骨髓基质细胞复合聚DL-乳酸/羟基磷灰石修复兔桡骨缺损的研究

目的探讨复合兔骨髓基质细胞（BMSCs）的聚DL-乳酸/羟基磷灰石（PDLLA/HA）在修复兔桡骨缺损中的效果。方法 36只健康新西兰白兔,共72侧前肢,造成1.0cm长的桡骨缺损,随机分成A、B、C3组,A组植入BMSCs＋PDLLA/HA,B组单纯植入PDLLA/HA,C组不做任何植入,于术后2、4、8、12周时行X线、大体和组织学观察。结果 A组成骨效果优于B组,12周时已完全骨性愈合,髓腔通畅,在各骨缺损修复时期,其成骨速度、成骨量和再生髓腔结构均优于B组、C组;A组X线评分也高于B组,差异均有统计学意义（均P〈0.01）。结论 BMSCs复合PDLLA/HA具有较强的成骨能力,有望成为临床修复骨缺损的治疗方法。     

Protective effects of trimetazidine on bone marrow mesenchymal stem cells viability in an ex vivo model of hypoxia and in vivo model of locally myocardial ischemia

Bone marrow mesenchymal stem cells (MSCs) have shown potential for cardiac repair following myocardial injury,but this approach is limited by their poor viability after transplantation.The present study was to investigate whether trimetazidine (TMZ) could improve survival of MSCs in an ex vitro model of hypoxia,as well as survival,differentiation,and subsequent activities of transplanted MSCs in rat hearts with acute myocardial infarction (AMI).MSCs at passage 3 were examined for their viability and apoptosis under a transmission electron microscope,and by using flow cytometry following culture in serumfree medium and exposure to hypoxia (5% CO2,95% N2) for 12 h with or without TMZ.Thirty Wistar rats were divided into 3 groups (n=10 each group),including groupⅠ(AMI control),groupⅡ (MSCs transplantation alone),and group Ⅲ (TMZ+MSCs).Rat MSCs (4×107) were injected into peri-infarct myocardium (MSCs group and TMZ+MSCs group) 30 min after coronary artery ligation.The rats in TMZ+MSCs group were additionally fed on TMZ (2.08 mg?kg-1?day-1) from day 3 before AMI to day 28 after AMI.Cardiac structure and function were assessed by echocardiography at 28th day after transplantation.Blood samples were collected before the start of TMZ therapy (baseline),and 24 and 48 h after AMI,and inflammatory cytokines (CRP,TNF-α) were measured.Then the sur-vival and differentiation of transplanted cells in vivo were detected by immunofluorescent staining.The cellular apoptosis in the peri-infarct region was detected by using TUNEL assay.Furthermore,apoptosis-related proteins (Bcl-2,Bax) within the post-infarcted myocardium were detected by using Western blotting.In hypoxic culture,the TMZ-treated MSCs displayed a two-fold decrease in apoptosis under serumfree medium and hypoxia environment.In vivo,cardiac infarct size was significantly reduced,and cardiac function significantly improved in MSCs and TMZ+MSCs groups as compared with those in the AMI control group.Combined treatment of TMZ with MSCs implantation demonstrated further decreased MSCs apoptosis,further increased MSCs viability,further decreased infarct size,and further improved cardiac function as compared with MSCs alone.The baseline levels of inflammatory cyto-kines (CRP,TNF-α) had no significant difference among the groups.In contrast,all parameters at 24 h were lower in TMZ+MSCs group than those in MSCs group.Furthermore,Western blotting indicated that the expression of antiapoptotic protein Bcl-2 was upregulated,while the proapoptotic protein Bax was down-regulated in the TMZ+MSCs group,compared with that in the MSCs group.It is suggested that implantation of MSCs combined with TMZ treatment is superior to MSCs monotherapy for MSCs viability and cardiac function recovery.     

