表达HGF的骨髓间充质干细胞促进小移植肝早期肝再生

目的 建立大鼠小体积肝移植模型,输注表达人肝细胞生长因子(human hepatocyte growth factor,hHGF)的骨髓间充质干细胞(mesenchymal stem cells,MSCs),研究其在移植早期对小移植肝促再生作用.方法 将已建立的表达hHGF和绿色荧光蛋白(green fluorescence protein,GFP)的MSCs,分别命名为HGF/MSCs,GFP/Mscs.建立大鼠30%肝移植模型.受体分为4组,实验组输注5×106HGF/MSCs;对照组则分别输注相同体积的生理盐水(PS),5×106 GFP/MSCs或1.0×109 pfu含hHGF的重组腺病毒液(Ad-HGF).分别于术后1,3,5,7 d各组随机抽取5只大鼠处死.取血检测血清ALT和hHGF.记录移植物湿重.取肝组织检测hHGF、c-met表达,以及肝细胞凋亡和增殖活性.另每组15只,分组同上,用于观察生存期.结果 PS组大鼠7 d生存率33.3%;组织学及血清学检查示术后肝脏损伤重,汇管区单核细胞浸润多;而实验组大鼠7 d生存率为73.3%.肝脏损伤轻,炎性细胞浸润少;实验组移植肝再生较PS组明显增加.结论 大鼠部分肝移植后,输注HGF/MSCs能够保护小体积移植肝,促进小移植肝再生,提高7 d生存率.     

骨髓间充质干细胞联合肝细胞生长因子基因治疗兔肢体缺血

目的探讨联合应用骨髓间充质干细胞(BMSC)和肝细胞生长因子(HGF)基因治疗兔肢体缺血的疗效。方法 32只新西兰兔切除右后肢股浅动脉并结扎股深动脉建立后肢缺血模型,成模后随机均分为空腺病毒对照组(对照组),BMSC细胞治疗组(BMSC组),HGF基因治疗组(HGF组)和联合治疗组(BMSC+HGF组),各组均采用术肢肌肉内原位注射。治疗28 d后通过动脉造影行侧枝血管计数,治疗30 d后用免疫组化和Western blot法检测注射点周围组织中CD31和HGF的表达。结果 BMSC组及HGF组的侧支血管计数与对照组间差异无统计学意义,而BMSC+HGF组侧支血管计数较其余各组明显增加(P<0.05或P<0.01);免疫组化显示,各处理组CD31表达量明显高于对照组(P<0.05或P<0.01),BMSC组和HGF组间CD31表达量无明显差异(P>0.05),但两者均明显低于BMSC+HGF组(均P<0.05);Western blot显示,各处理组HGF表达量均高于对照组(P<0.05或P<0.01),且HGF在BMSC组、HGF组和BMSC+HGF组中的表达量依次增高,差异均有统计学意义(均P<0.05)...     

骨髓间充质干细胞自体移植治疗心肌梗死的实验研究

目的探讨兔骨髓间充质干细胞(MSCs)移植至缺血心肌后的增殖分化情况,对缺血心肌细胞的修复重建能力及心功能改善情况。方法将20只新西兰白兔随机分为骨髓间充质干细胞移植组(MSCs组,n=10)和对照组(n=10),采用结扎冠状动脉左前降支(LAD)制备心肌梗死模型,2周后分别将Dil标记的1×106个细胞悬液400μl或等量L-DMEM培养基用微量注射器注入梗死灶边缘,于建模前、建模后2周、细胞移植后2、4周采用多普勒超声心动图检测左心室收缩期末内径(LVESD)、左心室舒张期末内径(LVEDD),计算左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)评价心脏收缩功能,同时进行心肌声学造影评价心肌组织的血流灌注情况。细胞移植后8周处死所有动物,病理学检查移植细胞在梗死区的生长状况。结果多普勒超声心动图检测结果显示:两组动物建模前、建模后2周LVEF、LVFS差异无统计学意义(0.72±0.08vs.0.71±0.04,0.56±0.11vs.0.55±0.09;0.35±0.06vs.0.35±0.04,0.24±0.08vs.0.23±0.03,P>0.05),细胞移植后2、4周MSCs组LVEF、LVFS值均明显高于对照组(0.71±0.05vs.0.60±0.05,0.72±0.07vs.0.62±0.08;0.34±0.03vs.0.29±0.01,0.35±0.06vs.0.27±0.05,P<0.05);病理学检查见自体MSCs移植8周后存活于梗死心肌中,表达肌细胞特异性标志,并且能显著增加瘢痕区毛细血管密度(38.6±7.6/mm2vs.21.4±3.9/mm2,P<0.05),心肌声学造影亦显示梗死局部血流灌注MSCs组较对照组明显改善。结论自体MSCs移植缺血心肌中可向心肌细胞分化,增加心肌血流灌注,改善心脏收缩功能。     

