pCD-rbFGF重组质粒的构建及其在成骨细胞中的表达

为了构建碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)真核表达质粒,并探讨应用bFGF基因治疗骨科各种疾患的可能性,将鼠碱性成纤维细胞生长因子cDNA克隆至真核表达载体pcDNA3中,构建了重组质粒pCD-rbFGF.通过Liofectin介导将pCD-rbFGF转染兔成骨细胞,经免疫组化检测证实兔成骨细胞获得了bFGF的瞬时表达.提示该bFGF真核表达质粒有效,可用于进一步基因治疗研究.     

BASIC FIBROBLAST GROWTH FACTOR GENE TRANSFECTION TO ENHANCE THE REPAIR OF AVASCULAR NECROSIS OF THE FEMORAL HEAD

A VASCULAR necrosis of the femoral head usually leads to destruction of the hip joint, increasing musculoskeletal morbidity in young and middle aged patients. Total joint replacement is commonly used to treat disabling secondary osteoarthrosis. Although most recent improvements in total hip replacement may decrease the failure, patients are usually young and the current hip prothesis function would not fulfill purpose for the remaining life ex-pectancy for these patients. So it is necessary…     

Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF func-tion in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoRⅠ, PstⅠ and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supernatants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfec-tion into tenocytes could significantly enhance the biological activity of bFGF, and increase the ex-pression of type Ⅰ and Ⅲ collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.     

碱性成纤维细胞生长因子真核荧光表达载体的构建

目的：构建可在真核细胞中表达碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的真核表达载体,为进一步开展bFGF基因治疗缺血性脑血管疾病奠定基础。方法：①采用线栓法建立SD大鼠大脑中动脉局灶性脑缺血模型,取缺血灶周围脑组织经逆转录聚合酶链反应(RT-PCR)扩增bFGF基因;②将bFGFDNA克隆到pGEM-TEasy质粒,经菌落PCR和HindⅢ和EcoRI双酶切鉴定;③然后将bFGF DNA亚克隆到pEGFP-N3质粒;通过抗性基因筛选阳性克隆,经菌落PCR、酶切和测序鉴定。结果：菌落PCR、酶切和DNA序列鉴定均证实插入片段与GenBank报道的bFGF基因序列一致。结论：经RT-PCR反应得到了特异的bFGF基因片段并成功构建了bF-GF荧光真核表达载体。     

bFGF真核表达质粒的构建及骨髓基质细胞转染

目的 探讨bFGF基因转染骨髓基质干细胞的方法 及可行性.方法 构建bFGF基因真核表达质粒,用脂质体法介导转染骨髓基质细胞,通过形态学观察、免疫组化、ELISA法、及RT-PCR方法 检测bFGF基因转染骨髓基质干细胞的成功性,以及转染后骨髓基质干细胞的生物学特性.结果 bFGF基因成功转染骨髓基质干细胞,并能持续稳定的分泌bFGF蛋白,可诱导骨髓基质干细胞向成软骨方向分化.结论 bFGF基因可以转染骨髓基质干细胞,并能诱导骨髓基质干细胞向成软骨方向分化.     

广谱细胞生长因子的表达纯化与活性测定

目的 对人工合成的人广谱细胞生长因子基因进行体外表达与纯化，并测定其活性。方法 将人广谱细胞生长因子基因克隆于pGEX 4T-1表达载体，转化于大肠杆菌BL21菌株进行诱导表达，利用亲和层析纯化表达产物，通过体外培养的大鼠成纤维细胞存活实验检验其活性。结果 携带重组质粒的菌株经诱导产生高水平的表达产物，纯化的表达产物具备较高的纯度，并基本保有其天然活性。结论 广谱细胞生长因子表达体系的成功建立，为该产品功能的进一步研究与临床应用创造了条件。     

