| IntroductionHigh-dose chemotherapy is mainstay in the treatment of malignant hematological diseases and also the most important strategies of preparative regimens of hematopoietic stem cell transplantation. However, regimens of chemotherapy due to its non-tissue and non-cell-specific characteristics may have great damage to the gastrointestinal mucosa, which is also highly proliferative, resulting in necrosis and ulceration. or eventually leading to infection and even sepsis because of bacterial migration. One of the urgent problems remained to be solved was how to effectively alleviate the injury to gastrointestinal mucosa by chemotherapy and facilitated recovery from injury. Recently, there were many polypeptides founded in gastrointestinal tract which involved in controlling the development, regeneration and repairing of gastrointestinal mucosa epithelia. Among which, intestinal trefoil factor (ITF). the third member of trefoil factor family, is the most likely polypeptide to be used to protect the gastrointestinal mucosa from diverse damage such as MTX, ethanol, indomethacin and so on. According to literature and outcome analyzed by protein analysis software such as ''ANTHEPRO verS.O", human ITF is low-molecular-weight polypeptide with its isoelectric point approximately 4.5 or 4.9. and is acid-resistance, proteolysin-resistance and thermal-resistance. It maintains integrity of mucosa by several mechanisms such-7-as promoting migration of epithelia. anti-oxidation, anti-apoptosis and involvement in inflammatory response. As a '"motogen", ITF only promotes cell migration compared with other growth factors involved in cell division and proliferation, the latter may be potential carcinogens. In fact, almost all our knowledge about hITF function conies from study on recombined product of gene engine till now. However, the nature of any naturally occurring posttranslational modifications is unknown. In the case of hITF, it is also unknown whether this peptide occurs in a monomer or in a dimmer form in vivo. There were rare formal reports regarding purification of normal human ITF from natural sources in literature, the causes may due to the lack of sources of natural hITF-rich. Based on the fact reported firstly by Dr. Lin. our co-worker, that ITF increased significantly during the late stage of embryo development and the expression of ITF was related to the normal development of gastrointestinal mucosa, we speculated that there was high concentration of hITF existing in meconium of mature newborn, whose composition was relatively less complex. According to the classic strategy of purification of calmodulin from tissue proposed by Cool Spring Harbor laboratory, we try to isolate and purify natural hITF from meconium taking advantage of its characteristic of acid-resistance and then to explore its biologic function.Material and methodsIdentification of proteins1. Concentration of total protein: The total protein concentrations were determined by Coomassie?Plus protein assay, in which human BSA as a standard.2. Immunodetection of hITF's antigenicity: The blotted nitrate fibrous membrane (0.45um) was blocked by 3% BSA, and then incubated with specific polyclonal rabbit anti-human ITF antibody, and then incubated with secondary alkaline phosphatase conjugated goat anti-rabbit IgG, and detectionwas performed by 1-Step?NBT/BCIP at the last.3. Relative molecular weight and its antigenicity of protein: The relative molecular-weight was measured by SDS-PAG electrophoresis (modified Laemmli discontinuous system, 20%T\2.6%C, 2*SDS electrophoresis buffer) utilizing Hoefer?relative mobility calculator, protein samples were prepared by precipitated in trichloracetic acid and treated by SDS in boiling wash for 3 minutes. Western blotting in semi-dry system in Towbin buffer and the immunodetection method mentioned above was used to identify antigenicity ofhITF.Optimizing the protocol of purification1. Material: Meconium of mature newborn excreted within 24 hours a...
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