【作者】 张建；

【导师】 周云；

【作者基本信息】 苏州大学 ， 儿科学， 2008， 硕士

【摘要】 目的:应用SD大鼠肾脏缺血再灌注损伤模型,探讨骨髓间充质干细胞(MSCs)移植及淮山药灌胃预处理在对肾脏缺血再灌注损伤后微血管方面的保护作用。方法:体外实验:采用梯度离心和贴壁筛选法分离纯化MSCs,取第三代的MSCs进行诱导。在MSCs的培养液中加入VEGF、bFGF对MSCs进行诱导,分别于0d、3d、7d、14d、21d时对诱导的MSCs细胞进行大体形态学观察、细胞组织化学染色、超微结构观察。体内试验:1.选用60只雌性SD大鼠(4～6周龄)随机分4组进行动物实验。正常对照组(N,n=6)常规麻醉后行剖腹手术下腔静脉采血和收获肾脏标本;缺血再灌注损伤组(I/R,n=18)常规麻醉后行剖腹手术,游离左侧肾蒂血管后无损伤动脉夹阻断左肾血流60min后恢复血流,关闭腹腔,再灌注后60min股静脉穿刺,输注生理盐水,再分别于再灌注6h,48h,2w后采血和收获肾脏标本;缺血再灌注损伤干细胞移植组( MSCs,n=18)游离左侧肾蒂血管后无损伤动脉夹阻断左肾血流60min后恢复血流,关闭腹腔,再灌注后60min股静脉穿刺,输注BrdU标记的MSCs细胞悬液1ml(1×106/ml)再分别于再灌注6h,48h,2w后采血和收获肾脏标本;缺血再灌注淮山药灌胃预处理联合干细胞移植组(淮山药MSCs组,n=18)动物造模前五天,用淮山药10g/kg体重灌胃,连续五天,游离左侧肾蒂血管后无损伤动脉夹阻断左肾血流60min后恢复血流,关闭腹腔,再灌注后60min股静脉穿刺,输注BrdU标记的MSCs细胞悬液1m(l1×10~6/ml)再分别于再灌注6h,48h,2w后采血和收获肾脏标本。2.应用全自动生化分析仪测定血清BUN、Scr,了解肾功能;内皮素放免药盒体外放射免疫分析法检测肾脏组织匀浆内皮素水平(ET);肾脏标本常规病理切片,H-E染色观察肾组织损伤情况;免疫组化S-P法检测细胞增殖情况, BrdU、FⅧ-RAg阳性细胞分布情况。主要结果:体外实验:MSCs细胞为纺锤形,梭形的成纤维细胞形态;加入VEGF、bFGF后可见到部分扁平宽大的长梭形细胞胞质向核收缩,变成不规则形,多角形或圆形,有些区域呈“铺路石”样征象。诱导后的MSCs做透射电镜检测,部分细胞胞浆内可观察到Weibel-Palade小体。体内实验:1﹒肾组织损伤情况:再灌注6h、48h、2w后,MSCs组、淮山药MSCs组的组织学评分(MSCs组分别为:6.63±0.54、6.20±0.28、5.48±0.49)(淮山药MSCs组分别为:5.10±0.47、5.06±0.37、4.93±0.53)较同时间点I/R组(I/R组分别为:7.35±0.56、7.50±0.93、6.23±0.33)明显减低,有显著性统计学差异(P﹤0.05);淮山药MSCs组的组织学评分于再灌注后6h、48h较同时间点MSCs组明显减低,有显著性统计学差异(P﹤0.05) ,缺血再灌注损伤2w后,两组的组织学评分比较相近,无显著性统计学差异(P>0.05)。2.肾功能情况:N组血清Scr、BUN水平(分别为43.24±2.58μmol/L、6.48±0.64mmol/L);肾脏缺血再灌注损伤6h后各组Scr、BUN水平均有上升,MSCs组(61.93±5.05μmol/L、11.05±1.32 mmol/L)、淮山药MSCs组(50.40±3.38μmol/L、8.82±0.49 mmol/L)与I/R组(75.38±4.53μmol/L、18.72±0.59 mmol/L)相较明显降低,有显著性统计学差异(P﹤0.05),且淮山药MSCs组与MSCs组比较也有显著性统计学差异(P﹤0.05);缺血再灌注损伤48h后仅I/R组(79.09±3.32μmol/L、15.37±1.69 mmol/L)较N组有明显上升(P﹤0.05),MSCs组(46.76±2.64μmol/L、5.63±0.82 mmol/L)、淮山药MSCs组(44.33±5.51μmol/L、5.70±1.06 mmol/L)较N组无显著性统计学差异(P>0.05);缺血再灌注损伤2w后I/R组(45.63±2.09μmol/L、6.10±0.53 mmol/L)、MSCs组(42.55±1.70μmol/L、5.88±0.52 mmol/L)、淮山药MSCs组(47.23±1.95μmol/L、5.80±0.59 mmol/L)较N组均无显著性统计学差异(P>0.05)。3.肾血管内皮功能情况:N组肾组织匀浆ET水平为158.25±15.13 pg/ml;缺血再灌注损伤6h后I/R组、MSCs组、淮山药MSCs组ET水平分别为258.30±25.04 pg/ml、203.40±6.01pg/ml、168.56±12.12pg/ml,I/R组、MSCs组肾组织匀浆ET值较N组显著增高(P﹤0.01),但淮山药MSCs组肾组织匀浆ET值却较N组无显著性统计学差异(P>0.05);缺血再灌注损伤48h后:I/R组、MSCs组、淮山药MSCs组ET水平分别为154.61±16.12 pg/ml、144.48±21.34pg/ml、158.25±22.91pg/ml较N组肾组织匀浆ET的水平无显著性统计学差异(P>0.05)。4.细胞增殖和BrdU、FⅧ-RAg阳性细胞分布情况:再灌注6h、48h、2w后,MSCs组、淮山药MSCs组的PCNA阳性细胞数分别为(MSCs组:22.42±0.68/HP、24.60±0.84/HP、22.36±0.85/HP)(淮山药MSCs组:30.17±0.63/HP、36.52±1.17/HP、32.01±0.42/HP)较同时间点I/R组(I/R组:7.06±0.28/HP、17.64±0.72/HP、19.03±0.41/HP)明显增多,有显著性统计学差异(P﹤0.05);淮山药MSCs组的PCNA阳性细胞数于再灌注后6h、48h、2w较同时间点MSCs组明显增多,有显著性统计学差异(P﹤0.05)。再灌注后6h、48h于肾间质可见散在BrdU阳性的细胞,再灌注后2w可见少量BrdU染色阳性细胞位于肾小球,肾间质微血管。再灌注6h、48h、2w后,MSCs组、淮山药MSCs组的FⅧ-RAg阳性细胞数分别为(MSCs组:23.98±0.44/HP、22.94±1.50/HP、22.10±0.78/HP)(淮山药MSCs组:34.96±0.56/HP、30.19±0.54/HP、27.42±0.77/HP)较同时间点I/R组(I/R组:17.82±0.58/HP、18.23±0.49/HP、20.40±0.66/HP)明显增多,有统计学差异(P﹤0.05);淮山药MSCs组的FⅧ-RAg阳性细胞数于再灌注后6h、48h、2w较同时间点MSCs组明显增多,有显著性统计学差异(P﹤0.05)。主要结论:在体外MSCs的培养液中加入VEGF、bFGF对MSCs进行诱导,可以使MSCs诱导为血管内皮细胞。体内实验结果表明MSCs移植对急性肾缺血再灌注损伤具有治疗作用;淮山药灌胃预处理合并MSCs移植对急性肾缺血再灌注损伤具有协同治疗作用。部分MSCs在体内可分化形成肾血管内皮细胞。更多还原