人碱性成纤维细胞生长因子腺病毒表达载体的构建及其在人脐静脉内皮细胞中的表达

目的:构建人碱性成纤维细胞生长因子(basic fibroblast cell growth factor,bFGF)腺病毒载体,并观察其在体外血管内皮细胞中的表达.方法:采用同源重组的方法,构建重组腺病毒Ad5-bFGF.携有绿色荧光蛋白(green fluorescence protein, GFP)的Ad5腺病毒转染人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),检测最佳转染复数.Ad5-bFGF体外转染HUVEC,免疫细胞化学法、Western印迹法检测bFGF蛋白的表达.结果:Ad5转染HUVEC的最佳转染复数为200,转染率为90%.免疫细胞化学法和Western印迹结果显示bFGF基因以Ad5为载体可以在HUVEC的胞质和胞核表达,并且表达量较未转染的HUVEC明显增加.结论:成功构建了携带bFGF基因的腺病毒载体,体外可以成功表达,为bFGF基因治疗及肿瘤发病机制的研究奠定了基础.     

人碱性成纤维细胞生长因子基因真核表达载体的构建

目的:构建人碱性成纤维细胞生长因子bFGF基因真核表达载体,探讨bFGF基因转移治疗视网膜病变的方法.方法:将bFGF和GFP基因克隆到真核表达载体pcDNA3中.结果:酶切及琼脂糖电泳分析 pcFG中含有bFGF和GFP基因.结论:成功的构建了含有绿色荧光蛋白的人碱性成纤维细胞生长因子基因的真核表达载体.     

VEGF基因真核表达载体的构建及其在骨髓间充质干细胞中的表达

目的探讨经腺病毒表达载体转染血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)165基因的骨髓间充质干细胞(mesenchymal stemcells,MSCs)体内外基因表达的特点。方法构建VEGF基因腺病毒表达载体,将VEGF165基因转染到MSCs中。用RT-PCR法检测转基因MSCs中基因表达情况。结果转基因MSCs及其分化的子代细胞均有VEGF165的表达,并持续2周左右时间。结论经腺病毒表达载体转染VEGF165基因的MSCs在体内外均有良好的基因表达。     

Cloning and Expression of Rat Transforming Growth Factor β1 cDNA in Osteoblasts

In order to study transforming growth factor81 (TGF51) in molecular level and investigate thefeasibility of TGF51 gene therapy for bone defects,an eukaryotic expression vector containing ratTGF51 cDNA was constructed. The expression ofTGF51 in the transfected osteoblasts was observed.1 MATERIALS AND METHODS1. 1 Animals and Cell CultureSprague--Dawley rats, I adult and 5 newborn,were purchased from Animal Center of Tong nMedical University. Lymphocytes, isolated fromperipheral…     

bFGF基因体外转染兔骨髓间充质干细胞及对其增殖的影响

目的：观察bFGF基因体外转染兔骨髓间充质干细胞（bMSCs）后细胞目的基因的表达及对其增殖的影响。方法：将兔骨髓来源的间充质干细胞分离培养扩增,用PCR扩增法获得绿色荧光蛋白-bFGF基因重组质粒,转染间充质干细胞,用荧光显微镜观察转染后绿色荧光蛋白基因的表达,计算转染率,用MTT检测转染细胞的增殖特性。结果：实验成功地构建了绿色荧光蛋白-bFGF基因重组质粒,倒置荧光显微镜下观察到转染后的bMSCs发出稳定的绿色荧光信号,转染率为（43.32±4.51）%,MTT法显示重组质粒组增殖速度显著高于空白质粒组和未转染细胞组（P〈0.05）。结论：运用转染的方法实现了两种基因在MSCs中的共同表达,为监控MSCs细胞的转染和表达过程提供了一种手段。为后期利用MSCs细胞进行组织工程骨组织制造和移植提供了规范化的监控和程序化生产手段。     

