血管内皮生长因子基因转染骨髓间充质干细胞移植于梗塞心肌的实验研究

目的 探讨骨髓间充质干细胞（MSCs）移植联合血管内皮生长因子（VEGF）基因转染对大鼠梗塞心肌组织的修复重建、血管再生及梗塞后心功能的影响。方法 体外分离、培养、纯化SD大鼠的MSCs，以BrdU标记MSCs，腺病毒介导VEGF基因转染MSCs。建立大鼠急性心肌梗死模型4周后，随机分为4组（每组10只），分别行梗塞心肌内注射：转染VEGF基因的MSCs移植组（组Ⅰ）、单纯MSCs移植组（组Ⅱ）、单纯VEGF基因治疗组（组Ⅲ）和以注射无血清IMDM培养液为对照组（组Ⅳ）。移植4周后观察移植细胞的分化和新生血管的形成，并通过超声多普勒检测心功能变化。结果 组Ⅰ和组Ⅱ中，梗塞心肌处可见BrdU标记的移植细胞，cTnT染色阳性。超声心动图检查发现，组Ⅰ和组Ⅱ的左室射血分数（LVEF）的改善显著高于对照组（P均〈0．01），而组Ⅰ的LVEF改善程度要明显高于组Ⅱ；部分BrdU染色阳性的细胞可以分化成为内皮细胞，参与构成了梗塞区域的新生毛细血管。相对于对照组，组Ⅰ和组Ⅲ都有明显的血管新生（P均〈0．01）。结论 MSCs移植联合VEGF基因转染可以通过促进心肌再生和新生血管的形成来重建缺血心肌，显著改善心功能。     

pEGFP-C1/Akt体外转染骨髓间充质干细胞后对血管内皮生长因子表达的影响

目的 克隆构建绿色荧光蛋白(GFP)-Akt表达载体,观察其在鼠骨髓间充质干细胞(MSCs)中的表达和定位及其对(VEGF)mRNA和蛋白表达的影响.方法 采用酶切法将Akt重组于GFP表达载体pEGFP-C1中,经酶切序列鉴定后,将该重组质粒GFP-Akt及GFP空质粒通过脂质体法转染骨髓MSCs.荧光显微镜观察其转染率及在细胞中分布.分别采用用Western blot和逆转录-聚合酶链反应(RT-PCR)方法检测pEGFP-C1和pEGFP-C1/Akt转染MSCs后蛋白和VEGF mRNA的表达.结果 GFP-Akt基因表达载体经酶切鉴定和测序分析确认构建成功,并在鼠MSCs中表达.荧光显微镜下pEGFP-C1转染后,绿色荧光均匀分布于细胞中,而pEGFP-C1/Akt转染后,绿色荧光分布于胞质中;与未转染组比较,pEGFP-C1转染组Akt mRNA和蛋白及VEGF mRNA和蛋白表达差异无统计学意义(P＞0.05);而pEGFP-C1/Akt转染组Akt mRNA和蛋白及VEGF mRNA和蛋白表达显著增加(P＜0.05).结论 成功构建GFP-Akt融合基因表达载体在鼠MSCs中表达;并可诱导AktmRNA及蛋白和VEGF mRNA和蛋白的表达.     

