电刺激调控胶质细胞活性促进损伤感光细胞存活的实验研究

发布时间：2018-05-13 04:13

本文选题：电刺激 + 光损伤诱导感光细胞变性　；参考：《复旦大学》2012年博士论文

【摘要】：第一部分：电刺激促进光损伤感光细胞存活的神经保护作用体外培养感光细胞系(661W),接种于六孔板贴壁后,放入光照培养箱中接受4-6h光照强度为15,0001ux的宽谱蓝光照射。在光照后的24h进行LDH细胞坏死测定实验、TUNEL凋亡染色及免疫荧光染色观察661W的凋亡情况以及形态学改变。结果显示：光照后,LDH实验显示59.7%±2.7%的661W细胞坏死,TUNEL显示70%左右的染色阳性即凋亡,而免疫荧光染色则发现正常661W呈扁平状,细胞之间连接紧密,但光损伤后661W突触变细长,细胞失去扁平形态,且细胞间隙明显变大,表明光损伤诱导感光细胞凋亡模型成功建立。利用1h双相方波、直流电刺激(3ms,20Hz,300～1600μA)预先作用于光损伤661W和小胶质细胞或Muller细胞共培养体系,并将其视为干预组；将未接受电刺激的此共培养体系视为未干预对照组；正常661W细胞和小胶质细胞或Muller细胞共培养体系视为正常对照组。在共培养后3h、6h、12h、24h进行LDH实验,并在共培养后24h进行TUNEL凋亡染色实验及免疫荧光染色。结果如下：LDH凋亡-时间曲线表明1000μA的电刺激预先作用于光损伤661W和小胶质细胞共培养组之后,相较于未干预组相对应时间点的细胞死亡率,从共培养后12h开始661W的细胞死亡率显著降低,并保持相对恒定直到共培养后24h；同样,1600M的电刺激预先作用于光损伤661W和Muller细胞共培养组之后,从共培养后3h开始661W的细胞死亡率显著降低,并逐渐加大降低幅度直到共培养后24h。 TUNEL染色则显示共培养后24h,电刺激干预组的TUNEL染色阳性细胞比率较未干预组明显减少。免疫荧光染色也提示了光损伤661W细胞和小胶质细胞干预组的661W较未干预组细胞形态改变程度轻,细胞间隙更小。另一方面,我们也将电刺激总用于光损伤后单独培养的661W细胞,并在相同时间节点进行LDH、TUNEL及免疫荧光染色实验,但结果与作用于共培养体系的电刺激不同,电刺激干预并未减轻661W细胞的凋亡。本部分的实验研究结果表明,电刺激能够减轻强光照射引起的感光细胞凋亡变性,但感光细胞本身对电刺激并没有反应,可能电刺激改变了胶质细胞的活性,而胶质细胞活性的改变转而影响了感光细胞的存活。第二部分：光损伤诱导感光细胞变性激活视网膜胶质细胞,激活的胶质细胞进而影响光损伤感光细胞凋亡的作用成功建立了光损伤诱导感光细胞变性的模型后,我们进一步利用胶质细胞与光损伤感光细胞共培养体系(未干预组)探索小胶质细胞和Muller细胞受光损伤感光细胞影响后的数量、形态学的改变以及相伴随的功能的变化,同时探索平行的661W细胞的生存状态变化。正常661W细胞和小胶质细胞或Muller细胞共培养组视为正常对照组。在共培养24h后利用免疫荧光染色观察两组小胶质细胞或Muller细胞数量及形态学的差异；同时利用Real-time PCR和Western blot法在共培养后3h,6h,12h,24h对两组小胶质细胞所分泌的促炎因子IL-1β、 TNF-α以及Muller细胞所分泌的神经营养因子BDNF、CNTF的基因表达量和蛋白分泌量两组差异以及时间趋势变化进行定量分析。并且运用LDH、TUNEL及免疫荧光染色等实验检测单独培养光损伤661W细胞和与小胶质细胞或Muller细胞共培养的661W细胞坏死和凋亡率的差异。结果显示：免疫细胞化学实验发现与正常661W细胞共培养的小胶质细胞成静息状态,形态成分支状,突触长,胞体较小,而与光损伤661W细胞共培养的小胶质细胞成激活状态,形态成阿米巴样或圆形,突触短,胞体大,且数量较正常对照组增多。两组Muller细胞的形态则无显著差异。另外,在共培养后的24h,Real time PCR和Western blot显示与光损伤661W共培养的小胶质细胞IL-1β和TNF-α的基因和蛋白表达量较正常对照组显著上升,与光损伤661W共培养的Muller细胞BDNF和CNTF的基因和蛋白表达量也较正常对照组明显上调；蛋白量-时间趋势曲线则发现在正常对照组中,小胶质细胞分泌的IL-1β和TNF-α在共培养后3h至24h时间段内基本保持稳定且较低的水平。而相反的,与光损伤661W共培养的小胶质细胞分泌的IL-1β蛋白量则随着时间进展逐步升高,在共培养后24h达到最高峰；同样,其所分泌的TNF-α蛋白量也随着时间上调,在共培养后12h达到最高峰,之后到24h时间段内略微下降。另一方面,正常对照组中的Muller细胞分泌的BDNF、CNTF蛋白量-时间曲线显示BDNF蛋白量随时间呈现逐渐衰减的趋势,而CNTF则相对保持稳定。然而与光损伤661W共培养的Muller细胞分泌的BDNF蛋白量迅速上调,在共培养后的6h达到最高峰,之后略有下降；其所分泌的CNTF则逐步平稳上升。LDH、TUNEL等实验结果显示相较单独培养的光损伤661W细胞,与小胶质细胞共培养的光损伤661W细胞的坏死率随着时间进展显著上升；而与Muller细胞共培养的光损伤661W细胞的坏死率则明显减低。