腺病毒介导的HGF转染骨髓间充质干细胞影响移植颗粒脂肪新生血管形成的初步研究

目的:研究腺病毒介导的肝细胞生长因子(HGF)转染骨髓间充质干细胞对移植颗粒脂肪新生血管形成的影响。方法:分离培养雄性大鼠MSCs,用Ad-GFP、Ad-HGF转染MSCs,流式细胞仪测定转染效率。Wistar雌性大鼠150只,随机分为5组:A组颗粒脂肪+0.4mlDMEM-LG;B组颗粒脂肪+0.4ml DMEM-LG+1×108pfuAd-HGF液;C组颗粒脂肪+0.4mlDMEM-LG+1×106MSC;D组颗粒脂肪+0.4ml DMEM-LG+1×106MSC(携带GFP基因);E组颗粒脂肪+0.4mlDME-LG+1×106MSC(携带HGF基因),均匀震动5min,移植于背部皮肤与肌肉之间。3、5、7、14、28、60天取移植的颗粒脂肪组织,HE染色观察病理变化,用免疫组化法检测移植体CD34表达,观察新生血管生成。结果:MOI=100时,Ad-GFP转染MSCs效率达86.4%,Ad-HGF转染MSCs,ELISA检测HGF分泌量在48h表达量最高。E组移植体新生血管7天左右达高峰,14天后趋于平稳,在5、7、14、28、60天血管数量多于其他组(P0.05)。结论:携带有HGF基因骨髓间充干细胞有效的能促进移植颗粒脂肪的血管形成。     

粒细胞集落刺激因子联合携带肝细胞生长因子基因的BMSCs移植对心肌梗死大鼠血管重建的影响

目的研究粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)联合携带肝细胞生长因子(hepatocyte growth factor,HGF)基因的BMSCs移植对心肌梗死大鼠血管重建的影响,初步探讨作用机制。方法取3周龄雄性SD大鼠骨髓分离培养BMSCs,取第3代BMSCs以携带HGF基因的5型复制缺陷型腺病毒(Ad-HGF)感染。成年雄性SD大鼠44只,体重200～250 g,结扎左冠状动脉建立心肌梗死模型。造模4周后心脏超声检查,以左室短轴缩短率(shorting fraction,FS)<30%作为造模成功标准。取其中12只大鼠,于梗死心肌边缘注射0.1 mL Ad-HGF感染的BMSCs(5×107个/mL),2、7、14 d后用Western blot方法检测大鼠体内HGF蛋白的表达。将其余32只大鼠随机分为4组,每组8只:对照组注射0.1 mL生理盐水;G-CSF组注射0.1 mL生理盐水并于腹腔注射G-CSF 100μg(/kg.d)共5 d;HGF组注射0.1 mL Ad-HGF感染的BMSCs(5×107个/mL);联合治疗组注射0.1 mL Ad-HGF感染的BMSCs(5×107个/mL)并于腹腔注射G-CSF 100μg/(kg.d)共5 d。细胞移植后2周,行心功能和血流动力学检测,然后处死大鼠取心肌组织行免疫荧光双染后激光共聚焦显微镜下评价血管生成情况,Western blot检测VEGF蛋白表达。结果感染Ad-HGF的BMSCs移植2、7 d时在大鼠体内表达HGF蛋白。心功能及血流动力学检测显示,G-CSF组左室收缩压(left ventricular systolic pressure,LVSP)、左室舒张末期压力(left ventricularend-diastolic pressure,LVEDP)、LVSP上升/降低时间(dP/dtmax)、FS与对照组相比差异均无统计学意义(P>0.05);HGF组和联合治疗组与对照组相比,LVEDP显著降低,LVSP、FS和dP/dtmax显著升高(P<0.05);与HGF组相比,联合治疗组的FS和dP/dtmax升高(P<0.05)。免疫荧光双染显示心肌梗死交界区增生细胞是血管内皮细胞。联合治疗组血管密度明显高于其他3组(P<0.05),VEGF蛋白表达较其他3组明显增加(P<0.05)。结论 在大鼠心肌梗死4周时给予G-CSF联合携带HGF基因的BMSCs移植治疗,可明显改善心功能,促进心肌梗死边缘缺血区域的血管生成,其作用机制之一是增加了缺血心肌VEGF蛋白的表达。     

