C57小鼠急性脊髓损伤微循环功能障碍机理及治疗机制研究

【作者】 武清斌；

【导师】 修瑞娟；

【作者基本信息】 北京协和医学院 ， 生物医学工程， 2014， 博士

【摘要】 第一部分Wistar大鼠脑和脊髓微血管周细胞的分离、培养、鉴定及其功能差异比较目的：分离培养并鉴定Wistar大鼠脑微血管周细胞(brain microvascular pericyte, BMP)和脊髓微血管周细胞(spinal cord microvascular pericyte, SCMP),系统比较两种周细胞的异同点。方法：3周龄雄性Wistar大鼠,取其大脑和脊髓组织,运用超高速离心方法使得微血管和其他杂质分成不同的层次,吸出微血管片段层,然后用含10%FBS的DMEM培养基培养,观察周细胞从微血管中爬出。用NG2和PDGFRβ鉴定周细胞、用划痕实验测定细胞迁移能力、用BrdU和细胞周期测定细胞增殖、用基质胶成管实验测定成管能力和血管新生能力和用免疫印记分析实验测定屏障相关蛋白。结果：本部分实验观察并比较了BMP和SCMP形态差异,发现BMP较SCMP连接更为紧密,并且BMP较SCMP拥有更大的细胞核,展开面积更大的胞浆；运用周细胞非特异性标记物NG2和PDGFRβ鉴定分离得到的细胞为周细胞,并用vWF和GFAP排除了内皮细胞和星形胶质细胞的污染；发现相同细胞数目的BMP表达F-actin显著多于SCMP (P<0.01);用划痕实验测定了两种周细胞的迁移能力,BMP的迁移数目为79±5,显著低于SCMP的140±4(P<0.01)；将周细胞种在基质胶上,发现两种周细胞均能够成管,且BMPs成管长度4.66±0.25mm显著大于SCMPs的2.56±0.49mm(P<0.05):用BrdU结合新生的周细胞,和所有的周细胞merge以后发现两种周细胞和BrdU的结合率并无显著性差异(P>0.05),说明两种周细胞的增殖能力无显著性区别；归类统计处于不同分裂时期的细胞,结果发现BMP:G1=71.0±2.5%；G2=12.0±1.2%；S=17.1±1.7%；SCMP: G1=74.9±2.8%；G2=10.4±1.3%;S=14.7±1.1％.两种周细胞在上述细胞周期的3个分裂时期无显著性差异,故认为它们的细胞增殖能力没有显著性区别；BMP和SCMP均表达a-SMA.connexin43.NG2.N-cadherin、PDGFRβ、VEGF、desmin和TLR4.BMP表达的α-SMA.connexin43、NG2、PDGFRβ和VEGF显著高于SCMP(P<0.05),而SCMP表达的N-cadherin和desmin显著高于BMP(P<0.05)。同时,两种周细胞均表达TLR4,表达量没有显著性差异(P>0.05)。结论：通过提取、分离、鉴定Wistar大鼠脑和脊髓微血管周细胞及比较两种周细胞之间的不同,我们首次从脊髓微血管中分离得到周细胞；发现BMPs和SCMPs均表达N-cadherin、connexin43、desmin、VEGF和TLR4；发现BMPs和SCMPs分别在单独培养状态下均能成管；发现BMPs相比SCMPs有更强的成管能力；发现SCMPs相比BMPs有更强的迁移能力。BMPs和SCMPs间的不同特征有助于我们认识由周细胞调节的防御机制和炎症反应,更广泛地反映BBB和BSCB的不同之处,这些不同可能与许多重大疾病的病理生理进程相关。