血管内皮生长因子对骨髓间充质干细胞向软骨细胞分化的影响

[目的]探讨外源性血管内皮生长因子（VEGF）对人骨髓来源间充质干细胞（MSCs）增殖、向成软骨方向定向分化等生物学行为的影响及其机理，为MSCs构建组织工程化软骨奠定基础．[方法]取人骨髓来源间充质干细胞体外培养，将传代后的细胞分别置于含有800ng／ml和1600ng／ml VEGF的诱导培养基中进行培养，将具有促进细胞增殖分化及抑制多种炎性介质活性等多重生物学效应的TGF-β1以10ng／ml浓度配置无血清培养基诱导同组MSCs作阳性对照，观察细胞的增殖，分化。用Ⅱ型胶原免疫组化染色评价不同诱导因素下的软骨基质合成情况。[结果]所有细胞显示了良好的增殖能力，暴露于外源性血管内皮生长因子下的MSCs保持了良好的生长活性，并开始向软骨表型分化，与未添加诱导因子组细胞相比差异有显著性意义，不同浓度VEGF组的细胞合成软骨基质的能力无差异，但均不如阳性对照组。[结论]外源性VEGF具有诱导体外培养的人MSCs向软骨表型分化的能力，但与TGF-β1的诱导能力相比有差距。提示VEGF在MSCs来源的软骨修复过程中充当诱导信息提供者的角色。     

转染血管内皮生长因子基因神经干细胞移植对大鼠脑缺血损伤的修复作用

目的 探讨转染血管内皮生长因子（VEGF）基因神经干细胞移植对大鼠脑缺血损伤的修复作用。方法 建立大鼠大脑中动脉梗塞（tMCAO）模型，将tMCAO模型随机分为对照组、注射磷酸盐缓冲液（PBS液）的PBS液对照组、接受神经干细胞（NSCs）移植的NSCs组、接受转染VEGF基因的NSCs移植的NSCs-VEGF组，采用立体定向法将相应移植物注射到tMCAO模型的右侧纹状体缺血半暗区。各组移植后2、4、6、8、10、12周进行神经功能损害程度（NSS）评分；移植后7d，采用免疫荧光染色法观察NSCs-VEGF组移植细胞的VEGF基因表达情况；12周时，采用免疫荧光染色法追踪NSCs-VEGF组移植细胞向神经元方向分化情况，并同时行各组移植区周围血管内皮细胞半定量计数。结果 移植后2～12周，NSCs-VEG组的NSS评分均低于其它3组，第12周时的NSS评分与其它3组比较，差异有统计学意义（P〈0．01）；移植后7d，NSCs-VEGF组的移植细胞从移植点向周围迁徙，部分表达VEGF基因；移植后12周，NSCs-VEGF组部分移植细胞分化成神经元，其移植区血管内皮细胞数显著高于其它3组（P〈0．05）。结论 转染VEGF基因的神经干细胞移植对脑缺血所致的损伤具有一定的修复作用。     

VEGF基因体外转染大鼠骨髓间充质干细胞的实验研究

目的:探讨脂质体介导血管内皮细胞生长因子(VEGF)基因转染大鼠骨髓间充质干细胞(MSCS)应用于基因治疗的可行性、安全性。方法:体外分离、培养、鉴定MSCs,PcDNA3.1(-)/VEGF165质粒转染MSCs,转染后用免疫荧光和ELISA检测MSCs表达VEGF蛋白的情况,MTT检测MSCs对VEGF质粒转染的敏感性。结果:骨髓中分离得到MSCs,流式细胞检测显示MSCs不表达CD34和CD45,但表达CD90。透射电镜观察可见细胞浆中含大量粗面内质网和分泌颗粒。VEGF基因转染MSCs后第5天抗VEGF免疫荧光染色约90%的MSCs呈阳性,ELISA检测结果显示PcDNA3.1(-)/VEGF165质粒转染组细胞培养上清液中VEGF含量明显高于对照组,并于转染后第5天达到峰值。MTT检测结果显示VEGF质粒转染对MSCs增殖无影响。结论:MSC可作为VEGF基因转染的靶细胞用于基因治疗。     