腹腔镜结直肠癌根治对机体免疫状态的影响

目的探讨腹腔镜手术治疗结直肠癌对机体免疫状态的影响。方法2004年3月至2004年12月,同一手术组将可手术治疗的结直肠癌患者60例,随机分为腹腔镜手术组和开放手术组,每组各30例。分别在术前1d和术后3、7d取外周静脉血,测定C反应蛋白(CRP)、免疫球蛋白(Ig)A、IgM、IgG,CD3+、CD4+、CD8+、自然杀伤细胞(NK细胞)和CD4+CD45RA+、CD4+CD45RO+细胞及血淋巴细胞数并进行比较。结果开放手术组术后3d外周血淋巴细胞数(1.09±0.29)×109/L,CD4+细胞(0.54±0.14)×109/L,CD8+细胞(0.31±0.08)×109/L,CD4+CD45RO+细胞(61.1±8.9)%,IgM(136.9±52.8)IU/ml,IgG(115.2±45.7)IU/ml;术后7dCD8+细胞(0.32±0.09)×109/L,CD4+CD45RO+细胞(63.2±9.1)%,均明显低于术前(P<0.05,P<0.01)。而腹腔镜手术组术后3d除CD4+CD45RO+细胞犤(62.7±12.5)%犦较术前降低(P<0.05)外,其余免疫学指标无明显降低;而淋巴细胞数犤(1.29±0.37)×109/L犦、IgM犤(164.5±48.2)IU/ml犦和CD8+细胞数犤(0.38±0.09)×109/L犦与开放手术组比较,差异有统计学意义(P<0.05)。结论腹腔镜手术治疗结直肠癌比开放手术在免疫功能保护上更具有优势。     

重组人粒细胞集落刺激因子促进骨髓间充质干细胞增殖

目的探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠骨髓间充质干细胞(MSCs)增殖的影响。方法将昆明小鼠随机分为2组(n=15),G-CSF组采髓前皮下注射重组人粒细胞集落刺激因子(rhG-CSF)80μg·kg-1·d-1,连用5d,对照组皮下注射等量生理盐水。每组分别于最后一次注射后6h、12h及7d(168h)取小鼠骨髓,分离、培养MSCs,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪检测单个核细胞(MNCs)细胞周期及CFU-F细胞表面抗原特征。结果应用rhG-CSF后,培养MNCs所获得的CFU-F数目增加(P<0.01),CFU-F数与取材时间(6h、12h、7d)呈负相关(P<0.05),但12h取材者与7d取材者比较,CFU-F数目的差异无统计学意义(P>0.05)。MNCs培养形成的CFU-F,其细胞表面抗原呈CD34-、CD133-、CD90+、CD105+,分别占2.5%、3.1%、67.0%和78.0%。G-CSF组各时间点获得的MNCs,其G0/G1期细胞百分率较对照组低(P<0.05),而S+G2/M期增高(P<0.05),且6h取材者S+G2/M期细胞百分率明显高于12h和7d取材者(P<0.05)。结论rhG-CSF可促进骨髓MNCs进入细胞增殖周期,增殖的高峰在应用G-CSF后6h左右。     

3种不同途径移植自体骨髓间充质干细胞治疗急性心肌梗死效果比较

目的比较3种不同途径移植自体骨髓间充质干细胞(MSCs)治疗急性心肌梗死效果。方法小型猪12只,建立心肌梗死(AMI)模型,纯化扩增并将DAPI标记的MSCs经冠脉、心内膜及心外膜注射移植,移植前后记录左室血流动力学指标及LVEF变化,3个月后取心肌行免疫组织化学检测结蛋白desmin和心肌特异性肌钙蛋白I(cTnI)的表达。结果冠脉组梗死心肌周围未见明确的移植细胞,仅见非特异的荧光染色,与心梗后比较心功能改善,但与对照组比较无明显差异;心内膜及心外膜组移植的MSCs分布较广,胞核为蓝色椭圆形,免疫组织化学检测胞浆心肌特异蛋白染色阳性,与冠脉移植组比较LVEDP降低(P<0．05)、LV±dp／dtmax增快(P<0．05)、 LVEF增高(P<0．05)、新生血管数增加及瘢痕面积缩小等指标改善更明显,且有统计学意义。结论 3种途径移植MSCs均可改善AMI后心功能,经冠脉移植不是最佳途径,心肌内注射效果更好。     

