Effect Of Blocking The SHH Signaling Pathway On Inflammation-actived Of Glioma Cell

The experimental application of lipopolysaccharide (LPS) is used to stimulate themalignant glioma cell line LN229cell line to establish a model of inflammatory activation ofglia cells, we observe the expression of LN229cell activation and inflammatory factors. whenthe SHH pathway blocker cyclopamine block the SHH pathway, and on further observe thecell protective of SHH pathway in LPS-stimulated glia-neuron sculptured. At last observehow the blocking SHH pathway influence the expression of Parkinson-related gene Nurr1,and how to provide new strategies for the clinical treatment of Parkinson’s disease. Thisexperiment is divided into three parts:Part one: Establishment of the LN229inflammatory activation modeland the influence of blocking SHH pathway on the expression ofinflammatory cytokinesObjective: Apply lipopolysaccharide(LPS) to stimulate the LN229cell to establish amodel of glia cells activation, on this basis we discuss the influence when cyclopamine blockthe SHH pathway glia cells activated.Methods: CCK method used to detect the relationship of concentration of LPS andcyclopamine and the cytotoxicity of LN229. Real-time quantitative PCR to determine the LPSconcentration. The result of experiment above apply lipopolysaccharide (LPS) to stimulate theLN229cell to establish a model of Glia cells activation, Real-time quantitative PCR is used todetect the mRNA expression of inflammatory factors iNOS, TNFα, IL-1β and Glia cellactivation associated with genes caspase8, caspase3. After12h pretreatment with cells withcyclopamine, LPS stimulate the LN229cell, real-time quantitative PCR applied to detect themRNA expression of inflammatory factors iNOS, TNFα, IL-1β and Glia cell activationassociated genes caspase8, caspase3.Results: The CCK experimental results show that, within24hours in concentrations ofLPS and cyclopamine-treated cells, there is no obvious difference compared with LPS groupand Blank control group, so is the cyclopamine group and Blank control group(P>0.05).Conclusions:1. LPS can induce inflammatory cytokines expression of malignant glioma cell LN229;2. LPS stimulate the LN229cell to establish a model of Glia cells activation;3.When the SHH pathway blocker cyclopamine block the SHH pathway, the expression ofLN229cell activated and inflammatory factors maybe enhanced. Part tow: Blocking SHH pathway enhance the damage of nerve cellinduced by LPSObjective: We discuss the Neuronal damage when cyclopamine block SHH pathway tostimulate LPS as well as Neuron-glia cells were cultured.Methods: Culturing the ancient cell of embryonic rat, training system of Neuron-gliacells and Glia cells. We established Immunofluorescence appraisal NESTIN and GFAP. LPSrespectively, cyclopamine dealed with various systems cells, effect of different group cellswas Observed.Results: In the glia cell culture system, there was no significant difference about cellviability between LPS group and blank control group(P>0.05). Neither between cyclopaminegroup and blank control group(P>0.05). In the Neuron-glia cell culture system, there wassignificant cell viability different between LPS group and blank control group(P<0.05). Therewas Obvious difference between LPS/Cyclopamine combination and LPS group, betweenLPS/Cyclopamine combination and cyclopamine group, and between LPS/Cyclopaminecombination and blank control group(P<0.05).Conclusions:1. Cyclopamine blocking SHH pathway can damage nerve cells culturedby neurons-glia cells;2. Cyclopamine blocked the SHH pathway can enhance nerve celldamage stimulated by LPS. Part three: The affect of expression about cyclopamine on LPS-induced Parkinson’s disease-related genes Nurr1under LN229cellsObjective: Search the affect of expression when cyclopamine on LPS-inducedParkinson’s disease-related genes Nurr1under LN229cells, on Further to investigate themechanism of the nerve damage. Methods: On the conditions of LPS stimulation LN229cells, real-time PCR was used todetect the mRNA expression affect of cyclopamine to block the SHH pathway Nurr1gene;western used to blot to detect expression by blocking the SHH pathway Nurr1gene protein.Results: The expression of mRNA in LN229cells Nurr1gene and protein under LPSstimulation is elevated. Cyclopamine strengthen the regulation of the Nurr1gene expressioninduced by LPS furtherly..Conclusions:1. The expression of LN229cells Nurr1gene stimulated by LPS iselevated;2. The nerve cell damaged through the SHH pathway blocked by Cyclopamine isnot achieved by inhibiting the Nurr1gene expression.