新型雌激素受体β信号通路调节因子的分离及鉴定

目的：利用酵母双杂交技术筛选与ERβ相互作用的蛋白，为进一步研究ERβ的功能奠定基础。方法：从乳腺cDNA文库中钓取ERβ-AF1 cDNA，构建诱饵蛋白载体pAS2-1-ERβ—AF1，利用酵母双杂交筛选技术，在乳腺cDNA文库中筛选与ERβ-AF1相互作用的蛋白。将含有pACT2-候选基因的酵母菌与含有pAS2—1-ERp—AF1的酵母菌进行交配实验，利用LacZ报告基因检测ERβ—AF1与候选蛋白之间是否存在相互作用。然后将ERp—AF1和候选蛋白分别构建到pGEx-KG和pET-28a上，并纯化GST-ERβ-AF1和His-侯选蛋白融合蛋白，利用GST沉淀实验进一步验证ERβ-AF1和候选蛋白的体外相互作用。结果：所获取的候选蛋白为CTGF，是CCN多肽家族成员。交配实验表明，CTGF与ERβ-AF1特异相互作用，而不与空载体或BRCT1对照相互作用。GST沉淀实验进一步验证，CTGF与ERβ、ERα均存在相互作用，但与CTGF同源性较高的NOV蛋白却不与ERβ、ERa结合。结论：CTGF和ERβ存在特异的相互作用。     

前列腺素E1脂微球载体制剂对多柔比星肾病大鼠结缔组织生长因子表达的影响

目的研究多柔比星肾病大鼠肾组织中结缔组织生长因子(CTGF)表达,同时探讨前列腺素E1脂微球载体制剂(Lipo-PGE1)对其表达的影响.方法 将24只雌性SD大鼠(体重180～200 g)随机分为对照组、多柔比星肾病模型组和多柔比星肾病Lipo-PGE1治疗组3组.采用尾静脉一次性注射多柔比星7.5 mg/kg的方法建立多柔比星肾病动物模型,第8周开始Lipo-PGE1治疗组给予尾静脉注射Lipo-PGE1,用量200 μg/(kg*d).10周后处死全部大鼠,并观察肾组织病理改变,应用免疫组织化学方法和原位杂交技术检测CTGF在肾组织中的表达. 结果 Lipo-PGE1治疗组大鼠肾小球硬化及基质增生程度比多柔比星肾病组明显减轻,免疫组织化学染色及原位杂交结果显示多柔比星肾病组较正常对照组肾小球和肾小管区CTGF蛋白及CTGFmRNA表达量明显增加(P<0.05).Lipo-PGE1治疗组肾小球和肾小管间质CTGF蛋白及CTGFmRNA表达量明显低于多柔比星肾病组(P<0.05).结论 多柔比星肾病组大鼠第10周肾小球及肾小管间质尤其是肾小管间质区CTGF蛋白及CTGFmRNA表达量明显增加.Lipo-PGE1延缓多柔比星肾病肾损害,其机制可能与通过下调CTGF的表达有一定关系,并且可能通过减少肾小球内细胞增生和细胞外基质(ECM)沉积,延缓慢性肾脏疾病的进展.     

结缔组织生长因子在脑胶质瘤组织中的表达和肿瘤级别的关系

目的通过检测人脑胶质瘤患者肿瘤组织中结缔组织生长因子的表达水平,探讨其和肿瘤级别的关系及作用机制。方法采用荧光实时定量聚合酶链式反应(RT-PCR)法检测结缔组织生长因子(CTGF)mRNA在47例脑胶质瘤组织和25例正常脑组织中的表达水平,统计分析表达水平和肿瘤级别之间的关系。结果CTGF在胶质瘤Ⅰ-Ⅱ级(高分化组)及胶质瘤Ⅲ~Ⅳ级(低分化组)中的表达均明显高于正常脑组织(P<0.01),而且在Ⅲ~Ⅳ级胶质瘤中的表达量显著高于胶质瘤Ⅰ~Ⅱ级(P<0.01),说明随着肿瘤级别的升高CTGF表达也增强。结论CTGF在胶质瘤组织中高表达,而且表达水平和恶性程度有密切联系。CTGF的检测可作为胶质瘤恶性程度判断的参考,为从基因水平上探讨胶质瘤的生物学行为、预后及治疗提供新的思路。     

