应用活细胞工作站测量大鼠脊髓星形胶质细胞氧糖剥夺/复氧后细胞体积的研究
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摘要
目的:探讨应用活细胞工作站测量大鼠脊髓星形胶质细胞氧糖剥夺/复氧(OGD/R)后细胞体积的效果。方法:取新生24h内Sprague-Dawley乳鼠,取出乳鼠脊髓采用胰酶消化及差速粘附法分离大鼠脊髓星形胶质细胞,并进行体外扩增培养。将第4代成熟的星形胶质细胞进行氧糖剥夺/复氧处理,并分为4组:A组(不做糖氧剥夺及复氧处理),B组(糖氧剥夺6h,复氧12h),C组(糖氧剥夺6h,复氧24h),D组(糖氧剥夺6h,复氧48h)。通过活细胞工作站扫描检测计算各组细胞的体积,利用光镜及透射电镜观察细胞形态和超微结构变化,通过Western blot测定其水通道蛋白4(Aquaporin 4,AQP4)表达变化来进一步验证活细胞工作站测量细胞体积的准确性。结果:利用活细胞工作站检测体积,与A组细胞2810.19±306.11μm~3相比,B组细胞体积4311.53±407.73μm~3明显高于A组(P<0.01),C组细胞体积6248.86±702.11μm~3为到最大值,D组细胞体积4541.33±503.17μm~3,细胞体积较C组有所下降,但仍明显高于A组,差异有统计学意义(P<0.01)。光镜下观察细胞B组出现肿胀、折光性增强,C组细胞水肿达到高峰,D组水肿虽有缓解,但仍很明显;透射电镜下观察B组可见细胞器开始肿胀,C组细胞肿胀更加明显,出现线粒体嵴发生断裂等表现,D组水肿稍有缓解,但仍很明显。Western blot结果显示,A组细胞AQP4蛋白表达为(12.37±0.85)%,B组细胞AQP4蛋白表达为(34.17±2.07)%,C组细胞AQP4蛋白表达(43.57±1.75)%,D组细胞AQP4蛋白表达(32.26±1.37)%,与A组细胞相比,B组、C组、D组明显高于A组(P<0.01),与C组细胞相比,B组、D组明显低于C组(P<0.01)。结论:活细胞工作站能较为准确地检测大鼠脊髓星形胶质细胞氧糖剥夺/复氧后细胞体积,可作为一种常规的检测手段运用于星形胶质细胞体积测量。
Objectives: To investigate the effect of oxygen-glucose deprivation/reoxygenation(OGD/R) by live cell imaging system on the volume of rat spinal cord astrocytes. Methods: The neonatal Sprague-Dawley rats within 24 hours were taken and the rat spinal cords were collected. Rat spinal cord astrocytes were isolated by trypsinization and differential adhesion ways and cultured in vitro. The fourth generation mature astrocytes were conducted with OGD/R and divided into four groups: group A(normal astrocytes), group B(OGD6 h/R12 h),group C(OGD6 h/R24 h), group D(OGD6 h/R 48 h) group. The astrocyte volumes were measured and calculated by live cell imaging system, and the morphological and ultrastructural changes of the astrocytes were observed by light microscopy and transmission electron microscopy, and the expression of AQP4 protein was measured by Western blot protein to further testify the accuracy of live cell imaging system in measuring cell volume.Results: Compared with group A(2810.19 ±306.11 μm~3), the cell volume of group B(4311.53 ±407.73 μm~3)was significantly increased(P<0.01), and the maximum increase was observed in group C(6248.86±702.11μm~3)(P<0.01). The astrocytes volume in group D(4541.33±503.17 μm~3) was reduced, and it was higher than group A(P<0.01). The astrocyte swelling and increased refraction were observed in group B with light microscope. The cells swelling reached the peak in group C and was obvious in group D although the swelling was alleviated. The swelling of astrocyte organelles was observed in group B under transmission electron microscope. The organelles swelling was more significant and the breakage of mitochondria was observed in group C. The swelling was slightly relieved, but it was evident in group D. The Western blot results showed that the AQP4 expression in group A was(12.37±1.25)%, the AQP4 expression in group B was(35.50±1.08)%, the AQP4 expression in group C was(43.57±1.75)%, the AQP4 expression in group D was(32.63±0.81)%. Compared with that in group A, the AQP4 expression was significantly increased in group B, group C and group D(P<0.01). Compared with that in group C, the AQP4 expression was reduced in group B and group D(P<0.01). Conclusions: Live cell imaging system is used to measure the swelling volume of rat spinal cord astrocytes after oxygen glucose deprivation/reoxygenation, and this method can be used as a routine detection method in the measurement of astrocytes volume. It can also be applied in the detection of other cell volumes.