血管内皮生长因子基因体外转染大鼠神经干细胞的研究

目的 探讨经脂质体介导转染血管内皮生长因子(VEGF)121基因的大鼠胚胎神经干细胞体内外基因表达的特点。方法脂质体介导法将VEGF121基因转染到大鼠神经干细胞中。经逆转录一聚合酶链反应(RT—PCR)及免疫荧光染色检测转基因神经干细胞及其分化细胞的基因表达情况。用立体定向法将BrdU标记的转基因神经移植到大鼠大脑中动脉梗死模型的纹状体缺血半暗区。免疫荧光染色观察移植后1周转基因神经干细胞在脑内的基因表达情况。结果转基因神经干细胞及其分化的子代细胞均有VEGF121的表达并持续2周左右。转基因神经干细胞移植后1周在宿主脑内迁徙并表达VEGF基因产物。结论经脂质体介导转染VEGF121基因的大鼠胚胎神经干细胞在体内外均有良好的基因表达。     

人血管内皮生长因子165基因转染人骨髓间充质干细胞的实验研究

Objective To establish an effective method of transfecting human marrow mesenchymal stem cells (MSC) with human vascular endothelial growth factor 165 ( VEGF 165) gene. Methods MSCs isolated and cultured in vitro were divided into transfection group (pShuttle-CMV/VEGF 165 plasmid was transfected into MSCs through liposome-mediating method), empty plasmid group (pShuttle-CMV vehi-cle was transfected into MSCs as control), liposome group (liposome was transfected into MSCs as control) and control group(normal culture). Expressions of Mrna and protein of MSCs were determined by RT-PCR, enzyme-linked immunosorbent assay and Western Blot. Sensitivity to MSCs on VEGF plasmid transfec-tion was detected by MTT test. Results Expression level of VEGF 165 gene Mrna in transfection group, empty plasmid group, liposome group, and control group was respectively 0.89 ± 0.03, 0. 34 ± 0.04, 0.40 ± 0.03, and 0.30 ± 0.03, and the difference between transfection group and the other three groups was statistically significant ( P <0. 01 ). Content of VEGF protein in transfection group, empty plasmid group, liposome group, and control group was respectively (778 ± 35 ), (543 ± 24), (561 ± 28), (571 ± 23) pg/Ml, and the difference between transfection group and the other three groups was statistically significant ( P <0.01 ). In the transfection group, expression level of VEGF protein peaked on 7th day after transfec-tion, which was decreased gradually later. In transfection group, expression level of V EGF 165 protein was obviously higher than that of the other three groups ( P <0. 01 ), and no inhibitory effect of VEGF plasmid transfection on MSCs proliferation was found. Conclusions The method for transfecting human VEGF 165 gene into MSCs is established in this research, through which target gene and protein can express effectively.     

人血管内皮细胞生长因子基因转染大鼠骨髓间充质干细胞对脂肪颗粒移植存活率的影响

目的 检测大鼠骨髓间充质干细胞(BMSCs)经人血管内皮细胞生长因子(VEGF165)基因转染后,对脂肪颗粒移植存活率的影响.方法　利用FuGENE HD介导VEGF165基因体外转染SD大鼠BMSCs,与取自SD大鼠的脂肪组织混合后移植于大鼠背部,类似方法设立未转染组及DMEM液空白对照组.检测脂肪组织存活率及再生血管密度.结果　转染组存在外源性基因和蛋白的表达,脂肪组织存活率及再生血管密度高于未转染组,且两者均高于对照组.结论　转染VEGF165基因的BMSCs具有更强的促血管再生作用,可提高游离移植脂肪的存活率.     