本部分的实验研究发现光损伤诱导的感光细胞凋亡引发了小胶质细胞的激活和Muller细胞的反应性胶质化,并伴随着小胶质细胞分泌的促炎因子IL-1β和TNF-α和Muller细胞分泌的神经营养因子BDNF、CNTF的上调,并且小胶质细胞的激活加重了661W细胞的损伤,而Muller细胞的反应性胶质化则减轻了661W细胞的凋亡,表明了胶质细胞活性改变在光损伤诱导感光细胞变性过程起着重要的作用。第三部分：电刺激干预光损伤感光细胞凋亡诱导的胶质细胞激活的作用将1h双相方波、直流电刺激(3ms,20Hz,300～1600μA)预先作用于光损伤661W和小胶质细胞或Muller细胞共培养体系,视为干预组；将未接受电刺激的此共培养体系视为未干预对照组；正常661W细胞和小胶质细胞或Muller细胞共培养体系视为正常对照组。采用免疫荧光染色在共培养24h后观察各组小胶质细胞及Muller细胞形态学、数量的差异；利用Real-time PCR和Western blot法在共培养后3h,6h,12h,24h对各组小胶质细胞所分泌的促炎因子IL-1β、TNF-α以及Muller细胞所分泌的神经营养因子BDNF、CNTF的基因表达量和蛋白分泌量两组差异以及时间趋势变化进行定量分析。结果显示：免疫学荧光染色显示的未干预对照组和正常对照组间的小胶质细胞和Muller细胞形态学差别如第一部分所述,电刺激干预组的小胶质细胞的阿米巴样的活化数量则较干预组明显减少,并且一部分细胞虽然没有出现像静息状态时的明显的分支状,但胞体呈扁平状态,我们将其视作“中间状态”；而电刺激干预组的Muller细胞则较未干预对照组和正常对照组的Muller细胞显得胞体更大,胞体内纤维组织更丰富,且细胞数量也明显增多。伴随着形态学的改变,Real-time PCR和Western blot结果显示电刺激干预组中的小胶质细胞IL-1β和TNF-α的基因和蛋白表达量较未干预组显著降低,Muller细胞BDNF和CNTF的基因和蛋白表达量较未干预组进一步升高；蛋白量-时间趋势曲线则显示电刺激干预抑制了未十预组中小胶质细胞IL-1β和TNF-α持续升高,分别在共培养后24h和12h降至最低最低点；而电刺激干预组中Muller细胞分泌的BDNF蛋白量较未干预组随时间上升幅度进一步加大；CNTF的分泌高峰则被提前至共培养后6h,上升幅度增大并保持至共培养后24h。这部分实验结果表明：电刺激能够有效抑制由光损伤感光细胞诱导的小胶质细胞激活,也能够显著提升Muller细胞反应性胶质化,从而减轻小胶质细胞的毒性作用,促进Muller细胞的神经保护作用,证明了电刺激保护光损伤感光细胞是通过调控胶质活性这一机理,使得电刺激作为一种促进感光细胞存活的治疗方法效果更为确切。
[Abstract]:Part I: neuroprotective effects of electrical stimulation on photoreceptor survival
The photoreceptor cell line (661W) was cultured in vitro. After inoculation on the six hole plate wall, a broad spectrum blue light irradiation with the intensity of 4-6h light intensity of 150001ux was received in a light irradiation incubator. The LDH cell necrosis was measured by 24h after light irradiation. The apoptosis and morphological changes of 661W were observed by TUNEL apoptosis and immunofluorescence staining. The results showed: light After the LDH experiment, the LDH experiment showed that 59.7% + 2.7% of 661W cell necrosis, and 70% of the positive staining was apoptosis, while the immunofluorescence staining found that the normal 661W was flat and the cells were closely connected. But after the light damage, the 661W synapses became elongated, the cells lost the flat shape, and the space between the cells became significantly larger, indicating that light damage induced the photoreceptor cells. The apoptosis model was successfully established.
