縫隙連接細(xì)胞間通訊對(duì)大鼠星形膠質(zhì)細(xì)胞缺氧后處理的影響
發(fā)布時(shí)間：2018-09-01 19:49
【摘要】：目的： 1.觀察原代星形膠質(zhì)細(xì)胞中縫隙連接(GJ)在缺氧/復(fù)氧損傷和缺氧后處理之間的差異。 2.運(yùn)用GJ工具藥，改變GJ功能后，觀察缺氧后處理對(duì)細(xì)胞保護(hù)作用的變化。 3.初步探討缺氧后處理保護(hù)作用的可能作用機(jī)制。 方法： 采用細(xì)胞培養(yǎng)技術(shù)，取24h新生SD大鼠大腦皮層，培養(yǎng)星形膠質(zhì)細(xì)胞，在細(xì)胞純化后，通過(guò)膠質(zhì)纖維酸性蛋白特異性免疫熒光鑒定星形膠質(zhì)細(xì)胞純度。建立星形膠質(zhì)細(xì)胞缺氧/復(fù)氧模型和缺氧后處理模型。采用細(xì)胞接種熒光示蹤法(Parachute Assay)檢測(cè)星形膠質(zhì)細(xì)胞間的熒光傳遞功能。MMT法檢測(cè)缺氧后處理對(duì)星形膠質(zhì)細(xì)胞存活率的影響，采用Annexin V/PI雙染法和Hochest33258熒光染色法觀察HPO對(duì)H/R損傷導(dǎo)致的細(xì)胞凋亡的影響。采用Western blot檢測(cè)缺氧后處理及調(diào)控GJ功能后，HPO對(duì)細(xì)胞凋亡蛋白Bcl-2、Bax、caspase-3表達(dá)的影響以及對(duì)細(xì)胞Cx43蛋白和Src激酶蛋白表達(dá)的影響。通過(guò)免疫熒光法(Immunofluorescence Assay)檢測(cè)HPO及調(diào)控GJ功能對(duì)星形膠質(zhì)細(xì)胞膜表面Cx43蛋白表達(dá)的影響。最后通過(guò)siRNA特異性干擾Cx43蛋白，觀察其對(duì)H/R損傷和HPO的影響。 結(jié)果： 對(duì)星形膠質(zhì)細(xì)胞進(jìn)行純度鑒定后發(fā)現(xiàn)，熒光顯微鏡下占光鏡下星形膠質(zhì)細(xì)胞比例為95%以上，說(shuō)明得到了高純度星形膠質(zhì)細(xì)胞。 熒光示蹤實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)，與正常對(duì)照組相比，H/R組熒光傳遞受體細(xì)胞數(shù)增加；較H/R組，HPO組熒光傳遞受體細(xì)胞數(shù)降低；較HPO組，用10μM GJ增強(qiáng)劑RA預(yù)處理24h后的HPO組熒光傳遞受體細(xì)胞數(shù)增加，用25μM GJ抑制劑oleamide預(yù)處理1h后的HPO組熒光傳遞受體細(xì)胞數(shù)減少(p0.01)。結(jié)果表明，在星形膠質(zhì)細(xì)胞中，HPO可顯著降低H/R所致的GJ功能增強(qiáng)。 通過(guò)MTT實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)，較正常對(duì)照組，H/R組細(xì)胞存活率降低0.370±0.027(p0.01)；較H/R組， HPO組細(xì)胞存活率增加0.180±0.096(p0.05)；而較HPO組，使用RA預(yù)處理的HPO組的細(xì)胞存活率降低0.188±0.049(p0.05)，使用oleamide預(yù)處理的HPO組的細(xì)胞存活率增加0.186±0.089(p0.05）。MTT結(jié)果說(shuō)明HPO可以減少由H/R所導(dǎo)致的細(xì)胞存活率降低，而通過(guò)調(diào)控GJ功能，可以影響HPO的這種作用。 Annexin V/PI雙染實(shí)驗(yàn)結(jié)果顯示，各組細(xì)胞早期凋亡率為0.082±0.009，0.357±0.027，0.217±0.008，0.278±0.021和0.150±0.019。較正常對(duì)照組，H/R組的凋亡率明顯增加，而HPO可明顯抑制由H/R所引起的細(xì)胞凋亡(p0.01)。在使用了GJ增強(qiáng)劑RA和GJ抑制劑oleamide預(yù)處理后，細(xì)胞凋亡率較HPO組分別增加和降低(p0.01)。 Hochest33258熒光染色實(shí)驗(yàn)結(jié)果顯示，與正常對(duì)照組相比，H/R組細(xì)胞凋亡率增加；進(jìn)行HPO后，細(xì)胞凋亡率較H/R組降低；而較HPO組，使用RA預(yù)處理的HPO組的細(xì)胞凋亡率增加，使用oleamide預(yù)處理的HPO組的細(xì)胞凋亡率降低。 