人碱性成纤维细胞生长因子基因转染大鼠骨髓间充质干细胞的研究

目的 观察人碱性成纤维细胞生长因子(bFGF)基因体外转染对大鼠骨髓间充质干细胞(MSCs)bFGF表达的影响.方法 密度梯度离心、贴壁法培养分离SD雄性大鼠MSCs,体外扩增,流式细胞仪检测MSCs表面抗原表达.利用慢病毒载体系统介导将具有人源性bFGF基因转染至第2代MSCs,在倒置荧光显微镜下观察转染后细胞形态和生长的变化,应用逆转录-聚合酶链反应(RT-PCR)、Western blot法鉴定bFGF在MSCs中的表达.结果 密度梯度离心、贴壁法培养分离可获得MSCs,P3代大鼠细胞利用流式细胞仪检测CD11b/c阳性细胞表达率为(13.2±0.6)%,CD34阳性细胞表达率为(1.2±0.5)%,CD44阳性细胞表达率(97.8±0.9)%,CD90阳性细胞表达率(96.8±1.4)%.MSCs转染48 h后,绿色荧光蛋白的表达明显增强.RT-PCR证实转基因MSCs表达bFGF mRNA明显增强,Western blot检测证实转基因MSCs在49 KDr出现特异性条带,而空白和空载组的MSCs则未见阳性条带.结论 采用慢病毒介导的基因转染技术可以将bFGF基因转染至MSCs中,并有外源性bFGFmRNA和蛋白的有效表达,MSCs可作为bFGF基因治疗的载体.     

血管内皮生长因子基因稳定转染兔骨髓基质干细胞的研究

目的 检测人血管内皮生长因子(VEGF)基因稳定转染对兔骨髓基质干细胞(BM-SC)VEGF表达的影响.方法 分离并培养兔骨髓基质干细胞,利用脂质体介导pcDNA3.1-VEGF165转染兔BMSC,G418筛选稳定表达pcDNA3.1-VEGFl65的细胞克隆,RT-PCR、Western blot法检测转染前后细胞中VEGF165 mRNA和蛋白的表达.结果 通过筛选获得了稳定表达pcDNA3.1-VEGF165的细胞克隆.逆转录-聚合酶链反应(RT-PCR)、Western blot结果显示:稳定转染组BMSC中可检测到VEGF165 mRNA和蛋白的表达,空白和空载体组未见VEGF表达.结论 pcDNA3.1-VEGF165载体转染的兔BMSC可稳定表达外源性VEGF165 mRNA和蛋白,可作为治疗骨缺损和骨缺血性坏死研究的种子细胞.     

Effect of bone morphogenetic protein-7 transfected bone marrow mesenchymal stem cells on renal injury in rats with chronic renal failure

Objective To observe the bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein-7 (BMP-7) gene on the expression of renal BMP-7, transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), further to explore its protective mechanism on renal injury in rats with chronic renal failure (CRF). Methods BMSCs with high expression of BMP-7 gene (BMSCs-BMP-7) and empty vector-BMSCs (BMSCs-EV) were obtained by lentiviral-mediated gene transfection. Thirty male Sprague-Dawley (SD) rats were randomly divided into 5 groups, 6 in each group: normal control (CON) group; PBS intervention (CRF with PBS infusion, CRF+PBS) group; BMSCs intervention (CRF with BMSCs infusion, CRF+BMSCs) group; BMSCs-EV intervention (CRF with BMSCs-empty vector infusion, CRF+BMSCs-EV) group and BMSCs-BMP-7 intervention (CRF with BMSCs-BMP-7 infusion, CRF+BMSCs-BMP-7) group. The CRF model was established by 5/6 nephrectomy. The CON group was a sham operation group. The corresponding 12-weeks interventions of each experimental group were performed after 2 weeks of modeling, the rats in the CON group and the CRF+PBS group were injected with 1 ml of PBS through the tail vein, and the other three groups were injected with 1 ml of the corresponding cell suspension once a week. At the time of sacrifice, blood and renal tissue samples were reserved. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by routine biochemical methods, and the expression of BMP-7, VEGF, TGF-β1 in kidney was assayed by Western blotting. Results At the time of sacrifice, the levels of Scr and BUN in the CRF+PBS group were significantly higher than those in the CON group (all P＜0.01); Compared with the CRF+PBS group, the Scr and BUN of the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were decreased to different extents, the differences were statistically significant (all P＜0.01); the Scr and BUN of the CRF+BMSCs-BMP-7 group were significantly lower than CRF+BMSCs group and CRF+BMSCs-EV group (all P＜0.05). The expression of BMP-7 and VEGF were the lowest in the CRF+PBS group. Compared with the CRF+PBS group, the expression of BMP-7 and VEGF in the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were significantly increased respectively (all P＜0.05). The expression of the BMP-7 and VEGF in the CRF+BMSCs-BMP-7 group were higher than those in the CRF+BMSCs group and CRF+BMSCs-EV group (P＜0.01). Compared with the CON group, the expression of TGF-β1 in the CRF+PBS group was significantly increased (P＜0.01); compared with the CRF+PBS group, the expression of TGF-β1 in the CRF+BMSCs group, CRF+BMSCs-EV and CRF+BMSCs-BMP-7 group was significantly decreased (all P＜0.01); the expression of TGF-β1 in the CRF+BMSCs-BMP-7 group was lower than the CRF+BMSCs and CRF+BMSCs-EV group (both P＜0.01). Conclusions BMSCs modified by BMP-7 has a protective effect on CRF rats; its protective mechanism may be related to antagonizing TGF-β1 and up-regulation of renal VEGF expression.     

