高糖对骨形态发生蛋白-2、胰岛素样生长因子-1基因转染大鼠骨髓基质干细胞增殖的影响

目的 观察高糖环境下骨形态发生蛋白-2(BMP-2)和胰岛素样生长因子-1(IGF-1)基因转染大鼠骨髓基质干细胞(BMSC)后BMSC的增殖.方法 用Ad-BMP-2和Ad-IGF-1转染大鼠BMSC,Wester blot检测蛋白表达.噻唑蓝(MTT)比色法及流式细胞术检测细胞增殖.结果 Western blot检测到转染组细胞中有目的 蛋白表达.MTT结果显示第5天细胞增殖能力达到高峰,5 d光吸收值:A～E组分别为0.324±0.027、0.319±0.017、0.622±0.028、0.626±0.020、0.778±0.031.流式细胞术结果显示A～E组细胞处于增殖期的比重分别为23.92±3.07、23.51±2.11、34.37±6.85、35.04±1.45、42.56±1.15.结论 高糖环境下BMP-2和IGF-1基因转染均能促进BMSC增殖,联合转染对BMSC增殖有协同效应.

Abstract：

Objective To observe the proliferation of bone marrow stromal cell (BMSC) transfected by bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 ( IGF-1 ) gene under high glucose condition.Methods Ad-BMP-2 and Ad-IGF-1 transfected rat BMSC,protein expression of BMSC were detected by Western blotting analysis.Methyl thiazol tetrazolium (MTT) assay and flow cytometry to observe the proliferation potential of BMSC.Results In the Western blotting analysis,positive signal lane of protein was observed in transfected group.MTT assay show that proliferation reached the peak in all groups on the fifth day,and the absorbency values of A to E group were 0.324 ± 0.027,0.319 ± 0.017,0.622 ±0.028,0.626 ± 0.020,0.778 ± 0.031.Flow cytometry show that the proliferative percentage from A to E group were 23.92 ±3.07,23.51 ±2.11,34.37 ±6.85,35.04 ± 1.45,42.56 ± 1.15.Conclusion BMP-2 or IGF-1 can promote the proliferation of BMSC under high glucose condition,but the combined has the synergy effect.     

骨形态发生蛋白-2对兔骨髓基质细胞向软骨细胞分化基因表达的影响

目的 观察骨形态发生蛋白-2(BMP-2)基因对兔骨髓基质细胞(MSCs)向软骨细胞分化mRNA表达的影响.方法 从兔骨髓细胞中分离提取BMP-2 mRNA,构建重组BMP-2真核表达质粒,并转染兔MSCs,采用荧光定量逆转录-聚合酶链反应(RT-PCR)方法检测各组MSCs内Ⅱ型胶原和蛋白多糖mRNA表达水平.结果 转染后2、4 d实验组MSCs内的Ⅱ型胶原和蛋白多糖mRNA表达量28.7±4.0、236.7±48.5及26.9±4.3、208.2±36.7,均明显高于转染后2、4 d的空白对照组(16.1±2.8、99.2±24.8及14.6±2.7、111.1±18.9)和空载体对照组(14.6±2.6、85.4±24.7及16.1±2.8、98.0±22.5)(P＜0.05).结论 重组真核表达载体BMP-2质粒能够诱导MSCs向软骨细胞分化.

Abstract：

Objective To observe the effect of bone morphogenetic protein-2 (BMP-2) on gene expression during the differentiation of marrow stromal cells (MSCs) into chondrocytes. Methods The BMP-2 mRNA was extracted from the rabbit bone marrow cells. The recombinant BMP-2 eukaryotic expression plasmids were constructed and transfected into MSCs. The mRNA expression of type Ⅱ collagen and proteoglycan was detected by using quantitative polymerase chain reaction (PGR) technique. Results At 2nd, and 4th day after transfection of plasmid pEGFP-C3-BMP-2 into MSCs, the mRNA expression levels of type Ⅱ collagen and proteoglycan in MSCs of experimental group were significantly higher than controls (P ＜ 0. 05 ). Conclusion Recombinant eukaryotic expression plastmid pEGFP-C3-BMP-2 can induce MSCs differentiation towards chondrocytes.     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

