【摘要】：目的： 1.觀察原代星形膠質(zhì)細胞中縫隙連接(GJ)在缺氧/復氧損傷和缺氧后處理之間的差異。 2.運用GJ工具藥，改變GJ功能后，觀察缺氧后處理對細胞保護作用的變化。 3.初步探討缺氧后處理保護作用的可能作用機制。 方法： 采用細胞培養(yǎng)技術，取24h新生SD大鼠大腦皮層，培養(yǎng)星形膠質(zhì)細胞，在細胞純化后，通過膠質(zhì)纖維酸性蛋白特異性免疫熒光鑒定星形膠質(zhì)細胞純度。建立星形膠質(zhì)細胞缺氧/復氧模型和缺氧后處理模型。采用細胞接種熒光示蹤法(Parachute Assay)檢測星形膠質(zhì)細胞間的熒光傳遞功能。MMT法檢測缺氧后處理對星形膠質(zhì)細胞存活率的影響，采用Annexin V/PI雙染法和Hochest33258熒光染色法觀察HPO對H/R損傷導致的細胞凋亡的影響。采用Western blot檢測缺氧后處理及調(diào)控GJ功能后，HPO對細胞凋亡蛋白Bcl-2、Bax、caspase-3表達的影響以及對細胞Cx43蛋白和Src激酶蛋白表達的影響。通過免疫熒光法(Immunofluorescence Assay)檢測HPO及調(diào)控GJ功能對星形膠質(zhì)細胞膜表面Cx43蛋白表達的影響。最后通過siRNA特異性干擾Cx43蛋白，觀察其對H/R損傷和HPO的影響。 結果： 對星形膠質(zhì)細胞進行純度鑒定后發(fā)現(xiàn)，熒光顯微鏡下占光鏡下星形膠質(zhì)細胞比例為95%以上，說明得到了高純度星形膠質(zhì)細胞。 熒光示蹤實驗結果發(fā)現(xiàn)，與正常對照組相比，H/R組熒光傳遞受體細胞數(shù)增加；較H/R組，HPO組熒光傳遞受體細胞數(shù)降低；較HPO組，用10μM GJ增強劑RA預處理24h后的HPO組熒光傳遞受體細胞數(shù)增加，用25μM GJ抑制劑oleamide預處理1h后的HPO組熒光傳遞受體細胞數(shù)減少(p0.01)。結果表明，在星形膠質(zhì)細胞中，HPO可顯著降低H/R所致的GJ功能增強。 通過MTT實驗結果發(fā)現(xiàn)，較正常對照組，H/R組細胞存活率降低0.370±0.027(p0.01)；較H/R組， HPO組細胞存活率增加0.180±0.096(p0.05)；而較HPO組，使用RA預處理的HPO組的細胞存活率降低0.188±0.049(p0.05)，使用oleamide預處理的HPO組的細胞存活率增加0.186±0.089(p0.05）。MTT結果說明HPO可以減少由H/R所導致的細胞存活率降低，而通過調(diào)控GJ功能，可以影響HPO的這種作用。 Annexin V/PI雙染實驗結果顯示，各組細胞早期凋亡率為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所引起的細胞凋亡(p0.01)。在使用了GJ增強劑RA和GJ抑制劑oleamide預處理后，細胞凋亡率較HPO組分別增加和降低(p0.01)。 Hochest33258熒光染色實驗結果顯示，與正常對照組相比，H/R組細胞凋亡率增加；進行HPO后，細胞凋亡率較H/R組降低；而較HPO組，使用RA預處理的HPO組的細胞凋亡率增加，使用oleamide預處理的HPO組的細胞凋亡率降低。 通過western blotting實驗發(fā)現(xiàn)，與空白對照組相比， H/R組Bax/Bcl-2的比例增加(p0.01)；在進行后處理后，較H/R組，Bax/Bcl-2的比例降低(p0.01)；與HPO組相比，用RA預處理的HPO組Bax/Bcl-2的比例增加(p0.05)，用Olea預處理的HPO組Bax/Bcl-2的比例降低(p0.05)。另外，對caspase-3蛋白表達進行統(tǒng)計分析后，與空白對照組相比，H/R組caspase-3剪切條帶表達增加(p0.01)；與H/R組相比，HPO組caspase-3剪切條帶表達減少(p0.01)；與HPO組相比，用RA預處理的HPO組caspase-3剪切條帶表達增加(p0.01)，用Olea預處理的HPO組caspase-3剪切條帶表達減少(p0.05)。與正常對照組相比，H/R組Src激酶表達增加(p0.01)，而經(jīng)過后處理后，Src激酶表達降低(p0.01)。較HPO組，使用RA預處理的HPO組Src激酶表達增加(p0.05)，使用oleamide預處理的HPO組的Src激酶表達減少(p0.05)。 通過免疫熒光檢測星形膠質(zhì)細胞膜表面Cx43蛋白表達可發(fā)現(xiàn)， H/R組，細胞膜Cx43表達較正常對照組異常增強，而HPO可顯著減少Cx43的表達。在用10μM GJ增強劑RA預處理24h后，HPO組的Cx43表達增加，而在用25μM GJ抑制劑oleamide預處理1h后，HPO組Cx43表達減少。通過western blotting檢測并對條帶灰度值掃描并進行統(tǒng)計學分析后，較空白對照組，H/R組Cx43蛋白表達增加。較H/R組，HPO組降低(p0.01)。而與HPO組相比，RA預處理組蛋白表達增加(p0.05)，Olea預處理組蛋白表達降低(p0.05)。這說明GJ功能的改變有可能與Cx43蛋白表達的變化有關。 采用siRNA干擾Cx43蛋白表達可以發(fā)現(xiàn)，陰性對照組對細胞GJ功能、細胞存活率和凋亡率無顯著影響(p0.05)，但與陰性對照組相比，Cx43-siRNA轉(zhuǎn)染組可顯著降低由H/R導致的GJ功能增加(p0.01)。與陰性對照組相比，Cx43-siRNA轉(zhuǎn)染組也顯著抑制了由H/R導致的細胞存活率降低(p0.01)，。Cx43-siRNA轉(zhuǎn)染組較陰性對照組，，可顯著降低H/R導致的細胞早期凋亡率增加(p0.01)。 Cx43-siRNA轉(zhuǎn)染組顯著降低H/R導致的細胞晚期凋亡率增加(p0.01)。以上結果說明干擾Cx43蛋白，抑制GJ功能，可以抑制由H/R所致的細胞凋亡率增加。 結論： 1.缺氧后處理可以降低由缺氧/復氧損傷導致的星形膠質(zhì)細胞間GJ功能增強。 2.缺氧后處理對缺氧/復氧損傷的保護作用可能與降低GJ功能有關。 3.改變GJ功能會影響缺氧后處理的保護作用。 4.降低GJ功能導致的缺氧后處理保護作用可能與Bax/Bcl-2、caspase3相關凋亡蛋白有關。
[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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.3

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