大鼠骨髓间充质干细胞侧脑室植入对SAH后CVS大鼠的影响
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摘要
一、大鼠骨髓间充质干细胞的分离、培养、鉴定及神经元条件培养基对其定向诱导分化
目的:掌握骨髓间充质干细胞(Bone Marrow Mesenchymal Stem cells, BMSCs)原代和传代培养方法,并对其进行鉴定,探讨应用海马神经元条件培养基对其进行定向诱导分化。
方法:体质量(125±25)g SD大鼠,取股骨、胫骨,骨髓腔用DMEM培养基冲出骨髓于离心管中,吹打制成单细胞悬液。接种于培养瓶中,37℃,5% CO2培养箱中培养。取P5细胞爬片多聚甲醛固定,加一抗(兔抗大鼠CD44、CD34),生物素化二抗,37℃孵育1 h,DAB染色,酒精脱水、二甲苯透明,中性树胶封片、显微镜下观察。取原代培养的海马神经元培养液,作为海马神经元条件培养基。另外一组加入碱性成纤维细胞生长因子(Basic-Fibroblast Growth Factor, b-FGF)和10%二甲基亚枫(Dimethl Sulfoxide, DMSO)无血清培养基,最后一组以神经元培养基(Neurobasal加B27)作为神经元基础培养基阳性对照,阴性对照为原培养基(DMEM加10% FBS)继续培养。第5代(Passage 5, P5)BMSCs接种在放有盖玻片的6孔培养板里,爬片后换为基础培养基,海马神经元培养基和无血清培养基(含b-FGF和10%DMSO的无血清L-DMEM培养基)进行诱导,分别将诱导12 h和1 d的细胞用4%多聚甲醛固定。加入小鼠抗大鼠微管相关蛋白(Microtubula association protein-2, MAP-2),兔抗鼠神经元特异性烯醇化酶(Neurospecific enolase, NSE)及兔抗鼠胶质纤维酸性蛋白(Glia fibrillary acidic protein, GFAP)一抗。光镜下观察MAP-2、NSE和GFAP表达阳性细胞,计数阳性细胞百分率并进行显微摄影。
结果:大鼠BMSCs原代培养,半量换液,悬浮的杂质细胞被清除,贴壁细胞的形态清晰可见并开始迅速增殖。7～9 d左右细胞可长满培养瓶底。大鼠BMSCs传代培养生长较原代快,5～7 d细胞达90%融合。H-E染色BMSCs表现为细胞核大,圆形,居中深染,嗜碱性;胞质着色浅,呈弱嗜酸性,细胞平行排列。兔抗大鼠CD34,CD44免疫细胞染色,BMSCs为CD34染色阴性细胞,而CD44表达阳性,这些CD44阳性细胞胞核和胞浆呈棕褐色。海马神经元条件培养液、b-FGF的无血清培养基和含B27的Neurobasal培养基对P5的BMSCs诱导12 h和1 d后,诱导后细胞体逐渐变成圆形和锥形,细胞向外伸出突起。随着时间延长,突起也逐渐延长,1 d后可见有些连接形成网状。诱导后的BMSCs免疫细胞化学染色,神经类标记物MAP-2,NSE和GFAP进行标记,计数细胞阳性率,以12 h后海马神经元条件培养基的诱导阳性率最高。
结论:1应用全骨髓结合贴壁分离法成功建立了一种体外简单、快速的大鼠BMSCs原代及传代培养方法,在体外能够进行迅速增殖,且性状稳定。2体外培养的BMSCs经免疫细胞化学染色,表达间充质干细胞表面标记,证明非造血干细胞。3应用含b-FGF的DMEM培养基、海马神经元条件培养基、无血清培养基诱导BMSCs 12 h与1 d,BMSCs均可分化成为神经元样细胞和神经胶质样细胞,经免疫细胞化学染色均表达NSE、MAP-2和GFAP。4三种培养基诱导BMSCs,以12 h的诱导率最高,可达到44.78%,其中海马神经元条件培养基的诱导效率高于其它两组,差异有显著性。
二、大鼠SAH后CVS模型制备及行为学、生物学指标测定
目的:大鼠自体血枕大池二次注血法制造蛛网膜下隙出血(Subarachnoid Hemorrhage, SAH)后脑血管痉挛(Cerebro Vascular Spasm, CVS)的动物模型,测定血和脑组织中一氧化氮合酶(Nitric Oxide synthesis, NOS)及内皮素-1(Endotheliam-1, ET-1)含量的变化,测量脑血管痉挛后的血管直径,结合行为学改变对脑血管痉挛动物模型进行评定。
方法:枕大池二次注血法建立蛛网膜下隙出血模型,尾动脉取血,脑立体定位仪下环枕膜处自体动脉血约5 min注完,于2 d后再次同样的方法二次注血。手术后大鼠参照神经功能等级评分表评定,在1 d、2 d、3 d、5 d、7 d对SAH和对照组大鼠分别评分。10%印度墨水血管灌注,45倍体视显微镜下,应用Nikon NIS-Elements BR 3.0和Image J图像分析系统,测量基底动脉、颈内动脉和大脑中动脉直径。另外应用同样灌注技术,海马组织切片于前囱后4 mm处切开,体视显微镜放大评价微血管充盈状态。于SAH成功后1 d、2 d、3 d、5 d及7 d时间取血,分离海马及皮层组织,取组织上清液测定血清及脑组织匀浆中NOS、ET-1含量。注射血液结束后14 d,进行Morris水迷宫测试。所有数据均用±s表示,应用SPSS14.0软件包进行分析,对照组和实验组间比较用Dunnett-t检验,组间比较用SNK-q检验,神经生物功能评分和脑血管直径,NOS和ET-1含量进行多元线性回归,Morris水迷宫中的平均潜伏时间和NOS、ET-1含量及脑血管直径的关系也进行多元线性回归。
结果:大鼠SAH后警觉性低,毛发杂乱,饮水摄食行为差,自洁性差,于次日后稍好转,但二次注血后加重,少数可出现偏瘫。术后脑内有血凝块,主要位于小脑延髓池内,1 d可见有陈旧性血液。在海马(前囟后4 mm)切片处可见大脑皮层血管在SAH组血管较少,血管变细。测量大脑中动脉,基底动脉和颈内动脉直径,在SAH组2 d和3 d组血管直径与生理盐水和对照组相比有明显差异(P<0.05)。SAH大鼠在1 d、2 d、3 d、5 d、7 d可见神经功能评分明显增高,与对照组、生理盐水组相比有明显的统计学差异(P<0.01)。SAH后1 d、2 d、3 d、5 d、7 d组脑组织和血液中ET-1均高于对照组和生理盐水组(P<0.05)。进行神经生物功能评分与脑血管直径、NOS和ET-1含量分析,建立回归方程为:Y=-3.586+0.059X1-0.55 X2,X1为血中ET-1的含量,X2为大脑中动脉的直径,可见神经生物功能评分与血中ET-1的含量呈正相关,与大脑中动脉的直径呈负相关,血中ET-1和大脑中动脉相比对神经生物学功能评分的密切程度没有明显统计学差异(P>0.05)。SAH组Morris水迷宫游泳距离明显增长,逃避潜伏期时间较长。在随后的几天实验中各组逃避潜伏期时间逐渐缩短,SAH组成绩虽有提高,但其逃避潜伏期始终维持在较高水平,平均潜伏期与血中ET-1含量呈正相关。SAH组大鼠跨越平台次数明显少于对照组。
结论:1大鼠自体尾动脉取血枕大池二次注血后,可见各脑池脑沟内有明显的陈旧血凝块聚积,说明蛛网膜下隙出血模型成功。2大鼠脑血管管径测量显示SAH血管变细,血管灌注显示有脑血管灌注不足,证明SAH后有脑血管痉挛的缺血表现。3 SAH组血和脑内NOS含量低于对照组,
ET-1含量升高,表明SAH后有脑血管痉挛。4 SAH组神经生物学功能评分均明显高于其它组,证明脑血管痉挛后有行为学表现功能下降。5 Morris水迷宫测试表明SAH后大鼠的空间学习记忆能力有一定下降。
三、BrdU标记的BMSCs侧脑室植入后对SAH后CVS大鼠行为学影响的研究
目的:通过BMSCs侧脑室植入SAH后CVS大鼠,观察标记的BMSCs在大鼠脑内生长情况,结合行为学检测评价BMSCs植入后效果。
