Mechanistic Investigation Of "Nourishing Kidney To Produce Marrow And Form Liver" Key Proteins Interaction

Mechanistic investigation of "nourishing kidney to produce marrow and form liver" key proteins interaction Major:Traditional Chinese Medicine, Internal Medicine Graduated:Gao Xiang Director:Li Han-min, chief physician, professorObject:This program researched the biological basis of "nourishing kidney to produce marrow and form liver" therapeutic principle, analyzed the key protein targets of the process that nourishing kidney (by Zuogui Wan) regulated bone marrow mesenchymal stem cells (MSCs) transformed into liver cells. It provided scientific experiment evidence for clinical application of bone marrow stem cells transformed into liver cells, and for the therapeutic principle of nourishing kidney to cure liver diseases. It further proclaimed the scientific connotation of "kidney manufacture marrow, marrow manufacture liver" and "liver and kidney being of same source", enriched the new etiology of "marrow form liver abnormally" and the therapeutic principle of "nourishing kidney to produce marrow and form liver".Method:This study based on the clinical and experimental research of "nourishing kidney to produce marrow and form liver" therapeutic principle, applied proteomics research method to analysis the protein expression difference of the process that nourishing kidney (by Zuogui Wan) regulated MSCs transformed into liver cells, to determine the key proteins, then using immune precipitation technology and bioinformatics analysis to study the key protein interaction.402months Wistar rats (SPF) were randomly divided into Zuogui Wan drug serum group and blank serum group. Zuogui Wan drug serum group was treated by Zuogui Wan with10ml/kg/d dose by gavage, blank serum group was given the same volume of saline. After treated15days, all the rats were sacrificed to take blood. The blood centrifuged at2500rpm in20minutes to get serum, then using0.22um pore film filtered sterilized and stored at4℃.2days Wistar rat was sacrificed to get liver tissue as cell resourses by using liver tissue culture method. OriCe11TM Wistar rats bone marrow mesenchynal stem cells and liver tissue cells were used to build co-culture system with Millicell Inserts. Co-culture system was added in10%Zuogui Wan drug serum and10%blank serum according to two groups. There were10%poly-lysine coated slides placed in Millicell Inserts before cell culture. The cell slides were collected in7,15and30d co-culture, and detected glycogen by PAS method and AFP, CK18, ALB expression by ICC method. Liver cells slides were used as the positive control, bone marrow mesenchymal stem cells slides were used as the negative control. All the slides were observed with microscope (200×), randomly selected10vision to count positive cells rate(%). The positive cells rate of positive control slides was counted as100%. Experimental data was analysised with the statistical software SPSS. In co-culture15d and30d, cells in Millicell Inserts were collected for proteomics research. Total protein samples were extracted by using RIPA lysis, separated with SDS-PAGE and analysised to determine the difference expression protein. Difference expression protein bands were cutted to analysis by mass spectrometry. The mass spectrometry results were analysised by bioinformatics to determine key proteins of "nourishing kidney to produce marrow and form liver". Normal Wistar rat liver tissue was used to extract total protein sample (non-denatured). Using immune precipitation technology, mass spectrometry and bioinformatics analysis detected proteins thate interacted with key proteins (14-3-3, GRP78and H4).Result:After co-cultured3days, there were a few cells of bone marrow mesenchymal stem cells in10%Zuogui Wan drug serum group shown as liver cells sample cells form that the cells were polygon, cytoplasm with big nuclear or multinuclear. After co-cultured15days, there were about50%bone marrow mesenchymal stem cells performances as liver cells. After co-cultured30days, there were about80%bone marrow mesenchymal stem cells performances as liver cells, and glycogen dyeing positive cells rate (81.39±3.12) significantly higher than co-cultured15days (19.88±2.07). After co-cultured30days, AFP positive cells rate (3.74±0.47) was significantly lower than co-cultured15days (19.36±2.66), CK18positive cells rate (91.14±7.03) was significantly higher than co-cultured15days (59.21±3.61), ALB positive cells rate (88.72±4.35) was significantly higher than co-cultured15days (60.27±5.13). Using gel imaging analysis and bioinformatics analysis determined key proteins14-3-3, GRP78and H4as bait protein of protein interaction research. The proteins interacted with H4were NPTX1, CATA, OGG1, IMA5, Vimentin, GSTA5, TM196, Nestin and DDX25. The proteins interacted with GRP78were AL1A7, ALIBI, AL1A1, AL1A2, UD2B2, UD2B8, ACTB, GRK5, DPYS, GSTM1, GSTA2, GSTA3, G3P, ACSL1, ACSL5, PYC, GRP78, ECHP, TRHDE, CES3, ODP2, ARNT2, HMCS1, Nestin, CP2CN and CP2CB. The proteins interacted with14-3-3were Neuromedin-S, IMA5, EF2, CARD9and RAF1.Conclusion:This program based on bone marrow mesenchymal stem cells-liver cells co-culture system combined with nourishing kidney (by Zuogui Wan drug serum), applied proteomics research idea and research technology to confirm "nourishing kidney to produce marrow and form liver" key proteins that at least included14-3-3protein, glucose regulation protein78(GRP78) and histone4(H4). The interaction proteins of key proteins were more than30kinds. All of these proteins expression changes and interaction formed the biological basis of "nourishing kidney to produce marrow and form liver". The key proteins and interacetion proteins were acting targets of that nourishing kidney (by Zuogui Wan drug serum) regulated bone marrow forming liver cells.