| Background and Objective:In Chinese Medicine,the physical function of Spleen involves many aspects:governs transport and transformation of nutrients from foodstuff,controls blood,is in charge of sending up essential substance,is the source of generating Qi and blood,provides the material basis for the acquired constitution,is in charge of thoughts,is responsible for the growth of muscles and four limbs,and mouth is the window of spleen,lip is the reflection of splenic conditions.Above its function determine that its pathology doesn't involve one kind of disease or one kind of syndrome or one organ.Meanwhile,the relation between of spleen and other organs,indicates if hurting spleen and stomach in the body,every kind of diseases would onset,and vice versa.To study on the essence of spleen from spleen deficiency syndrome,always is an important topic to investigate the essence of syndrome with method of integrated traditional Chinese and western medicine.Many scholars approach the essence from biochemistry,physiology, ultra structure and so on by lots of modern technology,even someone go on research from the net of nerve-endocrine secretion-immunity recently,and required several outcome. However,these researches reflect some change of known gene expression product(such as receptor,enzyme,cytokine,and peptide),these keep away from the essence of syndrome. Genome era and post-genome era have been coming,this provides new method to study syndrome from gene.Most scholars presume that there are similarities between Chinese Medicine on holistic approach,yin-yang theory,theory of five organs,etiological theory and genome;they think that the connotation of syndrome is change of gene expression,so they put forward definition of Chinese Medicine syndrome genome.At present,scholars mostly analyze the spectrum of differentially expressed genes with cDNA chip technology to go on research of Chinese Medicine syndrome genome.The representative syndromes are kidney-yang deficiency syndrome,deficiency-cold syndrome,heart-yang deficiency syndrome,lung deficiency syndrome,spleen deficiency syndrome,and so on. We researched the spectrum of differentially expressed genes before on gastritis patients with spleen-qi deficiency syndrome and normal volunteers,chronic superficial gastritis patients with spleen-qi deficiency syndrome and chronic superficial gastritis patients with damp-heat syndrome,ulcerative colitis patients with spleen-qi deficiency syndrome and normal volunteers,ulcerative colitis patients with spleen-qi deficiency syndrome and ulcerative colitis patients with damp-heat syndrome.Our results prove that there are differentially expressed genes on chronic superficial gastritis patients with spleen-qi deficiency syndrome,and most of the genes are down regulation;compared with chronic superficial patients with damp-heat syndrome,differentially expressed genes of chronic superficial gastritis patients with spleen-qi deficiency syndrome are also down regulation, this status consistent with condition which body function behaves repressed under the condition of spleen-qi deficiency,and excited under the condition of damp-heat syndrome. The tendency is same when we made repeated analysis.After analysis lots of data by bioinformatics,we presume that spleen-qi deficiency syndrome has tendency of genes related protein synthesis to down regulation,especially ribosomal protein genes.Ribosomal protein constitutes ribosome,and plays an act to promote protein synthesis.Although ribosomal protein is highly conserved,it will produce live but abnormal phenotype when ribosomal protein gene mutable or absence or unfit chemical modification,this will impact ribosome's function,lower activity of polypeptide, and impact physical function of ribosomal protein,lead to serious results.We choose RPS20,which expressed down regulation repeatedly,to identify its function.Our experiment observes effect on function of IEC-6 cells,after interference RPS20,in order to detect its function.Methods:1.Observation to growth conditions of IEC-6 cellsObserve cell morphology by phase contrast microscope;detect cell viability with trypan blue;draw growth curve by viable cell counts and MTT colorimetric assay.2.Optimization of transfect conditions of IEC-6 cells in the process of RNA interference For example,in 24 well plates,adjust the ratio between cationic liposome and FAM-siRNA, transfect FAM-siRNA into IEC-6 cells with cationic liposome,then detect fluorescence intensity after interference by fluorescence microscope and flow cytometry.3.Screen the best efficient siRNA to RPS203.1 Detect the expression of RPS20 mRNA by RT-PCRSet blank group,group siRNA1,group siRNA2,group siRNA3.As 6 well plate for an example,plant 8×10~5 cells every well,on the second day,transfect following this condition: whole transfection volume is 2.5mL,final concentration of siRNA is 160nM,the volume of Lipofectamine2000 is 4.2μL.Then detect the expression of RPS20 mRNA after transfection 24h,48h,72h by RT-PCR.3.2 Detect the expression of RPS20 mRNA by fluorescence quantitative RT-PCR Set blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1,Then detect the expression of RPS20 mRNA by fluorescence quantitative RT-PCR after transfection 48h.3.3 Detect the expression of RPS20 protein by western-blotSet blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1.Then detect the expression of RPS20 protein by western-blot after transfection 24h,48h,72h.4.Change of cellular morphology and function after RNA interference4.1 Change of cells morphology after RNA interferenceSet blank group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1.Observe cell morphology by phase contrast microscope after interference 24h, 48h,72h.Set blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Observe cell ultra structure with transmission electron microscope after interference 48h.4.2 Effect on migration of IEC-6 cells after RNA interferenceSet blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1.Establish migration model by scratch after transfection 48h,and after 24h,take five pictures per well,and count migrated cells by IPP software to observe change of cellular migration