血管内皮生长因子基因转染骨髓间充质干细胞对慢性肾衰竭大鼠血管新生的影响

目的：探讨血管内皮生长因子（ vascular endothelial growth factor , VEGF）基因转染骨髓间充质干细胞（ mesenchy-mal stem cells,MSCs）对慢性肾衰竭（chronic renal failure, CRF）大鼠肾小球内皮细胞修复和血管新生的影响。方法体外分离、培养大鼠MSCs,Ad5-hVEGF-EGFP转染MSCs并观察转染对细胞增殖,分泌VEGF的影响。50只雄性SD大鼠按数字表法随机分为假手术组、慢性肾衰竭组、MSCs移植组、Ad-VEGF注射组和VEGF基因转染的MSCs移植组,每组10只。采用分阶段5／6肾切除术制备大鼠慢性肾衰竭动物模型。假手术组与慢性肾衰竭组于切除右肾之前从该侧肾动脉注射不含血清的改良杜氏伊格尔（DMEM）培养液,其余3组分别给予MSCs,Ad-VEGF和VEGF基因转染的MSCs。8周后检测各组大鼠肾功能,并用免疫组化检测肾小球CD31表达情况。结果 Ad5-hVEGF-EGFP转染后对MSCs细胞增殖无影响,转染后分泌VEGF蛋白较空质粒转染组明显增加（P＜0.05）。 MSCs移植组和VEGF基因转染的MSCs移植组血肌酐（Scr）和尿素氮（BUN）均较慢性肾衰竭组降低（P＜0．05）;与MSCs移植组比较,VEGF基因转染的MSCs移植组Scr和BUN降低更显著（P＜0．05）。慢性肾衰竭组肾小球中CD31表达较假手术组显著降低（P＜0．05）,应用MSCs和VEGF165基因转染的MSCs干预后肾小球中CD31表达有所增加,VEGF基因转染的MSCs组增加更明显（P＜0．05）。结论慢性肾衰竭大鼠给予转染VEGF基因的MSCs移植后毛细血管内皮细胞数量增多,肾脏功能改善;其作用可能与基因转染增加VEGF表达及有利于肾小球毛细血管内皮修复、血管新生有关。     

血管内皮生长因子转染骨髓间充质干细胞心肌移植对心肌梗死后大鼠心功能及血管新生的作用

目的建立重组人血管内皮生长因子(VEGF)165基因转染大鼠骨髓间充质干细胞(MSC)的方法,探讨该细胞心肌移植对缺血性心脏病心功能及血管新生的影响,并比较联合治疗与单独基因或细胞治疗的疗效差异。方法采用密度梯度离心-贴壁培养法获取Wistar近交系大鼠MSC,用脂质体将pcDNA3.1-hVEGF165转入该细胞,通过ELISA、RT—PCR和Western印迹检测后者VEGF的表达情况;以Wistar近交系大鼠建立心肌缺血模型,随机分成4组(每组12只),心肌梗死模型建立2周后,联合组在心肌梗死区移植转染VEGF165基因的MSC,细胞组移植等量的MSC,基因组注射脂质体-pcDNA3.1-VEGF165DNA复合物,对照组注射等容积培养液;另取12只未结扎冠脉的大鼠为假手术组。细胞移植4周后,用Buxco系统检测心功能;然后处死动物,取心肌标本,测量心肌梗死面积;用5-溴脱氧尿嘧啶(Brdu)、肌钙蛋白T免疫组化双染法确定移植细胞的存活与分化;用Ⅷ因子染色法检测血管新生,RT-PCR法检测VEGF165基因的体内表达情况。结果(1)pcDNA3.1-hVEGF165基因通过脂质体转染大鼠MSC后获稳定表达;(2)移植4周后,联合组心肌梗死面积(27．8％±3．0％)明显低于细胞组(37．0％±10．1％)与基因组(37．1％±5．2％,均P<0．05),心功能改善优于细胞组与基因组;(3)联合组心肌梗死区毛细血管密度(每视野40．2个±5．5个)高于细胞组(27．2个±6．3个,P<0．01)和对照组(18．5个±5．8个,P<0．01),较基因组(35．8个±7．7个)亦有增加的趋势(P=0．189);(4)Brdu、肌钙蛋白T双染示各治疗组心肌梗死区心肌细胞数量不同程度的多于对照组;(5)联合组VEGF基因的体内表达(hVEGFmRNA相对含量0．18±0．04)高于基因组(0．10±0．03,P<0．01)。结论转染VEGF基因的MSC移植可使鼠冠脉结扎造成的心肌梗死面积缩小、心功能明显改善,其疗效优于单独应用基因或细胞治疗,为缺血性心脏病的细胞基因联合治疗提供了理论依据。     