Using 1H biphasic square wave, direct current stimulation (3MS, 20Hz, 300~1600 u A) pre acted on the co culture system of light damaged 661W and microglia or Muller cells, and regarded it as an intervention group. The co culture system without electrical stimulation was considered as a non intervention control group; the co culture system of normal 661W cells and microglia or Muller cells was used as a co culture system. As a normal control group, LDH experiments were carried out after co culture of 3H, 6h, 12h, 24h, and TUNEL apoptosis staining and immunofluorescence staining were carried out after co culture of 24h. The results were as follows: the apoptosis time curve of LDH showed that the 1000 micron electrical stimulation was preacted on the co culture of 661W and microglia in the light damage of 661W and microglia. The cell mortality of the cell was significantly reduced from 12h to 661W after co culture, and remained relatively constant until the co culture of 24h. Similarly, after the 1600M electrical stimulation was pre acted on the co culture group of 661W and Muller cells, the death rate of 661W from the co culture of 3H was significantly reduced and the decrease was gradually increased. After co culture, 24h. TUNEL staining showed 24h after co culture. The ratio of TUNEL staining positive cells in the electrical stimulation intervention group decreased significantly than that in the non intervention group. The immunofluorescence staining also indicated that the 661W of the light injured 661W cells and the microglia intervention group was lighter than that in the non intervention group, and the cell space was smaller. We also used electrical stimulation to stimulate the 661W cells that were cultured separately after light damage, and performed LDH, TUNEL and immunofluorescence staining at the same time nodes, but the results were different from the electrical stimulation that acted on the co culture system. The intervention of electrical stimulation did not reduce the apoptosis of 661W cells.
The experimental results in this part show that electrical stimulation can reduce the apoptosis and degeneration of photoreceptor cells caused by strong light irradiation, but the photoreceptor cells themselves do not respond to the electrical stimulation. Electrical stimulation may change the activity of glial cells, and the changes in the activity of glial cells affect the survival of the photoreceptor.
The second part: light induced photoreceptor degeneration can activate retinal glial cells and activate glial cells, thereby affecting the apoptosis of photoreceptor cells.
After successful establishment of a model of photoreceptor degeneration induced by light damage, we further explore the number of microglia and Muller cells affected by photoreceptor cells, morphologic changes and the changes associated with the associated function, and explore parallel, using the co culture system of glial cells and light damaged photoreceptor cells. Changes in the survival state of 661W cells. Normal 661W cells and microglia or Muller cell co culture group were considered as normal controls. After co culture of 24h, the number and morphology of two groups of microglia and Muller cells were observed by immunofluorescence staining, while Real-time PCR and Western blot were used in the co culture of 3H, 6h, 12h, and 24. H was used to quantitatively analyze the difference in the two groups of gene expression and protein secretion of CNTF secreted by two groups of microglia secreted by two microglia cells, the two groups of neurotrophic factor BDNF, the two groups of gene expression and protein secretion and the change of the time trend. And the individual cultured 661W cells were cultured by LDH, TUNEL and immunofluorescence staining. The difference in necrosis and apoptosis rate between 661W cells co cultured with microglia or Muller cells was observed.