CD8+T细胞在胃癌组织中的表达及其临床意义

目的 观察胃癌组织中浸润性淋巴细胞CD8+T的表达及临床作用.方法 应用免疫组织化学染色检测102例胃癌患者手术标本中浸润淋巴细胞CD8+T的表达.结果 在胃癌、胃炎及胃息肉组织中均可见浸润淋巴细胞CD8+T的表达,但胃癌组浸润淋巴细胞CD8+T的表达[(29.53±14.71)个/HPF]明显高于慢性胃炎组[(19.54±8.10)个/HPF]和胃息肉组[(14.26±6.83)个/HPF],差异有统计学意义(P＜0.05).CD8+T细胞的表达与胃癌患者的性别、年龄、肿瘤部位、肿瘤大小、淋巴结转移、复发、病理分级和TNM分期无明显相关(P＞0.05);但与组织学类型和浸润深度有关,其中分化型胃癌患者CD8+T细胞的表达[(33.9±18.4)个/HPF]高于低分化型患者的表达[(27.6±12.0)个/HPF],差异有统计学意义(t=2.04,P＜0.05);未侵入肌层组的CD8+T细胞表达[(38.8±13.8)个/HPF]明显高于侵入肌层组[(27.7±14.1)个/HPF],差异有统计学意义(t=2.45,P＜0.05).与CD8+T细胞低表达组比较,CD8+T细胞高表达组胃癌患者中位生存时间延长5个月,但差异无统计学意义(χ2=1.0274,P＞0.05).多因素COX模型分析显示,与浸润淋巴细胞CD8+T低表达组比较,高表达组有降低胃癌死亡风险的趋势(RR=0.82,95%CI=0.41～1.65).结论 浸润性淋巴细胞CD8+T的表达与胃癌的预后有关.

Abstract：

Objective To study the expression and clinical significance of infiltrating lymphocytes CD8 + T in gastric carcinoma tissues.Methods The expression of infiltrating lymphocytes CD8 + T in 102surgically resected specimens of gastric cancer tissues was detected by using immunohistochemistry.Results CD8+ T cells were expressed both in gastric carcinoma,gastritis and gastric polyp tissues,but the number of CD8 + T cells in gastric carcinoma tissues [(29.53 ± 14.71 )/high powered field (HPF)]wassignificantly greater than in gastritis tissues [( 19.54 ±8.10)/HPF]and polyp tissues [( 14.26 ±6.83)/HPF](P ＜0.05).No correlation was found between CD8 + T lymphocyte count and gender,age,tumor location,tumor size,lymph node metastasis,tumor relapse,pathological grade or TNM stage ( P ＞ 0.05 ).However,there was a statistically significant correlation between the number of CD8 + T cells and the histological type,invasive depth.The CD8 + T cell count was obviously higher in the differentiated group [(33.9 ± 18.4)/HPF]than in the undifferentiated group [(27.6 ± 12.0)/HPF]( t = 2.04,P＜0.05 ).And the number of CD8 + T cells was greater in the non-invasive group [(38.8 ± 13.8)/HPF]than in the invasive group [(27.7 ± 14.1 )/HPF]( t = 2.45,P＜0.05 ).The median survival time of gastric cancer patients with high expression of CD8 + T cells was 5 months longer than in those with low expression,but there was no significant difference.Multi-factor COX model analysis demonstrated that the risk of death in gastric cancer patients was lower in the CD8 + T cells high expression group than in the low expression group ( RR = 0.82,95%,CI = 0.41-1.65 ).Conclusion Detection of infiltrating lymphocytes CD8 + T expression in gastric carcinoma tissues will be beneficial to the judgment of the prognosis.     

梗阻性黄疸患者肠黏膜瘦素及其受体与肠黏膜免疫屏障损害的关系

目的研究梗阻性黄疸患者肠黏膜瘦素(Lp)、瘦素受体(Ob-R)表达水平与肠黏膜免疫屏障损害(CD4+、CD8+T细胞数量)的关系。方法收集2012年3月至2013年7月间因梗阻性黄疸住院手术治疗患者30例为试验组(OJ组),再根据血清总胆红素浓度分为轻度黄疸组(≤171μmol/L,OJ 1组,17例),中重度黄疸组(>171μmol/L,OJ 2组,13例);同期非黄疸患者21例为对照组。采用免疫组化法检测梗阻性黄疸患者小肠黏膜及正常小肠黏膜中Lp和Ob-R表达水平以及CD4+、CD8+T细胞数量,分析其相关性。结果梗阻性黄疸患者肠黏膜Lp和Ob-R表达水平、CD4+、CD8+T细胞的数量均低于对照组(Lp:OJ组1.63±1.25,对照组2.48±1.25,P<0.05;Ob-R:OJ组2.63±1.27,对照组3.90±1.00,P<0.05;CD4+:OJ组17.74±4.33,对照组28.33±4.53,P<0.05;CD8+:OJ组11.61±3.36;对照组15.95±3.69,P<0.05);肠黏膜Lp和Ob-R表达水平与肠黏膜CD4+、CD8+T细胞的数量呈正相关(P<0.05)。结论梗阻性黄疸患者肠黏膜Lp和Ob-R表达减少与肠黏膜CD4+、CD8+T细胞的数量减少相关,Lp可能对肠黏膜免疫屏障的稳定起一定作用。     