方法:第5代BMSCs,用5-溴脱氧尿核苷(Bromodeoxyuridine, BrdU)标记2 d。将标记后的细胞爬片进行免疫细胞化学染色,一抗为BrdU抗体。标记好的细胞以5×106的细胞悬液侧脑室注入SAH后的大鼠。SAH大鼠以其神经功能评分为参照,随机分为三组:SAH组、SAH后植入DMEM组和SAH后BMSCs植入组。SAH组不植入干细胞,而DMEM组于第二次注血2 d后立体定向仪下用微量注射器注入30μLDMEM培养基。BMSCs组在第二次注血后2 d立体定位仪下注入30μL的BMSCs悬液。BMSCs组中5只大鼠于14 d后进行Morris水迷宫检测,另外5只取脑进行免疫组化染色。脑立体定位仪下大鼠侧脑室穿刺取前囟定位,前囟后1.2 mm,中线旁2 mm处进针,接上已事先抽好BMSCs悬液或是DMEM的微量注射器,深度为4.5 mm,将30μL BMSCs悬液或DMEM培养基缓慢注入侧脑室。各组大鼠在注射后14 d麻醉,固定取脑,切片,进行免疫组化染色,H2O2灭活内源性酶,加入抗原修复液,生物微波炉加热抗原修复,滴加5% BSA封闭液,一抗(小鼠抗BrdU IgG),37℃1 h,生物素化二抗,37℃20 min,DAB染色,脱水透明,中性树胶封片。SAH组及DMEM组、BMSCs组1 d、2 d、3 d、5 d、7 d后行神经生物学功能评分,于14 d后进行水迷宫检测。
结果:BrdU标记后BMSCs,经免疫细胞化学染色为胞核着色,以胞核呈棕黄色者为阳性。脑立体定位仪下行侧脑室穿刺,以前囟定位,能准确注入侧脑室。SAH大鼠侧脑室穿刺注入干细胞悬液后无不良反应。脑石蜡切片DAB染色后,可见经BrdU标记的阳性细胞胞核呈棕褐色,阳性细胞在切片内无明显规律,散在分布于阴性细胞之间。DMEM组可见阴性细胞核仍呈蓝色。将SAH组、DMEM组及BMSCs组大鼠于注射后1 d、2 d、3 d、5 d、7 d进行神经生物功能评分,无论是SAH组还是DMEM、BMSCs组可见,在各组里以1 d神经生物学功能评分普遍高于其它组(P<0.05),以后评分逐渐下降,BMSCs组大鼠在7 d时与DMEM和SAH组相比,神经生物功能评分明显减低,感觉运动功能恢复(P<0.05)。Morris水迷宫检测BMSCs组大鼠平均逃避潜伏期时间比SAH组和DMEM组要短,有统计学差异,探索实验显示BMSCs组跨越平台的次数多于其它两组,差异有明显统计学意义(P<0.05)。
结论:1 BrdU可以用来标记BMSCs,标记后的细胞可用相应的抗体检测。2脑立体定位仪下以前囟后1.2 mm,中线旁2 mm处进针,深度为4.5 mm的位置能准确注入侧脑室,注射后大鼠未见明显不良反应。3注入BrdU标记过的BMSCs后,取前囟后4 mm的海马组织切片免疫组化染色显示有BrdU阳性细胞存在。4生物学功能评分可见BMSCs组在7 d时评分下降,表明感觉和运动功能有一定程度的恢复。5 Morris水迷宫显示BMSCs组在学习记忆能力方面也有一定的改善。
四、SAH后CVS大鼠移植BMSCs后海马神经元凋亡的研究
目的:大鼠SAH后CVS模型基础上植入BMSCs,大鼠感觉、运动功能有一定程度的恢复,进一步观察BMSCs植入后对大鼠脑内海马神经元凋亡相关蛋白Bcl-2和Bax表达的影响。
方法:在第三部分大鼠脑组织切片基础之上,取SAH组、DMEM组和BMSCs组14 d前囟后4 mm处大脑皮层及海马组织石蜡切片,以兔抗鼠Bcl-2和Bax为一抗,进行免疫组织化学染色。图像分析软件分析阳性细胞数目比率。无菌条件下取SAH及BMSCs14 d组大脑,剥出海马,按每100 mg脑组织加入1 mL裂解液,超声裂解后低温离心,取上清,考马斯亮兰测定蛋白含量,蛋白变性后上样,电泳、转硝酸纤维素膜(NC膜),脱脂奶粉封闭,分别加入兔抗大鼠Bcl-2、Bax一抗,4℃静置过夜。滴加辣根过氧化物酶标记二抗,免疫化学发光,X光片显影定影。使用Hema成像分析系统对显色条带进行半定量分析,测定各组蛋白相对表达水平。
结果:与SAH组相比,BMSCs组海马可见Bcl-2阳性细胞数目增加,细胞呈黄褐色,深染;Bax阳性细胞数目减少,着色较浅。SAH组大鼠海马可见Bcl-2阳性细胞数目较少,着色浅;相反Bax阳性细胞数目增加,细胞呈棕褐色较深。DMEM组可见Bax阳性细胞数目增加,而Bcl-2阳性细胞数目相对减低。Western blot显示,DMEM组、SAH组和BMSCs组大鼠海马GAPDH表达三组基本相似,Bcl-2在BMSCs组比DMEM组和SAH组表达高(P<0.05)。Bax的表达在SAH组与DMEM组高于BMSCs组,相比有明显统计学差异(P<0.05)。
结论:1 SAH后CVS模型大鼠侧脑室植入BMSCs后,大脑皮层和海马内可见神经元抑制凋亡相关基因表达增加,而促凋亡相关基因表达下降。2 SAH组14 d大鼠脑内促凋亡相关基因表达升高,抑制凋亡相关基因表达下降。3 DMEM组大鼠脑内仅有少量的抑制凋亡相关基因表达,促凋亡相关基因表达较高。
五、SAH后CVS大鼠侧脑室植入BMSCs脑血管超微结构观察与血中NOS、ET-1含量变化
目的:观察SAH后CVS大鼠侧脑室植入BMSCs后血中一氧化氮合酶、内皮素-1含量变化,同时透射电镜观察大鼠脑内血管和神经元超微结构改变,为细胞移植治疗提供理论和实验依据。
方法:实验动物分组、SAH模型制备及BMSCs移植同前面几部分,分为正常对照组、SAH组及BMSCs组。于移植BMSCs后1 d、3 d、7 d进行取血,测定大鼠血中NOS和ET-1含量,具体方法同前面第二部分。同时取前囟后4 mm大脑皮层脑组织约1 mm×1 mm×2 mm大小,用2.5%戊二醛磷酸盐缓冲液中后固定1 d,备透射电镜检查。将标本用0.1 M磷酸缓冲液洗3次,质量分数为1%锇酸固定,逐级梯度乙醇、丙酮脱水,环氧树脂812包埋,聚合,用超薄切片机作0.5μm厚度切片,经1%甲苯胺蓝-天青II染色,光镜下定位后制作超薄切片,片厚约50 nm,铜网捞片,醋酸铀、柠檬酸铅液染色。日立H-7500型透射电镜观察各组大鼠大脑皮层神经元及微血管的超微结构,照相,记录。组间比较采用单因素方差分析,各组间两两比较采用SNK-q检验,P<0.05表示差异有统计学意义。
结果:与对照组相比,SAH组后1 d、3 d、7 d血中NOS含量明显下降,而血中ET-1含量则明显上升,其中以SAH 3 d组血中ET-1升高最为明显,差异有统计学意义。与SAH组相比,BMSCs组可见血液中ET-1含量逐渐下降,而NOS含量上升,差异有统计学意义。与正常组相比,BMSCs组ET-1含量仍然较高,差异有明显统计学意义(P<0.05),血中NOS含量有一定下降,与对照组相比不能说明有差异(P>0.1)。透射电镜显示,对照组神经元细胞核大而圆,染色质密度均匀,核仁明显,核膜完整。大脑皮层毛细血管可见其内皮细胞内膜光滑,细胞核、细胞器、紧密连接清晰可见,内皮细胞的基质和基膜完整,层次清楚,管腔无狭窄。SAH组可见大脑皮层神经元和微血管内皮细胞的水肿程度达到高峰,神经元高度水肿,线粒体肿胀,可见部分凋亡、坏死神经元。BMSCs组大脑皮层神经元和微血管内皮细胞可见轻度水肿,血管内皮细胞的吞饮小泡略减少。微血管内皮细胞核固缩,管腔内微绒毛减少,管腔狭窄有一定缓解。
结论:1 SAH后CVS大鼠侧脑室移植BMSCs后ET-1下降,NOS含量升高。说明脑血管痉挛有一定程度的缓解。2脑组织及血管超微结构观察,SAH组大脑皮层神经元坏死加重,部分微血管闭塞。BMSCs移植后神经元的凋亡与死亡减少,微血管闭塞有一定的缓解。
1.Rat bone marrow mesenchymal stem cells isolation, cultivation, identification and neuronal conditioned medium induce it’s differentiation
Objective: To master the method of rat bone marrow mesenchymal stem cells primary cultivation, passage, and identification, to investigate use hippocampal neuron’s conditioned medium to induce it differentiation.