骨形态发生蛋白-2基因转染兔骨髓基质干细胞及复合纳米羟基磷灰石体外构建组织工程骨的研究

目的 探讨腺病毒介导的人骨形态发生蛋白-2表达载体(Ad-BMP-2)转染兔骨髓基质干细胞(rBMSCs)后复合到纳米羟基磷灰石(Nano-HA)体外构建仿生人工骨的可行性. 方法 采用GatewayTM技术构建腺病毒载体,溶胶-絮凝法制备Nano-HA,用Ad-BMP-2转染体外培养的rBMSCs,免疫组化、RT-PCR和蛋白印迹方法检测转染后细胞的BMP-2表达.将转染48 h的细胞均匀接种到Nano-HA支架上,第3、5天取细胞载体复合物扫描电镜观察细胞贴附、生长状况,消化收集贴附支架的细胞,蛋白印迹检测贴附支架细胞BMP-2的表达.结果 转染后BMP-2基金在mRNA水平和蛋白水平均有高表达;扫描电镜见转染细胞在支架材料分布均匀,黏附良好,转染后及复合后Western Blot检测BMP-2表达较高.结论 利用转基因技术,用含目的基因的腺病毒载体转染靶细胞复合到Nano-HA,细胞载体复合物稳定长效地分泌生长因子,体外成功地构建了仿生人工骨.     

pcDNA3.1-BMP-2真核表达载体构建及转染兔骨髓基质细胞的实验研究

目的 构建人骨形态发生蛋白2(BMP-2)真核表达载体，鉴定并转染兔骨髓基质细胞，为转基因治疗骨缺失疾病提供实验基础。方法 双酶切克隆载体将BMP-2目的基因与真核表达载体pcDNA3．1连接，免疫组化及Western blotting检测转染BMP-2基因骨髓基质细胞的表达效果。结果 成功构建BMP-2真核表达载体，转染骨髓基质细胞后，有BMP-2表达。结论 骨髓基质细胞经基因转染后可以表达BMP-2。为进一步实验研究提供基础。     

Effect of simvastatin on bone morphogenetic protein-2 expression and alkaline phosphatase activity of bone marrow stromal cell]

OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.     

转染重组人骨形态发生蛋白-7基因对骨髓基质细胞生物学行为的影响

目的 观察重组人骨形态发生蛋白-7(rhBMP-7)基因转染骨髓基质细胞(BMSCs)后的稳定表达及表达产物对BMSCs生物学行为的影响。方法利用脂质体介导法将rhBMP．7基因转染BMSCs，以G418筛选出阳性克隆并扩大培养，用免疫组织化学链霉抗生物素蛋白-生物素-过氧化物酶复合物(SABC)法检测转基因细胞的稳定表达；转染48h后，用转基因细胞的培养液上清刺激正常培养的BMSCs，利用^3H标记的胞腺嘧啶氧核苷(^3H-TdR)掺入法、Na2^35SO4掺入法以及氯胺T法检测基因表达产物对BMSCs增殖、合成蛋白多糖以及胶原的影响。结果 转基因细胞4周时仍能表达外源性基因；基因表达产物能明显促进BMSCs的增殖以及蛋白多糖和胶原的合成。结论 rhBMP-7基因转染BMSCs后可获得稳定表达，且基因表达产物能促进BMSCs增殖。诱导其向软骨细胞分化并增强其生物学活性。     

pcDNA_(3.1)-OGP基因转染兔骨髓基质干细胞的表达及生物学效应的观察

[目的]观察成骨生长肽(osteogenic growth peptid,OGP)基因转染兔骨髓基质干细胞后的表达及表达产物对骨髓基质干细胞向成骨细胞分化的影响.[方法]构建重组真核表达载体pcDNA_(3.1)-OGP,并在脂质体介导下,将其导入兔骨髓基质干细胞.通过G418筛选获得阳性克隆.用RT-PCR方法榆测OGP基因在骨髓基质干细胞内的表达.Ⅰ型胶原和碱性磷酸酶的检测观察转染pcDNA_(3.1)-OGP后骨髓基质干细胞向成骨细胞分化情况.[结果]成功构建真核表达载体pcDNA_(3.1)-OGP,用RT-PCR方法及碱性磷酸酶和Ⅰ型胶原检测证实OGP基因能在骨髓基质干细胞中表达并促进其向成骨细胞分化.[结论]经pcDNA_(3.1)-OGP转染的兔骨髓基质干细胞不仅可以表达OGP,而且具有向成骨细胞系分化的特性.     