Objectives: To investigate the effect of oxygen-glucose deprivation/reoxygenation(OGD/R) by live cell imaging system on the volume of rat spinal cord astrocytes. Methods: The neonatal Sprague-Dawley rats within 24 hours were taken and the rat spinal cords were collected. Rat spinal cord astrocytes were isolated by trypsinization and differential adhesion ways and cultured in vitro. The fourth generation mature astrocytes were conducted with OGD/R and divided into four groups: group A(normal astrocytes), group B(OGD6 h/R12 h),group C(OGD6 h/R24 h), group D(OGD6 h/R 48 h) group. The astrocyte volumes were measured and calculated by live cell imaging system, and the morphological and ultrastructural changes of the astrocytes were observed by light microscopy and transmission electron microscopy, and the expression of AQP4 protein was measured by Western blot protein to further testify the accuracy of live cell imaging system in measuring cell volume.Results: Compared with group A(2810.19 ±306.11 μm~3), the cell volume of group B(4311.53 ±407.73 μm~3)was significantly increased(P<0.01), and the maximum increase was observed in group C(6248.86±702.11μm~3)(P<0.01). The astrocytes volume in group D(4541.33±503.17 μm~3) was reduced, and it was higher than group A(P<0.01). The astrocyte swelling and increased refraction were observed in group B with light microscope. The cells swelling reached the peak in group C and was obvious in group D although the swelling was alleviated. The swelling of astrocyte organelles was observed in group B under transmission electron microscope. The organelles swelling was more significant and the breakage of mitochondria was observed in group C. The swelling was slightly relieved, but it was evident in group D. The Western blot results showed that the AQP4 expression in group A was(12.37±1.25)%, the AQP4 expression in group B was(35.50±1.08)%, the AQP4 expression in group C was(43.57±1.75)%, the AQP4 expression in group D was(32.63±0.81)%. Compared with that in group A, the AQP4 expression was significantly increased in group B, group C and group D(P<0.01). Compared with that in group C, the AQP4 expression was reduced in group B and group D(P<0.01). Conclusions: Live cell imaging system is used to measure the swelling volume of rat spinal cord astrocytes after oxygen glucose deprivation/reoxygenation, and this method can be used as a routine detection method in the measurement of astrocytes volume. It can also be applied in the detection of other cell volumes.
引文
1. Ahmed A, Patil AA, Agrawal DK. Immunobiology of spinal cord injuries and potential therapeutic approaches[J]. Mol Cell Biochem, 2018, 441(1-2):181-189.
2 . Anwar MA, Al Shehabi TS, Eid AH. Inflammogenesis of secondary spinal cord injury[J]. Front Cell Neurosci, 2016, 10(14):98-121.
3 . Okada S, Hara M, Kobayakawa K, et al. Astrocyte reactivity and astrogliosis after spinal cord injury[J]. Neurosci Res,2018, 126:39-43.
4 . Leonard AV, Thornton E, Vink R. The relative contribution of edema and hemorrhage to raised intrathecal pressure after traumatic spinal cord injury[J]. J Neurotrauma, 2015, 32(6):397-402.
5 . Hu AM, Li JJ, Sun W, et al. Myelotomy reduces spinal cord edema and inhibits aquaporin-4 and aquaporin-9 expression in rats with spinal cord injury[J]. Spinal Cord, 2015, 53(2):98-102.
6 . Xiao M, Hu G. Involvement of aquaporin 4 in astrocyte function and neuropsychiatric disorders[J]. CNS Neurosci Ther,2014, 20(5):385-390.
7 . Kletzien RF, Pariza MW, Becker JE, et al. A method using 3 -O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture[J]. Anal Biochem, 1975, 68(2):537-544.
8 . Kerstetter AE, Miller RH. Isolation and culture of spinal cord astrocytes[J]. Methods Mol Biol, 2012, 814:93-104.
9 . Takebayashi H, Nabeshima Y, Yoshida S, et al. The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages[J]. Curr Biol,2002, 12(13):1157-1163.
10 . de Pablo Y, Nilsson M, Pekna M, et al. Intermediate filaments are important for astrocyte response to oxidative stress induced by oxygen-glucose deprivation and reperfusion[J].Histochem Cell Biol, 2013, 140(1):81-91.
11 . Agre P, Sasaki S, Chrispeels MJ. Aquaporins:a family of water channel proteins[J]. Am J Physiol, 1993, 265(3 Pt 2):F461.
12 . Oklinski MK, Skowronski MT, Skowronska A, et al. Aquaporins in the spinal cord[J]. Int J Mol Sci, 2016, 17(12).pii:E2050.
13 .王东旭,许卫兵.水通道蛋白与脊髓水肿的研究进展[J].脊柱外科杂志, 2015, 13(4):250-253.
14 . Nagelhus EA, Ottersen OP. Physiological roles of aquaporin-4 in brain[J]. Physiol Rev, 2013, 93(4):1543-1562.
15 . Li Y, Hu H, Liu J, et al. Effects of aquaporin 4 and inward rectifier potassium channel 4.1 on medullospinal edema after methylprednisolone treatment to suppress acute spinal cord injury in rats[J]. Acta Cir Bras, 2018, 33(2):175-184.