骨髓间质干细胞移植对心肌再生的影响

目的研究移植骨髓间质干细胞(MSCs)对梗死区心肌和血管再生的影响。方法抽取香猪骨髓,体外分离MSCs,经5-氮胞苷(5-aza)转化。结扎冠状动脉左前降支(LAD),经LAD和梗死区注射MSCs;对照组注射培养液。3周和6周后,行单光子发射型计算机断层(SPECT)心肌显像检查。结果SPECT显像,对照组心肌有明显的充盈缺损;实验组细胞移植3周后在梗死区内有岛状的灌注显像区,6周后这些区域相互之间以及与正常心肌之间发生融合。心肌灌注显像,实验组评分高于对照组(P<0.01)。结论MSC移植可再生心肌组织和血管。经LAD注射结合多点局部注射的方法可使移植细胞均匀分布于整个心梗区,促进再生的心肌组织与宿主心肌组织之间产生融合。     

三维多孔聚DL-乳酸／碱性成纤维细胞生长因子修复长骨缺损

OBJECTIVE: To investigate the osteoinductive ability of the composites consisting of basic fibroblast growth factor (bFGF) and porous poly-DL-lactide (PDLLA) for the development of a new absorbable osteosynthesis material. METHODS: Highly porous foams of PDLLA with the pore size ranging from 150 to 300 microm were prepared by a solvent-casting, particulate-leaching technique with NaCl as the leachable component. Animal models of radial diaphyseal defects of 1.0 cm with complete removal of the periosteum were induced in 45 rabbits, which were randomly divided into 3 groups to receive the defect repair with PDLLA and PDLLA/bFGF respectively, leaving one group untreated to serve as the control group. The implant specimens were harvested at 2, 4, 8, and 12 weeks respectively after the surgery and X-ray, histological and scanning electron microscopic (SEM) examinations were performed to evaluate the effectiveness of defect repair. At 8 and 12 weeks after implantation, biomechanical test (for three-point bending strength) was employed to study the quality of bone formation. RESULTS: PDLLA/bFGF composite stimulated more bone formation and had higher bending strength than PDLLA (P<0.05), and the bone formation induced by both materials was significantly more than that observed in the control group in every postoperative stage (P<0.05). CONCLUSION: PDLLA possesses good biocompatibility and absorbability, and when prepared into a porous material, it exhibits good osteoconductibility. As a good bFGF carrier, the foam of PDLLA with three- dimensional structure shows good osteoinductive ability with regard to the rapidity, quantity and quality of the bone formation.     

血管内皮生长因子基因在兔骨髓间充质干细胞中的转染和表达

目的：探讨应用人血管内皮生长因子165 (hVEGF₁₆₅)进行基因修饰的可行性，为血管化组织工程组织的构建及缺血性疾病的治疗奠定实验基础。方法：构建pcDNA3.0-VEGF₁₆₅真核表达载体，利用脂质体介导转染兔骨髓间充质干细胞（MSCs），ELISA方法检测转基因MSCs的hVEGF蛋白表达情况，MTT法检测转基因MSCs表达物对血管内皮细胞增殖活性的影响，设单纯培养MSCs及pcDNA3.0转染MSCs组为对照组。结果：成功构建hVEGF真核表达载体，并成功地将其转入MSCs中；MSCs细胞在转染 pcDNA3.0-VEGF₁₆₅ 质粒24、48和72 h后，其培养上清中hVEGF蛋白表达量均较对照组显著升高（P<0.05），至G418筛选后12代，转基因MSCs培养上清中仍有hVEGF蛋白的表达，含2%、4%、8%、16%和32%转基因细胞培养上清的培养液均明显增加血管内皮细胞的增殖率，与对照组比较，差异均具有显著性（P<0.05）。 结论： hVEGF₁₆₅基因成功地转染至MSCs中，并可进行有效地表达。     

消旋聚乳酸/羟基磷灰石/脱钙骨基质人工骨修复兔桡骨大段骨缺损的实验研究

目的研制理想的,能够修复大段骨缺损的人工骨材料。方法采用乳液共混法将消旋聚乳酸(PDLLA)、羟基磷灰石(HA)、脱钙骨基质(DBM)结合,制成PDLLA/HA/DBM人工骨,并将PDLLA/HA/DBM和PDLLA进行兔桡骨大段骨缺损修复的对比研究。术后2、4、8和12周时摄X片及病理形态学观察及新骨形成定量分析。结果PDLLA/HA/DBM人工骨内新骨形成量明显多于PDLLA及空白对照组(P<0.01),且能够有效修复骨缺损。结论PDLLA/HA/DBM人工骨能促进长骨大段骨缺损的修复,是一种较理想的骨修复材料。     

rAAV2-slug-siRNA对原位胰腺癌移植瘤转移和血管生成影响的实验研究

目的:研究slug基因的RNA干扰重组腺病毒载体(rAAV2-slug-siRNA)对原位胰腺癌移植瘤的转移和血管生成的影响。方法:建立胰腺癌细胞株AsPC-1裸鼠原位胰腺癌移植瘤模型,随机分成3组,每组8只,分为空白对照组(腹腔注射生理盐水)、阴性对照组(腹腔注射rAAV2-GFP)、实验组(腹腔注射rAAV2-slug-siRNA)。10周后利用CO2麻醉法处死裸鼠,观察原位胰腺癌移植瘤的重量,抑瘤率,肝、胃肠、腹腔转移,腹水等情况,及肿瘤细胞微血管密度(MVD)。RT-PCR检测移植瘤的slug mR-NA表达,Western blot检测slug蛋白的表达。结果:胰腺癌细胞株AsPC-1裸鼠原位胰腺癌移植瘤模型建立成功,成瘤率100%。实验组原位胰腺癌移植瘤瘤重明显低于阴性对照组(P<0.05),抑瘤率为70.83%。阴性对照组诱发的裸鼠胰腺癌质地硬,肿块向四周浸润并形成癌性粘连,与阴性对照组相比,实验组腹膜、肝、毗邻脏器转移率和形成腹水率均明显下降(P<0.05)。实验组MVD明显少于阴性对照组(P<0.05),slug mRNA相对表达量(RT-PCR)和slug蛋白相对表达量(Western blot)明显低于阴性对照组(P<0.05)。结论:rAAV2-slug-siRNA可能通过抑制肿瘤血管生成而抑制原位胰腺癌移植瘤转移。     

Effects of heme oxygenase-1 gene modulated mesenchymal stem cells on vasculogenesis in ischemic swine hearts

Background  Mesenchymal stem cells (MSCs) transplantation may partially restore heart function in the treatment of acute myocardial infarction (AMI). The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 (HO-1) on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells.

Methods  In vitro, MSCs were divided into four groups: (1) non-transfected MSCs (MSCs group), (2) MSCs transfected with the pcDNA3.1-Lacz plasmid (Lacz-MSCs group), (3) MSCs transfected with pcDNA3.1-hHO-1 (HO-1-MSCs group), and (4) MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin (SnPP) (HO-1-MSCs+SnPP group). Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at <1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor (TGF-β) and fibroblast growth factor (FGF-2) in the supernatant. In vivo, 28 Chinese mini-pigs were randomly allocated to the following treatment groups: (1) control group (saline), (2) Lacz-MSCs group, (3) HO-1-MSCs group, and (4) HO-1-MSCs + SnPP group. About 1×10⁷ of autologous stem cells or an identical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging (MRI) assay and postmortem analysis were assessed four weeks after stem cell transplantation.

Results  Post hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs ((874.88±68.23) pg/ml) compared with Lacz-MSCs ((687.81±57.64) pg/ml, P <0.001). FGF-2 was also significantly increased in the HO-1-MSCs ((1106.48±107.06) pg/ml) compared with the Lacz-MSCs ((853.85±74.44) pg/ml, P <0.001). In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area compared with the Lacz-MSCs group (14.24±1.66/HPFs vs. 11.51±1.34/HPFs, P <0.001). Arteriolar density was also significantly higher in HO-1-MSCs group than in the Lacz-MSCs group (7.86±2.00/HPFs vs. 6.45±1.74/HPFs, P=0.001). At the same time, the cardiac function was significantly improved in the HO-1-MSCs group compared with the Lacz-MSCs group ((53.17±3.55)% vs. (48.82±2.98)%, P <0.05). However, all these effects were significantly abrogated by SnPP.

Conclusion  MSCs provided a beneficial effect on cardiac function after ischemia/reperfusion by the induction of therapeutic angiogenesis, and this effect was amplified by HO-1 overexpression.

     

利用骨髓间质干细胞修复小鼠颅骨缺损的实验研究

目的探讨小鼠骨髓间质干细胞(MSCs)修复小鼠颅骨缺损的能力。方法分离培养扩增小鼠MSCs,并在其同窝小鼠颅骨上造约5.0 mm圆形缺损,分别植入煅烧骨+藻酸钙(载体组)和MSCs细胞+煅烧骨+藻酸钠(实验组)及空白组,4个月后,取出标本进行病理分析。结果空白组未见骨组织形成;载体组仅见有少量骨组织形成;实验组有较多的新生骨组织形成,并且新生骨和宿主骨有连接。结论小鼠骨髓间质干细胞能促进骨组织形成,具有修复骨缺损的潜能。     

两种重组腺相关病毒介导增强型绿色荧光蛋白转染大鼠成骨细胞效率的体外研究

目的 比较2种不同重组腺相关病毒(rAAV)介导的增强型绿色荧光蛋白(EGFP)对大鼠成骨细胞的转染效率,评价其作为成骨细胞病变基因治疗载体的可行性.方法 采用Ⅰ型胶原酶阶段消化法分离培养大鼠成骨细胞并鉴定,rAAV-EGFP按转染复数(MOI) 1×10~3、1×10~4、1×10~5、5×10~5分为只加rAAV和rAAV与腺病毒(ADV)共同转染组转染成骨细胞,倒置荧光显微镜观察转染后荧光强度随转染时间的变化,流式细胞仪检测rAAV2/6-EGFP和rAAV2/9-EGFP对成骨细胞的转染效率及荧光强度,确定转染的较佳MOI值,以此值用MTT法描绘细胞生长曲线,观察rAAV对细胞的毒性.结果 分离培养的细胞具有体内成骨细胞的生物学行为,rAAV对成骨细胞的转染效率随MOI值的增加而提高,ADV(-)组荧光强度在第5 d达到高峰,当MOI为1×10~5时,rAAV2/6-EGFP和rAAV2/9-EGFP的转染效率分别为90.2%、66.1%,MOI值增到5×10~5时转染效率无显著提高;ADV(+)组荧光强度在第3 d即达高峰,MOI值为5×10~5时,rAAV2/6-EGFP和rAAV2/9-EGFP的转染效率为47.6%、30.5%.细胞生长正常,rAAV对细胞活性影响小.结论 两种病毒载体对成骨细胞转染效率均较高,其中rAAV2/6高于rAAV2/9,是一种理想的基因治疗载体.     

兔骨髓基质细胞与聚DL-乳酸/羟基磷灰石生物相容性的实验研究

目的检测聚DL-乳酸/羟基磷灰石（PDLLA/HA）复合材料的特性,探讨骨髓基质细胞（BMSCs）与PDLLA/HA的生物相容性,为筛选骨组织工程的支架材料提供依据.方法将BMSCs与PDLLA/HA复合体外培养,通过形态学的观察、细胞增殖率及ALP活性的测定,观察PDLLA/HA对培养细胞的影响.结果复合培养时BMSCs能在PDLLA/HA上贴附、繁殖,其生长及功能不受影响.结论 PDLLA/HA具有良好的细胞相容性,能作为骨组织工程种子细胞的支架材料.     

人精子细胞膜结合型透明质酸酶促进人乳腺癌细胞增殖的实验观察

目的　探讨人精子细胞膜结合型透明质酸酶 (PH2 0 )促进乳腺癌细胞增殖的机制。方法　将人PH2 0cDNA转染到人乳腺癌细胞株MDA2 31中 (MDA2 31 PH2 0 ) ,对照组用pcDNA3空载体替代PH2 0重组体。将MDA2 31 PH2 0和MDA2 31 pcDNA3分别以同样数量种植到鸡胚绒毛尿囊膜 (CAM)上 ,种植后第 4天切取肿瘤称重 ,并用免疫组织化学方法研究肿瘤组织中血管形成的变化。另外 ,用Trans well细胞培养法研究MDA2 31 PH2 0和MDA2 31 pcDNA3细胞对牛主动脉内皮细胞 (ABAE)生长的影响 ;用Western印迹法观察这两组细胞的成纤维细胞生长因子 2 (FGF 2 )表达水平 ,并用酶联免疫吸附试验测定两组细胞的FGF 2和透明质酸 (HA)分泌量。结果　MDA2 31 PH2 0组平均肿瘤重量为4 4 7mg± 10 2mg ,MDA2 31 pcDNA3组为 2 1 3mg± 2 8mg,两组比较差异有显著意义 (t=2 4 18,P =0 0 38)。MDA2 31 PH2 0肿瘤组织中新生血管明显增多。MDA2 31 PH2 0细胞FGF 2蛋白质表达水平增高 ,同时细胞FGF含量 (8 10pg/ml± 1 5 6pg/ml)和HA含量 (12 2 0ng/ml± 2 5 4ng/ml)也均高于对照组(分别为 3 94pg/ml± 0 82pg/ml和 4 6 2ng/ml± 96ng/ml,均P <0 0 1)。结论　PH2 0可能通过促进癌细胞释放FGF 2和分解HA成小片段 ,刺激新生血管生长并促进肿瘤     

Study on biocompatibility of PDLLA／HA／DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDIZA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen I expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of bio-material and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.     