骨髓间充质干细胞联合血管内皮生长因子基因治疗肢体缺血的实验研究

目的 观察骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)联合血管内皮生长因子(vascular endothelial growth factor,VEGF)基因治疗对家兔肢体缺血模型的疗效.方法 切除新西兰兔右后肢全长股浅动脉并结扎股深动脉以建立兔后肢缺血模型,随机分为空质粒对照组(EP组)、骨髓间充质干细胞组(BMSC组)、VEGF基因治疗组(VEGF组)及联合治疗组(BV组),每组各8只.分别于治疗后28 d及30 d进行动脉造影及VEGF免疫组化染色.结果 EP组、BMSC组及VEGF组的新生血管计数组间比较差异无统计学意义(P＞0.05).BV组的新生血管计数较其余3组明显增加,差异有统计学意义(F=35.47,P＜O.01).BMSC组及VEGF组的VEGF免疫组化染色呈阳性表达,与EP组比较差异有统计学意义(F=764.32,P＜0.01).BV组的VEGF免疫组化染色呈强阳性表达,与其余3组比较差异有统计学意义(F =764.32,P＜0.01).结论 BMSC联合VEGF基因治疗兔肢体缺血可使VEGF获得稳定而有效的表达,从而改善肢体缺血.     

骨髓间充质干细胞对重症急性胰腺炎大鼠肾损害干预的研究

目的 探讨骨髓间充质干细胞对重症急性胰腺炎(SAP)大鼠肾损害的干预效果及机制.方法 将鉴定过的骨髓间充质干细胞注射到SAP大鼠体内,观察血清淀粉酶、肌酐、尿素氮及肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1变化;透射电镜下观察肾脏间质毛细血管超微结构变化;免疫组织化学对肾脏AQP1定位;荧光定量聚合酶链反应(FQ-PCR)检测肾脏水通道蛋白1(AQP1) mRNA变化,Western blot检测肾脏AQP1蛋白变化.结果 (1)培养的细胞经流式细胞术检测,CD29阳性细胞率为99.30％,CD90阳性细胞率为93.50％,CD34阳性细胞率为0.82％,CD45阳性细胞率为2.22％;(2)间充质干细胞干预后,SAP大鼠6、12、24h血清淀粉酶、肌酐、尿素氮及TNF-α、IL-1低于对应时间点未干预的SAP大鼠,两者差异有统计学意义(P＜0.05);(3)间充质干细胞干预后,SAP大鼠6、12、24 h肾脏间质毛细血管变化均轻于对应时间点未注射SAP大鼠;(4)间充质干细胞干预后,SAP大鼠12、24 h AQPl mRNA均高于对应时间点未干预的SAP大鼠,两者差异有统计学意义(P＜0.05);6、12、24 h AQP1蛋白均高于对应时间点未干预的SAP大鼠,两者差异有统计学意义(P＜0.05).结论 骨髓间充质干细胞能减轻SAP大鼠肾损害,其机制可能同抑制炎性因子表达,减轻间质毛细血管损害,阻滞肾脏AQP1表达下调有关.     

