脂肪干细胞/Ⅰ型胶原凝胶复合体的构建及其体内外成骨分化研究

目的探讨利用脂肪干细胞与Ⅰ型胶原凝胶复合构建新型骨组织工程复合体的可行性并对其体内外成骨分化情况进行分析。方法取3月龄日本大耳白兔肩胛部皮下脂肪，Ⅰ型胶原酶消化获得细胞，于体外依次进行骨、软骨、脂肪多向诱导分化鉴定；取第3代细胞与Ⅰ型胶原凝胶复合，于成骨诱导条件下体外培养，相差显微镜、扫描电镜观察复合情况并对其碱性磷酸酶、细胞外基质矿化程度进行检测，同时设立单层贴壁培养细胞作为对照（n=4）；两周后移植于裸鼠皮下，12周后处死动物，依次行放射学、组织学检测观察成骨情况（n=3）。结果（1）所获细胞具备骨、软骨、脂肪多向分化潜能，为多能干细胞。（2）形态学观察显示脂肪干细胞呈三维立体生长状态，均匀、高密度悬浮于Ⅰ型胶原凝胶中。在体外成骨诱导条件下，其碱性磷酸酶活性以及外基质矿化程度均显著高于单层贴壁培养细胞（P〈0．01，n=4）。（3）于裸鼠皮下移植12周后，放射学、组织学检测显示脂肪干细胞．Ⅰ型胶原凝胶复合体成骨明显（n=3）。结论（1）IⅠ型胶原凝胶可显著促进脂肪干细胞的成骨分化。（2）作为一种新型的骨组织工程复合物，脂肪干细胞-Ⅰ型胶原凝胶复合体可应用于骨组织工程，特别是腔隙性骨缺损的修复过程。     

三羟基异黄酮对增生性瘢痕成纤维细胞增殖与胶原合成作用的影响

目的探讨5,7,4′-三羟基异黄酮对体外培养的人增生性瘢痕成纤维细胞增殖及胶原合成作用的影响。方法以不同浓度三羟基异黄酮处理体外培养的人增生性瘢痕成纤维细胞分为25、50、100μmol/L三组,以加DMSO溶剂为对照组,MTT法测定成纤维细胞的增殖,3H-脯氨酸掺入法测定细胞胶原合成情况,逆转录聚合酶链式反应（RT-PCR）检测Ⅰ、Ⅲ型前胶原mRNA的表达。结果三羟基异黄酮能有效抑制增生性瘢痕成纤维细胞的增殖及胶原合成（P〈0.05）,且具有剂量-效应关系;100μmol/L浓度组作用后,与对照组相比,差异有显著意义（P〈0.01）;三羟基异黄酮作用后成纤维细胞Ⅰ、Ⅲ型前胶原mRNA的表达显著下调（P〈0.05）。结论三羟基异黄酮能抑制增生性瘢痕成纤维细胞的增殖与活性,可能成为治疗瘢痕及纤维化疾病的有效药物。     

A reasonable mechanism for visible light-induced skin rejuvenation

In recent years, much research has been done in the field of non-ablative skin rejuvenation. This comes as a response to the continuous demand for a simple method of treating rhytides, UV exposure, and acne scars. Numerous researches involve visible light-pulsed systems (20–30 J/cm²). The mechanism of action is believed to be a selective heat-induced denaturalization of dermal collagen that leads to subsequent reactive synthesis (Bitter Jr., Dermatol. Surg., 26:836–843, 2000; Fitzpatrick et al., Arch. Dermatol., 132:395–402, 1996; Kauvar and Geronemus, Dermatol. Clin., 15:459–467, 1997; Negishi et al., Lasers Surg. Med., 30:298–305, 2002; Goldberg and Cutler, Lasers Surg. Med., 26:196–200, 2000; Hernandez-Perez and Ibeitt, Dermatol. Surg., 28:651–655, 2002). In this study, we suggest a different mechanism for photorejuvenation based on light-induced reactive oxygen species (ROS) formation. We irradiated collagen in vitro with a broadband of visible light (400–800 nm, 24–72 J/cm²) and used the spin trapping coupled with electron paramagnetic resonance spectroscopy to detect ROS. Irradiated collagen resulted in hydroxyl radicals formation. We propose, as a new concept, that visible light at the energy doses used for skin rejuvenation (20–30 J/cm²) produces high amounts of ROS, which destroy old collagen fibers, encouraging the formation of new ones. On the other hand, at inner depths of the skin, where the light intensity is much weaker, low amounts of ROS are formed, which are well known to stimulate fibroblast proliferation.     