腺病毒载体介导的bFGF基因体外转染大鼠平滑肌细胞的实验研究

目的观察携带bFGF基因的重组腺病毒体外转染平滑肌细胞的效率及转染后目的基因的表达。方法应用磷酸钙共沉淀法分别构建携带bFGF基因和大肠杆菌LacZ报道基因的重组腺病毒载体Ad．bFGF和Ad．LacZ,并以Ad．bFGF（组Ⅰ）、Ad．LacZ（组Ⅱ）、PBS（组Ⅲ,对照组）转染体外培养的大鼠平滑肌细胞（SMCs）。X—gal染色检测腺病毒载体的转染效率,噻唑蓝（MTT）检测各组SMCs的增殖情况,酶联免疫吸附（ELISA）检测各组培养液中bFGF蛋白的浓度。结果当MOI值为100时,X-gal核蓝染细胞比率达95％以上;组ⅠSMCs的增殖水平显著高于组Ⅱ和组Ⅲ（P〈0．01）;ELISA方法检测,在转染后第1天组Ⅰ培养波中即有bFGF蛋白的分泌表达,第3天达峰值后分泌量逐渐下降,第8天仍能检测到少量分泌,而组Ⅱ和组Ⅲ培养液中均未检测到bFGF蛋白。结论成功地将所构建的Ad. bFGF转染体外培养的SMCs,并在培养液中检测到目的基因的蛋白表达。     

重组人bFGF蛋白的克隆、表达、纯化及鉴定

【目的】构建人碱性成纤维细胞生长因子（bFGF）基因的原核表达载体,并对其进行诱导表达和蛋白质纯化,以获得大量重组人bFGF蛋白。【方法】人工合成bFGF基因,进行测序分析,将该基因克隆入原核表达载体pET22b中,转化感受态细胞大肠杆菌RPX,经IPTG诱导表达重组bFGF蛋白,对表达产物进行SDS-PAGE电泳分析和Western blotting检测分析。【结果】成功构建了人重组bFGF表达质粒pET22b-rhbFGF,表达的蛋白经SDS-PAGE电泳分析,分子量约在18×103Da处出现了一条新生的蛋白条带,经灰度扫描检测,表达量约占菌体总蛋白的28%,纯化后的蛋白质灰度扫描检测,纯度为95%。【结论】成功构建了重组人bFGF原核表达载体,并成功纯化了该基因的原核表达产物,为下一步人bFGF的产业化打下了基础。     

碱性成纤维细胞生长因子真核表达载体转染骨髓间充质干细胞的表达

目的：探讨碱性成纤维细胞生长因子(bFGF)真核表达载体转染骨髓间充质干细胞(MSCs)后的表达情况。方法：应用脂质体介导的基因转移技术将bFGF真核表达载体导入MSCs,用抗药性标志选择方法筛选出阳性克隆后,用免疫细胞化学检测bFGF表达分泌情况。结果与结论：经转染bFGF真核表达载体的MSCs细胞呈阳性,说明经转染的MSCs表达并分泌bFGF蛋白。MSCs有望成为bFGF基因治疗中枢神经系统疾病的理想载体。     

Cloning and expression of rat transforming growth factor β1 cDNA in osteoblasts

Summary Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gene was confirmed to code rat TGFβ1 by restriction enzyme analysis. pcDNA3-TGFβ1 plasmid was transfected into rat osteoblasts by using liposome-mediated gene transfer technique and the expression of TGFβ1 was detected by using immunohistochemical staining assay. It was found that the rat TGFβ1 expression product was obviously detectable in the transfected osteoblasts in 48 h. High expression of TGFβ1 was obtained in the rat osteoblasts in which the constructed TGFβ1 expression vector was transfected.     