骨髓间充质干细胞移植抗大鼠肝纤维化的作用及机制

目的：探讨骨髓间充质干细胞（ MSCs ）移植对大鼠肝纤维化模型的作用与机制。方法分离大鼠MSCs培养传代至第4代。采用大鼠腹腔注射四氯化碳制造肝纤维模型。将造模成功SD大鼠60只,随机分为3组,每组20只：（1）对照组：鼠尾静脉注射等量的生理盐水;（2） MSCs 组：鼠尾静脉注射MSCs 悬液;（3）诱导组：鼠尾静脉注射经肝细胞生长因子（HGF）诱导14 d后的MSCs悬液。于移植后第1周、2周、3周、4周分别处死大鼠5只,检测大鼠血清透明质酸（ HA）、层黏蛋白（ LN）、Ⅳ型胶原水平;光镜下观察肝组织纤维化程度;分别用PCR法及Western检测大鼠肝脏Col-Ⅰ、RhoA、Cdc42、Rac1 mRNA基因及其蛋白质表达水平。结果（1）诱导组与MSCs组的纤维化程度评分随着时间延长逐渐下降,且在第4周诱导组评分明显低于MSCs组（ P＜0．05）。（2）移植3周后各组血清HA、LN、Ⅳ型胶原含量均显著下降,4周时诱导组和MSCs组各指标含量明显低于对照组,并且诱导组低于MSCs组（ P＜0．05）。（3） MSCs移植后诱导组、MSCs组大鼠肝脏组织Col-Ⅰ、RhoA、Cdc42、Rac1 mRNA和蛋白表达均随时间延长而下降,移植4周时诱导组各项指标明显低于MSCs组和对照组（ P＜0．05）。结论 MSCs移植可抑制肝纤维化大鼠肝组织中HA、LN、Ⅳ型胶原的分泌,并可下调肝纤维化大鼠肝组织Rho信号通路相关因子RhoA,Cdc42,Rac1 mRNA及蛋白表达。 HGF诱导MSCs后抗大鼠肝纤维化作用显著增强。     

GATA4基因修饰骨髓间充质干细胞对心肌梗死大鼠心功能的影响

目的: 研究锌指转录因子(GATA4)基因修饰骨髓间充质干细胞（MSCs）对心肌梗死大鼠心功能的影响,为进一步探讨其机制提供依据。方法: 构建GATA4-pEGFP基因质粒,体外分离培养大鼠MSCs,利用脂质体将质粒转染入MSCs。40只SD大鼠制备大鼠心肌梗死模型,于术后10 d测定心功能指标。将模型鼠随机分为MSCs 组和DMEM组,每组各20只。MSCs 组将GATA4-pEGFP基因修饰的MSCs植入心肌梗死后10 d大鼠心肌梗死区,DMEM组同时注射等体积无血清DMEM于心肌梗死区。移植注射后4周,检测左心室射血分数（LEVF）和左室缩短分数（FS）。结果: 体外分离培养的MSCs呈梭形或条形,贴壁生长 。 GATA4-pEGFP重组基因质粒转染MSCs 2周后,可见细胞中有绿色荧光蛋白表达。移植4周后,MSCs组大鼠LEVF（85%±7%）明显高于DMEM组(47%±4%)（P<0.01）; MSCs组大鼠FS(52%±6%)明显高于DMEM组(35%±8%)（P<0.01）。结论: GATA4基因修饰的MSCs移植能够改善心肌梗死大鼠心功能。     