Gene transfer of hepatocyte growth factor improves angiogenesis and function of chronic ischemic myocardium in canine heart

BACKGROUND: Hepatocyte growth factor (HGF) induces angiogenesis in myocardium. In the present study, its effects in chronic ischemic myocardium were tested. METHODS: Four weeks after left anterior descending coronary artery ligation in canine hearts, HVJ-liposome containing either human HGF gene (160 microg; HGF group, n = 7) or nothing (control group, n = 6) was directly injected into ischemic myocardium. Four weeks after gene transfection, the thickness fraction (TF), an index of regional myocardial contractility (assessed by epicardial pulse-Doppler crystals), the myocardial perfusion flow (assessed by color microspheres), and the capillary density (assessed by immunostaining of vessels) were evaluated in ischemic myocardium. RESULTS: Thickness fraction (percent of nonischemic myocardium) was significantly improved in the HGF group (80 +/- 15 from 52 +/- 16 of pregene; p < 0.05) whereas it was not changed in the control group (52 +/- 10 from 50 +/- 8 of pregene). The perfusion flow (% of nonischemic myocardium) was significantly improved in the HGF group (98 +/- 17 from 51 +/- 14 of pregene; p < 0.05) while it was not changed in the Control group (58 +/- 13 from 62 +/- 18 of pregene). The capillary density was significantly higher in the HGF group (894 +/- 211/mm2; p < 0.05) than that in the control group (511 +/- 127/mm2). CONCLUSIONS: Gene transfection of HGF improved angiogenesis, thereby improved regional myocardial function and perfusion in chronic ischemic myocardium. It indicates a potent therapeutic value of HGF gene transfection for chronic ischemic heart diseases such as myocardial infarction.     

Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion.

OBJECTIVE: The effect of transplanted mesenchymal stem cells (MSCs) on the left ventricular (LV) function and morphology in a rat myocardial infarct heart with reperfusion model were analyzed. METHODS: One week after 60 min of myocardial ischemia and reperfusion by left anterior descending artery (LAD) occlusion, 1.0x10(7) 6-diamidino-2-phenylindole (DAPI)-labeled MSCs were injected into the infarcted myocardium and compared with controls, and sham-operated rats, in which a cell-free serum medium was injected into the infarcted region or the myocardial wall, respectively. Measurement of vascular endothelial growth factor (VEGF) expression 1 week after MSC injection using Western blot analysis (n=5), and immunohistochemical staining using HE staining and fluorescent microscopy of the DAPI-positive regions from MSC implantation, cTnT immunostaining of potential myocardial-like cells, and SM-actin and CD31 immunostaining demonstrating neovascular transformation of implanted MSCs 1 week, 2 weeks and 4 weeks after transplantation (n=5). Hemodynamic measurements were performed after 4 weeks in vivo. Subsequently, hearts were quickly removed and cut for histological analysis using HE staining with measurement of the infarcted LV-area, the LV-wall thickness within the scar segment compared to non-infarcted scar segments, and the capillary density counting capillary vessels with 400x light microscopy (n=10). RESULTS: Measurement of hemodynamics 4 weeks after transplantation in vivo showed LV function to be significantly greater in MSCs than in the control group. Semi-quantitative histomorphometric examinations showed a significantly lower infract size, a greater LV-wall thickness, and a lower Hochman-Choo expansion index in the MSC-treated group compared to the control group. Immunofluorescence demonstrated that transplanted MSCs were positive for cTnT, suggesting that a small number of transplanted MSCs can differentiate into cardiomyocytes. Other MSCs were positive for CD31 and SM-actin. The transplanted MSCs in MI area had significantly higher expression rates of cTnT, CD31 and SM-actin 2 weeks after transplantation. HE staining showed marked augmentation of neovascularization in the MSC group. Semi-quantitative analysis demonstrated that capillary density was significantly higher in the MSC group than in the control group. CONCLUSION: Implanted MSCs could improve cardiac structure and function through the combined effect of myogenesis and angiogenesis.     

