表达神经营养因子Neurturin的骨髓间充质干细胞的构建及体外活性检测

研究神经营养因子Neurturin(NTN)在由于神经元损伤而造成的神经退行性疾病中对神经元的保护和修复作用。利用重组腺病毒载体将NTN基因转入恒河猴骨髓间充质干细胞(rMSC),通过RT-PCR、IF及Western blot方法检测NTN的转录和表达,并采用鸡胚背根神经节体外培养实验和胚胎大鼠中脑多巴胺能神经元存活实验对NTN进行体外活性检测。结果表明NTN在rMSC中稳定表达和分泌,并具有体外生物学活性,为由于神经元损伤造成的神经退行性疾病的干细胞移植治疗奠定了一定的基础。     

VEGF165 induces differentiation of hair follicle stem cells into endothelial cells and plays a role in in vivo angiogenesis

Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi‐directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs’ directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host‐derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases.     

人白血病抑制因子在原核细胞中的表达与纯化

研究证明人白血病抑制因子(hLIF)是一种对多种不同类型的细胞和组织具有重要功能的细胞因子,其独特的生物学特性使其被广泛应用。介绍了自制的具有生物活性的hLIF,将hLIF基因克隆到pET32a中,并利用硫氧还蛋白(Trx)作为融合配体,在大肠杆菌中成功表达可溶性融合蛋白Trx-hLIF。亲和层析纯化后利用SDS-PAGE和Western blot对纯化结果进行检验。利用肠激酶(EK酶)切割融合蛋白,释放hLIF,随后通过简单的阳离子交换得到纯度高达98.1%的hLIF4.75mg。小鼠M1髓系白血病细胞增殖分析测定纯化的rhLIF的功能与hLIF具有相似的生物活性,EC50为5ng/ml,对应的比活性为0.5×107 IU/mg。     

阴道镜直视宫颈多点活检与传统宫颈活检在宫颈疾病诊断中的应用价值比较

目的:探讨阴道镜直视宫颈多点活检与传统宫颈活检对宫颈疾病诊断的临床应用及影响因素研究。方法:选择2015年5月至2016年3月我院妇科收治的宫颈疾病患者86例,按照随机数表法,将患者分为对照组和研究组,每组43例。对照组患者进行常规的宫颈活检,研究组患者采用阴道镜直视宫颈多点活检。对比两种检查方法与病理检查结果的检出率,并比较两种检查方法的漏诊率,另外考察影响阴道镜直视宫颈多点活检漏诊率的相关因素。结果:与病理检查结果相比,除宫颈炎和宫颈癌外,研究组对其他类型宫颈疾病的检出率及总检出率明显高于对照组(P0.05);除宫颈癌外,研究组对各种宫颈疾病的漏诊率与对照组相比明显下降(P0.05);年龄超过50岁,高级别的CIN类型、标本个数的减少及宫颈微小病变都可能升高阴道镜直视宫颈多点活检漏诊率。结论:与传统宫颈活检相比,阴道镜直视宫颈多点活检对宫颈疾病的诊断具有较高的诊断率,并且漏诊率较低,可以适当增加活检的样本个数来降低漏诊率,从而尽早发现并进行有效治疗,值得在临床上推广应用。     

小鼠成纤维细胞凋亡与bcl-2结合蛋白

为检测细胞凋亡过程中bcl-2基因的结合蛋白,用PCR技术扩增小鼠bcl-2基因(mbcl-2)调控区,经亚克隆后的序列分析证实其准确性.用5-氟尿嘧啶(5-Fu)诱导小鼠成纤维细胞(C3H 10 T1/2 Cl 8)凋亡,以此PCR产物作探针,对上述细胞的核蛋白质粗提物进行迁移位移法(EMSA)与DNA-蛋白质印迹分析.结果表明,细胞核内一分子质量为53 ku的结合蛋白与mbcl-2调控区的结合信号在5-Fu刺激12 h后明显增强,而一种100 ku的DNA结合蛋白与该区的结合信号在5-Fu刺激12 h后明显减弱.因而,p53蛋白有可能是bcl-2的负转录因子,100 ku的DNA结合蛋白有可能是bcl-2的正转录因子.     

一个Ⅰ型遗传性淋巴水肿中国家系的VEGFR3基因的新突变方式

通过对国人Ⅰ型遗传性淋巴水肿一家系分子遗传学检测,报告VEGFR-3基因新突变。首先在Ⅰ型遗传性淋巴水肿对该家系进行致病基因的连锁分析,然后用DNA直接测序方法进行基因突变分析。连锁分析和单倍体分析确定该家系致病基因位于5q35.3,与Ⅰ型遗传性淋巴水肿连锁。VEGFR-3基因突变分析发现了一个新的错义突变D1055V,该错义突变在家系中共分离,且在100个正常对照组中未发现该序列改变。本研究首次报告了国内Ⅰ型遗传性淋巴水肿VEGFR-3基因新的错义突变D1055V,丰富了VEGFR-3基因基因突变谱,为今后开展遗传性淋巴水肿的基因诊断和遗传咨询奠定基础。     

Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling

Laminin alpha chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin alpha5 in mouse submandibular glands (SMGs). Lama5(-/-) SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs alpha5 and alpha1 are present in epithelial clefts but as branching begins alpha5 expression increases while alpha1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-beta1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that beta1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3(-/-):Itga6(-/-) SMGs have a similar phenotype to Lama5(-/-). Our findings suggest that laminin alpha5 controls SMG epithelial morphogenesis through beta1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling.