缝隙连接细胞间通讯对大鼠星形胶质细胞缺氧后处理的影响
发布时间：2018-09-01 19:49
【摘要】：目的： 1.观察原代星形胶质细胞中缝隙连接(GJ)在缺氧/复氧损伤和缺氧后处理之间的差异。 2.运用GJ工具药，改变GJ功能后，观察缺氧后处理对细胞保护作用的变化。 3.初步探讨缺氧后处理保护作用的可能作用机制。 方法： 采用细胞培养技术，取24h新生SD大鼠大脑皮层，培养星形胶质细胞，在细胞纯化后，通过胶质纤维酸性蛋白特异性免疫荧光鉴定星形胶质细胞纯度。建立星形胶质细胞缺氧/复氧模型和缺氧后处理模型。采用细胞接种荧光示踪法(Parachute Assay)检测星形胶质细胞间的荧光传递功能。MMT法检测缺氧后处理对星形胶质细胞存活率的影响，采用Annexin V/PI双染法和Hochest33258荧光染色法观察HPO对H/R损伤导致的细胞凋亡的影响。采用Western blot检测缺氧后处理及调控GJ功能后，HPO对细胞凋亡蛋白Bcl-2、Bax、caspase-3表达的影响以及对细胞Cx43蛋白和Src激酶蛋白表达的影响。通过免疫荧光法(Immunofluorescence Assay)检测HPO及调控GJ功能对星形胶质细胞膜表面Cx43蛋白表达的影响。最后通过siRNA特异性干扰Cx43蛋白，观察其对H/R损伤和HPO的影响。 结果： 对星形胶质细胞进行纯度鉴定后发现，荧光显微镜下占光镜下星形胶质细胞比例为95%以上，说明得到了高纯度星形胶质细胞。 荧光示踪实验结果发现，与正常对照组相比，H/R组荧光传递受体细胞数增加；较H/R组，HPO组荧光传递受体细胞数降低；较HPO组，用10μM GJ增强剂RA预处理24h后的HPO组荧光传递受体细胞数增加，用25μM GJ抑制剂oleamide预处理1h后的HPO组荧光传递受体细胞数减少(p0.01)。结果表明，在星形胶质细胞中，HPO可显著降低H/R所致的GJ功能增强。 通过MTT实验结果发现，较正常对照组，H/R组细胞存活率降低0.370±0.027(p0.01)；较H/R组， HPO组细胞存活率增加0.180±0.096(p0.05)；而较HPO组，使用RA预处理的HPO组的细胞存活率降低0.188±0.049(p0.05)，使用oleamide预处理的HPO组的细胞存活率增加0.186±0.089(p0.05）。MTT结果说明HPO可以减少由H/R所导致的细胞存活率降低，而通过调控GJ功能，可以影响HPO的这种作用。 Annexin V/PI双染实验结果显示，各组细胞早期凋亡率为0.082±0.009，0.357±0.027，0.217±0.008，0.278±0.021和0.150±0.019。较正常对照组，H/R组的凋亡率明显增加，而HPO可明显抑制由H/R所引起的细胞凋亡(p0.01)。在使用了GJ增强剂RA和GJ抑制剂oleamide预处理后，细胞凋亡率较HPO组分别增加和降低(p0.01)。 Hochest33258荧光染色实验结果显示，与正常对照组相比，H/R组细胞凋亡率增加；进行HPO后，细胞凋亡率较H/R组降低；而较HPO组，使用RA预处理的HPO组的细胞凋亡率增加，使用oleamide预处理的HPO组的细胞凋亡率降低。 通过western blotting实验发现，与空白对照组相比， H/R组Bax/Bcl-2的比例增加(p0.01)；在进行后处理后，较H/R组，Bax/Bcl-2的比例降低(p0.01)；与HPO组相比，用RA预处理的HPO组Bax/Bcl-2的比例增加(p0.05)，用Olea预处理的HPO组Bax/Bcl-2的比例降低(p0.05)。另外，对caspase-3蛋白表达进行统计分析后，与空白对照组相比，H/R组caspase-3剪切条带表达增加(p0.01)；与H/R组相比，HPO组caspase-3剪切条带表达减少(p0.01)；与HPO组相比，用RA预处理的HPO组caspase-3剪切条带表达增加(p0.01)，用Olea预处理的HPO组caspase-3剪切条带表达减少(p0.05)。与正常对照组相比，H/R组Src激酶表达增加(p0.01)，而经过后处理后，Src激酶表达降低(p0.01)。较HPO组，使用RA预处理的HPO组Src激酶表达增加(p0.05)，使用oleamide预处理的HPO组的Src激酶表达减少(p0.05)。 通过免疫荧光检测星形胶质细胞膜表面Cx43蛋白表达可发现， H/R组，细胞膜Cx43表达较正常对照组异常增强，而HPO可显著减少Cx43的表达。在用10μM GJ增强剂RA预处理24h后，HPO组的Cx43表达增加，而在用25μM GJ抑制剂oleamide预处理1h后，HPO组Cx43表达减少。通过western blotting检测并对条带灰度值扫描并进行统计学分析后，较空白对照组，H/R组Cx43蛋白表达增加。较H/R组，HPO组降低(p0.01)。而与HPO组相比，RA预处理组蛋白表达增加(p0.05)，Olea预处理组蛋白表达降低(p0.05)。这说明GJ功能的改变有可能与Cx43蛋白表达的变化有关。 采用siRNA干扰Cx43蛋白表达可以发现，阴性对照组对细胞GJ功能、细胞存活率和凋亡率无显著影响(p0.05)，但与阴性对照组相比，Cx43-siRNA转染组可显著降低由H/R导致的GJ功能增加(p0.01)。与阴性对照组相比，Cx43-siRNA转染组也显著抑制了由H/R导致的细胞存活率降低(p0.01)，。Cx43-siRNA转染组较阴性对照组，，可显著降低H/R导致的细胞早期凋亡率增加(p0.01)。 Cx43-siRNA转染组显著降低H/R导致的细胞晚期凋亡率增加(p0.01)。以上结果说明干扰Cx43蛋白，抑制GJ功能，可以抑制由H/R所致的细胞凋亡率增加。 结论： 1.缺氧后处理可以降低由缺氧/复氧损伤导致的星形胶质细胞间GJ功能增强。 2.缺氧后处理对缺氧/复氧损伤的保护作用可能与降低GJ功能有关。 3.改变GJ功能会影响缺氧后处理的保护作用。 4.降低GJ功能导致的缺氧后处理保护作用可能与Bax/Bcl-2、caspase3相关凋亡蛋白有关。
[Abstract]:Objective:
1. To observe the difference of gap junction (GJ) in primary astrocytes between hypoxia/reoxygenation injury and hypoxia postconditioning.
2. using GJ tool to change the function of GJ, we observed the changes of cell protection after hypoxic postconditioning.
3. to explore the possible mechanism of hypoxic postconditioning protection.
Method:
Astrocytes were cultured from the cerebral cortex of newborn SD rats for 24 hours. After purification, the purity of astrocytes was determined by glial fibrillary acidic protein specific immunofluorescence. The models of hypoxia/reoxygenation and hypoxia postconditioning of astrocytes were established. The effects of hypoxic postconditioning on the survival rate of astrocytes were examined by MMT method, and the effects of HPO on apoptosis induced by H/R injury were observed by Annexin V/PI double staining and Hochest33258 fluorescence staining. After hypoxic postconditioning and GJ function regulation, the effects of HPO on fine cells were detected by Western blot. Effects of apoptotic proteins Bcl-2, Bax and caspase-3 on the expression of Cx43 protein and SRK protein were studied. The effects of HPO and GJ on the expression of Cx43 protein on the surface of astrocytes were detected by immunofluorescence Assay. Finally, Cx43 protein was specifically interfered with by siRNA to observe its effect on H/R damage. Injuries and HPO effects.
Result:
The purity of astrocytes was identified and the proportion of astrocytes under fluorescence microscope was more than 95%, indicating that high purity astrocytes were obtained.
The results of fluorescence tracing showed that the number of fluorescent transfer receptor cells in H/R group increased, the number of fluorescent transfer receptor cells in HPO group decreased, the number of fluorescent transfer receptor cells in HPO group increased 24 hours after pretreatment with 10 mu GJ enhancer RA, and the number of fluorescent transfer receptor cells in HPO group 1 hour after pretreatment with 25 mu GJ inhibitor oleamide. The number of transfer receptor cells decreased (p0.01). The results showed that HPO significantly decreased the enhancement of GJ function induced by H/R in astrocytes.
MTT assay showed that compared with the normal control group, the cell survival rate of H/R group decreased by 0.370 (+ 0.027) (p0.01); compared with H/R group, the cell survival rate of HPO group increased by 0.180 (+ 0.096) (p0.05); compared with HPO group, the cell survival rate of HPO group pretreated with RA group decreased by 0.188 (+ 0.049) (p0.05); the cell survival rate of HPO group pretreated with oleamide group increased by 0.18 (+). MTT results showed that HPO could decrease the cell survival rate induced by H/R, and could affect the effect of HPO by regulating GJ function.
The results of Annexin V/PI double staining showed that the early apoptosis rate of cells in each group was 0.082+0.009, 0.357+0.027, 0.217+0.008, 0.278+0.021 and 0.150+0.019. Compared with the normal control group, the apoptosis rate in H/R group was significantly increased, while HPO could significantly inhibit the apoptosis induced by H/R (p0.01). Pretreatment with GJ enhancer RA and GJ inhibitor oleamide was used. The apoptotic rate was increased and decreased compared with HPO group (P0.01).
The results of Hochest33258 fluorescence staining showed that compared with the normal control group, the apoptosis rate of H/R group was increased, that of H/R group was decreased after HPO, that of RA pretreated HPO group was increased, and that of oleamide pretreated HPO group was decreased.
Western blotting showed that the ratio of Bax/Bcl-2 increased in H/R group (p0.01), decreased in post-treatment group (p0.01), increased in RA pretreated HPO group (p0.05), and decreased in Olea pretreated HPO group (p0.05). After statistical analysis of caspase-3 protein expression, the expression of Caspase-3 shear bands increased in H/R group (p0.01), decreased in HPO group (p0.01), increased in HPO group pretreated with RA (p0.01), and increased in HPO group pretreated with Olea (p0.01). Compared with the normal control group, the expression of Src kinase increased in H/R group (p0.01), but decreased in post-treatment group (p0.01). Compared with HPO group, the expression of Src kinase increased in HPO group pretreated with RA (p0.05), and decreased in HPO group pretreated with oleamide (p0.05).
The expression of Cx43 on the surface of astrocyte membrane was detected by immunofluorescence. The expression of Cx43 on the surface of astrocyte membrane was abnormally increased in H/R group compared with normal control group, while the expression of Cx43 was significantly decreased in HPO group. The expression of Cx43 in HPO group increased 24 hours after pretreatment with 10 mu GJ enhancer RA, while the expression of Cx43 in HPO group increased after pretreatment with 25 mu GJ inhibitor oleamide for 1 hour. Compared with the blank control group, the expression of Cx43 protein in the H/R group was increased. Compared with the H/R group, the expression of Cx43 protein in the HPO group was decreased (p0.01). Compared with the HPO group, the expression of Cx43 protein in the RA pretreatment group was increased (p0.05) and that in the Olea pretreatment group was decreased (p0.05). It may be related to the change of Cx43 protein expression.
Using siRNA to interfere with Cx43 protein expression, we found that the negative control group had no significant effect on GJ function, cell survival rate and apoptosis rate (p0.05), but compared with the negative control group, the Cx43-siRNA transfection group could significantly reduce the increase of GJ function induced by H/R (p0.01). Compared with the negative control group, the Cx43-siRNA transfection group also significantly inhibited the increase of GJ function induced by H/R. Cx43-siRNA transfection group significantly decreased the rate of early apoptosis induced by H/R (p0.01). Cx43-siRNA transfection group significantly decreased the rate of late apoptosis induced by H/R (p0.01). These results suggest that interfering with Cx43 protein and inhibiting GJ function can inhibit the cell induced by H/R. The apoptosis rate increased.
Conclusion:
1. hypoxic postconditioning can reduce the GJ function of astrocytes induced by hypoxia / reoxygenation injury.
2. the protective effect of hypoxic postconditioning on hypoxia / reoxygenation injury may be related to the reduction of GJ function.
3. changes in GJ function will affect the protection of hypoxic postconditioning.
4. The protective effect of hypoxic postconditioning induced by decreasing GJ function may be related to Bax/Bcl-2 and caspase-3 related apoptotic proteins.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
本文编号：2218172
[Abstract]:Objective:
1. To observe the difference of gap junction (GJ) in primary astrocytes between hypoxia/reoxygenation injury and hypoxia postconditioning.
2. using GJ tool to change the function of GJ, we observed the changes of cell protection after hypoxic postconditioning.
3. to explore the possible mechanism of hypoxic postconditioning protection.
Method:
Astrocytes were cultured from the cerebral cortex of newborn SD rats for 24 hours. After purification, the purity of astrocytes was determined by glial fibrillary acidic protein specific immunofluorescence. The models of hypoxia/reoxygenation and hypoxia postconditioning of astrocytes were established. The effects of hypoxic postconditioning on the survival rate of astrocytes were examined by MMT method, and the effects of HPO on apoptosis induced by H/R injury were observed by Annexin V/PI double staining and Hochest33258 fluorescence staining. After hypoxic postconditioning and GJ function regulation, the effects of HPO on fine cells were detected by Western blot. Effects of apoptotic proteins Bcl-2, Bax and caspase-3 on the expression of Cx43 protein and SRK protein were studied. The effects of HPO and GJ on the expression of Cx43 protein on the surface of astrocytes were detected by immunofluorescence Assay. Finally, Cx43 protein was specifically interfered with by siRNA to observe its effect on H/R damage. Injuries and HPO effects.
Result:
The purity of astrocytes was identified and the proportion of astrocytes under fluorescence microscope was more than 95%, indicating that high purity astrocytes were obtained.
The results of fluorescence tracing showed that the number of fluorescent transfer receptor cells in H/R group increased, the number of fluorescent transfer receptor cells in HPO group decreased, the number of fluorescent transfer receptor cells in HPO group increased 24 hours after pretreatment with 10 mu GJ enhancer RA, and the number of fluorescent transfer receptor cells in HPO group 1 hour after pretreatment with 25 mu GJ inhibitor oleamide. The number of transfer receptor cells decreased (p0.01). The results showed that HPO significantly decreased the enhancement of GJ function induced by H/R in astrocytes.
MTT assay showed that compared with the normal control group, the cell survival rate of H/R group decreased by 0.370 (+ 0.027) (p0.01); compared with H/R group, the cell survival rate of HPO group increased by 0.180 (+ 0.096) (p0.05); compared with HPO group, the cell survival rate of HPO group pretreated with RA group decreased by 0.188 (+ 0.049) (p0.05); the cell survival rate of HPO group pretreated with oleamide group increased by 0.18 (+). MTT results showed that HPO could decrease the cell survival rate induced by H/R, and could affect the effect of HPO by regulating GJ function.
The results of Annexin V/PI double staining showed that the early apoptosis rate of cells in each group was 0.082+0.009, 0.357+0.027, 0.217+0.008, 0.278+0.021 and 0.150+0.019. Compared with the normal control group, the apoptosis rate in H/R group was significantly increased, while HPO could significantly inhibit the apoptosis induced by H/R (p0.01). Pretreatment with GJ enhancer RA and GJ inhibitor oleamide was used. The apoptotic rate was increased and decreased compared with HPO group (P0.01).
The results of Hochest33258 fluorescence staining showed that compared with the normal control group, the apoptosis rate of H/R group was increased, that of H/R group was decreased after HPO, that of RA pretreated HPO group was increased, and that of oleamide pretreated HPO group was decreased.
Western blotting showed that the ratio of Bax/Bcl-2 increased in H/R group (p0.01), decreased in post-treatment group (p0.01), increased in RA pretreated HPO group (p0.05), and decreased in Olea pretreated HPO group (p0.05). After statistical analysis of caspase-3 protein expression, the expression of Caspase-3 shear bands increased in H/R group (p0.01), decreased in HPO group (p0.01), increased in HPO group pretreated with RA (p0.01), and increased in HPO group pretreated with Olea (p0.01). Compared with the normal control group, the expression of Src kinase increased in H/R group (p0.01), but decreased in post-treatment group (p0.01). Compared with HPO group, the expression of Src kinase increased in HPO group pretreated with RA (p0.05), and decreased in HPO group pretreated with oleamide (p0.05).
The expression of Cx43 on the surface of astrocyte membrane was detected by immunofluorescence. The expression of Cx43 on the surface of astrocyte membrane was abnormally increased in H/R group compared with normal control group, while the expression of Cx43 was significantly decreased in HPO group. The expression of Cx43 in HPO group increased 24 hours after pretreatment with 10 mu GJ enhancer RA, while the expression of Cx43 in HPO group increased after pretreatment with 25 mu GJ inhibitor oleamide for 1 hour. Compared with the blank control group, the expression of Cx43 protein in the H/R group was increased. Compared with the H/R group, the expression of Cx43 protein in the HPO group was decreased (p0.01). Compared with the HPO group, the expression of Cx43 protein in the RA pretreatment group was increased (p0.05) and that in the Olea pretreatment group was decreased (p0.05). It may be related to the change of Cx43 protein expression.
Using siRNA to interfere with Cx43 protein expression, we found that the negative control group had no significant effect on GJ function, cell survival rate and apoptosis rate (p0.05), but compared with the negative control group, the Cx43-siRNA transfection group could significantly reduce the increase of GJ function induced by H/R (p0.01). Compared with the negative control group, the Cx43-siRNA transfection group also significantly inhibited the increase of GJ function induced by H/R. Cx43-siRNA transfection group significantly decreased the rate of early apoptosis induced by H/R (p0.01). Cx43-siRNA transfection group significantly decreased the rate of late apoptosis induced by H/R (p0.01). These results suggest that interfering with Cx43 protein and inhibiting GJ function can inhibit the cell induced by H/R. The apoptosis rate increased.
Conclusion:
1. hypoxic postconditioning can reduce the GJ function of astrocytes induced by hypoxia / reoxygenation injury.
2. the protective effect of hypoxic postconditioning on hypoxia / reoxygenation injury may be related to the reduction of GJ function.
3. changes in GJ function will affect the protection of hypoxic postconditioning.
4. The protective effect of hypoxic postconditioning induced by decreasing GJ function may be related to Bax/Bcl-2 and caspase-3 related apoptotic proteins.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
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