胶原凝胶复合骨髓间充质干细胞对小鼠 急性肝衰竭的疗效与机制研究

目的 探讨移植胶原凝胶复合骨髓间充质干细胞（bone marrow mesenchymal stem cells,MSCs） 对小鼠急性肝功能衰竭（acute liver failure,ALF）的治疗效果与机制。方法 105只雄性C57BL/6 小鼠随机 分为5组：对照组、ALF组、ALF+胶原凝胶组、MSCs组和胶原凝胶-MSCs组,每组21只。ALF组、ALF+ 胶原凝胶组、MSCs组和胶原凝胶-MSCs组采用腹腔注射D-氨基半乳糖苷（D-Gal）12 h后,分别于肝脏多 点注射200 μL PBS、200 μL胶原凝胶、1×106 MSCs和1×106胶原凝胶-MSCs。记录各组小鼠7 d存活率, 全自动生化仪检测肝功能水平,HE染色检测肝脏组织病理学改变,免疫组化检测肝细胞凋亡和增殖情况, 利用实时荧光定量PCR（RT-qPCR）检测肝脏炎症因子水平,Western blotting检测PI3K-AKT信号通路表达。 结果 扫描电镜示胶原凝胶与MSCs具有较好的相容性,动物活体成像检测显示胶原凝胶-MSCs移植的 信号强于单独MSCs移植的ALF小鼠。D-Gal处理后,胶原凝胶-MSCs组和MSCs组的小鼠生存率明显提高 （P＜0.05）,小鼠血清AST、ALT在胶原凝胶-MSCs组和MSCs组的小鼠明显优于ALF组和ALF+胶原凝胶 组（P＜0.05）,且肝功能指标明显改善,胶原凝胶-MSCs组要更优于MSCs组（P＜0.05）。 HE染色提示胶原 凝胶-MSCs组肝脏的病理改变（肝细胞坏死部位和范围）轻于MSCs组,Western blotting、Ki-67和cleavedCaspase3免疫组化结果显示,胶原凝胶-MSCs组肝细胞增殖能力强于MSCs组,同时肝细胞凋亡程度要低 于MSCs组,相应的胶原凝胶-MSCs组肝脏内IL-1β、IL-6、TNF-α、iNOS和CCL2等炎性因子水平也低于 MSCs组。且胶原凝胶-MSCs移植能明显激活肝脏内的PI3K-AKT通路。结论 胶原凝胶复合MSCs移植能 增强MSCs的肝脏定植能力,通过抑制细胞凋亡和促进细胞增殖减轻肝损伤,并调控AKT信号通路改善肝 细胞功能,因此胶原凝胶复合MSCs移植优于单纯MSCs移植治疗ALF。     

Myocardial protective effect of human recombinant hepatocyte growth factor for prolonged heart graft preservation in rats

BACKGROUND: In heart transplantation, myocardial apoptosis during hypothermic storage contributes to graft dysfunction. On the other hand, hepatocyte growth factor (HGF) has been reported to be an antiapoptotic factor in the heart. Therefore, we assessed whether the administration of recombinant human HGF (rh-HGF) prevents apoptosis in the prolonged preserved myocardium, resulting in an improvement in the cardiac function of the graft. METHODS: Isolated rat hearts were subjected to 4 hr (group A), 6 hr (group B), and 8 hr (group C: without rh-HGF vs. group D: with 100 microg of rh-HGF) of hypothermic storage followed by 60 min of normothermic reperfusion (n=5 in each group). RESULTS: Compared with non-HGF-treated hearts (group C), HGF-treated hearts (group D) showed a significantly higher recovery rate of left ventricular developed pressure (38+/-5% vs. 58+/-6%, P<0.01) and maximum dp/dt (53+/-7% vs. 74+/-4%, P<0.01) and a lower rate of TUNEL-positive cardiomyocytes (7.8+/-6.0% vs. 25.3+/-8.9%, P<0.05) after 60 min of reperfusion. Western blot analysis revealed that c-Met/HGF receptor expression was stronger in the HGF-treated myocardium than in the non-HGF-treated myocardium after 8 hr of storage and was associated with a weaker expression of caspase-3 and a stronger expression of Bcl-xL after 60 min of reperfusion. CONCLUSION: The administration of rh-HGF before storage improved cardiac function after prolonged myocardial preservation by preventing apoptosis through the c-Met/HGF receptor. Thus, the addition of rh-HGF in the storage solution may be a promising strategy for prolonged heart graft preservation.     

小鼠骨髓间充质干细胞减轻胰岛移植物排斥反应的作用

目的 探讨同种异基因小鼠骨髓间充质干细胞(MSC)与胰岛联合移植对胰岛移植物的免疫调节作用.方法 将18只糖尿病模型小鼠随机分成3组:(1)糖尿病组,不进行任何移植;(2)胰岛移植组,在无菌操作下将10μl纯化后的200个胰岛移植于受者的左肾包膜下;(3)胰岛+MSC移植组,除与胰岛移植组进行相同的移植外,还在胰岛移植前3、2、0d经受者尾静脉分别注射1×106个MSC.移植后,连续监测非空腹血糖至第30 d;第14和28 d对移植部位的左肾进行组织病理学观察;采集外周血进行免疫荧光染色,流式细胞术分析TH1/TH2、Tc1/Tc2细胞的比值、初始和记忆T淋巴细胞的变化、以及骨髓来源的树突状细胞(DC)成熟度和功能的变化.结果 与胰岛移植组比较,胰岛+MSC移植组血糖明显降低,移植部位炎症细胞浸润明显减轻,移植物的存活时间延长;TH1和Tc1细胞明显下降,TH2和Tc2细胞升高,TH1/TH2和Tc1/Tc2细胞比值显著下降;初始T淋巴细胞和记忆T淋巴细胞下调;DC成熟度降低,分泌白细胞介素12(IL-12)的能力下降.结论 MSC与胰岛联合移植可通过对T淋巴细胞和树突状细胞的免疫调节作用,减轻胰岛移植物的排斥反应,从而延长移植胰岛的存活时间.     