第二部分褪黑素治疗C57BL/6小鼠急性脊髓损伤机制研究目的：通过研究褪黑素对血脊髓屏障的保护作用,探讨其保护C57小鼠脊髓损伤的相关机制。方法：C57BL/6小鼠被随机分成三组：假手术组(n=36),仅摘除椎板不打击,每只小鼠腹腔注射0.4mL生理盐水(内含5%乙醇)；SCI组(n=36),既摘除椎板又打击,每只小鼠腹腔注射0.4mL生理盐水(内含5%乙醇)；褪黑素组(n=60),既摘除椎板又打击后腹腔给药(剂量为5、10、25、50和100mg/kg)。模型组和给药组是用Aliens打击装置对小鼠脊髓造成50g/mm的中度打击(重量为5g的打击棒从10mm高处坠落)。给褪黑素或给生理盐水时间三组一致,为打击后或摘除椎板后10min、24h和48h三个时间点。使用通透性示踪剂Evans blue (EB)和荧光素钠(NaFlu)检测脊髓损伤及给药以后损伤部位的EB和NaFlu渗透量变化,对通透性进行定量分析；取注射EB小鼠的脊髓做冰冻切片,于荧光显微镜下观察,比较渗出EB的荧光面积,对损伤进行定量分析。用LEA标注有灌注功能的微血管,检测微血管在损伤前后血流灌注情况。用CD31标注内皮细胞,检测微血管的变化。用CD31和PDGFRP共同标记内皮细胞/周细胞,分析微血管上内皮细胞和周细胞的变化。用CD31和claudin-5共标,以检测主要由内皮细胞分泌的紧密连接蛋白claudin-5变化。用TUNEL测定损伤前后细胞凋亡情况,分析给药对细胞的保护作用。用免疫组化方法(IHC)检测紧密连接蛋白ZO-1,occludin和claudin-5,观察损伤前后蛋白的变化。应用免疫印记分析方法(Western blot),检测通透性相关蛋白：MMP3, AQP4, HIF-1a, VEGF和VEGFR2等。结果：本实验通过运用Evans Blue (EB)和NaFlu两种荧光示踪剂,定量测定SCI给药前后通透性的变化,发现给药后测得EB渗透量0.26±0.04μg/mg显著低于损伤组的0.50±0.08μg/mg(P<0.05)；给药后测得NaFlu渗透量822.1±153.7ng/mg显著低于损伤组的447.9±102.3ng/mg(P<0.05)；通过TUNEL实验,给药组后角部位细胞凋亡数目为82.3±6.9显著低于损伤组的151.0±9.7(P<0.05)；通过IF实验,发现有灌注功能的微血管和总微血管,在给药后均得到保护；微血管上最重要的两种细胞周细胞和内皮细胞,在给药后也得到保护；通过IHC实验,定性观测SCI给药前后连接蛋白的变化,发现给药后连接蛋白的表达量较损伤组均增加；用Western blot来进行BSCB相关蛋白检测,发现褪黑素均能够显著减少脊髓损伤后MMP3、AQP4、VEGF、VEGFR2和HIF-1α的表达水平(P<0.05)。结论：脊髓损伤后脊髓屏障的通透性升高,组织水肿,屏障中紧密连接蛋白ZO-1,occludin和claudin-5被破坏,微血管被破坏,其灌注水平下降,细胞凋亡,周细胞和内皮细胞大量死亡,AQP4表达增多,VEGF表达量增多,以上这些病理现象均表明脊髓损伤后血脊髓屏障被破坏。用褪黑素治疗后可以降低脊髓损伤时血脊髓屏障的通透性,提高屏障中紧密连接蛋白的含量,保护原有的微血管不被破坏,提高微血管灌注水平,减少组织内细胞凋亡,抑制血管新生,减少组织水肿。这些结果均提示褪黑素可以通过保护脊髓损伤后被破坏的微血管和血脊髓屏障,改善微循环来达到治疗脊髓损伤的目的。更多还原

