| Background:Because of the complex pathogenesis of the NNP, traditional analgesics such as anti-inflammatory drugs?calcium channel antagonist and tricyclic antidepressants are less effective. So the treatment of NNP is becoming a urgent clinical problem to be solved. Bone marrow mesenchymal stem cells (BMMSCs)are directly used in the treatment of neuropathic pain, in virtue of biological advantages of BMMSCs, such as high proliferation rate, hereditary stability, low immunogenic, and immune regulating function. However, the mechanisms by which BMMSCs palys its therapeutic effects are unclear. Researche suggests that microglia can be activated by a small amount of proinflammatory cytokines, then activated microglia produce a great deal of proinflammatory cytokines and lead the occurrence and progress of neuropathic pain. Thus, the regulation of microglial excessive activation is an important approach to the treatment of neuropathic pain.Studies show that stem cells can be activated by inflammatory cytokines in transplanted microenvironment and inhibite microglial activation to relieve neuropathic pain by paracrine secretion. However, less research of pre-treatment of BMMSCs with inflammatory cytokines to alleviate neuropathic pain has been done.So it is of great significance to study the role of pre-treatment of BMMSCs in regulating the activity of microglia in the treatment of neuropathic pain.Objective:To investigate the analgesic effect and possible mechanism of BMMSCs pretreated by TNF- a transplanted in rats with neuropathic pain through intrathecal injection.Contents:1. Culturing and identifying BMMSCs.2. Effects of microglia activation and pain behaviors in rats with neuropathic pain through intrathecal injection of BMMSCs pretreated by TNF- a3. Effects of the inhibition of microglial activation of BMMSCs pretreated by TNF- a in vitro.Methods:1. BMMSCs is separated from bone marrow of male SD rats (3-4 weeks old)and incubated by cell adherence method; Morphological characters of BMMSCs were observed under light microscope; Flow cytometry was used to detect the cell surface markers to identify BMMSCs;2. 120 male SD rats (6-8 weeks old) were selected to establish chronic constrictive injury (CCI) pain model then randomly divided into 3 groups(n=40):?The CCI group: 7 days after modeling, intrathecal injection of PBS(20ul) without BMMSCs was performed; ?The BMMSC transplantation group (B group): 7 days after modeling, intrathecal injection of PBS(20ul) with BMMSCs (1 x 106)was performed; ?The BMMSCs pretreated by TNF-a transplantation group (T+B group): 7 days after modeling, intrathecal injection of PBS(20ul) with BMMSCs pretreated by TNF- ?(1 x 106)was performed;3. Observe the mechanical pain threshold and thermal hyperalgesia latency of rats in each group at the day of one day before the transplantation and D3, D7, D14,D21, D28 after transplantation;4. In day before the transplantation maked D1 and D3, D7, D14, D21, D28 after transplantation , rats were killed by cervical dislocation, and collected lumbar spinal cord and dorsal root ganglion;detect the levels of TNF-a, IL-1?, IL-6,IL-10 byELISA; detect the levels of CD68 by Immunofluorescence method.5. To further verify the effect of BMMSCs on the activity of microglia, LPS(lOug/ml) induced activation of microglia (MG) and verify the effect of BMMSCs on activated MG by coculture; pretreated the BMMSCs with complete culture medium containing TNF-? (1000ng/ml); Purified microglia after separation with 1x105/ hole planted in 6 well plates and coculture microglia and BMMSCs according to the following groups: groupB (BMMSCs), groupL+B (LPS+BMMSCs), groupT+B (BMMSCs pretreated by TNF-a), groupL+T+B (LPS+BMMSCs pretreated by TNF-a), groupM(MG), groupL+M(LPS+MG),groupB+L+M(BMMSCs +LPS+MG), groupT+B+L+M(BMMSCs pretreated by TNF-a+LPS+MG);6. Take the supernatant after 24hours of coculture, then detect the levels of TNF-a, IL-6, IL-10 by ELISA; detect the levels of CD68 by Immunofluorescence method.Results:1. BMMSCs cultured by whole bone marrow adherent culture method were growing as a long spindle and beam pattern. The surface markers of BMMSCs like as CD90(83.95%); CD29(99.59%); CD45(7%); CD34(0.1%);2. Compared with the CCI group and B group: the PWT and PWL of T+B group has no significant difference on the day before the transplantation and the D28 after the transplantation(p>0.05); thePWT and PWL of T+B group is in a decrease trend from D3 to D7, then increased slowly from D7 to D28; on the D7,D14,D21 the value of the PWT and PWL of T+B group is higher(p<0.05); the mental state of T+B group is better and the wound healed faster (p < 0.05);3. Compared with the CCI group and B group: deteced by ELISA after BMMSCs transplantation, the level of TNF- ?? IL-6 of T+B group is significantly increased on the D3,D7 then become in a decrease trend onD14,D21 and reached the basic line on D28(p < 0.05); the expression of IL-10 of T+B group keep in a low level on D3,D7and reach the highest level onD14 (p < 0.05);4. Compared with the CCI group and B group: theexpression of CD68 of T+B group is significantly decreased (p < 0.05);5. Coculture in vitro: the expression of CD68 of group L+M increased, and the secretion of TNF- a and IL-6increased significantly; compared with the L+M group,the expression of CD68, TNF-?,IL-6 of groupB+ L+M and groupT+B+ L+M.Conclusion:1. BMMSCs transplantation can regulate the pain behavior of CCI rats and produce analgesic effect. BMMSCs pretreated by TNF-? have a stronger analgesic effect.2. BMMSCs pretreated by TNF-a transplantation can inhibit microglial activation and reduce the pro-inflammatory factor and improve the anti-inflammatory factors, reduce the neuropathic pain in CCI rats.3. BMMSCs and BMMSCs pretreated by TNF-a can both inhibit microglial activation induced by LPS in vitro and the inhibitory effect of BMMSCs pretreated by TNF-a is stronger. |
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