肝细胞生长因子基因转染骨髓间充质干细胞对脂肪颗粒移植存活率的影响

目的研究携带人肝细胞生长因子基因的重组腺病毒（Ad—HGF）修饰骨髓间充质干细胞（MSCs）对大鼠脂肪颗粒移植存活率的影响。方法用携带绿色荧光蛋白的重组腺病毒（Ad-GFP）、Ad—HGF转染雄性Wistar大鼠MSCs,测定转染效率与感染上清中肝细胞生长因子（HGF）的表达。用雌性Wistar大鼠150只,随机分为5组：空白对照组（A组）、Ad—HGF组（B组）、MSCs组（C组）、Ad—GFP感染MSCs组（D组）和Ad—HGF感染MSCs组（E组）,每组30只。各组均匀振动5分钟,将颗粒脂肪移植于自体背部皮下;移植后3、5、7、14、28、60d,每组各取5只大鼠脱颈处死,取出移植物;测体积、HE、Masson染色观察病理改变,免疫组织化学法测定组织中HGF表达和血管生成。结果积分吸光度＝100时,Ad—GFP转染MSCs效率可达89．6％;移植后3、5、7、14d,E组移植物中HGF的表达高于其他各组（P〈0．05）;移植后5、7、14、28、60d,E组血管数量也多于其他各组（P〈0．05）;28d和60d时,E组脂肪颗粒体积保持率明显好于其他4组（P〈0．05）。结论携带HGF基因的MSCs在移植物中能够持续表达HGF,有效地减少脂肪颗粒移植后的吸收,从而提高移植颗粒脂肪的存活率。     

基因修饰的骨髓间充质干细胞移植于慢性心肌梗死模型的实验研究

目的 研究血管内皮生长因子（VEGF）165基因修饰的骨髓间充质干细胞（MSCs）移植于慢性心肌梗死模型后的血管新生及对心功能的作用。方法 同源重组法构建含有VEGF。基因的重组腺病毒载体（rAd—VEGF165）;密度梯度离心法分离骨髓单个核细胞,贴壁法培养兔MSCs;rAd—VFGF165转染MSCs,并用4,6-联脒-2-苯基吲哚（DAPI）标记MSCs;结扎前降支法建立兔心肌梗死模型,存活6周的实验动物36只随机分为3组：移植rAd—VFGF165转染的MSCs组（Ⅰ组）12只、单纯移植MSCs组（Ⅱ组）12只和只注射无血清培养基的对照组（Ⅲ组）12只。移植术后4周,超声法检测心脏功能,并同术前比较;荧光显微镜下观察MSCs分布情况;免疫组化法检测梗死区微血管密度。结果 移植4周后Ⅰ 组的MSCs存活率大于其他2组;Ⅰ组的左室射血分数、E／A比值和梗死区微血管密度与其他2组比较差异有统计学意义（P〈0．05）。结论 VEGF165基因和MSCs联合策略是治疗心肌梗死的有效方法,所产生的协同治疗效果大于单纯的MSCs移植。     

蛛网膜下腔移植骨髓间充质干细胞对大鼠慢性神经病理性痛的影响

目的 探讨蛛网膜下腔移植骨髓间充质干细胞(MSCs)对大鼠慢性神经病理性痛的影响.方法 雌性SD大鼠140只,体重180～200 g,采用坐骨神经分支选择损伤(SNI)法制备慢性神-经病理性痛模型,术后2周,随机分为4组(n=35):模型组(NP组)、MSCs组、PBS组和骨髓未贴壁单核细胞组(BNMCs组).MSCs组、PBS组、BNMCs组蛛网膜下腔分别注射1×105/μl的第3代MSCs悬液10μl、PBS 10μl、1×105/μl的BNMCs悬液10μl.于术前、术后2周、移植后1、2、3、4、5周(T0-6)时测定机械痛阈.同时于T1～6时测定痛阈后随机选择5只大鼠取脊髓腰膨大组织,使用RT-PCR法测定脑源性神经营养因子(BDNF)mRNA表达.结果 与T0时比较,T1时各组机械痛阈降低,T2～6时NP组、PBS组、BNMCs组机械痛阈降低(P＜0.05).与T1时比较,MSCs组T2～6时机械痛阈升高,T2-4时BDNF mRNA表达上调(P＜0.05),PBS组和BNMCs组其余时点各指标差异无统计学意义(P＞0.05).与NP组比较,MSCs组机械痛阈升高,BDNFmRNA表达上调(P＜0.05),PBS组和BNMCs组各指标差异无统计学意义(P＞0.05).结论 蛛网膜下腔移植MSCs可减轻大鼠慢性神经病理性痛,其机制可能与上调脊髓BDNF mRNA表达有关.     