Quantitative Monitoring of Extracellular Matrix Production in Bone Implants by <Superscript>13</Superscript>C and <Superscript>31</Superscript>P Solid-State Nuclear Magnetic Resonance Spectroscopy

We used ³¹P and ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy to detect and analyze the major organic and inorganic components (collagen type I and bioapatite) in natural rabbit bone and β-tricalcium phosphate implants loaded with osteogenically differentiated mesenchymal stem cells. High-resolution solid-state NMR spectra were obtained using the magic-angle spinning (MAS) technique. The ³¹P NMR spectra of bone specimens showed a single line characteristic of bone calcium phosphate. ¹³C cross-polarization (CP) MAS NMR spectra of bone exhibited the characteristic signatures of collagen type I with good resolution for all major amino acids in collagen. Quantitative measurements of ¹³C-¹H dipolar couplings indicated that the collagen segments are very rigid, undergoing only small amplitude fluctuations with correlation times in the nanosecond range. In contrast, directly polarized ¹³C MAS NMR spectra of rabbit bone were dominated by signals of highly mobile triglycerides. These quantitative investigations of natural bone may provide the basis for a quality control of various osteoinductive bone substitutes. We studied the formation of extracellular bone matrix in artificial mesenchymal stem cell-loaded β-tricalcium phosphate matrices that were implanted into the femoral condyle of rabbits. The NMR spectra of these bone grafts were acquired 3 months after implantation. In the ³¹P NMR spectra, β-tricalcium phosphate and bone calcium phosphate could be distinguished quantitatively, allowing recording of the formation of the natural bone matrix. Further, ¹³C CPMAS allowed detection of collagen type I that had been produced in the implants. Comparison with the spectroscopic data from natural bone allowed assessment of the quality of the bone substitute material. J. S. and M. P. contributed equally to this study     

术前MRI判断垂体腺瘤质地及其与胶原含量的对照

目的探讨术前MR I判断垂体腺瘤质地的可能性,及其与肿瘤胶原含量的关系。方法57例经手术证实的垂体腺瘤标本,根据术中肿瘤硬度分为质地韧(纤维化)组8例与质地软(非纤维化)组49例,所有病例术前均行MR I检查并测量T1加权像(T1W I)、T2加权像(T2W I)、T1增强像(T1 W I)上肿瘤与灰、白质的信号强度比(瘤体/灰质、瘤体/白质);标本行天狼猩红染色检测其胶原含量。结果纤维化与非纤维化组T W I瘤体/灰质分别为1.31±0.31、1.16±0.26;T1W I瘤体/白质分别为1.01±0.25、0.91±0.18,两组间无显著统计学差异(P>0.05)。T1 W I瘤体/灰质分别为1.83±0.28、1.84±0.51;T1 W I瘤体/白质分别为1.56±0.24、1.46±0.37两组间无显著统计学差异(P>0.05)。T2W I瘤体/灰质分别为1.15±0.26、1.57±0.46(P<0.05);T2W I瘤体/白质分别为1.48±0.39、2.10±0.61(P<0.01)两组间有显著统计学差异。胶原含量分别为20.03%±7.99%、7.87%±4.82%,两者有显著统计学差异(P<0.01);且T2W I瘤体/灰质、T2W I瘤体/白质与胶原含量呈显著负相关(r=-0.531、r=-0.726,P均<0.01)。结论术前MR I可以预测垂体腺瘤的质地;其中以T2W I瘤体/白质作预测指标为最佳,T2W I瘤体/白质小于1.9者提示腺瘤质地较硬。胶原含量影响垂体腺瘤质地,MR I上T2W I瘤体/白质、T2W I瘤体/灰质可以反映其胶原含量的多少。     