新型活性修饰对聚乳酸组织工程骨支架上种子细胞生物学行为的影响

目的探讨氨等离子体改性、酰胺键接枝甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)短肽的活性修饰方法对消旋聚乳酸(PDLLA)组织工程骨上慢病毒转染红色荧光蛋白的骨髓间充质干细胞生物学行为的影响。方法制备圆片状直径8 mm、厚1 mm的PDLLA三维多孔支架,分为3组:表面氨基化PDLLA(A组),接枝肽A/PDLLA(PA组),以未经处理的PDLLA(P组)作为对照组。CyQuant NF和AlamarBlue分别检测支架上细胞的增殖和代谢活性;钙黄绿素进行矿化荧光染色,荧光显微镜观察支架上钙盐沉积以及RFP-BMSCs的贴附与增殖;扫描电镜观察细胞的贴附。结果接种细胞后各时间点,3组材料上细胞均能增殖,组间细胞数量差别均有统计学意义(P<0.001),PA组>A组>P组。除第10天(P=0.077)和第12天(P=0.491)PA组和A组间细胞数量差异无统计学意义外,其余各时间点,PA组数量均明显高于A组。随着时间的延长,两组间细胞数量逐渐接近。细胞代谢活性检测与增殖检测结果近似,骨支架上细胞增殖程度越高,其代谢越活跃。荧光显微镜观察,A组和PA组材料上RFP-BMSCs较P组增殖活跃;钙盐沉积荧光染色显示,第14天和第21天,绿色荧光强度PA组>A组>P组。扫描电镜显示,PA组材料能获得较A组更好的细胞贴附,P组细胞较稀疏。结论氨等离子体改性接枝GRGDS肽的新型活性修饰PDLLA骨支架,能明显促进RFP-BMSCs为种子细胞的贴附、增殖、代谢与矿化。     

谷氨酰胺联合脐血间充质干细胞移植在大鼠肠缺血再灌注损伤中的作用

目的探讨谷氨酰胺联合脐血间充质干细胞(MSCs)移植在大鼠肠缺血再灌注损伤中作用。方法体外复苏并培养脐血间充质干细胞移植前备用,观察CM-Di I荧光标记后脐血间充质干细胞的去向。80只SD大鼠随机分为正常对照组,缺血再灌注损伤组,谷氨酰胺组,MSCs移植组及联合组每组各15只。对照组采用生理盐水灌肠,损伤组采用TNB(S乙醇稀释)灌肠,在TNBS建模后1 h,谷氨酰胺组于尾静脉输入谷氨酰胺0.45 g/kg、MSCs移植组于尾静脉输入1×10~(10)/L脐血间充质干细胞悬液,联合组尾静脉输入谷氨酰胺0.45 g/kg+脐血间充质干细胞悬液1×10~(10)/L。通过ELISA法检测各组大鼠血清中肠脂肪酸结合蛋白(IFABP)、白介素-6(IL-6)、超氧化物歧化酶(SOD)的含量;各组于再灌注1 h、3 h后检测肠组织含水率;通过RT-PCR、Western blot观察大鼠肠黏膜上皮细胞caspase-3、NF-k B、Bcl-2在谷氨酰胺联合MSCs移植后的mRNA和蛋白的表达情况。结果通过荧光示踪法观察到移植的MSCs细胞分布于肠粘膜淋巴组织内和腺上皮细胞间,表明MSCs可能参与了肠缺血再灌注损伤的修复过程。各组大鼠血清中SOD、IFABP、IL-6的含量变化比较,损伤组血清中IFABP、IL-6的含量较对照组显著增加,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著减少,联合组减少更为明显,损伤组血清中SOD的含量较对照组显著减少,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著增高,联合组增高更为明显(P0.05)。再灌注1 h和3 h,损伤组肠组织含水率均明显高于对照组;与损伤组相比,谷氨酰胺组、MSCs移植组及联合组肠组织含水率值均显著降低,联合组降低更为明显,而谷氨酰胺组、MSCs移植组差异无统计学意义(P0.05)。与对照组比较,损伤组肠黏膜上皮细胞caspase-3、NF-k B的mRNA和蛋白表达明显上调,Bcl-2的mRNA和蛋白表达明显下调(P0.05),而谷氨酰胺组、MSCs移植组及联合组与之比较,caspase-3、NF-k B的mRNA和蛋白表达明显下调,Bcl-2的mRNA和蛋白表达明显上调(P0.05),谷氨酰胺组及MSCs移植组之间无统计学差异(P0.05),但两组与联合组比较,差异明显(P0.05)。结论谷氨酰胺组及MSCs移植后,明显减轻了大鼠肠缺血再灌注损伤程度,其可能通过抑制caspase-3、NF-k B表达和促进Bcl-2表达减轻肠黏膜缺血再灌注损伤。