通過(guò)western blotting實(shí)驗(yàn)發(fā)現(xiàn)，與空白對(duì)照組相比， H/R組Bax/Bcl-2的比例增加(p0.01)；在進(jìn)行后處理后，較H/R組，Bax/Bcl-2的比例降低(p0.01)；與HPO組相比，用RA預(yù)處理的HPO組Bax/Bcl-2的比例增加(p0.05)，用Olea預(yù)處理的HPO組Bax/Bcl-2的比例降低(p0.05)。另外，對(duì)caspase-3蛋白表達(dá)進(jìn)行統(tǒng)計(jì)分析后，與空白對(duì)照組相比，H/R組caspase-3剪切條帶表達(dá)增加(p0.01)；與H/R組相比，HPO組caspase-3剪切條帶表達(dá)減少(p0.01)；與HPO組相比，用RA預(yù)處理的HPO組caspase-3剪切條帶表達(dá)增加(p0.01)，用Olea預(yù)處理的HPO組caspase-3剪切條帶表達(dá)減少(p0.05)。與正常對(duì)照組相比，H/R組Src激酶表達(dá)增加(p0.01)，而經(jīng)過(guò)后處理后，Src激酶表達(dá)降低(p0.01)。較HPO組，使用RA預(yù)處理的HPO組Src激酶表達(dá)增加(p0.05)，使用oleamide預(yù)處理的HPO組的Src激酶表達(dá)減少(p0.05)。 通過(guò)免疫熒光檢測(cè)星形膠質(zhì)細(xì)胞膜表面Cx43蛋白表達(dá)可發(fā)現(xiàn)， H/R組，細(xì)胞膜Cx43表達(dá)較正常對(duì)照組異常增強(qiáng)，而HPO可顯著減少Cx43的表達(dá)。在用10μM GJ增強(qiáng)劑RA預(yù)處理24h后，HPO組的Cx43表達(dá)增加，而在用25μM GJ抑制劑oleamide預(yù)處理1h后，HPO組Cx43表達(dá)減少。通過(guò)western blotting檢測(cè)并對(duì)條帶灰度值掃描并進(jìn)行統(tǒng)計(jì)學(xué)分析后，較空白對(duì)照組，H/R組Cx43蛋白表達(dá)增加。較H/R組，HPO組降低(p0.01)。而與HPO組相比，RA預(yù)處理組蛋白表達(dá)增加(p0.05)，Olea預(yù)處理組蛋白表達(dá)降低(p0.05)。這說(shuō)明GJ功能的改變有可能與Cx43蛋白表達(dá)的變化有關(guān)。 采用siRNA干擾Cx43蛋白表達(dá)可以發(fā)現(xiàn)，陰性對(duì)照組對(duì)細(xì)胞GJ功能、細(xì)胞存活率和凋亡率無(wú)顯著影響(p0.05)，但與陰性對(duì)照組相比，Cx43-siRNA轉(zhuǎn)染組可顯著降低由H/R導(dǎo)致的GJ功能增加(p0.01)。與陰性對(duì)照組相比，Cx43-siRNA轉(zhuǎn)染組也顯著抑制了由H/R導(dǎo)致的細(xì)胞存活率降低(p0.01)，。Cx43-siRNA轉(zhuǎn)染組較陰性對(duì)照組，，可顯著降低H/R導(dǎo)致的細(xì)胞早期凋亡率增加(p0.01)。 Cx43-siRNA轉(zhuǎn)染組顯著降低H/R導(dǎo)致的細(xì)胞晚期凋亡率增加(p0.01)。以上結(jié)果說(shuō)明干擾Cx43蛋白，抑制GJ功能，可以抑制由H/R所致的細(xì)胞凋亡率增加。 結(jié)論： 1.缺氧后處理可以降低由缺氧/復(fù)氧損傷導(dǎo)致的星形膠質(zhì)細(xì)胞間GJ功能增強(qiáng)。 2.缺氧后處理對(duì)缺氧/復(fù)氧損傷的保護(hù)作用可能與降低GJ功能有關(guān)。 3.改變GJ功能會(huì)影響缺氧后處理的保護(hù)作用。 4.降低GJ功能導(dǎo)致的缺氧后處理保護(hù)作用可能與Bax/Bcl-2、caspase3相關(guān)凋亡蛋白有關(guān)。
[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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3
本文編號(hào)：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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3
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