Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

^60Coγ射线全身照射后大鼠空肠亮脑啡肽的变化

应用免疫细胞化学(ICC)和放射免疫分析(RIA)方法观察了大鼠4、25Gy~(60)Coγ射线照射后24,48和,72h空肠亮脑啡肽(L-ENK)样免疫反应性神经的分布和L-ENK含量的变化。结果:4Gy照射后24h以及25Gy照射后24,48和72h光镜下见空肠L-ENK样免疫反应性神经的分布密度稍有增加,4Gy照射后24h以及25Gy照射后24,48和72h空肠L-ENK含量分别为29.81±0.84pg/mg,34.96±4.38pg/mg,40.71±3.62pg/mg和38.93±2.31pg/mg,与正常对照组(18.26±1.95pg/mg)相比明显升高(P<9.01)。     

Anti-anti-idiotypic (Ab3) antibodies that bind progesterone-11alpha-bovine serum albumin differ in their combining sites from antibodies raised directly against the antigen

Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antiprogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone response in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-50-times lower than that of DB3, their steroid-binding specificity showed considerable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal antibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progesterone conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound free progesterone, although with low affinity and very broad cross-reactivity. Like DB3, 1A4 is encoded by a heavy-chain variable region (VH) gene segment from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11alpha-BSA. In contrast, 3B11 binds progesterone-11alpha-BSA but not free progesterone and is encoded by an unrelated VH gene from the J558 family. The light chain variable region (VL) of 1A4 lacks the intradomain disulphide bridge owing to replacement of CysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short complementarity determining region (CDR) H3 loops, comprising three and four amino acids, respectively. Modelling of the combining site of 1A4 from the X-ray crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.     

A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts

The bilayer structure of chitosan film and sponge was designed as a scaffold of skin tissue engineering and a dermis substitute. It was processed successively via the formation of a dense chitosan film by casting method and a porous chitosan sponge by lyophilization. The dry thickness of the film layer was 19.6 microm and that of the sponge layer was controlled at 60-80 microm. Porogens such as sodium chloride, glucose, and sucrose were used to create large pores of the chitosan sponge layer. Human neofetal dermal fibroblasts were seeded in the chitosan sponge layer and cultured for 4 weeks. It was found that the cells could grow and proliferate well in an extended shape on the flat bottom of large pores with 15-100 microm width and in spherical form on the rough pore walls or at the edges of micropores less than 5 microm. Fibroblasts after the culture could bind tightly with the sponge layer via newly formed extracellular matrices to give a living cell-matrix-chitosan composite. The bilayer chitosan material remained stable in shape and size during the cell culture. The results suggested that the bilayer chitosan material would be an alternative of collagen materials which was obviously contracted during cell culture.     

Delayed angiogenesis in aging rats and therapeutic effect of adenoviral gene transfer of VEGF

We have studied an age-related impairment in angiogenesis and evaluated the effect of overexpressing VEGF in this situation. Polyvinyl alcohol sponges were implanted subcutaneously into aged (24-month), adult (12-month), and young (2-month) rats. Blood vessel ingrowth and proliferative activity in the sponges were assessed by histology with immunostaining for von Willebrand's factor and proliferating cell nuclear antigen (PCNA), respectively. The percentage of total sponge area filled with ingrowing fibrovascular tissue was minimal in aged rats, intermediate in adult rats and highest in young rats. A similar pattern was observed for the total blood vessel numbers in the sponges from old to young animals. The percentage of total sponge endothelial cells (ECs) showing proliferative activity (PCNA positive) was lowest in the aged animals, intermediate in the adult rats and highest in the young rats. To further explore the mechanism of impaired angiogenesis in aged animals, we investigated and found a reduced level of endogenous VEGF protein expression in 12-month-old rats compared to that in 2-month-old rats. VEGF121 gene transfer significantly enhanced blood vessel and fibrovascular tissue ingrowth in adult/aged rats. Adenoviral-VEGF gene transfer also significantly stimulated EC proliferation in aged and adult rats. However, identical treatment failed to further stimulate the already more robust angiogenesis in young animals. The different angiogenic response in adult vs. young rats was not due to differences in gene transfer efficiency, since similar levels of human VEGF121 protein was detected in adult and young rats. Our results indicate that the decreased angiogenic response with aging is associated with reduced EC proliferation and reduced endogenous VEGF production. Adenoviral-VEGF121 gene transfer is effective in augmenting angiogenesis, particularly in older animals.     

Association between thyroid cancer of cribriform variant and familial adenomatous polyposis.