Methods: Use weight about (100-150) g SD rat, get the tibia and femur with germ free condition, expose the bone cavity, flush the cavity with antibiotic (penicillion 100 u/mL, streptomycin 100u/mL) DMEM medium, collect it in centrifuge tube, susupension it and cultured with 10% FBS medium into the cultured bottle, incubate in 37℃、5% CO2 incubator. 3d change the half medium, 5 d change the medium completely, then change the medium every 3 d. use CD34 and CD44 as the primary antibody to do the immunocytochemistry staining, PBS as the negative control, add the biotinize secondary antibody, 37℃, 1 h, PBS wash 3 times, add horseradish peroxidase, staining with DAB, stop with water, ethanol dehydration, xylene transparent, fix it with gum, observe under microscope. Use primary cultured hippocampal neuron’s medium as conditioned medium, b-FGF group add 10% DMSO, b-FGF and L-DMEM, the last group use Neurobasal plus B27 as serum free medium, negative control is DMEM medium(with 10%FBS). P5 BMSCs planted on the cover slip wih covering 0.1% PLL ( Poly-L-Lysine ) in 6 wells plate, when the BMSCs growth on the cover slip and change with Neurobasal medium, hippocampal conditioned medium and serumfree medium, induced the cell for 12 h and 1 d, after that fixed with paraformaldehyde. BMSCs following induce with different medium, use immunocytochemistry by MAP-2, NSE and GFAP antibody, observe the positive expression of MAP-2, NSE and GFAP, count the positive ratio of different groups and photographed.
Results: BMSCs primary culture for 1 d, few of them adhere to the bottom, 2 d cleavage and proliferate, show oval ship or multiform, often form cell cluster, 4 d prolife rapidally, 3 d change the medium half, the impurity is clear, the cell morphology is obvious, the body is larger and prolife. 7-9 d is full on the bottom, about 95% emerge. BMSCs after passage growth fast,4h is adhere to the substance, 24h adhere completely and prolife. 5-7d can growth about 90% of the bottom. After 1’st passage, the purity is about 95%, passage it for 6 generation, each passage is the same in morphology and growth. BMSCs H-E staining show spindle and multiform, the nuclei is large, round and heavy dye, basophilic, cytoplasma is light, acidophilic, cell paralled. Rabbit anti rat CD34 show negative and colorless, CD44 cell adhere molecular positive, nuclei and cytoplasma show brown. Hippocampal neuron conditioned medium, b-FGF medium and B27 plus neurobasal (serum free) medium induced the BMSCs for 12 h and 1 d, after inducing the cell body changed into round and pyramid gradually, process elongate, 1 d some of them can connect each other. With immunocytochemistry stainin, 12 h and 1 d labeled with MAP-2, NSE and GFAP, count the positive showed conditioned medium induced BMSCs positive ratio is the highest, express NSE, MAP-2, GFAP higher than others, have the significant statistically difference(P<0.05)
Conclusions: 1.Full bone marrow culture method and adhere way is a successful method to isolation, purify the BMSCs in vitro, and it can prolife rapidly and stable. 2. BMSCs in vitro with immunocytochemistry staining, it’s express mesenchymal stem cells characteristic, not the hemotopoitic stem cells. 3. With b-FGF DMEM medium, hippocampal conditioned medium, serum free medium induced BMSCs 12 h and 1 d, BMSCs can differentiate into neuronal like cell and glial like cell, with immunocytochemistry identification it’s express NSE, MAP-2 and GFAP positive characteristic. 3. With b-FGF DMEM medium, hippocampal medium, serum free medium induce BMSCs, 12 h is the highest ratio, it’s about 44.78%, conditioned medium compare with others, there are statistically differences.
2. CVS model following SAH and ethological, biological detection
Objective: Observe rat autologous blood cisterna magna injection to produce CVS model following SAH, detect blood and brain NOS and ET-1 content varation, the diameter of cerebro vessel, value the model of CVS from ethological performance.
Methods: Use double hemorrhage cistern magna injection method to produce SAH model, separate the atlanto-occipital membrane and cut anterio the end of tail about 5-6 cm, get the arterial blood 0.2-0.3 mL from tail artery, with stereotaxica coordinate inject the arterial blood from atlanto-occipital membrane about 5 min, and 2 d later inject the blood again the same way. Rat was valued with neurological functional score, in 1 d, 2 d, 3 d, 5 d, 7 d compare SAH and control group. After vessel perfusion with 10% of india ink detect the diameter of cerebral artery with magnification of 45, with Nikon NIS BR 3.0 and Image J analysis system, detect the basal artery, introcarotid artery, and middle cerebral artery diameter. With the same perfusion way, filling the microvessel, 1 mm thick slice around the hippocampus 4 mm posterior bregma, valure the vessel filling under 45 magnification. SAH 1 d, 2 d, 3 d, 5 d, 7 d extract the eyeball, get 2 mL blood, get the hippocampus and cortex of brain, use serum and detect the ET-1 and NOS according to it’s manual, after injection 14 d, do Morris mazer test. All data process with SPSS14.0 soft ware and use one way annova, compare control and experimental group with Dunnett-t method, among groups with SNK-q test, with neurological functional score and cerebral diameter, NOS and ET-1 use multiple linear regression, Morris mazer mean escape time and NOS, ET-1, cerebral diameter also use multiple linear regression.
Results: after SAH rat show decrese in eating, some kind of abnormal ethology, show lower reaction, lower alartness, sleepy, bloom, lack of diat, it can be better on 2’nd day, but double hemorrhage aggrevate, some of them can be paralysis. After injection there are blood clot in cerebullar medullary cistern, 1 d can see obsolete blood. Posterior 4 mm of bregma cortex vessel attenuate and lessen, the branches to medullary is lessen, but the control group show normal and abundant. With image analysis detect the vessel, SAH 2 d and 3 d the diameter have statistically difference versus control group. For neurological function score, SAH 1 d, 2 d, 3 d, 5 d have higher score, versus control group have significant difference. SAH 1 d, 2 d, 3 d, 5 d, 7 d blood and brain ET-1 were higher than sham. With multiple linear regression, neurological function score is related with ET-1 and middle cerebral artery. The average swimming speed is the same, while the swimming distance and escaping time of SAH group is longer than others, SAH span the flat less than control group.