【Abstract】 Part OneIsolated, cultivated, identified and compared of brain and spinal cord microvascular pericytes from Wistar ratsObjective:To compare the migrating rate, tube formation, proliferation and BBB and BSCB associated proteins of brain and spinal cord microvacular pericytes, they were isolated, cultured and identified.Methods:Pericytes were isolated from brain and spinal cord from3-week-old male rat. High-speed centrifugal method was used in this experiment and the capillaries and other impurities were separated into different layers. Then the microvessels were cultivated in the pericyte-specific medium. Pericyte was isolated from the microvessels. The cultured pericyte was performed in the following experiments: identification using NG2and PDGFRβ, migration ability using scratched test assay, proliferation using BrdU incorporation assay and the cell cycle assay, tubular formation ability using matrigel tubular assay and detected the associated proteins of BBB and BSCB using western blot assay. At last, these results were used to compare BMP to SCMP in their characteristic.Results:It was observed and compared the different morphology of BMPs and SCMPs. The results indicated that BMPs possessed bigger cell nucleus and more unfolded cytoplasm than SCMPs. BMPs and SCMPs were identified by using two markers NG2and platelet-derived growth factor receptorβ (PDGFRβ). Meanwhile, BMP and SCMP had the negative results with von Willebrand Factor (vWF) and Glial fibrillary acidic protein (GFAP) markers. Thus, it was indicated that the isolated BMPs and SCMPs did not contaminate with endothelial cells and astrocytes. The result showed that BMPs expressed much more F-actin than SCMPs (P<0.01). The scratch test was used to determine the migration of BMPs and SCMPs. The number of BMPs that migrated into the sound area was79±5, while that of SCMPs was140±4, P<0.01. There was a significant difference between the two types of pericytes. It was found that both the two types of pericytes could form vascular tube on the matrigel. However, we observed the tube length of BMPs (4.66±0.25mm) was larger than SCMPs (2.56±0.49mm). There was a significant difference between them (P<0.01). Using BrdU incorporation assay, the result indicated that there was no significant difference between BMPs and SCMPs. The cell cycle of BMPs and SCMPs was detected by Flow Cytometer. The result listed as follows: BMP:Gi=71.0±2.5%; G2=12.0±1.2%; S=17.1±1.7%; SCMP:Gi=74.9±2.8%; G2=10.4±1.3%; S=14.7±1.1%. There was no significant difference between the two types of pericytes in cell cycle. The results of BrdU incorporation and cell cycle indicated the proliferation of BMPs was no significant difference compared to that of SCMPs. Both BMPs and SCMPs expressed a-SMA, connexin43, NG2, VEGF, desmin, N-cadherin, PDGFRβ and TLR4. BMPs expressed more a-SMA, connexin43, NG2and VEGF. However, SCMPs expressed more N-cadherin, PDGFRβ and desmin. There were significant differences in the above results of WB. Meanwhile, both the two types of pericytes expressed TLR4, and there was no significant difference.Conclusion:This report showed that there were some different characteristics between BMPs and SCMPs when they were cultured alone. It was observed at the first time:a. Pericyte was successfully isolated from spinal cord microvessel. b. Both BMPs and SCMPs expressed N-cadherin, connexin43, desmin and TLR4. c. Monoculture BMPs and SCMPs formed vascular-tube. d. BMPs had stronger tube-formation ability. e. SCMPs possessed stronger ability of migration. These results strongly supported that the BBB integrity and stability could be perfective than BSCB from the angle of pericyte. The different characteristics in pericytes will help us to understand the defense mechanisms and inflammatory response mediated by pericytes in brain and spinal cord. These distinguishing features may reflect the more widespread differences between the BBB and BSCB which directly impact pathophysiological processes in various major diseases. Part TwoStudy on the mechanism of melatonin treated spinal cord injury in C57BL/6miceObjective:To investigate the mechanism of melatonin treated spinal cord injury in mice, the research studied the affection of melatonin on blood spinal cord barrier.Methods:Mice were randomized into the following three groups:(a) Sham+saline containing5%ethanol group (n=30), only removing vertebral plate but no impacting;(b) SCI+saline containing5%ethanol group (n=30), removing vertebral plate and impacting;(c) melatonin group (n=54), removing vertebral plate, impacting and administrating melatonin. The animals were subjected to an impact of50g/mm (5g weight from10mm height) to the dorsal surface of the spinal cord. Melatonin at the doses of5,10,25,50and100mg/kg was administered within30min,24h and48h after spinal cord injury. The permeability of different groups was determined using two tracers (Evans blue and NaFlu). The perfused micro vessels were labeled by LEA. Labeling CD31was used to analyze the endothelial cells surrounding the perfused microvessels. Double-labeling PDGFRβ and CD31immunofluorescence was used to analyze endothelial cells and pericytes on the microvessels. Double-labeling claudin5and CD31immunofluorescence was used to analyze the tight junction protein claudin5mainly secreted by endothelial cells. TUNEL assay was used to determine the apoptotic cells. Immunohistochemistry assay was used to analyze the tight junction proteins ZO-1, claudin5and occludin. Western blot assay was used to analyze the permeability and angiogenesis associated proteins, which included MMP3, AQP4, HIF-la, VEGF and VEGFR2. Results:The permeability of spinal cord injury was determined by two tracers Evans blue (EB) and NaFlu. The results listed as follows: the amount of EB0.4997±0.0729μg/mg and NaFlu822.1±153.7ng/mg in the injured groups. EB0.2643±0.0386μg/mg, NaFlu447.9±102.3ng/mg in the melatonin treated group. There were significant differences between the injured groups and the melatonin-treated groups (P<0.05). The apoptotic cell was detected by TUNEL assay. The result showed that the number of apoptotic cells in melatonin treated group (151.0±9.7) decreased more than those in the injured group (82.3±6.9)(P<0.05). LEA/CD31and CD31/claudin5were co-labeled perfused microvessels and endothelial cells and the tight junction protein claudin5that mainly secreted by endothelial cells using immunofluorescence assay. The results showed that the microvessels and endothelial cells were disrupted and claudin5was destroyed after impacted. After melatonin treatment, the amounts of perfused microvessels, endothelial cells and the expression of claudin5were markly increased. The change of tight junction proteins ZO-1, occludin and claudin5was tested using immunohistochemistry. The expression of tight junction proteins was increased after melatonin treated. The BSCB associated proteins were determined by western blot assay. It was found that melatonin treatment could decrease the expression of MMP3, AQP4, VEGF, VEGFR2and HIF-la (P<0.05).Conclusion:After SCI, the permeability of BSCB increased. Edema formation was found in spinal cord tissue. Tight junction proteins ZO-1, occludin and claudin-5were disrupted. Microvessels were destroyed. The amount of perfused microvessels was decreased. A large number of apoptosis cells appeared, which partly contained endothelial cells and pericytes. The expression of AQP4was increased, which indicated that the edema generated in spinal cord. The up-regulated expression of VEGF suggested that angiogenesis was in progress. All of the above pathological changes indicated that BSCB was disrupted after SCI. While, the permeability of BSCB was decreased after melatonin treated. This therapeutic measure could increase the expression of tight junction proteins, protect the microvessels from disruption, increase the perfusing microvessels, decrease the number of apoptosis cells, inhibit angiogenesis and decrease the edema in the spinal cord. All of the above results in the melatonin treatment indicated that melatonin could protect the disrupted microvessels and BSCB after SCI.更多还原