不同途径神经干细胞移植对肌萎缩侧索硬化神经炎症的影响.

BACKGROUND: Neuroinflammation affects the progression of amyotrophic lateral sclerosis. In vitro studies have shown that neural stem cells have antiinflammatory effect, but whether they can be anti-inflammatory in amyotrophic lateral sclerosis mouse, and the mechanism and optimal transplantation route are still unclear. OBJECTIVE: To investigate the anti-inflammatory effect and mechanism of neural stem cell transplantation in different ways on amyotrophic lateral sclerosis model G93A-SOD1 mice. METHODS: Neural stem cells were isolated and identified, transfected with green fluorescent protein. Totally 78 G93A-SOD1 mice aged 70 days were divided into lateral ventricle group, tail vein group, and control group. After adaptive feeding, 5×105 green fluorescent protein-transfected neural stem cells were transplanted into the lateral ventricle, and 1×106 green fluorescent protein-transfected neural stem cells were injected through the tail vein at the age of 84 days. From the age of 85 days, modified Wrathall motor score and rotation test were performed every week to evaluate motor function. Onset time, disease duration and survival time were recorded. At the age of 105 days, immunofluorescence method was conducted to detect survival, migration and differentiation of neural stem cells transfected with green fluorescent protein. The number of motor neurons was measured by Nissl staining. The expression of ChAT was detected by western blot assay. Immunofluorescence was used to detect the expression of NeuN in spinal cord anterior horn motor neurons. The protein and mRNA levels of tumor necrosis factor alpha, interleukin 1 beta, interleukin 6 and transforming growth factor beta in cerebrospinal fluid were detected by ELISA and RT-PCT. Western blot assay was used to detect the expression of iNOS, CD206 and NF-κB pathway proteins in the spinal cord. The pathological changes of gastrocnemius muscle were detected by hematoxylin-eosin staining. RESULTS AND CONCLUSION: (1) The extracted neural stem cells grew and differentiated well and could be transfected by green fluorescent protein. (2) The lateral ventricle pathway was more favorable for neural stem cells to enter the central nervous system in the super-early stage after the onset of disease, and decreased the levels of tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, increased the level of tumor necrosis factor-β, reduced the expression of inducible nitric oxide synthase and increased the expression level of CD206, inhibited the activation of NF-κB pathway, slowed down the progression of motor function damage, and reduced the degree of pathological damage of skeletal muscle. (3) In the super-early stage after the onset of the disease, the effects of two ways of transplanting neural stem cells to protect the anterior horn motor neurons and prolong the course of disease and survival time of mice were limited. (4) Results show that in the super early stage after the onset of amyotrophic lateral sclerosis, the lateral ventricle may be a better way of transplanting neural stem cells, which has a good anti-inflammatory effect. The mechanism may be regulating the polarization direction of microglia and inhibiting the activation of NF-κB pathway. BACKGROUND: Neuroinflammation affects the progression of amyotrophic lateral sclerosis. In vitro studies have shown that neural stem cells have antiinflammatory effect, but whether they can be anti-inflammatory in amyotrophic lateral sclerosis mouse, and the mechanism and optimal transplantation route are still unclear. OBJECTIVE: To investigate the anti-inflammatory effect and mechanism of neural stem cell transplantation in different ways on amyotrophic lateral sclerosis model G93A-SOD1 mice. METHODS: Neural stem cells were isolated and identified, transfected with green fluorescent protein. Totally 78 G93A-SOD1 mice aged 70 days were divided into lateral ventricle group, tail vein group, and control group. After adaptive feeding, 5×105 green fluorescent protein-transfected neural stem cells were transplanted into the lateral ventricle, and 1×106 green fluorescent protein-transfected neural stem cells were injected through the tail vein at the age of 84 days. From the age of 85 days, modified Wrathall motor score and rotation test were performed every week to evaluate motor function. Onset time, disease duration and survival time were recorded. At the age of 105 days, immunofluorescence method was conducted to detect survival, migration and differentiation of neural stem cells transfected with green fluorescent protein. The number of motor neurons was measured by Nissl staining. The expression of ChAT was detected by western blot assay. Immunofluorescence was used to detect the expression of NeuN in spinal cord anterior horn motor neurons. The protein and mRNA levels of tumor necrosis factor alpha, interleukin 1 beta, interleukin 6 and transforming growth factor beta in cerebrospinal fluid were detected by ELISA and RT-PCT. Western blot assay was used to detect the expression of iNOS, CD206 and NF-κB pathway proteins in the spinal cord. The pathological changes of gastrocnemius muscle were detected by hematoxylin-eosin staining. RESULTS AND CONCLUSION: (1) The extracted neural stem cells grew and differentiated well and could be transfected by green fluorescent protein. (2) The lateral ventricle pathway was more favorable for neural stem cells to enter the central nervous system in the super-early stage after the onset of disease, and decreased the levels of tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, increased the level of tumor necrosis factor-β, reduced the expression of inducible nitric oxide synthase and increased the expression level of CD206, inhibited the activation of NF-κB pathway, slowed down the progression of motor function damage, and reduced the degree of pathological damage of skeletal muscle. (3) In the super-early stage after the onset of the disease, the effects of two ways of transplanting neural stem cells to protect the anterior horn motor neurons and prolong the course of disease and survival time of mice were limited. (4) Results show that in the super early stage after the onset of amyotrophic lateral sclerosis, the lateral ventricle may be a better way of transplanting neural stem cells, which has a good anti-inflammatory effect. The mechanism may be regulating the polarization direction of microglia and inhibiting the activation of NF-κB pathway. Key words: neural stem cell; amyotrophic lateral sclerosis; inflammation; transplantation pathway; microglia; NF-κB pathway; motor neuron; inflammatory cytokine [ABSTRACT FROM AUTHOR]