CTGF反义寡核苷酸治疗肺纤维化的实验研究

目的:观察CGTF反义寡核苷酸对体外培养的人肺成纤维细胞的抗增殖作用。方法:将人肺成纤维细胞随机分为对照组、空载体转染组、CTGF反义寡核苷酸(CTGF-ASODN)组、CTGF正义寡核苷酸(CTGF-SODN)组。CTGF错义寡核苷酸(CT-GF-SCDN)组,转染后观察细胞的增殖程度以及CTGF mRNA的表达。结果:转染了CTGF反义寡核苷酸(CTGF-ASODN)组人成纤维细胞的增殖明显受到抑制,其CTGF mRNA的表达明显减少。结论:应用CGTF反义寡核苷酸可以起到对人肺成纤维细胞的抗增殖作用。     

Meniscus ECM‐functionalised hydrogels containing infrapatellar fat pad‐derived stem cells for bioprinting of regionally defined meniscal tissue

Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)‐derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM‐functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad‐derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad‐derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM‐functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM‐functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox‐9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM‐functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.     

Sustained cardiomyocyte apoptosis and left ventricular remodelling after myocardial infarction in experimental diabetes

Aims/hypothesis Diabetes is known to reduce survival after myocardial infarction. Our aim was to examine whether diabetes is associated with enhanced cardiomyocyte apoptosis and thus interferes with the post-infarction remodelling process in myocardium in rat.Methods Four weeks after intravenous streptozotocin (diabetic groups) or citrate buffer (controls) injection, myocardial infarction was produced by ligation of left descending coronary artery. Level of cardiomyocyte apoptosis was quantified by TUNEL and caspase-3 methods. Collagen volume fraction and connective tissue growth factor were determined under microscope. Left ventricular dimensions were evaluated by echocardiography and planimetry.Results The number of apoptotic cardiomyocytes was equally high in diabetic and non-diabetic rats after 1 week from infarction. At 12 weeks after infarction the number of apoptotic cells was higher in the diabetic as compared to non-diabetic rats both in the border zone of infarction and in non-infarcted area. Correspondingly, left ventricular end diastolic diameter, relative cardiac weight, connective tissue growth factor-expression and fibrosis were increased in diabetic compared with non-diabetic rats with myocardial infarction.Conclusion/interpretation Sustained cardiomyocyte apoptosis, left ventricular enlargement, increased cardiac fibrosis and enhanced profibrogenic connective tissue growth factor expression were detected after myocardial infarction in experimental diabetes. Apoptotic myocyte loss could be an important mechanism contributing to progressive dilatation of the heart and poor prognosis after myocardial infarction in diabetes.Abbreviations STZ streptozotozin - MI myocardial infarction - CTGF connective tissue growth factor - LV left ventricular - LVEDD LV end-diastolic diameter - BNP B-type natriuretic peptide     

血清Cyr61及CTGF水平对胃癌进展的诊疗意义

目的 评估血清Cyr61及CTGF水平变化与胃癌进展的关系,为早期检测胃癌进展提供依据.方法 选择自2012年6月至2014年6月于铜川矿务局中心医院就诊的胃癌患者132例,其中Ⅰ期45例、Ⅱ期36例、Ⅲ期28例、Ⅳ期23例,对各组Cyr61及CTGF、胃癌肿瘤标志物进行检测.结果 Ⅱ期较Ⅰ期Cyr61及CTGF均有升高,差异有统计学意义(P＜0.05),Ⅲ期Cyr61及CTGF较Ⅱ期、Ⅰ期升高,差异有统计学意义(P＜0.05),Ⅳ期Cyr61及CTGF较Ⅰ期、Ⅱ期、Ⅲ期均有升高,差异有统计学意义(P＜0.05).Ⅱ期较Ⅰ期CEA、CA125均有升高,差异有统计学意义(P＜0.05);Ⅲ期CEA、CA199、CA125较Ⅱ期、Ⅰ期升高,差异有统计学意义(P＜0.05),Ⅳ期CEA、CA199、CA125较Ⅰ期、Ⅱ期、Ⅲ期均有升高,差异有统计学意义(P＜0.05).Cyr61与CEA、CA125显著正相关(P＜0.05),与CA199无相关性(P＞0.05).CTGF与CEA、CA125显著正相关(P＜0.05).结论 Cyr61及CTGF与胃癌肿瘤标志物等指标具有较好的相关性,是反映胃癌恶性程度及侵袭能力的重要指标.