【Abstract】 Objectives:To investigate the protective effects of Rhiizoma Dioscoreae pretreatment and Bone Marrow Mesenchymal Stem Cells in easing renal ischemia-reperfusion injury and promoting the Endothelial cell regeneration in renal ischemia-reperfusion in rat.Methods:In Vitro Experiment:after isolation and purification,Rat MSCs were induced by VEGF,bFGF. The morphological and ultra-structure changes of the MSCs were observed with the inverted microscope and transmission electron microscope on the 0th,3d,5th,7th day respectively.In Vivo Experiment:1. 60 Spargue-Dawley rats (4-6 week-old) were randomly assigned into 4 group: (1) normal control group (N, n=6),(2) the I/R group(n=18),rats were subjected to 60 min of the left renal ischemia-reperfusion injury with microvascular clips ,after 60 min of reperfusion they were injected saline intravenously and then 6h, 48h, and 2w after reperfusion six rats each were randomly euthanized for blood sampling and kidney harvesting, (3)the MSCs group(n=18), rats were subjected to 60 min of the left renal ischemia-reperfusion injury with microvascular clips, after 60 min of reperfusion they were injected in BrdU positive MSCs ( 1×10~6/ml )intravenously and then 6h, 48h, and 2w after reperfusion six rats each were randomly euthanized for blood sampling and kidney harvesting, (4) the Rhiizoma Dioscoreae and MSCs group(n=18), the rats with pretreated with Rhiizoma Dioscoreae for five days before being subjected to 60 min of the left renal renal ischemia and reperfusion then the step is the same as the third group.2. The blood urea nitrogen(BUN) and serum creatinine(Scr) were measured with automatic biochemistry analyzer. The renal tissue homogenate content of endothelin (ET) was determined with radio immunoassay methods. Renal morphologic changes were scored with Paller’s criterion on hematoxylin and eosin(H&E) stained sections. PCNA positive cells of renal tubular epithelia and glomerulus were detected immunohistochemically as proliferation index. The distribution of BrdU positive MSCs and FⅧ-RAg positive cells were identified by immunohistochemically.Results:In Vitro Experiment: within days of induction, MSCs changed shape from spindle alike cells into slabstone-like cells. Weibel-Palade bodies can be found in the cytoplasm of some cells by transmission electron microscope.In Vivo Experiment:1.Histological examination:6h,48h,2w after renal ischemia-reperfusion injury, the renal morphologic score in the Rhiizoma Dioscoreae and MSCs group and the MSCs group were (5.10±0.47,5.06±0.37,4.93±0.53), (6.63±0.54,6.20±0.28,5.48±0.49) respectively, significantly lower than that in the I/R group (7.35±0.56,7.50±0.93,6.23±0.33)(P < 0.05);they were also significantly difference between the the Rhiizoma Dioscoreae and MSCs group and the MSCs group at 6h,48h after renal ischemia-reperfusion injury(P < 0.05),but there were no difference between them at two weeks after renal ischemia-reperfusion injury(P>0.05).2. The change of serum creatinine and urea nitrogen level: the Scr and BUN value in N group were 43.24±2.58μmol/L, 6.48±0.64mmol/L. 6h after renal ischemia-reperfusion injury, the Scr and BUN value in each group were increased, the I/R group (75.38±4.53μmol/L,18.72±0.59 mmol/L) were significantly higher than that in the MSCs group (61.93±5.05μmol/L, 11.05±1.32 mmol/L)and the Rhiizoma Dioscoreae and MSCs group(50.40±3.38μmol/L,8.82±0.49 mmol/L) (P < 0.05),and there were also significantly difference between the MSCs group and the Rhiizoma Dioscoreae and MSCs group. 