16 . Saadoun S, Papadopoulos MC. Aquaporin-4 in brain and spinal cord oedema[J]. Neuroscience, 2010, 168(4):1036-1046.
17 . Sturdivant NM, Smith SG, Ali SF, et al. Acetazolamide mitigates astrocyte cellular edema following mild traumatic brain injury[J]. Sci Rep, 2016, 6:33330. doi:10.1038/srep33330.
18 .邓江山,赵飞,俞晓燕,等.低糖对星形胶质细胞水通道蛋白4表达及分布的影响[J].中国神经免疫学和神经病学杂志, 2015, 22(4):263-268.
19 . Morris RT, Laye MJ, Lees SJ, et al. Exercise-induced attenuation of obesity, hyperinsulinemia, and skeletal muscle lipid peroxidation in the OLETF rat[J]. J Appl Physiol(1985),2008, 104(3):708-715.
20 . Lachmann V, G觟rg B, Bidmon HJ, et al. Precipitants of hepatic encephalopathy induce rapid astrocyte swelling in an oxidative stress dependent manner[J]. Arch Biochem Biophys,2013, 536(2):143-151.
2 . Anwar MA, Al Shehabi TS, Eid AH. Inflammogenesis of secondary spinal cord injury[J]. Front Cell Neurosci, 2016, 10(14):98-121.
3 . Okada S, Hara M, Kobayakawa K, et al. Astrocyte reactivity and astrogliosis after spinal cord injury[J]. Neurosci Res,2018, 126:39-43.
4 . Leonard AV, Thornton E, Vink R. The relative contribution of edema and hemorrhage to raised intrathecal pressure after traumatic spinal cord injury[J]. J Neurotrauma, 2015, 32(6):397-402.
5 . Hu AM, Li JJ, Sun W, et al. Myelotomy reduces spinal cord edema and inhibits aquaporin-4 and aquaporin-9 expression in rats with spinal cord injury[J]. Spinal Cord, 2015, 53(2):98-102.
6 . Xiao M, Hu G. Involvement of aquaporin 4 in astrocyte function and neuropsychiatric disorders[J]. CNS Neurosci Ther,2014, 20(5):385-390.
7 . Kletzien RF, Pariza MW, Becker JE, et al. A method using 3 -O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture[J]. Anal Biochem, 1975, 68(2):537-544.
8 . Kerstetter AE, Miller RH. Isolation and culture of spinal cord astrocytes[J]. Methods Mol Biol, 2012, 814:93-104.
9 . Takebayashi H, Nabeshima Y, Yoshida S, et al. The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages[J]. Curr Biol,2002, 12(13):1157-1163.
10 . de Pablo Y, Nilsson M, Pekna M, et al. Intermediate filaments are important for astrocyte response to oxidative stress induced by oxygen-glucose deprivation and reperfusion[J].Histochem Cell Biol, 2013, 140(1):81-91.
11 . Agre P, Sasaki S, Chrispeels MJ. Aquaporins:a family of water channel proteins[J]. Am J Physiol, 1993, 265(3 Pt 2):F461.
12 . Oklinski MK, Skowronski MT, Skowronska A, et al. Aquaporins in the spinal cord[J]. Int J Mol Sci, 2016, 17(12).pii:E2050.
13 .王东旭,许卫兵.水通道蛋白与脊髓水肿的研究进展[J].脊柱外科杂志, 2015, 13(4):250-253.
14 . Nagelhus EA, Ottersen OP. Physiological roles of aquaporin-4 in brain[J]. Physiol Rev, 2013, 93(4):1543-1562.
15 . Li Y, Hu H, Liu J, et al. Effects of aquaporin 4 and inward rectifier potassium channel 4.1 on medullospinal edema after methylprednisolone treatment to suppress acute spinal cord injury in rats[J]. Acta Cir Bras, 2018, 33(2):175-184.
16 . Saadoun S, Papadopoulos MC. Aquaporin-4 in brain and spinal cord oedema[J]. Neuroscience, 2010, 168(4):1036-1046.
17 . Sturdivant NM, Smith SG, Ali SF, et al. Acetazolamide mitigates astrocyte cellular edema following mild traumatic brain injury[J]. Sci Rep, 2016, 6:33330. doi:10.1038/srep33330.
18 .邓江山,赵飞,俞晓燕,等.低糖对星形胶质细胞水通道蛋白4表达及分布的影响[J].中国神经免疫学和神经病学杂志, 2015, 22(4):263-268.
19 . Morris RT, Laye MJ, Lees SJ, et al. Exercise-induced attenuation of obesity, hyperinsulinemia, and skeletal muscle lipid peroxidation in the OLETF rat[J]. J Appl Physiol(1985),2008, 104(3):708-715.
20 . Lachmann V, G觟rg B, Bidmon HJ, et al. Precipitants of hepatic encephalopathy induce rapid astrocyte swelling in an oxidative stress dependent manner[J]. Arch Biochem Biophys,2013, 536(2):143-151.