Experimental Study Of Differences In MRNA Expression In Rat Ileum Spleen Deficiency Syndrome

Purpose：This experiment aimed at testing Desmin, Krt8, Ezrin, pyruvate kinase,glyceraldehyde-3phosphate dehydrogenase protein which related with spleen-Yang on theapplication of proteomics technology identification by RT-PCR. Thus, detecting mRNAexpression difference of these proteins, papering the physiological and pathologicalmechanism of spleen-Yang deficiency syndrome and the functional mechanism of warmlyinvigorating spleen-Yang medicine from gene level, so as to provide basis for clinicaldiagnosis and treatment.Material and method：The method was applying SPF level36in SD rats divided intothree groups which adopt improper diet (feeding cabbage on odd-numbered days, feeding lardon even-numbered days) and overwork (swimming to the endurance limit), with Senna andintragastric administration method for flooding to shape in the spleen-Yang deficiency rats,Fuzi Lizhong decoction of Chinese medicine, saline intragastric administration ofinterventions. Proteomics differential protein in rat ileum tissue identification of spleen-Yangdeficiency. Used RT-PCR method, after Primer Design, RNA extraction, detection, RTsystem, analysis of the PCR reaction, electrophoresis and gel imaging system, detected thedifference of mRNA expression from gene level.Results：1. Proteomic identification of differential proteins: Spleen-Yang deficiency model groupcompared with healthy control group and the Chinese drugs therapeutic group has6associated differences in protein, including4up and2down proteins. The4proteins for upsincluding Desmin, Krt8, pyruvate kinase, Ezrin.And2downs including glyceraldehyde-3phosphate dehydrogenase and Actin, Desmin, Krt8and Actin belong to the cytoskeletonproteins; glyceraldehyde-3phosphate dehydrogenase and pyruvate kinase are involved inglucose metabolism,Ezrin belongs to the the signaling pathway proteins.2. Using RT-PCR molecular biology techniques, the role of the reverse transcriptase enzymesynthesis of cDNA from RNA, then cDNA as template, amplified fragment synthesispurposes. Associated syndromes of spleen-Yang deficiency model rats6proteome differences in gene expression levels: the level of mRNA expression in rats with spleen-Yang deficiencyof protein groups are not significantly different than normal (P>0.05). There are significantdifferences in mRNA expression levels of Krt8, pyruvate kinase, Ezrin, glyceraldehyde-3phosphate dehydrogenase of spleen-Yang deficiency rat compared with normal group(P<0.05).3. Ileal tissue of rats with spleen-Yang deficiency of glyceraldehyde-3phosphatedehydrogenase expression of mRNA compared with the normal group significantly decreased,consistent with up-regulation of identification and proteomics results, so this proteins relatedwith spleen-Yang deficiency.4. Spleen-Yang deficiency group of Krt8, pyruvate kinase, Ezrin significantly improvedcompared with the normal group, consistent with the increase-line results of expression ofproteomic identification,, so these proteins related with spleen-Yang deficiency.5. While Krt8, pyruvate kinase and Ezrin mRNA expression of Chinese medicine group ofspleen-Yang deficiency significantly reduced compared with the spleen-Yang deficiencymodel group, glyceraldehyde-3phosphate dehydrogenase mRNA expression of Chinesemedicine group of spleen-Yang deficiency significantly increased in comparison.Conclusion：1. Proteomic identification of differential proteins：The4proteins for upregulation includingDesmin, Krt8, pyruvate kinase and Ezrin; The2proteins for downregulation includingglyceraldehyde-3phosphate dehydrogenase and Actin.2. The cytoskeletal proteins including Desmin mRNA expression levels are not significantlydifferent from normal group. Krt8mRNA expression levels than normal group differences,can be verified as targets of spleen-Yang.3. The signal transduction protein including Ezrin, glyceraldehyde-3phosphatedehydrogenase and pyruvate kinase participating in carbohydrate metabolism mRNAexpression levels than normal of group differences, which can be verified as targets ofspleen-Yang deficiency.4. Correlation between ileal tissue of rats with spleen-Yang deficiency protein Krt8, pyruvatekinase, Ezrin, glyceraldehydes3-phosphate dehydrogenase upregulation of mRNA expression and proteomic identification result is consistent with downregulation.5. The group of invigoring spleen TCM taking Aconitum Fuzi Lizhong decoction has obviouseffect.