人胚胎骨髓间充质干细胞和单个核细胞静脉移植治疗大鼠脑梗死疗效的比较

Comparsion between Intravenous Delivered Human Fetal Bone Marrow Mesenchymal Stromal Cells and Mononuclear Cells in the Treatment of Rat Cerebral Infarct

目的比较人胚胎骨髓间充质干细胞(MSC)和单个核细胞(MNC)经静脉移植后对脑梗死的疗效。方法采用大脑中动脉远端凝断法制造大鼠脑梗死模型。将大鼠分为假手术组、缺血对照组(造模后经尾静脉给予生理盐水)、MSC组(造模后经尾静脉给予MSC)和MNC组(造模后经尾静脉给予MNC)。移植后2 d,取脑进行梗死体积测定;移植后1、3、5、7 d,采用肢体放置测验和平衡木实验进行行为学评价;移植后7 d,采用免疫荧光法测量梗死周边纹状体区激活的小胶质细胞Iba-1的染色丰度;移植后3、7 d,采用ELISA检测移植后梗死灶周边纹状体区胶质源性神经营养因子(GDNF)和脑源性神经营养因子(BDNF)的蛋白水平。结果缺血对照组、MSC组和MNC组的相对梗死体积分别为(37.85±4.40)%、(33.41±3.82)%和(30.23±3.63)%,其中,MSC组(t=2.100,P=0.034)和MNC组(t=2.109,P=0.0009)显著低于缺血对照组,MNC组显著低于MSC组(t=1.743,P=0.043)。移植后1 d,缺血对照组的肢体放置成功率为(4.32±0.71)%,明显低于假手术组的(9.73±0.36)%(t=2.178,P=8.61×10-11);MSC组和MNC组分别为(5.09±0.62)%(t=2.1009,P=0.024)和(5.90±0.68)%(t=2.1008,P=0.0001),明显高于缺血对照组;MNC组又明显高于MSC组(t=2.1009,P=0.0165)。MSC组和MNC组损伤对侧前肢错步数占总步数的百分比分别为(5.56±0.86)%(t=2.120,P=0.020)和(5.13±0.95)%(t=2.131,P=0.003),明显低于缺血对照组的(6.47±0.61)%。移植后3 d,MNC组的肢体放置成功率为(6.91±1.10)%,明显高于缺血对照组的(5.80±0.82)%(t=2.110,P=0.027);MSC组为(6.30±0.77)%,与缺血对照组差异无统计学意义(t=2.101,P=0.199)。MNC组损伤对侧前肢错步数占总步数的百分比为(4.34±0.58)%,明显低于缺血对照组的(5.31±0.65)%(t=2.100,P=0.006)和MSC组的(4.92±0.53)%(t=2.100,P=0.041),MSC组与缺血对照组差异无统计学意义(t=2.109,P=0.139)。假手术组、缺血对照组、MSC组和MNC组的Iba-1相对丰度分别为1.00+0.00、1.72±0.21、1.23±0.08和1.48±0.06,其中,缺血对照组显著升高,为假手术组的(1.72±0.21)倍(t=2.262,P=2.9×10-6),MSC组则降低至假手术组的(1.23±0.08)倍,比缺血对照组显著降低(t=2.178,P=3.91×10-5),MNC组降低至假手术组的(1.48±0.06)倍,比缺血对照组显著降低(t=2.200,P=0.007),MSC组和MNC组间差异也有统计学意义(t=2.120,P=7.09×10-6)。移植后3 d,MSC组的BDNF和GDNF分别为(531.127±73.176)pg/mg蛋白(t=2.109,P=0.003)和(127.780±16.733)pg/mg蛋白(t=2.100,P=2.76×10-5),均明显高于缺血对照组的(401.988±89.006)pg/mg蛋白和(86.278±14.832)pg/mg;MNC组分别为(627.429±65.646)pg/mg蛋白(t=2.144,P=0.017)和(153.117±20.443)pg/mg蛋白(t=2.144,P=0.017),均明显高于MSC组。移植后7 d,MSC组的BDNF和GDNF分别为(504.776±83.282)pg/mg蛋白(t=2.101,P=0.005)和(81.641±11.019)pg/mg蛋白(t=2.100,P=0.002),均明显高于缺血对照组的(389.257±70.440)pg/mg蛋白和(64.322±9.855)pg/mg;MNC组分别为(589.068±63.323)pg/mg蛋白(t=2.100,P=0.027)和(102.161±19.932)pg/mg蛋白(t=2.144,P=0.017),也均明显高于MSC组。结论脑梗死后1 h静脉移植人骨髓MSC和MNC都能够减小梗死体积、改善动物行为。二者的疗效产生机制可能均与抑制脑内炎症反应,促进脑内神经营养因子的产生有关。MNC减小梗死体积和改善动物行为比MSC更有效,推测可能与MNC诱导脑内产生的细胞因子GDNF和BDNF的量比MSC更高有关。