慢病毒介导骨形态发生蛋白-7基因的干细胞用于组织工程骨构建的实验研究

目的 建立能够稳定表达骨形态发生蛋白-7(BMP-7)的骨髓基质干细胞(BMSCs),观察其成骨分化,并与纳米羟基磷灰石胶原(nHAC)材料复合培养体外构建组织工程骨的可行性.方法 实验分4组:BMP-7和eGFI基因转染组(A组)、eGFP基因转染组(B组)、常规成骨诱导组(C组)、未转染组(D组).用G418筛选获得阳性细胞后接种于nHAC支架体外复合培养.荧光显微镜下观察eGFP表达,判断转染效率;以逆转录-聚合酶链反应(RTPCR)、ELISA检测目的基因表达,四唑盐(MTT)实验检测细胞活力,碱性磷酸酶(ALP)活性和Gomori染色榆测成骨情况;扫描电镜观察细胞在支架材料中的黏附、生长状况.结果 慢病毒24 h对BMSCs的转染率约为90%.RTPCR检测到A组在1300 bp处出现特异性条带,其他组结果阴性;ELISA检测24 h BMP-7蛋白含量为(150.2±18.3)pg/mL,种植支架复合培养8周后BMP-7含量为(76.6±7.4)pg/mL;MTT实验显示细胞活性与未转染组比较差异无统计学意义(P>0.05);ALP活性以16 d最强;扫描电镜见细胞种植nHAC支架后黏附、生长良好.结论 BMP-7可在BMSCs内稳定表达,并诱导其向成骨分化;与nHAC复合培养可构建组织工程骨.     

活化T细胞核因子-2可介导高迁移率族蛋白B1促进白细胞介素-2转录表达

目的 观察活化T细胞核因子-2(NFAT2)在高迁移率族蛋白B1(HMGB1)促进白细胞介素-2(IL-2)转录表达中的介导作用.方法 将构建好的HMGB1和NFAT2质粒单独或共同转染人胚胎肾细胞株293T,同时转染IL-2报告基因,并在此基础上应用小分子干扰RNA(siRNA)质粒对内源性及外源性NFAT2表达进行特异性抑制,观察对IL-2报告基因活性的影响.结果 在293T细胞中,内源性转染实验促使IL-2报告基因活性增加到17.81±1.49,但随着siRNA质粒转染剂量从0 μg/孔逐渐增加到4.8μg/孔,IL-2报告基因活性在内源性抑制实验中降低最小到1.42±0.17,在外源性抑制实验中活性最高为54.16±5.49,受抑制后降低最小到2.97±0.34.结论 NFAT2介导HMGB1促进IL-2报告基因转录表达.     

转化生长因子－β1基因修饰的不成熟树突状细胞的免疫生物学功能

目的 探讨转化生长因子(TGF) -β1基因转染后树突状细胞(DC)细胞表型和免疫生物学功能的变化.方法 利用大鼠骨髓细胞诱导、培养不成熟树突状细胞(imDC),以脂质体介导的pIRES2-EGFP-hTGF-β1转染imDC细胞,通过酶联免疫吸附试验(ELISA)和Western blot法检测转染后各组imDC培养上清TGF-β1蛋白表达;通过流式细胞法、免疫细胞化学法及混合淋巴细胞反应检测各组imDC表面分子的分子表型和免疫功能变化.结果 TGF-β1基因转染的imDC上清中均检测相应蛋白的高表达,TGF-β1基因转染组TGF-β1蛋白表达(252.75±11.31) μg/L均高于mDC组(11.19±2.29) μg/L、imDC组(35.18±6.17) μg/L、空载体组(33.67±4.61)μg／L;基因转染组的表面分子CD86、CD80及MHCⅡ表达明显低于空载体组和无任何转染的imDC组;基因转染组的imDC对T的增殖研究结果显示有显著抑制作用,低于空载体和无任何转染的imDC组.结论 TGF-β1基因成功导人imDC,基因改造的imDC不但能维持不成熟状态同时分泌免疫耐受抑制蛋白,致免疫耐受作用显著增强.