血管内皮细胞生长因子(VEGF)基因转染骨髓间充质干细胞的表达

目的检测兔骨髓间充质干细胞(BMSc)经血管内皮细胞生长因子(VEGF165)基因转染后外源基因的表达，为进一步利用经基因转染的BMSc构建血管化组织工程骨组织打下基础。方法构建VEGF真核细胞表达载体，利用脂质体介导转染兔BMSc，使用原位杂交、免疫组织化学的方法检测VEGF165在BMSc中的表达。结果成功构建VEGF真核细胞表达载体，原位杂交、免疫组化方法显示经基因转染的BMSc中有阳性棕黄色颗粒出现，而未转染组呈现阴性结果。结论采用基因转移技术可以将VEGF转染至．BMSc中，并有外源性基因和蛋白的表达。     

大鼠骨髓间充质干细胞与神经生长因子的体外构建的实验研究

目的探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)的体外构建表达神经生长因子(nerve growth factor,NGF)的能力,同时使MSCs被绿色荧光蛋白(green fluorescent protein,GFP)稳定标记,通过病毒载体将NGF和GFP基因高效转染到MSCs中。方法取2月龄100～150 g Wistar大鼠,全骨髓法分离培养纯化大鼠MSCs,利用携带人NGFβ基因的修复缺陷性重组腺病毒(Ad- hNGFβ)和携带GFP基因的复制缺陷性逆转录病毒(Rt-GFP)转染MSCs,荧光显微镜观察GFP阳性的MSCs,免疫细胞化学和Western-blot检测hNGFβ的表达。结果Rt-GFP转染后,80%～90%MSCs可激发出明亮的绿色荧光,且荧光不随培养时间延长和传代次数增加而衰减。Ad-hNGFβ的转染MSCs中hNGFβ阳性率(90．17±2．14)显著高于阴性对照组(2．17±0．75)和空白对照组(1．83±0．98,P＜0．01),转染后MSCs中NGFβ表达量(188．67±8．71)显著高于阴性对照组(25．67±4．08)和空白对照组(27．50±3．33,P＜0．01)。结论大鼠间充质干细胞的体外构建中Ad-hNGFβ可以高效转染MSCs,实现NGF在MSCs中高效表达,Rt-GFP可以使MSCs获得长效标记。     

神经生长因子基因修饰的骨髓间充质干细胞在糖尿病大鼠膀胱中的生长及表达

目的 将转入神经生长因子(NGF)的大鼠骨髓间充质干细胞(MSC)移植于糖尿病大鼠膀胱平滑肌组织内,观察MSC在膀胱组织内的存活和NGF基因的表达情况.方法 糖尿病组(30只)大鼠按链脲佐菌素(STZ)60 mg/kg行单次腹腔注射,对照组(15只)腹腔注射等体积的柠檬酸缓冲液.实验分组:对照组(正常大鼠膀胱)、发病组(糖尿病大鼠膀胱)、治疗组(糖尿病大鼠膀胱内移植转染NGF基因的MSC).溴苷法示踪NGF基因修饰的MSC在大鼠膀胱内的存活情况;RTPCR、ELISA法检测NGF基因在糖尿病大鼠膀胱内的表达情况.结果 单次腹腔注射STZ造模成功,8周后血糖仍处高位.NGF基因修饰的大鼠MSC移植入糖尿病大鼠膀胱内4周仍存活.ELISA检测结果显示对照组、发病组、治疗组大鼠膀胱NGF蛋白含量分别为(114±3)、(70±2)、(110±2)pg/ml,RT-PCR检测mRNA表达量分别为0.183±0.004、0.032±0.139、0.130±0.165.发病组与对照组相比NGF表达下降(P＜0.05),治疗组与发病组相比NGF表达上升(P＜0.05).结论转入NGF基因的大鼠MSC能在糖尿病大鼠膀胱中存活并稳定表达.