Therapeutic implications of mesenchymal stem cells transfected with hepatocyte growth factor transplanted in rat kidney with unilateral ureteral obstruction

Purpose

The purpose of the study was to establish whether bone marrow mesenchymal stem cells (MSCs) transfected with hepatocyte growth factor (HGF) can migrate and localize in the rat's kidney with unilateral ureteral obstruction (UUO) and contribute to repair of renal fibrosis.

Methods

We separated and cultured bone marrow-derived MSCs of male rats in vitro and transfected them with adenovirus-mediated HGF (Ad-HGF). The expression of HGF was measured with enzyme-linked immunosorbent assay. Sixty female rats were sham operated (n = 24) or subjected to left UUO: Ad-HGF-transfected MSCs, uninfected MSCs, or saline was injected into the rat's tail vein. Kidney tissue was collected at the end of the seventh or 14th day after operation. The distribution of Y chromosome in the kidney after Ad-HGF-transfected MSCs transplantation was determined by an in situ hybridization method. As the hallmark of myofibroblasts, α-smooth muscle actin (expression of which significantly increases in the presence of renal fibrosis) was detected by immunohistochemistry in all UUO rats' left kidney tissue.

Results

Y chromosome-positive cells were found only in the obstructed kidney of the transplantation group. The positive cells were mainly distributed in the tubular cells. The average intensity of immunolabeling for α-smooth muscle actin in the transplanted group significantly decreased compared with sham-transplanted group (P < .05), and the expression in the rats injected with uninfected MSCs was higher than that in the rats with MSCs transfected with HGF (P < .05).

Conclusions

Mesenchymal stem cells transfected with HGF can migrate to the rat kidney with UUO and are mainly distributed in the region of renal tubular epithelial cells. The data indicate that MSCs transfected with HGF contribute to a reduction of renal fibrosis after ureteral obstruction and suggest that this may be exploited therapeutically.     

不同数目骨髓间充质干细胞移植对大鼠肺动脉高压的作用及内皮素-1表达的影响

目的探讨不同数目骨髓间充质干细胞(mesenchymal stem cells,MSCs)移植对野百合碱(MCT)诱导大鼠肺动脉高压的治疗作用,以及对内皮素-1(endothelin-1,ET-1)表达的影响。方法成年雄性Wistar大鼠40只(体重180～250 g),按随机数字表法分为4组,每组10只。A组:大鼠腹腔注射MCT 60 mg/kg,经颈外静脉注入1×106MSCs;B组:大鼠腹腔注射MCT 60 mg/kg,经颈外静脉注入5×105MSCs;MCT组:大鼠腹腔注射MCT60 mg/kg和等量磷酸盐缓冲液(PBS);对照组:大鼠腹腔注入等量生理盐水和等量PBS。MSCs移植4周后测定右心室收缩压(RVSP),计算心室比,即右心室/(左心室+室间隔)[RV/(LV+VS)];观察肺组织形态学改变;检测肺组织ET-1基因表达和血清ET-1的含量。结果 MSCs移植4周后,A组RVSP和RV/(LV+VS)与MCT组比较明显降低[(35.8±4.2)mm Hg vs.(47.2±10.1)mm Hg,P<0.01;(0.357±0.032)vs.(0.452±0.056),P<0.01];而B组与MCT组比较差异无统计学意义(P>0.05)。A组肺小动脉中膜厚度较MCT组明显变薄[(19.7%±3.0%)vs.(26.8%±3.6%),P<0.01];而B组则差异无统计学意义。逆转录酶-聚合酶链反应(RTase-PCR)检测结果显示,MCT组肺组织ET-1 mRNA表达最强,A组肺组织ET-1 mRNA与MCT组比较明显减弱,B组表达与MCT组接近。A组血清ET-1含量与MCT组比较明显减少。结论 MSCs静脉移植对MCT诱导的肺动脉高压具有抑制作用,并能减少肺组织ET-1的mRNA表达及血清ET-1浓度。采用1×106MSCs移植具有较好的治疗作用。