基于DNA甲基化途径同型半胱氨酸对大鼠神经干细胞增殖作用机制的研究

发布时间：2018-05-27 08:45

本文选题：同型半胱氨酸 + 神经干细胞　；参考：《天津医科大学》2014年硕士论文

【摘要】：目的研究同型半胱氨酸(homocysteine, Hcy)对体外培养神经干细胞(neural stem cells, NSCs)增殖作用的影响,以及通过建立大脑中动脉栓塞(middle cerebral artrty occlusion, MCAO)模型,观察Hcy对局灶性脑缺血损伤大鼠神经干细胞增殖的作用,探讨Hcy基于DNA甲基化途径调控NSCs增殖的作用机制。方法采用无血清培养原代培养新生大鼠NSCs,将细胞随机分成4组：正常对照组(培养液中添加Hcy0μmol/l, Hcy-C组)、Hey低剂量组(培养液中添加Hey30μmol/l, Hcy-L组)、Hcy中剂量组(培养液中添加Hey300μmol/l, Hcy-M组)、Hcy高剂量组(培养液中添加Hey1mmol/l,Hcy-H组,)。采用Cell Dimension软件对神经球直径进行测量；采用免疫荧光(Immunofluorescent, IF)检测NSCs标记物Brdu和Sox2的表达：采用乳酸脱氢酶(lactate dehydrogenase, LDH)测定试剂盒检测不同浓度Hcy作用下培养液中LDH的活力；采用DNA甲基化定量试剂盒检测不同Hcy浓度下细胞总甲基化水平；采用甲基转移酶活性检测试剂盒检测DNMTs总活性以及Western Blot检测DNA甲基转移酶(DNA methyltransferases, DNMTs)的蛋白表达；采用高效液相法(HPLC)检测细胞内S-腺苷甲硫氨酸(s-adenosylmethionine, SAM)、S-腺苷同型半胱氨酸(s-adenosylhomocysteine, SAH)的水平。成年雄性SD(sprague-nawley)大鼠随机分假手术组(sham operation group, SO组)、大脑中动脉栓塞模型(middle cerebral artery occlusion group, MCAO组)以及大脑中动脉栓塞模型+同型半胱氨酸(middle cerebral artery occlusion+Hcy group, MCAO+Hcy组)3组。SO组和MCAO组腹腔注射生理盐水5ml/kg·bw·d, MCAO+Hcy组腹腔注射2%Hcy溶液5ml/kg·bw·d,注射28d。通过Morris水迷宫实验检测大鼠的空间学习记忆能力；采用免疫组化法以及Western blot检测Sox2蛋白的表达；采用DNA甲基化定量试剂盒检测总甲基化水平；采用甲基转移酶活性检测试剂盒检测DNMTs总活性；采用Western Blot检测DNA甲基转移酶的蛋白表达。采用高效液相法(HPLC)检测大鼠血浆内Hey、SAM、SAH的水平。结果用无血清培养原代培养新生大鼠NSCs6d后,细胞聚集成球形团块,呈悬浮状态,Hcy添加组细胞神经球直径较对照组小,且差异均有统计学意义(P0.05)。Brdu和Sox2经免疫荧光双标记法鉴定均呈双阳性表达,Hcy添加组Brdu/Sox2双标阳性率较对照组小,且差异均有统计学意义(P0.05)。经LDH测定试剂盒检测,Hcy添加组细胞培养液中LDH活性较对照组升高,且差异均有统计学意义(P0.05)。DNA总甲基化水平检测结果显示：与对照组相比,Hey添加组细胞总甲基化水平降低,差异均有统计学意义(P0.05)。DNMTs总活性检测结果显示：与对照组相比,Hcy添加组较对照组DNMTs总活性均下降,差异均有统计学意义(P0.05)。Western Blot结果显示,与对照组相比,Hcy添加组DNMT1蛋白表达均下降,差异均有统计学意义(P0.05),Hcy-H组较Hcy-L组和Hcy-M组DNMT1蛋白表达下降,差异均有统计学意义(P0.05)；与对照组和Hcy-L组相比,Hcy-M组和Hcy-H组DNMT3a蛋白表达均下降,差异均有统计学意义(P0.05)；与对照组和Hcy-L组相比,Hcy-M组和Hcy-H组DNMT3b蛋白表达均下降,差异均有统计学意义(P0.05)。HPLC检测结果显示：与对照组和Hcy-L组相比,Hcy-M组和Hcy-H组SAH的水平升高,差异均有统计学意义(P0.05)；与对照组和Hcy-L组相比,Hcy-M组和Hcy-H组SAM/SAH比值降低,差异均有统计学意义(P0.05)。腹腔注射及造模成功后Morris水迷宫实验显示,与MCAO组相比,MCAO+Hcy组大鼠逃避潜伏期明显缩短,差异均有统计学意义(P0.05)；与MCAO组相比,及MCAO+Hcy组单位时间内穿越平台区域次数明显减少,差异均有统计学意义(P0.05)。DNA,总甲基化水平检测结果显示：与MCAO组相比,MCAO+Hcy组总甲基化水平下降,差异均有统计学意义(P0.05)。