The results showed that the immunocytochemical experiments found that the microglia co cultured with normal 661W cells were in a state of rest, with a branched form, a long synapse and a smaller cell body. The microglia co cultured with light damaged 661W cells was activated by a form of amoeba like or round shape, short synapses and large bodies, and the number was increased in number than that of a normal control group. There was no significant difference in the form of Muller cells in the two groups. In addition, the 24h, Real time PCR and Western blot after co culture showed that the gene and protein expression of IL-1 beta and TNF- a in the microglia co cultured with light damaged 661W was significantly higher than that in the normal control group. The expression of IL-1 beta and TNF- alpha secreted by microglia in the normal control group was basically stable and low in the time period of 3H to 24h in the normal control group. On the contrary, the amount of IL-1 beta protein secreted by the microglia cultured with light damaged 661W was the amount of IL-1 beta protein As time progresses gradually, 24h reaches the highest peak after co culture. Similarly, the amount of TNF- alpha protein secreted by the 12h is up to the highest peak after co culture, and then slightly decreases in the 24h time period. On the other hand, the BDNF of Muller cells in the normal control group and the CNTF protein quantity time curve show BDNF protein. While the amount of CNTF was gradually declining with time, while the amount of BDNF protein secreted by Muller cells co cultured with light damage 661W increased rapidly, and the 6h reached the highest peak after co culture, and then decreased slightly, and the CNTF secreted by the co culture was gradually increased by.LDH, TUNEL and other experimental results showed that it was compared to the individual culture. Light damage of 661W cells and light injury co cultured with microglia cells, the necrosis rate of 661W cells increased significantly with time, while the necrosis rate of 661W cells co cultured with Muller cells decreased significantly.
The experimental study in this part found that photoreceptor apoptosis induced by light damage induces the activation of microglia and the reactive glia of Muller cells, and is accompanied by the increase of the neurotrophic factor BDNF, CNTF, secreted by microglia secreted by pro-inflammatory cytokines and TNF- A and Muller cells, and the activation of microglia is aggravated. The damage of 661W cells and the reactive glialization of Muller cells reduce the apoptosis of 661W cells, indicating that the change of glial cell activity plays an important role in the process of photoreceptor degeneration induced by light damage.
The third part: the effect of electrical stimulation on glial cell activation induced by photoreceptor apoptosis.
1H biphasic square wave, direct current stimulation (3MS, 20Hz, 300~1600 u A) were pre acted on the co culture system of light damage 661W and microglia or Muller cells, and the co culture system without electrical stimulation was considered as untreated control group; normal 661W cells and microglia or Muller cell co culture system were regarded as normal. In the control group, the morphologic and quantitative differences of microglia and Muller cells were observed by immunofluorescence staining after co culture of 24h, and 3h, 6h, 12h and 24h were used in the co culture of 3H, 6h, 12h, and 24h on the microglia and the secretion of neurotrophic factors secreted by Real-time PCR and Western blot. F, CNTF gene expression and protein secretion in two groups of differences and time trend changes were quantitatively analyzed.
The results showed that the morphological differences between the microglia and Muller cells between the non intervention control group and the normal control group showed that the number of amoeba like activation in the microglia in the electrical stimulation intervention group was significantly lower than that in the intervention group, and some of the cells did not appear to be resting like resting form in the electrical stimulation intervention group. The obvious branch of the state, but the cell body is flat, we regard it as the "middle state", and the Muller cells in the electrical stimulation intervention group are larger than the Muller cells in the control group and the normal control group, and the fibrous tissue in the cell is more abundant, and the number of cells is also significantly increased. With the morphological changes, Real- The results of time PCR and Western blot showed that the gene and protein expression of IL-1 beta and TNF- alpha in the microglia in the electrical stimulation intervention group was significantly lower than that in the non intervention group. The gene and protein expression of BDNF and CNTF in Muller cells was further higher than that in the unintervened group; the protein quantity time trend curve showed that the electrical stimulation intervention inhibited the pre ten preconditioning. The IL-1 beta and TNF- alpha of primary and middle glial cells in the group continued to rise, and 24h and 12h decreased to the lowest lowest point after co culture, and the amount of BDNF protein secreted by Muller cells in the electrical stimulation intervention group was further increased as compared with those in the non intervention group. The peak of CNTF secretion was advanced to the co culture of 6h, and the increase was increased and kept to the same level. 24h. after culture
The experimental results show that electrical stimulation can effectively inhibit the activation of microglia induced by light damaged photoreceptor cells, and can significantly enhance the reactive gliosis of Muller cells, thus mitigate the toxicity of microglia and promote the neuroprotective effect of Muller cells. It is proved that the electrical stimulation of light damaged photoreceptor cells can be used to protect the photoreceptor cells. Over regulation of glial activity makes electrical stimulation more effective as a therapeutic method to promote the survival of photoreceptors.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【相似文献】