ability.4.3 Effect on proliferation of IEC-6 cells after RNA interferenceSet blank group,group siRNA1,group siRNA2,group siRNA3.Count cell number after interference 24h,48h,72h by cell counting.Set blank group,transfection control group,group siRNA1,group siRNA2,group siRNA3. Transfection conditions are same to 3.1,As 96 well plate for an example,plant 2.5×10~4cells every well,on the second day,transfect following this condition:whole transfection volume is 0.15mL,final concentration of siRNA is 160nM,the volume of Lipofectamine2000 is 4.2μL.Then detect the proliferation efficiency of cells after transfection 24h,48h,72h by MTT colorimetric assay.4.4 Effect on apoptosis of IEC-6 cells after RNA interference 48h Set blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 2,observe effect on apoptosis of IEC-6 cells after RNA interference 48h by Annexin V-FITC.Results1.Observation to growth conditions of IEC-6 cellsIEC-6 cells had typically morphological characteristic of normal epithelial crypt cell,high viability;the third day after planted,IEC-6 cells got into exponential phase of growth, period from 7th day to 10th day is platform,then IEC-6 cells grew slowly.2.Optimization of transfect conditions of IEC-6 cells in the process of RNA interference Detected transfection efficiency by fluorescence microscope and scored to go on rank sum test;we found,the score was 0 when there was only transfection agent or FAM-siRNA;the groups with transfection agent and FAM-siRNA,had strong or weak fluorescence intensity. Detected transfection efficiency by flow cytometry,the combination of Lipofectamine2000 1.0μL+ FAM-siRNA 160nM,had the highest transfection efficiency,but blank group was only 1%.3.Screen the best efficient siRNA to RPS203.1 Detect the expression of RPS20 mRNA by RT-PCRComparison to blank group,the expression of RPS20 mRNA in group siRNA1 decreased significantly(P＜0.05)after interference 24h,however,group siRNA2 and group siRNA3 didn't suppress expression of RPS20 mRNA significantly;After interference 48h,72h, group siRNA1,group siRNA2 and group siRNA3 didn't suppress the expression of RPS20 mRNA significantly.3.2 Detect the expression of RPS20 mRNA by fluorescence quantitative RT-PCRComparison to blank group,the expression of RPS20 mRNA in group siRNA1,siRNA2, siRNA3 was interfered successfully,interference efficiency was about 80%-90%.3.3 Detect the expression of RPS20 protein by western-blotComparison to blank group,after interference 24h,the expression of RPS20 protein in group siRNA1,siRNA2 decreased significantly(P＜0.028,P＜0.049),the expression of RPS20 protein in group siRNA3 trended to low,but the variance wasn't significantly(P＜0.065);Comparison to blank group,after interference 48h,the expression of RPS20 protein in group siRNA2,siRNA3 decreased significantly(P＜0.028,P＜0.006),the expression of RPS20 protein in group siRNA1 trended to low,but the variance wasn't significantly(P＜0.619);Comparison to blank group,after interference 72h,the expression of RPS20 protein in group siRNA2,siRNA3 decreased significantly(P＜0.019,P＜0.015),the expression of RPS20 protein in group siRNA1 trended to low,but the variance wasn't significantly(P＜0.056).4.Change of cell morphology and function after RNA interference4.1 Change of cells morphology after RNA interferenceUnder the detection of phase contrast microscope,comparison to blank group,the cell number of group siRNA1,siRNA2,siRNA3 decreased,but there was no change on cell morphology.Under the detection of transmission electron microscope after interference 48h, cellular membrane and nucleus were complete in blank group,there were abundant cell organelles;transfection control group and negative control group had integrated nucleus, and a few of vacuoles and some plasmid;group siRNA1,siRNA2,siRNA3 had no integrated membrane,and there were lots of vacuoles,cytolysosome number increased, vacuoles in group siRNA2,siRNA3 maybe comprise high electron density content.Cell organelle decreased,and some of them were obscure.4.2 Effect on migration of IEC-6 cells after RNA interferenceComparison to blank group,after interference 48h,group siRNA1,siRNA2,siRNA3 suppressed cell migration significantly(P＜0.01,P＜0.01,P＜0.01).4.3 Effect on proliferation of IEC-6 cells after RNA interferenceComparison to blank group,after interference 24h,48h,72h,the growth cell number of group siRNA1,siRNA2,siRNA3 was suppressed significantly(P＜0.01)by cell counting. To detect proliferation of IEC-6 cells by MTT,comparison to blank group,after interference 24h,group siRNA2,siRNA3 suppressed cell proliferation significantly(P＜0.05),after interference 48h,group siRNA2 could suppress cell proliferation significantly(P＜0.05),after interference 72h,group siRNA2,siRNA3 could suppress significantly cell proliferation(P＜0.01,P＜0.05).4.4 Effect on apoptosis of IEC-6 cells after RNA interference 48hComparison to blank group,viable cells of group siRNA1 decreased,the difference was significantly(P＜0.05),and early apoptotic cells increased,but the difference was not significantly.Comparison to blank group,cells dead normally and late apoptotic cells of group siRNA1,siRNA2,siRNA3,had no significantly difference.Conclusion:1.After planted IEC-6 cells 10 days,growth curve changed from platform to the period increased slowly.Due to begin to present differentiation,IEC-6 cells grew slowly.2.This study design and compose three pairs siRNAs to RPS20 of rats,and they interference the expression of RPS20 on the aspect of mRNA and protein successfully,and prove their validity.3.After interference,IEC-6 cells cytoplasm present vacuolization,and its migration,and proliferation were suppressed,but there was no apoptosis to happen.These results indicate that interfering expression of RPS20 may affect the function of digestion and absorption and gastrointestinal mucosa healing,which is in accordance with condition of low level on digestive and absorptive function and gastrointestinal mucosa healing.This result support characteristic from clinical spectrum of gene expressed differently,support the hypothesis that essence of pi-deficiency syndrome maybe relate with RPS20 lowly expression.
|
PDF Full Text Request