经免疫组化及Western blot检测：与MCAO组相比,MCAO+Hcy组Sox2蛋白表达降低,差异均有统计学意义(P0.05)。DNMTs,总活性检测结果显示：与MCAO组相比,MCAO+Hcy组Sox2蛋白表达降低DNMTs活性下降,差异均有统计学意义(P0.05)。Western blot结果显示：与MCAO组相比,MCAO+Hcy组DNMT1、 DNMT3a蛋白表达降低,差异均有统计学意义(P0.05)。经HPLC检测显示,与SO组及MCAO组相比,MCAO+Hcy组Hcy浓度下降,差异均有统计学意义(P0.05)；与SO组及MCAO组相比,MCAO+Hcy组SAM/SAH比值下降,差异均有统计学意义(P0.05)。免疫荧光染色结果显示,Sox2呈阳性表达,且MCAO+Hcy组表达较SO组及MCAO组降低,差异均有统计学意义(P0.05)。结论本实验成功的分离培养大鼠新生鼠NSCs以及建立大鼠大脑中动脉栓塞模型。Hcy抑制NSCs的增殖其机制可能是：Hcy通过改变甲基化途径中关键代谢物SAH的浓度,降低SAM/SAH的比值,同时降低甲基化转移酶的活性及蛋白的表达,抑制了甲基化反应的发生,进而抑制了NSCs的增殖。
[Abstract]:objective
To investigate the effect of homocysteine (Hcy) on the proliferation of neural stem cells (NSCs) in vitro, and to observe the effect of Hcy on the proliferation of neural stem cells in rats with focal cerebral hemorrhage injury (middle cerebral artrty occlusion, MCAO) by establishing the model of the middle cerebral artery embolism (middle cerebral artrty occlusion, MCAO). Methylation pathway regulates the mechanism of NSCs proliferation.
Method
The primary cultured rat NSCs was cultured in serum-free culture, and the cells were randomly divided into 4 groups: normal control group (Hcy0 mol/l, Hcy-C group), low dose Hey group (Hey30 mol/l, Hcy-L group), Hcy medium dose group (Hey300 mu mol/l, Hcy-M group). Y-H group, Cell Dimension software was used to measure the diameter of the nerve bulb; the expression of NSCs markers Brdu and Sox2 were detected by Immunofluorescent (IF): the assay kit was used to determine the vitality in the incubated liquid with different concentrations in the incubated liquid with the lactic dehydrogenase (lactate dehydrogenase, LDH). The total methylation level of cells under different Hcy concentrations was detected by the agent box; the total activity of DNMTs and the expression of DNA methyltransferase (DNA methyltransferases, DNMTs) were detected by the methyl transferase activity detection kit and Western Blot, and the intracellular S- adenosine methionine (S-adenosylmethionine, SAM) was detected by high performance liquid phase (HPLC) method. The level of S- adenosine homocysteine (S-adenosylhomocysteine, SAH).
The adult male SD (sprague-nawley) rats were randomly divided into 3 groups, including the sham operation group, the SO group, the middle cerebral artery embolism model (middle cerebral artery occlusion group, the MCAO group) and the middle cerebral artery embolism model + homocysteine group. 5ml/kg / BW / D was injected into physiological saline, group MCAO+Hcy was injected with 2%Hcy solution 5ml/kg BW. D, and 28d. was injected by Morris water maze test to detect the spatial learning and memory ability of rats. Immunohistochemistry and Western blot were used to detect the expression of the protein. The total activity of DNMTs was detected by the enzyme activity detection kit and the protein expression of DNA methyltransferase was detected by Western Blot. The level of Hey, SAM and SAH in rat plasma was detected by high performance liquid phase (HPLC).
Result
After the serum-free culture of the primary culture of neonatal rat NSCs6d, the cells were aggregated into spherical mass and suspended. The diameter of the cell nerve ball in the Hcy addition group was smaller than that of the control group, and the difference was statistically significant (P0.05).Brdu and Sox2 were both positive and positive by immunofluorescence double labeling, and the positive rate of Brdu/Sox2 double standard in Hcy addition group was more than that of the control group. The difference was statistically significant (P0.05). The activity of LDH in the Hcy added group was higher than that of the control group by LDH assay, and the difference was statistically significant (P0.05).DNA total methylation level detection results showed that compared with the control group, the total methylation level of Hey added group decreased, the difference was statistically significant (P). 0.05).DNMTs total activity detection results showed that compared with the control group, the total activity of DNMTs in the Hcy addition group decreased compared with the control group, and the difference was statistically significant (P0.05).Western Blot results showed that the expression of DNMT1 protein in Hcy added group decreased, the difference was statistically significant (P0.05), Hcy-H group was more than Hcy-L group and Hcy-M group eggs. The difference of white expression was statistically significant (P0.05). Compared with the control group and the Hcy-L group, the expression of DNMT3a protein in the Hcy-M group and the Hcy-H group decreased, and the difference was statistically significant (P0.05). Compared with the control group and the Hcy-L group, the expression of DNMT3b protein in Hcy-M and Hcy-H groups decreased, and the difference was statistically significant (P0.05).HPLC detection results showed a significant difference. Compared with the control group and the Hcy-L group, the level of SAH in group Hcy-M and group Hcy-H was higher, the difference was statistically significant (P0.05). Compared with the control group and the Hcy-L group, the SAM/SAH ratio of the Hcy-M group and the Hcy-H group decreased, the difference was statistically significant (P0.05).
After the intraperitoneal injection and the success of the Morris water maze test, the escape latency of the MCAO+Hcy group was significantly shorter than that of the MCAO group, and the difference was statistically significant (P0.05). Compared with the MCAO group, the number of crossing platform areas in the unit time of MCAO+Hcy group decreased significantly (P0.05).DNA, and the total methylation level was significant. The results showed that compared with the MCAO group, the total methylation level of MCAO+Hcy group decreased, the difference was statistically significant (P0.05). After immunohistochemistry and Western blot, the Sox2 protein expression in the MCAO+Hcy group decreased, the difference was statistically significant (P0.05).DNMTs, the total activity detection results showed that the MCAO+Hcy group was compared with the MCAO group. The expression of ox2 protein decreased DNMTs activity, and the difference was statistically significant (P0.05).Western blot results showed that the expression of DNMT3a protein in MCAO+Hcy group was lower than that of MCAO group, and the difference was statistically significant (P0.05). 05): compared with group SO and group MCAO, the SAM/SAH ratio of group MCAO+Hcy decreased, and the difference was statistically significant (P0.05). The results of immunofluorescence staining showed that Sox2 was positive, and the expression of MCAO+Hcy group was lower than that of SO and MCAO groups, and the difference was statistically significant (P0.05).
conclusion
The successful separation and cultivation of neonatal rat NSCs and the establishment of rat middle cerebral artery embolization model.Hcy inhibit the proliferation of NSCs may be: Hcy by changing the concentration of the key metabolite SAH in the methylation pathway, reducing the ratio of SAM/SAH, reducing the activity of methyltransferase and the expression of protein, and inhibiting the methylation. The occurrence of the reaction and the inhibition of the proliferation of NSCs.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

【参考文献】