| Background and Objective: Scorpion venom compose of proteins and non-proteins.The main active elements are proteins. These polypeptides have different biological activities, such as anti-tumor, analgesic, anti-epileptic.Radiation therapy kills tumor cells while normal tissue is often injured, and bone marrow sensitive to radiation damage. Hematopoietic system injury after radiation includes not only early acute bone marrow suppression, but also the remnants of hematopoietic stem cells late dysfunction, these cause a serious impact on cancer treatment and patient quality of life. Current clinical treatment is given to conventional cytokine therapy, but radiation survival of hematopoietic stem / progenitor cell proliferation and cytokine response capacity decreased significantly, and sustained cytokine combination therapy can cause side effects such as inflammation and immune responses. Previous study in our laboratory showed that scorpion venom can significantly increased number of nucleated cells in bone marrow,thenumber of granulocyte progenitor colony forming units and the number of bone marrow stromal cell colonies of mice after radiation, spleen nodules and the proliferation of bone marrow cells Index also increased, so it indicated that SVP could reduce the radiation bone marrow suppression, based on these results, our group was first proposed, scorpion venom has the role of cytokines. can we separated and purified a proliferative peptide to instead of cytokines, which is natural and can effective protect the hematopoietic system in the same time reduce the other side effects? So, our aim is: First, using Gel filtration chromatography, ion exchange chromatography and RT-HPLC to separated and purified a single peptide; Second, using the bone marrow cells of C57BL/6 mouse as amodel to choose the most effective proliferative componts; Last, using M-NFS-60 cells, the mouse-derived promyelocytic leukemia cell line with the feature of hematopoietic factor-dependentcy, research the possible mechanisms and signal transduction of proliferation.Materials and Methods:1.The method of isolation and purification,authentication of polypeptide of scorpion venom. 1.1 Scorpion venom was separated with Sephadex G-50 chromatography, (according to molecular size of that). Then the whole elution peak of each group were collected and freeze-dried.1.2 The polypeptide of scorpion venom was separated with CM Sepharose FF ion chromatography, (according to the size of ionic strength), each elution peak were collected, freeze-dried after desalination membrane package.1.3 The purity of the various components was detected by RP-HPLC. Got the bone marrow cells from C57BL/6 mouse, obtained colony and cobblestone area forming cells in culture, selected the better components of proliferation.1.4 High purity components were detected molecular weight and amino acid sequence by Using mass spectrometry and Edman degradation.2. The selection of effective proliferative components of scorpion venom2.1 Obtained bilateral femoral bone marrow cells from C57BL/6 mice .After red blood cells were cracked. The remaining cells were divided into eight groups: normal control group, IL-3 factor group, components of scorpion venom B4, B5 group, culture 2weeks to observed GM-CFU and culture 4-7weeks to observe CAFC formation.2.2 Using M-NFS-60 cells as a model, firstly, the M-NFS-60 cells in the logarithmic phase were washed three times, respectively seeded in 96-well plates with a certain density. Secondly, after 24 hours starvation factor dosing, and then into the conventional incubator for 24 hours, 48 hours, then using microplate reader to detect the OD value of each group;3. Discussion of its proliferation mechanism.3.1 The method used immunofluorescence to detect the influence of components exerting the cell membrane expression of IL-3R;3.2 Detection of the expression of IL-3R with the method of Western Blotting.3.3 Detection of the expression of P-Jak2,P-Stat5,Jak2 and Stat5 protein with the method of Western Blotting.Results:1. Scorpion venom were divided into three components by gel chromatography, named peak I, peak II, peakâ…¢;The peak II of scorpion venom was divided into seven separate components with ion-exchange chromatography, named as B1, B2, B3, B4, B5, B6, B7;The purity of each components were identified by RP-HPLC , and the results show that B4, B5 were high purity, as a single peak, in which the highest purity was B5;The results of Bone marrow cells cultured in vitro displayed that B4, B5 group formed by the number of GM-CFU and CAFC more than the other groups;The results of adding CCK-8 displayed that the proliferation rate of B4, B5 groups were consistent with cell counting results. The results of immunofluorescence showed that the B4, B5 groups had higher cell surface fluorescence intensity than other groups;7. The results of Western Blotting showed that expression of IL-3R of B4, B5 groups increased at 24 hours and 48 hours; And 10 minutes, 30 minutes and 60 minutes time points showed B4, B5 groups allowed the expression of P-Jak2, P-Stat5 increased, too. However, little change in total protein.Conclusions:1.It's the first time that the single component in promoting proliferation were isolated and purified from scorpion venom, which has been patented, the patent number is: ZL201010508153.9.2.The initial inference the mechanism of promoting proliferation, which may be related to Jak2-Stat5 signaling pathway. |
PDF Full Text Request