A case of a 20 year old Japanese woman who developed thyroid cancer exhibiting unusual cribriform structures while being followed up for familial adenomatous polyposis/Gardner's syndrome is reported. The patient presented with osteomas, pigmented retinal lesions, and adenomas of the duodenum and the papilla of Vater, in addition to numerous adenomatous polyps in the colorectum. On ultrasonography, the thyroid cancer was localised to the right lobe and was identified as an irregular, internal echo tumour with a peripheral hypoechoic zone, measuring 1.8 cm in diameter. Histological examination of the resected tumour showed a concomitance of papillary proliferation and cribriform structures with follicles of varying sizes. These features can be distinguished from sporadic thyroid cancer.     

脂质、血红蛋白、胆红素标本对乙肝病毒DNA荧光定量测定的影响

目的:探讨脂血、高胆红素和溶血标本对乙肝病毒DNA(HBVDNA)荧光定量测定结果的影响。方法:将乙肝大三阳高脂血和非脂血、溶血血清和未溶血血清同时作HBVDNA荧光定量检测;将HBVDNA阴性黄疸血清和HBVDNA阴性正常血清与来自同一份乙肝大三阳血清混合,在相同条件下进行HBVDNA荧光定量。结果:乙肝大三阳溶血与未溶血样本HBVDNA含量都在同一数量级。乙肝大三阳高脂血的HBVDNA含量明显低于对照标本。高黄疸血清、正常对照血清与相同的HBVDNA阳性模板组合后所测得的HBVDNA结果无差异。结论:脂血对HBVD-NA定量测定有严重干扰;溶血样本、高胆红素样本对HBVDNA测定结果无影响。     

IL-1β诱导体外培养的大鼠中脑黑质神经元c-jun表达

应用细胞原位杂交技术，观察经重组小鼠白细胞介素-19（IL-1β）处理后的体外培养的新生1d大鼠中脑黑质神经元c－jun基因的表达．结果显示，培养的黑质细胞多为酪氨酸羟化酶阳性神经元，IL-1β可诱导体外培养的黑质神经元c－junmRNA表达，高水平的表达出现在IL-1β处理后2～4h。说明IL－1β有兴奋黑质神经元的作用，并提示黑质神经元上可能存在IL－1β受体．     

IL-12p40-overexpressing immature dendritic cells induce T cell hyporesponsiveness in vitro but accelerate allograft rejection in vivo: role of NK cell activation and interferon-gamma production

Infusion of genetically modified dendritic cells (DC) expressing immunosuppressive molecules is a potential therapy for organ rejection. IL-12p70, a cytokine produced mainly by DC and macrophages, consists of two subunits, p40 and p35. IL-12p70 is an activator of T cells, while the IL-12p40 subunit serves as a natural antagonist for IL-12p70 action. The primary aim of this study was to evaluate the effect of IL-12p40 gene-modification on both the T-cell stimulatory activity of immature DC (imDC) and their ability to prolong cardiac allograft survival. IL-12p40 gene-modified imDC (DC-p40) exhibited a phenotype characteristic of imDC and displayed impaired T-cell allostimulatory ability in vitro. However, to our surprise, for murine vascularized heterotopic heart transplantation (HHT), administration of donor-derived DC-p40 7 days prior to transplantation did not prolong allograft survival but instead significantly exacerbated cardiac allograft rejection. Further study showed that DC-p40 augmented NK cell activity both in vitro and in vivo and enhanced interferon-gamma (IFN-gamma) production in vivo, which might be due to the increased IL-23 production by DC-p40. Our data suggested that although IL-12p40 gene-modified immature DC can induce T cell hyporesponsiveness in vitro, their ability to activate NK cells and induce IFN-gamma production counterbalances this, exacerbating cardiac allograft rejection. The unexpected effects of DC-p40 limit their value in promoting allograft survival in vivo and likely reflect the complexity of IL-12p40 biology.     

KOCH三角的解剖

本实验解剖了110例人心标本(成人70、儿童40)的Koch三角区,观察和测量了房室结的形态、大小、毗邻和标志。房间隔及冠状窦口上、下方均有肌束连于房室结。Todaro腱在儿童多全部为腱性;在成人,其后部为肌性。三角区的深面为左、右心房壁和室间隔顶所构成的锥形间隙,其内为进入房室结区的血管和神经。根据构成不同,可将三角区分为5个区,即前上角的纤维支架区和房室结区,其余部分自上向下分别为房间隔区、右房壁区和室间隔区。本文还讨论了这些形态结构的功能及外科意义。