Conclusions: 1. Rat autologous tail blood cistern magna injection double hemorrhage model, there are clot around the sulcus and gyrus, with microperfusion shows clearly brain vessel hypoperfusion. 2. Compare with control group, SAH rat diameter of BA, MCA, ICA is thinning, especially for MCA. 3. SAH brain and blood ET-1, NOS show NOS lower than control group, ET-1 higher than the others. 4. SAH neurological score higher than others. 5. Morris mazer average escaping time of SAH is longer than control group, span times lower than sham.
3. BrdU Labelled BMSCs transplant to CVS rat following SAH from lateral ventricle injection
Objective: To transplant labeled BMSCs in to SAH following CVS from lateral ventricle, investigate the labeled cell development in brain, valure the cell transplantation effect with ethological method.
Methods: P5 BMSCs, with BrdU to labelled for 2 d, after that check it by immnunocytochemistry staining with BrdU antibody. Then use labelled cell suspension, as 5×106 cell density to transplant to rat brain from lateral ventricle. By neurological score valure the 3 groups: SAH group, BMSCs group, and only transplant DMEM group. Sandomize 3 group, SAH didn’t transplant cell, DMEM only inject 30μL DMEM medium after 2’nd blood injecton 2 d, BMSCs group after SAH 2 d under stereotaxic coordinate inject 30μL BMSCs suspension as previous describe density. The Morris mazer and ethological exam after 14 d. Under stereotaxic coordinate, rat lateral ventricle localize is: posterior to bragma 1.2 mm, median line 2 mm, and the deep is 4.5 mm, after injection 14 d, fixed with paraformadehyde and get the brain slices, by immunohistochemistry staining method to show the positive BrdU labeled BMSCs. Detect neurological score at 1 d, 2 d, 3 d, 5 d, 7 d after SAH inject DMEM and BMSCs, 14 d do the Morris mazer experiment.
Results: BrdU labelled BMSCs, immnunohistochemistry staining show nuclei brown is positive, negative is baby blue. Localize lateral ventricle under stereotaxic coordinate with bragam, it’s accurate into lateral ventricle and have no other untoward reaction, compare with DMEM group, BMSCs group sensory and motor disable no aggravate. With DAB staining of brain slice, the positive cell of BrdU labelled show brown color, positive cell irregular line within the negative cells. Neurological score show 1 d is the highest, with time it’s decrease little by little. DMEM group and SAH group have no statistically difference, Morris mazer BMSCs escaping time is shorter than SAH and DMEM group, spaning flat times show BMSCs more than others, have statistically difference.
Conclusions: 1. BrdU can labelled BMSCs, with BrdU antibody immnunocytochemistry staining show brow color. 2. Under stereotaxic coordinate localize posterior bragma 1.2 mm, median line 2 mm, depth 4.5 mm can accurate inject into lateral ventricle, have no other side effect. 3.Labelled BrdU BMSCs, show positive BrdU cell in the brain slice. 4. Rat neurological score show BMSCs group 7 d has recover in motor and sensory in certain degree. 5. Morris mazer show BMSCs group have some kind of recover in learning and memory.
4. The apoptosis investigation of hippocampus neuron after transplantation of BMSCs to CVS rat following SAH
Objective: after transplantation BMSCs to CVS rat following SAH, observe the expression of Bcl-2 and Bax protein which are related with apoptosis in brain and hippocampus.
Methods: With the SAH group, DMEM group and BMSCs group brain slices, use rabbit anti rat Bcl-2 and Bax as primary antibody, immunohistobiochemistry staining method to observe the morphological change. With Image analysis soft ware count the positive cell ratio of each groups. Get the SAH, DMEM and BMSCs group rat brain in 14 d, dissect the hippocampus, add the lysate about 1 mL per 100 mg brain tissue, ultrasonic clearage and centrifuge hypothermia, get the supernatant, detect it’s protein content with Coomassie agent. Protein denaturation and spotting, electrophoresis and transfer to nitrocellulose membrane. Blocking with defatted milk powder, add primary antibody of Bcl-2 and Bax, 4℃over night, add horseradish peroxidase, immune-developing, with Hema analysis system to detect each group protein expression level.
Results: Compare with SAH, BMSCs at hippocampal slice Bcl-2 positive expression increase, Bax expression decrease, SAH group at the hippocampal slice Bcl-2 decrease and Bax increase. DMEM group Bcl-2 decrease and Bax increase. Western blot show, control group, SAH group, BMSCs group GAPDH expression is the same, BMSCs group Bcl-2 is higher than the others, and Bax expression is lower. Have statistically difference.
Conclusions: 1. SAH following CVS transplant BMSCs, the express of inhibit apoptosis gene is decrease and promote apoptosis gene is increase. 2. SAH 14 d rat brain promote apoptosis gene expression increase and inhibit apoptosis gene decrease. 3.There are a little expression of promote apoptosis gene expression in DMEM group.
5. The blood NOS, ET-1 content change and micro-ultral structure change after BMSCs transplantation to CVS rat following SAH
Objective: To investigate blood NOS, ET-1 changes of BMSCs transplantation to CVS rat following SAH, transmission electron microscope observe the cerebrovascular and neuron’s ultra-micro-structural change.
Methods: Animal grouping, SAH model and cell transplantation as previous subscribe, collect blood after BMSCs transplantation 14 d, detect the content of NOS and ET-1, as previous. Get the blood at 1 d, 3 d, and 7 d. Detect the NOS and ET-1 content in blood, as previous describe. Get the brain cortex posterior bregma about 1mm×1mm×2mm, fix it with 2.5% glutaral phosphates about 24h, ultral thin slices for TEM, wash it with 0.1mol/L phosphate 3 times, 1% osmic acid fix, ethanol and acetone dehydration, bed with epoxy resins, polymerization, 0.5μm thickness slicing the tissue, 1% toluidin blue-azure thickness about 50 nm, dredge with copper screen, uranyl acetate and citric acid staining, Hitachi H-7500 TEM observe ultra-structure of the brain cortex neuron and microvascular, photography. Comparasion between groups use one way ANOVA, among groups use SNK-q test, P<0.05 represent difference.
Results: Compare with control group, SAH 1 d, 3 d and 7 d blood NOS decrease and ET-1 increase, as SAH 3 d increase for typical, have statistically difference. BMSCs group blood ET-1 decerease versus SAH group, NOS increase, have statistically difference. Compare with control group, BMSCs group ET-1 is still in high level, have statistically difference, NOS have certain level decrease, but have no statistically difference. TEM ultra-micro-structure show control group neuclus is big and round, Chromosome density is even, karyosome is clear, nuclear membrane is intergreted. Endothelial cell endomembrane is smooth, cell neuclear, apparatus, tight injunction is clear, endothalia matrix and basal lamina is integrated, stratification is clear, vessel have no narrow. SAH group cortex neuron and microvascular endothelial edema is the highest, mitochondria swelling, most of crest and membrane emerge, disappear, some of them medulla change, there are vaculous in cytoplasma, neuron nuclear chromosome concentrate, heterochromatin increase, mitochnodia, rough endoplasm reticulum swelling, even collapse. BMSCs transplantation can see little bit of edema in cortex neuron and microvasular endothelial, pinocytosis bulb attenuate in vascular endothelial cell.
Conclusions: BMSCs lateral ventricle transplantation to SAH following CVS rat blood ET-1 decreas, NOS increase, SAH group neuron necrosis deeply and some of microvessel stenosis, after transplant BMSCs is attenuate the effect of CVS, imply cerebral vascular have a certain degree of release.