48h after renal ischemia-reperfusion injury,there was only the I/R group (79.09±3.32μmol/L,15.37±1.69 mmol/L) have significantly higher than that in the N group(P < 0.05). The Scr and BUN value were no difference among the I/R group (45.63±2.09μmol/L,6.10±0.53 mmol/L), the MSCs group(42.55±1.70μmol/L,5.88±0.52 mmol/L) and the Rhiizoma Dioscoreae and MSCs group ( 47.23±1.95μmol/L,5.80±0.59 mmol/L)(P>0.05) at two weeks after renal ischemia-reperfusion injury.3. The change of endothelin (ET) : the ET value in N group was 158.25±15.13 pg/ml. 6h after renal ischemia-reperfusion injury, the ET value in the I/R group, the MSCs group and the Rhiizoma Dioscoreae and MSCs group were 258.30±25.04 pg/ml,203.40±6.01pg/ml, 168.56±12.12pg/ml;The I/R group and the MSCs group were significantly higher than that in the N group(P < 0.05), but there were no difference between the Rhiizoma Dioscoreae and MSCs group and the N group. The ET value were no difference among the I/R group (154.61±16.12 pg/ml), the MSCs group(144.48±21.34pg/ml) and the Rhiizoma Dioscoreae and MSCs group(158.25±22.91pg/ml)(P>0.05) at 48h after renal ischemia-reperfusion injury.4.Cell proliferation and distriburion of the BrdU and the FⅧ-RAg positive cells in the kidney: 6h,48h,2w after renal ischemia-reperfusion injury, the PCNA-positive cells in the Rhiizoma Dioscoreae and MSCs group and the MSCs group were ( 30.17±0.63/HP,36.52±1.17/HP,32.01±0.42/HP ) , (22.42±0.68/HP,24.60±0.84/HP,22.36±0.85/HP) respectively, significantly higher than that in the I/R group (7.06±0.28/HP,17.64±0.72/HP,19.03±0.41/HP)(P < 0.05);there were also significantly difference between the the Rhiizoma Dioscoreae and MSCs group and the MSCs group at 6h,48h, 2w after renal ischemia- reperfusion injury(P < 0.05).After 6h and 48h of reperfusion, BrdU-positive cell were located diffusely at the renal interstitium. After 2w of reperfusion, a few BrdU-positive cells were located at the renal capillary and glomerulus.6h,48h,2w after renal ischemia-reperfusion injury, the FⅧ-RAg positive cells in the Rhiizoma Dioscoreae and MSCs group and the MSCs group were (34.96±0.56/HP,30.19±0.54/HP,27.42±0.77/HP),(23.98±0.44/HP,22.94±1.50/HP,22.10±0.78/HP)respectively, significantly higher than that in the I/R group (17.82±0.58/HP,18.23±0.49/HP,20.40±0.66/HP)(P < 0.05);there were also significantly difference between the the Rhiizoma Dioscoreae and MSCs group and the MSCs group at 6h,48h, 2w after renal ischemia-reperfusion injury(P < 0.05).Conclusions:A proportion of MSCs can be induced to vascular endothelial cells in vitro in culture medium including of VEGF, bFGF. MSCs is of therapeutical value to renal ischemia-reperfusion injury,ameliorating the tissue damage,contributing to renal function recovery,and MSCs may also differentiate into renal vascular endothelial cells in vivo. Rhiizoma Dioscoreae and MSCs have synergistic action in easing renal ischemia-reperfusion injury and promoting the Endothelial cell regeneration in renal ischemia-reperfusion.更多还原