouse model was identified and evaluated to determine the best AR model creation method.2.hUCMSC culture,identification and GFP labeling in vitro:Take the umbilical cord of healthy full-term newborn,cut it into 1 mm3 tissue block and isolate it,culture it,and after 3 passages,observe the cells under inverted phase contrast microscope.The growth characteristics,flow cytometry analysis of hUCMSC positive expression ratio of CD73,CD90,CD105,CD34 and CD45 antigen,induced hUCMSC differentiation into fat-like,osteogenic and chondrogenic cells,analysis of their differentiation potential.hUCMSC were labeled in vitro using GFP,and the labeling rate of GFP was analyzed by fluorescence microscopy and flow cytometry.3.Treatment of hUCMSC:30 BALB/c female mouse models of 4-6 weeks old were established according to the best AR model creation method defined in the previous experiment.They were randomly divided into model treatment group A,B group and model control group M,each group 10 only.Mice in groups A and B were respectively treated with 200 ?l of hUCMSC suspension at a concentration of 2×105 cells/ml.The mice were treated with intraperitoneal and tail vein injections once every 3 days for 6 times;no treatment was given in group M.Add a group of healthy control N groups,the number is 10,without any treatment.Four groups of mice were housed in an SPF environment,and on the second day of the last injection treatment,the mice were sacrificed.4.Efficacy and mechanism evaluation of hUCMSC:? The sneezing and runny nose were observed every day after the start of treatment,and the score was calculated.The mouse nasal mucosa was stained with HE staining to observe the changes of histological structure.ELISA was used to detect the concentration of IL-4 and IFN-y in the serum of each group of mice.Determination of total protein content in serum of each group by BCA method.The mRNA levels of IL-4,6,10 and IFN-y in spleen tissues of each group were determined by PCR,and the therapeutic effect of hUCMSC on AR model was evaluated.?GFP-labeled hUCMSC was injected into the mice in three groups by intranasal,intraperitoneal injection and tail vein injection.After harvesting,the migration and colonization of hUCMSC in mice were observed by fluorescence standard of tissue section and flow cytometry,and the cells were observed to enter the nasal cavity situation.5.The data obtained from each experiment used the meanąstandard deviation to indicate the significance of the differences between the experimental groups.SPSS statistical software was used to compare the two groups by repeated measures and analysis of variance.P<0.05 was used as the significant difference.Results:1.Model establishment and evaluation results:Compared with N group,the symptom scores of mice in group A and B were significantly increased(p<0.05),among which group A was more obvious.The results of HE staining of nasal mucosa in group A and group B showed local inflammatory cell infiltration was obvious compared with the N group;the serum levels of IFN-?,IL-4 and total protein were significantly increased in group A(p<0.05),serum IFN-? levels in group B also increased(p<0.05),but serum IL-4 and total protein levels in group B only increased slightly(p>0.05).The transcription levels of IL-6 in group B and IL-4,6 mRNA in group A were significantly increased(p<0.05),and the mRNA levels of other factors were slightly increased(p>0.05).2.Culture,identification and GFP labeling of hUCMSC:hUCMSC were cultured by tissue block-cut direct adherence method.After about 1 week,cells were found to climb out of the tissue block.After repeated tissue adherent culture,the cells were fibrillar-like and had a typical vortex.Growth characteristics;the expression of surface antigens in the fourth generation of hUCMSC,the positive expression ratios of CD73,CD90 and CD105 were 100.0%,99.1%and 99.2%,respectively,while the positive expression ratios of CD34 and CD45 were 0.05%and 0.30%;induced adipogenic differentiation at 2 weeks,oil red O staining was positive;induced osteogenic differentiation for 3 weeks,alizarin red staining was positive;induced cartilage for 4 weeks,azinc blue staining was positive.hUCMSC was successfully labeled with GFP in vitro,and the cells showed obvious green fluorescence under fluorescence microscope.The positive rate of GFP labeling by flow cytometry was 100%.3.Efficacy and mechanism evaluation of hUCMSC:? Compared with the M group,the mouse AR model gradually decreased from the beginning to the end of treatment,and the scores of the A and B groups decreased gradually from the 4th day(p<0.05),and the 15th day was close to 5 points;The cilia structure of group A and group B was complete and the infiltration of inflammatory cells was decreased.The serum total protein content of group A decreased slightly(p>0.05),and the decrease of group B was more obvious(p<0.05);The serum levels of IFN-y and IL-4 in group A and group B decreased(p<0.05).The levels of IL-10 and IFN-y mRNA in spleen tissue of group A decreased significantly(p<0.05).Group A IL-4 mRNA transcription level decreased slightly(p>0.05),and the IL-4,10 and IFN-y mRNA transcription levels in group B decreased significantly(p<0.05),but the IL-6 mRNA transcription levels in group A and B increased,and group A was more obvious(p<0.05).?The hUCMSC were traced in mice,and the tissue sections were observed by immunofluorescence.The fluorescent cells were found in the heart,lung,ovary and nasal mucosa,and the fluorescent cells in the nasal mucosa of each group were the most in the second week;The cytometric analysis further confirmed that the fluorescent cells of the nasal mucosa of each group were the most in the second week.Conclusions:1.The mouse AR model was established by intraperitoneal injection of OVA plus nasal stimulation,and the model was effective and reproducible.2.hUCMSC with morphological,growth characteristics,cell surface expression markers and induced differentiation potential were obtained by repeated direct adherence culture of multiple tissue blocks.GFP-labeled hUCMSC has good effect and stable cells,which can be used for tracing in animals.3.Both intraperitoneal and tail vein injections of hUCMSC in the treatment of AR model of mice have a certain effect,and the effect of tail vein injection is better.In vivo tracing of hUCMSC animals showed that the mice in the three groups had the most hUCMSC in the nose at the 2nd week. |
PDF Full Text Request