目的:掌握骨髓间充质干细胞(Bone Marrow Mesenchymal Stem cells, BMSCs)原代和传代培养方法,并对其进行鉴定,探讨应用海马神经元条件培养基对其进行定向诱导分化。
方法:体质量(125±25)g SD大鼠,取股骨、胫骨,骨髓腔用DMEM培养基冲出骨髓于离心管中,吹打制成单细胞悬液。接种于培养瓶中,37℃,5% CO2培养箱中培养。取P5细胞爬片多聚甲醛固定,加一抗(兔抗大鼠CD44、CD34),生物素化二抗,37℃孵育1 h,DAB染色,酒精脱水、二甲苯透明,中性树胶封片、显微镜下观察。取原代培养的海马神经元培养液,作为海马神经元条件培养基。另外一组加入碱性成纤维细胞生长因子(Basic-Fibroblast Growth Factor, b-FGF)和10%二甲基亚枫(Dimethl Sulfoxide, DMSO)无血清培养基,最后一组以神经元培养基(Neurobasal加B27)作为神经元基础培养基阳性对照,阴性对照为原培养基(DMEM加10% FBS)继续培养。第5代(Passage 5, P5)BMSCs接种在放有盖玻片的6孔培养板里,爬片后换为基础培养基,海马神经元培养基和无血清培养基(含b-FGF和10%DMSO的无血清L-DMEM培养基)进行诱导,分别将诱导12 h和1 d的细胞用4%多聚甲醛固定。加入小鼠抗大鼠微管相关蛋白(Microtubula association protein-2, MAP-2),兔抗鼠神经元特异性烯醇化酶(Neurospecific enolase, NSE)及兔抗鼠胶质纤维酸性蛋白(Glia fibrillary acidic protein, GFAP)一抗。光镜下观察MAP-2、NSE和GFAP表达阳性细胞,计数阳性细胞百分率并进行显微摄影。
结果:大鼠BMSCs原代培养,半量换液,悬浮的杂质细胞被清除,贴壁细胞的形态清晰可见并开始迅速增殖。7～9 d左右细胞可长满培养瓶底。大鼠BMSCs传代培养生长较原代快,5～7 d细胞达90%融合。H-E染色BMSCs表现为细胞核大,圆形,居中深染,嗜碱性;胞质着色浅,呈弱嗜酸性,细胞平行排列。兔抗大鼠CD34,CD44免疫细胞染色,BMSCs为CD34染色阴性细胞,而CD44表达阳性,这些CD44阳性细胞胞核和胞浆呈棕褐色。海马神经元条件培养液、b-FGF的无血清培养基和含B27的Neurobasal培养基对P5的BMSCs诱导12 h和1 d后,诱导后细胞体逐渐变成圆形和锥形,细胞向外伸出突起。随着时间延长,突起也逐渐延长,1 d后可见有些连接形成网状。诱导后的BMSCs免疫细胞化学染色,神经类标记物MAP-2,NSE和GFAP进行标记,计数细胞阳性率,以12 h后海马神经元条件培养基的诱导阳性率最高。
结论:1应用全骨髓结合贴壁分离法成功建立了一种体外简单、快速的大鼠BMSCs原代及传代培养方法,在体外能够进行迅速增殖,且性状稳定。2体外培养的BMSCs经免疫细胞化学染色,表达间充质干细胞表面标记,证明非造血干细胞。3应用含b-FGF的DMEM培养基、海马神经元条件培养基、无血清培养基诱导BMSCs 12 h与1 d,BMSCs均可分化成为神经元样细胞和神经胶质样细胞,经免疫细胞化学染色均表达NSE、MAP-2和GFAP。4三种培养基诱导BMSCs,以12 h的诱导率最高,可达到44.78%,其中海马神经元条件培养基的诱导效率高于其它两组,差异有显著性。
二、大鼠SAH后CVS模型制备及行为学、生物学指标测定
目的:大鼠自体血枕大池二次注血法制造蛛网膜下隙出血(Subarachnoid Hemorrhage, SAH)后脑血管痉挛(Cerebro Vascular Spasm, CVS)的动物模型,测定血和脑组织中一氧化氮合酶(Nitric Oxide synthesis, NOS)及内皮素-1(Endotheliam-1, ET-1)含量的变化,测量脑血管痉挛后的血管直径,结合行为学改变对脑血管痉挛动物模型进行评定。
方法:枕大池二次注血法建立蛛网膜下隙出血模型,尾动脉取血,脑立体定位仪下环枕膜处自体动脉血约5 min注完,于2 d后再次同样的方法二次注血。手术后大鼠参照神经功能等级评分表评定,在1 d、2 d、3 d、5 d、7 d对SAH和对照组大鼠分别评分。10%印度墨水血管灌注,45倍体视显微镜下,应用Nikon NIS-Elements BR 3.0和Image J图像分析系统,测量基底动脉、颈内动脉和大脑中动脉直径。另外应用同样灌注技术,海马组织切片于前囱后4 mm处切开,体视显微镜放大评价微血管充盈状态。于SAH成功后1 d、2 d、3 d、5 d及7 d时间取血,分离海马及皮层组织,取组织上清液测定血清及脑组织匀浆中NOS、ET-1含量。注射血液结束后14 d,进行Morris水迷宫测试。所有数据均用±s表示,应用SPSS14.0软件包进行分析,对照组和实验组间比较用Dunnett-t检验,组间比较用SNK-q检验,神经生物功能评分和脑血管直径,NOS和ET-1含量进行多元线性回归,Morris水迷宫中的平均潜伏时间和NOS、ET-1含量及脑血管直径的关系也进行多元线性回归。
结果:大鼠SAH后警觉性低,毛发杂乱,饮水摄食行为差,自洁性差,于次日后稍好转,但二次注血后加重,少数可出现偏瘫。术后脑内有血凝块,主要位于小脑延髓池内,1 d可见有陈旧性血液。在海马(前囟后4 mm)切片处可见大脑皮层血管在SAH组血管较少,血管变细。测量大脑中动脉,基底动脉和颈内动脉直径,在SAH组2 d和3 d组血管直径与生理盐水和对照组相比有明显差异(P<0.05)。SAH大鼠在1 d、2 d、3 d、5 d、7 d可见神经功能评分明显增高,与对照组、生理盐水组相比有明显的统计学差异(P<0.01)。SAH后1 d、2 d、3 d、5 d、7 d组脑组织和血液中ET-1均高于对照组和生理盐水组(P<0.05)。进行神经生物功能评分与脑血管直径、NOS和ET-1含量分析,建立回归方程为:Y=-3.586+0.059X1-0.55 X2,X1为血中ET-1的含量,X2为大脑中动脉的直径,可见神经生物功能评分与血中ET-1的含量呈正相关,与大脑中动脉的直径呈负相关,血中ET-1和大脑中动脉相比对神经生物学功能评分的密切程度没有明显统计学差异(P>0.05)。SAH组Morris水迷宫游泳距离明显增长,逃避潜伏期时间较长。在随后的几天实验中各组逃避潜伏期时间逐渐缩短,SAH组成绩虽有提高,但其逃避潜伏期始终维持在较高水平,平均潜伏期与血中ET-1含量呈正相关。SAH组大鼠跨越平台次数明显少于对照组。
结论:1大鼠自体尾动脉取血枕大池二次注血后,可见各脑池脑沟内有明显的陈旧血凝块聚积,说明蛛网膜下隙出血模型成功。2大鼠脑血管管径测量显示SAH血管变细,血管灌注显示有脑血管灌注不足,证明SAH后有脑血管痉挛的缺血表现。3 SAH组血和脑内NOS含量低于对照组,
ET-1含量升高,表明SAH后有脑血管痉挛。4 SAH组神经生物学功能评分均明显高于其它组,证明脑血管痉挛后有行为学表现功能下降。5 Morris水迷宫测试表明SAH后大鼠的空间学习记忆能力有一定下降。
三、BrdU标记的BMSCs侧脑室植入后对SAH后CVS大鼠行为学影响的研究
目的:通过BMSCs侧脑室植入SAH后CVS大鼠,观察标记的BMSCs在大鼠脑内生长情况,结合行为学检测评价BMSCs植入后效果。
