| Chinese traditional medicine has a long long history and it is proved that Chinese traditional medicine has great therapeutic power on clinic. Though it is one of the representatives of traditional Chinese culture, it has not been accepted worldwide. So the modernization of Chinese traditional medicine is necessary, one of the essential contents of which is the study of therapeutic mechanism. It's difficult to study the mechanism of Chinese traditional medicine because it has a lot of components. Thus the Chinese traditional medicine genomics, involving modern biotechnology and genomic theory, seems to be an effective method for the mechanism research.In this paper, oligonucleotide microarrays were prepared and optimized, by which the blood enriching mechanism of Chinese traditional medicine Si-Wu-Tang(SWT) was studied. The aim of this research is to investigate the changes of genes about hematopoietic related cytokines after administration of Si-Wu-Tang, and to implore the therapeutic mechanism.1. Preparation of and optimization of the hematopoietic associated oligonucleotide microarraysThe results of the optimization indicate that the bifunctional thiophosphyl-amino-oligonuleotide probes get the strongest hybridization signal when printed on the bromide slide. The bifunctional probes are proper for any kinds of slides. But the higher cost is a shortcoming. By contrast the microarrays by printing the amino-oligonucleotide probes on the bromide slide are better and cost less. Finally the amino-probe with PEG linker was selected.The hematopoietic cytokine probes were designed by software Mprobe and blastedby Blast program on internet. The probes were synthesized by PE8909 DNA synthesor and printed on the slides by Cartisen Microarray Printer. Microarrays couldn't be used till it is 24 hours after probe pringting.2. Study of the enriching blood mechanism of SWT using hematopoietic associated cytokines expression profiles microarraysThe mouse bone marrow cell lines QXMSC1 were irradiated by 60Co y with dose of 15 Gy then cultured with SWT and its extractions B,C with 2 mg-mr'for 6~8h. The total RNAs were isolated and reverse-transcripted. The cDNAs were hybridized to the microarrays. The differential expressed genes were detected. After the treatment of SWT and its extractions B and C, the cytokine genes GM-CSF, M-CSF, IL-3, PDGF-P , IL-9, IL-17, EPO-R and IL-4R expressed higher while MIP lower. The findings show that SWT and its extractions B and C can promote enriching blood in bone marrow directly, enhancing immunity and activating signal transduction. After clustering the results, it was found that the extractions B and C had the similar mechanism but different from SWT.The Balb/c mice were irradiated by 5.5 Gy 60Co Y then administered SWT with 5 mg.kg-1.d-1 and 10 mg.kg-1.d-1 respectively. At the 3rd and 7th day, the bone marrow, kidneys, thymuses, spleens and livers were scalpeled out. The total RNAs were isolated and reverse-transcripted into cDNAs. The cDNAs were hybridized to the microarrays. IL-3, GM-CSF, CSF1, G-CSF, IL-6, EPO, IL-4, IL-5and IL-11 expressed highly in bone marrow, all of which could promote enriching blood in bone marrow. SWT can also bring the other organs into playing an important role in enriching the blood byinfluencing the cytokines expression, for example, SWT promotes the expression of EPO in kidney, GM-CSF, CSF1, G-CSF, IL-6 in thymus and spleen. So it could be concluded that SWT activates the positive hematopoietic cytokine genes and immunity gene expressing, suppresses the negative hematopoietic cytokine genes expressing, promotes the hematopoietic associated signal transducing factor excretion, affects the regulating hematopoietic net, keeps its blood enriching power by many targets androutes. IL-17 and IL-4R may be the target site of SWT which were not discovered before.3. Application of bioinformatics in microarrays design and data analysisBioinformatics played an important role in this research. The hematopoietic associated cytokines oligo... |
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