Abstract：

Objective To confirm that rat bone marrow mesenchymal stem cells (MSC) transfected with nerve growth factor (NGF) gene in the bladder tissue of diabetic rats bladder tissues can survive and stably express NGF. Methods A diabetic rat model was constructed. The BrdU-labelled MSC transfected with NGF gene were transplanted into the diabetic rats bladder tissues. BrdUlabelled immunohistochemistry was used to observe the growth of MSC transfected with NGF gene in the diabetic rats bladder tissues. The expression of NGF mRNA and protein were checked by RT-PCR and ELISA. Results A diabetic rat model was successfully built by a single intraperitoneal injectionof STZ. The blood glucose was still high after 8 weeks. NGF gene modified MSC could be detected in the bladder of diabetic rats by BrdU-labelled immunohistochemistry. The concentration of NGF in the control group, disease group and treatment group were ( 114 ± 3), ( 70 ± 2), ( 110 ± 2) pg/ml by ELISA and mRNA quantity by RT-PCR were 0. 183±0. 004, 0. 032±0. 139, 0. 130±0. 165, respectively. Compared with the control group, the expression of NGF gene was decreased (P＜0. 05) in the incidence group. The expression of NGF gene was increased (P＜0. 05) in the treatment group compared with the disease group. Conclusions The NGF gene-modified MSC could survive in diabetic rats bladder tissues. The NGF gene in MSC could stably express in diabetic rats bladder tissues.     

VEGF165基因修饰脂肪干细胞对糖尿病大鼠骨缺损的修复研究

目的 :探讨采用基因转染大鼠脂肪干细胞构建血管化组织工程的方法对糖尿病骨质疏松性骨缺损的修复效果。方法:选取雄性Wistar大鼠78只,体重180~220 g,其中72只通过化学药物(STZ)诱导法建立糖尿病动物模型,成模大鼠血糖值均≥16.7 mmol/L。将实验动物随机分为5组,正常对照组6只,其他实验组各18只。正常对照组:在正常大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞;糖尿病组:单纯糖尿病骨缺损大鼠;生长因子组:在糖尿病大鼠骨缺损内单纯植入VEGF生长因子;干细胞组:在糖尿病大鼠骨缺损内单纯植入脂肪干细胞;实验组:在糖尿病大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞。将5×106个VEGF165-ADSCs细胞与凝胶海绵结合后,植入到糖尿病大鼠骨缺损模型中,在植入后第4周时,采用光学显微镜观察缺损修复组织大体形态;采用免疫组化SP法测定骨缺损区修复后局部微血管密度;应用美国IRIS IntrepidⅡXSP电感耦合等离子体发射光谱仪对修复骨痂内钙/磷含量和碱性磷酸酶(ALP)含量测定;统计分析上述测量结果验证VEGF165-ADSCs对糖尿病大鼠骨缺损的修复作用。结果:荧光染色结果显示,VEGF165表达定位于ADSCs的细胞浆,表达率在87﹪以上;大体组织学观察结果显示:实验组修复区内骨痂生成范围和质量接近正常组,糖尿病组、生长因子组、干细胞组修复效果欠佳。植入后第4周,实验组单位体积的修复组织钙、磷含量和ALP含量明显高于生长因子组、干细胞组(P0.05),与正常对照组组比较差异无统计学意义(P0.05);第4周时,实验组修复局部的血管密度低于正常对照组(P0.05),而显著高于其他组(P0.05)。结论 :VEGF165基因修饰的脂肪干细胞在糖尿病大鼠体内具有良好的成骨及成血管作用,有望成为修复糖尿病特定骨质条件下骨缺损的一种有效手段。