方法:第5代BMSCs,用5-溴脱氧尿核苷(Bromodeoxyuridine, BrdU)标记2 d。将标记后的细胞爬片进行免疫细胞化学染色,一抗为BrdU抗体。标记好的细胞以5×106的细胞悬液侧脑室注入SAH后的大鼠。SAH大鼠以其神经功能评分为参照,随机分为三组:SAH组、SAH后植入DMEM组和SAH后BMSCs植入组。SAH组不植入干细胞,而DMEM组于第二次注血2 d后立体定向仪下用微量注射器注入30μLDMEM培养基。BMSCs组在第二次注血后2 d立体定位仪下注入30μL的BMSCs悬液。BMSCs组中5只大鼠于14 d后进行Morris水迷宫检测,另外5只取脑进行免疫组化染色。脑立体定位仪下大鼠侧脑室穿刺取前囟定位,前囟后1.2 mm,中线旁2 mm处进针,接上已事先抽好BMSCs悬液或是DMEM的微量注射器,深度为4.5 mm,将30μL BMSCs悬液或DMEM培养基缓慢注入侧脑室。各组大鼠在注射后14 d麻醉,固定取脑,切片,进行免疫组化染色,H2O2灭活内源性酶,加入抗原修复液,生物微波炉加热抗原修复,滴加5% BSA封闭液,一抗(小鼠抗BrdU IgG),37℃1 h,生物素化二抗,37℃20 min,DAB染色,脱水透明,中性树胶封片。SAH组及DMEM组、BMSCs组1 d、2 d、3 d、5 d、7 d后行神经生物学功能评分,于14 d后进行水迷宫检测。
结果:BrdU标记后BMSCs,经免疫细胞化学染色为胞核着色,以胞核呈棕黄色者为阳性。脑立体定位仪下行侧脑室穿刺,以前囟定位,能准确注入侧脑室。SAH大鼠侧脑室穿刺注入干细胞悬液后无不良反应。脑石蜡切片DAB染色后,可见经BrdU标记的阳性细胞胞核呈棕褐色,阳性细胞在切片内无明显规律,散在分布于阴性细胞之间。DMEM组可见阴性细胞核仍呈蓝色。将SAH组、DMEM组及BMSCs组大鼠于注射后1 d、2 d、3 d、5 d、7 d进行神经生物功能评分,无论是SAH组还是DMEM、BMSCs组可见,在各组里以1 d神经生物学功能评分普遍高于其它组(P<0.05),以后评分逐渐下降,BMSCs组大鼠在7 d时与DMEM和SAH组相比,神经生物功能评分明显减低,感觉运动功能恢复(P<0.05)。Morris水迷宫检测BMSCs组大鼠平均逃避潜伏期时间比SAH组和DMEM组要短,有统计学差异,探索实验显示BMSCs组跨越平台的次数多于其它两组,差异有明显统计学意义(P<0.05)。
结论:1 BrdU可以用来标记BMSCs,标记后的细胞可用相应的抗体检测。2脑立体定位仪下以前囟后1.2 mm,中线旁2 mm处进针,深度为4.5 mm的位置能准确注入侧脑室,注射后大鼠未见明显不良反应。3注入BrdU标记过的BMSCs后,取前囟后4 mm的海马组织切片免疫组化染色显示有BrdU阳性细胞存在。4生物学功能评分可见BMSCs组在7 d时评分下降,表明感觉和运动功能有一定程度的恢复。5 Morris水迷宫显示BMSCs组在学习记忆能力方面也有一定的改善。
四、SAH后CVS大鼠移植BMSCs后海马神经元凋亡的研究
目的:大鼠SAH后CVS模型基础上植入BMSCs,大鼠感觉、运动功能有一定程度的恢复,进一步观察BMSCs植入后对大鼠脑内海马神经元凋亡相关蛋白Bcl-2和Bax表达的影响。
方法:在第三部分大鼠脑组织切片基础之上,取SAH组、DMEM组和BMSCs组14 d前囟后4 mm处大脑皮层及海马组织石蜡切片,以兔抗鼠Bcl-2和Bax为一抗,进行免疫组织化学染色。图像分析软件分析阳性细胞数目比率。无菌条件下取SAH及BMSCs14 d组大脑,剥出海马,按每100 mg脑组织加入1 mL裂解液,超声裂解后低温离心,取上清,考马斯亮兰测定蛋白含量,蛋白变性后上样,电泳、转硝酸纤维素膜(NC膜),脱脂奶粉封闭,分别加入兔抗大鼠Bcl-2、Bax一抗,4℃静置过夜。滴加辣根过氧化物酶标记二抗,免疫化学发光,X光片显影定影。使用Hema成像分析系统对显色条带进行半定量分析,测定各组蛋白相对表达水平。
结果:与SAH组相比,BMSCs组海马可见Bcl-2阳性细胞数目增加,细胞呈黄褐色,深染;Bax阳性细胞数目减少,着色较浅。SAH组大鼠海马可见Bcl-2阳性细胞数目较少,着色浅;相反Bax阳性细胞数目增加,细胞呈棕褐色较深。DMEM组可见Bax阳性细胞数目增加,而Bcl-2阳性细胞数目相对减低。Western blot显示,DMEM组、SAH组和BMSCs组大鼠海马GAPDH表达三组基本相似,Bcl-2在BMSCs组比DMEM组和SAH组表达高(P<0.05)。Bax的表达在SAH组与DMEM组高于BMSCs组,相比有明显统计学差异(P<0.05)。
结论:1 SAH后CVS模型大鼠侧脑室植入BMSCs后,大脑皮层和海马内可见神经元抑制凋亡相关基因表达增加,而促凋亡相关基因表达下降。2 SAH组14 d大鼠脑内促凋亡相关基因表达升高,抑制凋亡相关基因表达下降。3 DMEM组大鼠脑内仅有少量的抑制凋亡相关基因表达,促凋亡相关基因表达较高。
五、SAH后CVS大鼠侧脑室植入BMSCs脑血管超微结构观察与血中NOS、ET-1含量变化
目的:观察SAH后CVS大鼠侧脑室植入BMSCs后血中一氧化氮合酶、内皮素-1含量变化,同时透射电镜观察大鼠脑内血管和神经元超微结构改变,为细胞移植治疗提供理论和实验依据。
方法:实验动物分组、SAH模型制备及BMSCs移植同前面几部分,分为正常对照组、SAH组及BMSCs组。于移植BMSCs后1 d、3 d、7 d进行取血,测定大鼠血中NOS和ET-1含量,具体方法同前面第二部分。同时取前囟后4 mm大脑皮层脑组织约1 mm×1 mm×2 mm大小,用2.5%戊二醛磷酸盐缓冲液中后固定1 d,备透射电镜检查。将标本用0.1 M磷酸缓冲液洗3次,质量分数为1%锇酸固定,逐级梯度乙醇、丙酮脱水,环氧树脂812包埋,聚合,用超薄切片机作0.5μm厚度切片,经1%甲苯胺蓝-天青II染色,光镜下定位后制作超薄切片,片厚约50 nm,铜网捞片,醋酸铀、柠檬酸铅液染色。日立H-7500型透射电镜观察各组大鼠大脑皮层神经元及微血管的超微结构,照相,记录。组间比较采用单因素方差分析,各组间两两比较采用SNK-q检验,P<0.05表示差异有统计学意义。
结果:与对照组相比,SAH组后1 d、3 d、7 d血中NOS含量明显下降,而血中ET-1含量则明显上升,其中以SAH 3 d组血中ET-1升高最为明显,差异有统计学意义。与SAH组相比,BMSCs组可见血液中ET-1含量逐渐下降,而NOS含量上升,差异有统计学意义。与正常组相比,BMSCs组ET-1含量仍然较高,差异有明显统计学意义(P<0.05),血中NOS含量有一定下降,与对照组相比不能说明有差异(P>0.1)。透射电镜显示,对照组神经元细胞核大而圆,染色质密度均匀,核仁明显,核膜完整。大脑皮层毛细血管可见其内皮细胞内膜光滑,细胞核、细胞器、紧密连接清晰可见,内皮细胞的基质和基膜完整,层次清楚,管腔无狭窄。SAH组可见大脑皮层神经元和微血管内皮细胞的水肿程度达到高峰,神经元高度水肿,线粒体肿胀,可见部分凋亡、坏死神经元。BMSCs组大脑皮层神经元和微血管内皮细胞可见轻度水肿,血管内皮细胞的吞饮小泡略减少。微血管内皮细胞核固缩,管腔内微绒毛减少,管腔狭窄有一定缓解。
结论:1 SAH后CVS大鼠侧脑室移植BMSCs后ET-1下降,NOS含量升高。说明脑血管痉挛有一定程度的缓解。2脑组织及血管超微结构观察,SAH组大脑皮层神经元坏死加重,部分微血管闭塞。BMSCs移植后神经元的凋亡与死亡减少,微血管闭塞有一定的缓解。
1.Rat bone marrow mesenchymal stem cells isolation, cultivation, identification and neuronal conditioned medium induce it’s differentiation
Objective: To master the method of rat bone marrow mesenchymal stem cells primary cultivation, passage, and identification, to investigate use hippocampal neuron’s conditioned medium to induce it differentiation.