BC023882基因克隆及其对细胞周期的影响

目的获得基因BC023882全长真核表达载体及检测该基因对培养细胞生长的影响.方法利用逆转录-多聚酶链反应(RT-PCR)扩增基因BC023882的编码区序列,克隆到pcDNA3.1(-)/Myc-His( )/lac Z真核表达载体中,构建基因BC023882的真核表达载体;脂质体法转染小鼠胚胎成纤维细胞和COS-7细胞,并且通过细胞生长曲线测定和流式细胞仪观察该基因对细胞生长的影响.结果酶切并测序鉴定证明获得的重组子符合基因BC023882的编码序列;转染该基因后细胞增殖减慢,细胞增殖指数减低.结论成功构建了基因BC023882的真核表达载体,并且观察到该基因对细胞生长起到一定的抑制作用.     

siRNA沉默新生大鼠肺成纤维细胞转化生长因子-β1的表达及临床意义

目的利用RNA干扰技术（RNAi）抑制转化生长因子β1（TGF—β1）在新生大鼠肺成纤维细胞中的表达并探讨其临床意义。方法构建TGF—β1小分子干扰RNA（siRNA）重组表达载体,测序鉴定后转染新生大鼠肺成纤维细胞,应用RT—PCR检测其对TGF—β1mRNA表达的影响。结果实验组与对照组相比,TGF—β1mRNA的表达明显下调（P〈0．01）,其中以T1实验组的沉默效率最为显著（P〈0．05）。结论成功构建并筛选出能高效抑制TGF—β1表达的siRNA真核表达载体,有望成为临床治疗肺间质纤维化的新靶点。     

低氧诱导因子-1α对毛囊细胞的作用

目的探讨外源性人低氧诱导因子-1α（HIF-1α）在成纤维细胞中表达的可行性及其对毛囊周围细胞活性的影响。方法通过脂质体将含有HIF-1αcDNA的真核表达载体pcDNA3.0稳定转染成纤维细胞,应用RT-PCR及Westernblot方法检测HIF-1α在成纤维细胞中的表达,ELISA法检测转染细胞上清液中血管内皮细胞生长因子（VEGF）的表达,RT-PCR方法检测转染后细胞中成纤维细胞生长因子（bFGF）的表达。将该上清加入成纤维细胞及真皮鞘细胞中,MTT法检测加入上清后成纤维细胞及真皮鞘细胞活性。结果RT-PCR及Westernblot可检测出转染后细胞中HIF-1α的表达,MTT检测加入转染上清后成纤维细胞及真皮鞘细胞活性增强（P〈0.05）,并且该上清液VEGF的表达显著高于未转染组（P〈0.01）。转染后成纤维细胞的bFGF的mRNA表达显著高于未转染组（P〈0.01）。结论应用脂质体能够成功地将外源性人HIF-1α基因转染成纤维细胞,并进行有效表达,其表达的HIF-1α可增强细胞活性且可诱导转染细胞上清液中VEGF的表达,并增加bFGF的mRNA表达,且转染细胞上清可增强成纤维细胞及真皮鞘细胞活性。推测HIF-1α通过其下游因子VEGF及bFGF促进毛囊相关细胞的活性,为HIF-1α对毛囊作用的进一步研究打下了基础。     

人碱性成纤维细胞生长因子真核表达载体的构建及表达

目的: 构建研究人碱性成纤维细胞生长因子(bFGF)真核表达载体,并在体外进行表达和检测.方法: 从骨髓基质干细胞中分离总RNA,进行RT-PCR 获得人bFGF cDNA.测序证实其结果正确后,构建真核表达载体.并经BamHⅠ/EcoRⅠ双酶切、PCR及测序鉴定.利用Blast程序,搜索NCBI GenBank中与重组质粒(pVAX1-bFGF)中编码bFGF基因同源的序列.通过脂质体将重组质粒转染入骨髓基质干细胞,使用间接免疫荧光法进行表达产物检测.结果: 经EcoRⅠ/Pst BamHⅠ/EcoRⅠ双酶切、PCR及测序鉴定证实,人bFGF基因成功地克隆到真核表达载体pVAX1中;pVAX1- bFGF中编码bFGF的序列与GenBank中所报道的bFGF编码序列完全一致;间接免疫荧光结果显示转染重组质粒的细胞表面有绿色荧光.结论: 成功地构建了人bFGF真核表达载体,其可以在体外进行表达.     

Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3. 1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3. 1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5% and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3. 1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.