Objective To compare the effecacy of human mesenchymal stromal cell（ hM SC） with human mononuclear cell（ hM NC） in treating rat cerebral infarct.Methods The SD rat models of cerebral infarct were established by distal middle cerebral artery occlusion（ dM CAO）.Rats were divided into four groups：sham,ischemia vehicle,MSC,and MNC transplantation groups.For the transplantation group,1 × 106 hM SCs or hM NCs were intravascularly transplanted into the tail vein 1 hour after the ischemia onset.The ischemia vehicle group received dM CAO surgery and intravascular saline injection 1,3,5,and 7 days after the ischemia onset,and then behavioral tests were performed.At 48 h after the ischemia onset,the abundance of Iba-1,the symbol of activated microglia,was evaluated in the peri-ischemia striatum area; meanwhile,the neurotrophic factors such as glial cell line-derived neurotrophic factor（ GDNF） and brain-derived neurotrophic factor（ BDNF） in ipsilateral peri-ischemia striatum area were also measured.Results The relative infarct volume in ischemia vehicle group,hM SC group,and hM NC transplantation group were（ 37.85 ± 4.40） %,（ 33.41 ± 3.82） %,and（ 30.23 ± 3.63） %,respectively.The infarct volumes of MSC group（ t = 2.100,P = 0.034） and MNC group（ t = 2.109,P = 0.0009） were significantly smaller than that of ischemia vehicle group,and that of MNC group was significantly smaller than that of MSC group（ t = 1.743,P = 0.043）.One day after transplantation,the score of ischemia vehicle group in limb placing test was（ 4.32 ± 0.71） %,which was significantly lower than that in sham group（ 9.73 ± 0.36） %（ t =2.178,P = 8.61 × 10-11）.The scores of MSC and MNC group,which were（ 5.09 ±0.62） %（ t =2.1009,P =0.024） and（ 5.90 ± 0.68） %（ t = 2.1008,P = 0.0001）,respectively,were significantly higher than that of ischemia vehicle group; also,the score of MNC group was significantly higher than that of MSC group（ t = 2.1009,P = 0.0165）.The contralateral forelimb scores of MSC and MNC groups in beam walking test were（ 5.56 ±0.86） %（ t = 2.120,P = 0.020） and（ 5.13 ± 0.95） %（ t = 2.131,P = 0.003）,were both significantly lower than that of ischemia vehicle group [（ 6.47 ± 0.61） %].Three days after the transplantation,the limb placing test score of MNC group [（ 6.91 ± 1.10） %] was significantly higher than that of ischemia vehicle group（ 5.80 ±0.82） %（ t = 2.110,P = 0.027）.The score of MSC group [（ 6.30 ± 0.77） %] showed no statistic difference with that of ischemia vehicle group（ t = 2.101,P = 0.199）.The contralateral forelimb scores of MNC group in beam walking test [（ 4.34 ± 0.58） %] was significantly lower than that of ischemia vehicle group [（ 5.31 ±0.65） % ]（ t = 2.100,P = 0.006） and MSC group [（ 4.92 ± 0.53） % ]（ t = 2.100,P = 0.041）; there was no statistic difference between MSC group and ischemia vehicle group（ t = 2.109,P = 0.139）.The relative abundance of Iba-1 in sham,ischemia vehicle,MSC,and MNC groups was 1.00 ＋ 0.00,1.72 ± 0.21,1.23 ± 0.08,and 1.48 ± 0.06,respectively.The Iba-1 relative abundance of ischemia vehicle group was significantly higher than that of sham group（ t = 2.262,P = 2.9 × 10-6）.The Iba-1 relative abundances of both MSC（ t =2.178,P =3.91 × 10-5） and MNC（ t = 2.200,P = 0.007） groups were significantly lower than that of ischemia vehicle group.It was also significantly lower in MNC group than in MSC group also（ t = 2.120,P = 7.09 × 10-6）.Three days after transplantation,the BDNF and GDNF levels of MSC group,which were（ 531.127 ± 73.176）pg/mg（ t = 2.109,P = 0.003） and（ 127.780 ± 16.733） pg/mg（ t = 2.100,P = 2.76 × 10-5）,respectively,were significantly higher than those of ischemia vehicle group,which were（ 401.988 ± 89.006） pg/mg and（ 86.278 ± 14.832） pg/mg, respectively.The BDNF and GDNF levels of MNC group, which were（ 627.429 ± 65.646） pg/mg（ t = 2.144,P = 0.017） and（ 153.117 ± 20.443） pg/mg（ t = 2.109,P =0.010）,respectively,were all significantly higher than that of MSC group.At day 7,the BDNF and GDNF levels of MSC group, which were（ 504.776 ± 83.282） pg/mg（ t = 2.101, P = 0.005） and（ 81.641 ±11.019） pg/mg（ t = 2.100,P = 0.002）,respectively,were significantly higher than those of ischemia vehicle group,which were（ 389.257 ± 70.440） pg/mg and（ 64.322 ± 9.855） pg/mg,respectively.The BDNF and GDNF levels of MNC group, which were（ 589.068 ± 63.323） pg/mg（ t = 2.100, P = 0.027） and（ 102.161 ± 19.932） pg/mg（ t = 2.144,P = 0.017）,respectively,were all significantly higher than that of MSC group.Conclusions Both h MSC and h MNC are beneficial to the ischemia-damaged brain when they are intravascularly transplanted within 1 h after the onset of ischemia.The anti-inflammation ability and secretion of neurotrophic factors are the underlying mechanisms of the therapeutic effects.MNC is more effective than MSC in reducing infarct area and improving behaviors,which might be explained by the fact that MNC induces more GDNF and BDNF in brain than MSC.