Methods: Use weight about (100-150) g SD rat, get the tibia and femur with germ free condition, expose the bone cavity, flush the cavity with antibiotic (penicillion 100 u/mL, streptomycin 100u/mL) DMEM medium, collect it in centrifuge tube, susupension it and cultured with 10% FBS medium into the cultured bottle, incubate in 37℃、5% CO2 incubator. 3d change the half medium, 5 d change the medium completely, then change the medium every 3 d. use CD34 and CD44 as the primary antibody to do the immunocytochemistry staining, PBS as the negative control, add the biotinize secondary antibody, 37℃, 1 h, PBS wash 3 times, add horseradish peroxidase, staining with DAB, stop with water, ethanol dehydration, xylene transparent, fix it with gum, observe under microscope. Use primary cultured hippocampal neuron’s medium as conditioned medium, b-FGF group add 10% DMSO, b-FGF and L-DMEM, the last group use Neurobasal plus B27 as serum free medium, negative control is DMEM medium(with 10%FBS). P5 BMSCs planted on the cover slip wih covering 0.1% PLL ( Poly-L-Lysine ) in 6 wells plate, when the BMSCs growth on the cover slip and change with Neurobasal medium, hippocampal conditioned medium and serumfree medium, induced the cell for 12 h and 1 d, after that fixed with paraformaldehyde. BMSCs following induce with different medium, use immunocytochemistry by MAP-2, NSE and GFAP antibody, observe the positive expression of MAP-2, NSE and GFAP, count the positive ratio of different groups and photographed.
Results: BMSCs primary culture for 1 d, few of them adhere to the bottom, 2 d cleavage and proliferate, show oval ship or multiform, often form cell cluster, 4 d prolife rapidally, 3 d change the medium half, the impurity is clear, the cell morphology is obvious, the body is larger and prolife. 7-9 d is full on the bottom, about 95% emerge. BMSCs after passage growth fast,4h is adhere to the substance, 24h adhere completely and prolife. 5-7d can growth about 90% of the bottom. After 1’st passage, the purity is about 95%, passage it for 6 generation, each passage is the same in morphology and growth. BMSCs H-E staining show spindle and multiform, the nuclei is large, round and heavy dye, basophilic, cytoplasma is light, acidophilic, cell paralled. Rabbit anti rat CD34 show negative and colorless, CD44 cell adhere molecular positive, nuclei and cytoplasma show brown. Hippocampal neuron conditioned medium, b-FGF medium and B27 plus neurobasal (serum free) medium induced the BMSCs for 12 h and 1 d, after inducing the cell body changed into round and pyramid gradually, process elongate, 1 d some of them can connect each other. With immunocytochemistry stainin, 12 h and 1 d labeled with MAP-2, NSE and GFAP, count the positive showed conditioned medium induced BMSCs positive ratio is the highest, express NSE, MAP-2, GFAP higher than others, have the significant statistically difference(P<0.05)
Conclusions: 1.Full bone marrow culture method and adhere way is a successful method to isolation, purify the BMSCs in vitro, and it can prolife rapidly and stable. 2. BMSCs in vitro with immunocytochemistry staining, it’s express mesenchymal stem cells characteristic, not the hemotopoitic stem cells. 3. With b-FGF DMEM medium, hippocampal conditioned medium, serum free medium induced BMSCs 12 h and 1 d, BMSCs can differentiate into neuronal like cell and glial like cell, with immunocytochemistry identification it’s express NSE, MAP-2 and GFAP positive characteristic. 3. With b-FGF DMEM medium, hippocampal medium, serum free medium induce BMSCs, 12 h is the highest ratio, it’s about 44.78%, conditioned medium compare with others, there are statistically differences.
2. CVS model following SAH and ethological, biological detection
Objective: Observe rat autologous blood cisterna magna injection to produce CVS model following SAH, detect blood and brain NOS and ET-1 content varation, the diameter of cerebro vessel, value the model of CVS from ethological performance.
Methods: Use double hemorrhage cistern magna injection method to produce SAH model, separate the atlanto-occipital membrane and cut anterio the end of tail about 5-6 cm, get the arterial blood 0.2-0.3 mL from tail artery, with stereotaxica coordinate inject the arterial blood from atlanto-occipital membrane about 5 min, and 2 d later inject the blood again the same way. Rat was valued with neurological functional score, in 1 d, 2 d, 3 d, 5 d, 7 d compare SAH and control group. After vessel perfusion with 10% of india ink detect the diameter of cerebral artery with magnification of 45, with Nikon NIS BR 3.0 and Image J analysis system, detect the basal artery, introcarotid artery, and middle cerebral artery diameter. With the same perfusion way, filling the microvessel, 1 mm thick slice around the hippocampus 4 mm posterior bregma, valure the vessel filling under 45 magnification. SAH 1 d, 2 d, 3 d, 5 d, 7 d extract the eyeball, get 2 mL blood, get the hippocampus and cortex of brain, use serum and detect the ET-1 and NOS according to it’s manual, after injection 14 d, do Morris mazer test. All data process with SPSS14.0 soft ware and use one way annova, compare control and experimental group with Dunnett-t method, among groups with SNK-q test, with neurological functional score and cerebral diameter, NOS and ET-1 use multiple linear regression, Morris mazer mean escape time and NOS, ET-1, cerebral diameter also use multiple linear regression.
Results: after SAH rat show decrese in eating, some kind of abnormal ethology, show lower reaction, lower alartness, sleepy, bloom, lack of diat, it can be better on 2’nd day, but double hemorrhage aggrevate, some of them can be paralysis. After injection there are blood clot in cerebullar medullary cistern, 1 d can see obsolete blood. Posterior 4 mm of bregma cortex vessel attenuate and lessen, the branches to medullary is lessen, but the control group show normal and abundant. With image analysis detect the vessel, SAH 2 d and 3 d the diameter have statistically difference versus control group. For neurological function score, SAH 1 d, 2 d, 3 d, 5 d have higher score, versus control group have significant difference. SAH 1 d, 2 d, 3 d, 5 d, 7 d blood and brain ET-1 were higher than sham. With multiple linear regression, neurological function score is related with ET-1 and middle cerebral artery. The average swimming speed is the same, while the swimming distance and escaping time of SAH group is longer than others, SAH span the flat less than control group.
Conclusions: 1. Rat autologous tail blood cistern magna injection double hemorrhage model, there are clot around the sulcus and gyrus, with microperfusion shows clearly brain vessel hypoperfusion. 2. Compare with control group, SAH rat diameter of BA, MCA, ICA is thinning, especially for MCA. 3. SAH brain and blood ET-1, NOS show NOS lower than control group, ET-1 higher than the others. 4. SAH neurological score higher than others. 5. Morris mazer average escaping time of SAH is longer than control group, span times lower than sham.
3. BrdU Labelled BMSCs transplant to CVS rat following SAH from lateral ventricle injection
Objective: To transplant labeled BMSCs in to SAH following CVS from lateral ventricle, investigate the labeled cell development in brain, valure the cell transplantation effect with ethological method.
Methods: P5 BMSCs, with BrdU to labelled for 2 d, after that check it by immnunocytochemistry staining with BrdU antibody. Then use labelled cell suspension, as 5×106 cell density to transplant to rat brain from lateral ventricle. By neurological score valure the 3 groups: SAH group, BMSCs group, and only transplant DMEM group. Sandomize 3 group, SAH didn’t transplant cell, DMEM only inject 30μL DMEM medium after 2’nd blood injecton 2 d, BMSCs group after SAH 2 d under stereotaxic coordinate inject 30μL BMSCs suspension as previous describe density. The Morris mazer and ethological exam after 14 d. Under stereotaxic coordinate, rat lateral ventricle localize is: posterior to bragma 1.2 mm, median line 2 mm, and the deep is 4.5 mm, after injection 14 d, fixed with paraformadehyde and get the brain slices, by immunohistochemistry staining method to show the positive BrdU labeled BMSCs. Detect neurological score at 1 d, 2 d, 3 d, 5 d, 7 d after SAH inject DMEM and BMSCs, 14 d do the Morris mazer experiment.
Results: BrdU labelled BMSCs, immnunohistochemistry staining show nuclei brown is positive, negative is baby blue. Localize lateral ventricle under stereotaxic coordinate with bragam, it’s accurate into lateral ventricle and have no other untoward reaction, compare with DMEM group, BMSCs group sensory and motor disable no aggravate. With DAB staining of brain slice, the positive cell of BrdU labelled show brown color, positive cell irregular line within the negative cells. Neurological score show 1 d is the highest, with time it’s decrease little by little. DMEM group and SAH group have no statistically difference, Morris mazer BMSCs escaping time is shorter than SAH and DMEM group, spaning flat times show BMSCs more than others, have statistically difference.
Conclusions: 1. BrdU can labelled BMSCs, with BrdU antibody immnunocytochemistry staining show brow color. 2. Under stereotaxic coordinate localize posterior bragma 1.2 mm, median line 2 mm, depth 4.5 mm can accurate inject into lateral ventricle, have no other side effect. 3.Labelled BrdU BMSCs, show positive BrdU cell in the brain slice. 4. Rat neurological score show BMSCs group 7 d has recover in motor and sensory in certain degree. 5. Morris mazer show BMSCs group have some kind of recover in learning and memory.
4. The apoptosis investigation of hippocampus neuron after transplantation of BMSCs to CVS rat following SAH
Objective: after transplantation BMSCs to CVS rat following SAH, observe the expression of Bcl-2 and Bax protein which are related with apoptosis in brain and hippocampus.
Methods: With the SAH group, DMEM group and BMSCs group brain slices, use rabbit anti rat Bcl-2 and Bax as primary antibody, immunohistobiochemistry staining method to observe the morphological change. With Image analysis soft ware count the positive cell ratio of each groups. Get the SAH, DMEM and BMSCs group rat brain in 14 d, dissect the hippocampus, add the lysate about 1 mL per 100 mg brain tissue, ultrasonic clearage and centrifuge hypothermia, get the supernatant, detect it’s protein content with Coomassie agent. Protein denaturation and spotting, electrophoresis and transfer to nitrocellulose membrane. Blocking with defatted milk powder, add primary antibody of Bcl-2 and Bax, 4℃over night, add horseradish peroxidase, immune-developing, with Hema analysis system to detect each group protein expression level.
Results: Compare with SAH, BMSCs at hippocampal slice Bcl-2 positive expression increase, Bax expression decrease, SAH group at the hippocampal slice Bcl-2 decrease and Bax increase. DMEM group Bcl-2 decrease and Bax increase. Western blot show, control group, SAH group, BMSCs group GAPDH expression is the same, BMSCs group Bcl-2 is higher than the others, and Bax expression is lower. Have statistically difference.
Conclusions: 1. SAH following CVS transplant BMSCs, the express of inhibit apoptosis gene is decrease and promote apoptosis gene is increase. 2. SAH 14 d rat brain promote apoptosis gene expression increase and inhibit apoptosis gene decrease. 3.There are a little expression of promote apoptosis gene expression in DMEM group.
5. The blood NOS, ET-1 content change and micro-ultral structure change after BMSCs transplantation to CVS rat following SAH
Objective: To investigate blood NOS, ET-1 changes of BMSCs transplantation to CVS rat following SAH, transmission electron microscope observe the cerebrovascular and neuron’s ultra-micro-structural change.
Methods: Animal grouping, SAH model and cell transplantation as previous subscribe, collect blood after BMSCs transplantation 14 d, detect the content of NOS and ET-1, as previous. Get the blood at 1 d, 3 d, and 7 d. Detect the NOS and ET-1 content in blood, as previous describe. Get the brain cortex posterior bregma about 1mm×1mm×2mm, fix it with 2.5% glutaral phosphates about 24h, ultral thin slices for TEM, wash it with 0.1mol/L phosphate 3 times, 1% osmic acid fix, ethanol and acetone dehydration, bed with epoxy resins, polymerization, 0.5μm thickness slicing the tissue, 1% toluidin blue-azure thickness about 50 nm, dredge with copper screen, uranyl acetate and citric acid staining, Hitachi H-7500 TEM observe ultra-structure of the brain cortex neuron and microvascular, photography. Comparasion between groups use one way ANOVA, among groups use SNK-q test, P<0.05 represent difference.
Results: Compare with control group, SAH 1 d, 3 d and 7 d blood NOS decrease and ET-1 increase, as SAH 3 d increase for typical, have statistically difference. BMSCs group blood ET-1 decerease versus SAH group, NOS increase, have statistically difference. Compare with control group, BMSCs group ET-1 is still in high level, have statistically difference, NOS have certain level decrease, but have no statistically difference. TEM ultra-micro-structure show control group neuclus is big and round, Chromosome density is even, karyosome is clear, nuclear membrane is intergreted. Endothelial cell endomembrane is smooth, cell neuclear, apparatus, tight injunction is clear, endothalia matrix and basal lamina is integrated, stratification is clear, vessel have no narrow. SAH group cortex neuron and microvascular endothelial edema is the highest, mitochondria swelling, most of crest and membrane emerge, disappear, some of them medulla change, there are vaculous in cytoplasma, neuron nuclear chromosome concentrate, heterochromatin increase, mitochnodia, rough endoplasm reticulum swelling, even collapse. BMSCs transplantation can see little bit of edema in cortex neuron and microvasular endothelial, pinocytosis bulb attenuate in vascular endothelial cell.
Conclusions: BMSCs lateral ventricle transplantation to SAH following CVS rat blood ET-1 decreas, NOS increase, SAH group neuron necrosis deeply and some of microvessel stenosis, after transplant BMSCs is attenuate the effect of CVS, imply cerebral vascular have a certain degree of release.
引文
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