MR评价垂体腺瘤质地及与其胶原含量的关系

目的利用MR测量垂体腺瘤不同质地的信号强度,并分析其与肿瘤胶原含量间的关系。方法57例经手术证实的垂体腺瘤标本,术前均行MR检查,根据术中肿瘤硬度分为质地韧组(8例)与质地软组(49例),标本行天狼猩红染色检测其胶原含量。结果纤维化与非纤维化组T1WI瘤体/灰质信号强度比分别为1.31±0.31、1.16±0.26;T1WI瘤体/白质信号强度比分别为1.01±0.25、0.91±0.18,两组间差异无统计学意义(P(0.05);T1+WI瘤体/灰质信号强度比分别为1.83±0.28、1.84±0.51;T1+WI瘤体/白质信号强度比分别为1.56±0.24、1.46±0.37,两组间差异无统计学意义(P(0.05);T2WI瘤体/灰质信号强度比分别为1.15±0.26、1.57±0.46(P(0.05);T2WI瘤体/白质信号强度比分别为1.48±0.39、2.10±0.61,两组间有统计学意义(P(0.01);胶原含量分别为20.03%±7.99%、7.87%±4.82%,两组间有统计学意义(P<0.01);且T2WI瘤体/灰质、T2WI瘤体/白质与胶原含量呈显著负相关(r=-0.531、r=-0.726,P均(0.01)。结论术前MR可以预测垂体腺瘤的质地;其中以T2WI瘤体/白质为预测指标为最佳。胶原含量影响垂体腺瘤质地,MRI上T2WI可以反映其胶原含量的多少。     

暹罗鳄鱼鳞胶原蛋白多肽对小鼠免疫功能的影响

目的 观察暹罗鳄鱼鳞胶原蛋白多肽对小鼠免疫功能的影响.方法 采用环磷酰胺为免疫抑制剂,获得免疫功能低下的小鼠动物模型.比较小鼠服用胶原蛋白前后,小鼠T淋巴细胞增殖功能、NK细胞的杀伤功能以及小鼠碳粒廓清能力.结果 胶原蛋白多肽对免疫功能低下小鼠T淋巴细胞的增殖功能、NK细胞的杀伤活性,均具有免疫增强作用（P ＜0.05）,并呈剂量依赖性.结论鳄鱼鳞胶原蛋白多肽具有正向调节小鼠免疫功能的能力.     

大鼠慢性皮肤溃疡创面愈合中转化生长因子-β1、胶原Ⅰ、胶原Ⅲ的蛋白表达

目的 观察大鼠慢性皮肤溃疡创面愈合过程中转化生长因子-β1( TGF-β1)、胶原Ⅰ和胶原Ⅲ的蛋白表达。方法 将24只8周龄雌性Wister大鼠分为单纯创面组(A组)和皮瓣+创面组即缺血模型组(B组),每组各12只;苏木素-伊红(HE)染色法观察创面1、3、7、10d上皮化率、收缩率及中性粒细胞;采用酶联免疫吸附试验(ELISA)方法测定创面1、3、7、10 d TGF-β1、Ⅰ型和Ⅲ型胶原的蛋白表达。结果 A组上皮化率在各个时间段均高于B组,且在第7天差异有统计学意义(P＜0.05)。A组收缩率明显低于B组。A组中性粒细胞第1、3天逐渐增加,第3天增加到最多,随后逐渐减少;B组在1、3、7d出现增加趋势,第7天增加到最多,第10天减少。TGF-31含量A组于术后1、3、7、10d呈曲线上升趋势,B组在术后1、3、7d逐渐减低,10 d较7d略有回升,且在第1天两组差异有统计学意义(P＜0.05)。胶原Ⅰ蛋白的含量两组随着术后时间的延长均呈减少趋势,在第10天两组差异有统计学意义(P＜0.05)。胶原Ⅲ蛋白的含量两组随术后时间的延长也呈减少的趋势,但在第3天A组比B组明显增加,差异有统计学意义(P＜0.05)。结论 在缺血的干预因素作用下TGF-β1、Ⅰ型和Ⅲ型胶原蛋白表达的减少可能延迟了慢性创伤的正常愈合。     

铜绿假单胞菌感染大鼠皮肤创面愈合过程中TGF-β1及胶原蛋白含量的变化

目的 探讨大鼠慢性皮肤溃疡创面感染铜绿假单胞菌后,TGF-β1和胶原Ⅰ、Ⅲ蛋白表达的变化.方法 24只8周龄雌性Wister大鼠随机分为单纯创面组(A组)和创面+铜绿假单胞菌接种组(B组).分别在术后第1、3、7、10天观察创面上皮化率、收缩率及中性粒细胞情况;并采用ELISA方法测定创面第1、3、7、10天TGF-β1和胶原Ⅰ、Ⅲ蛋白的表达情况.结果 A组上皮化率在第7天高于B组,收缩率低于B组.随着时间的延长,A组中性粒细胞在第3天增加到最多,随后逐渐减少,而B组中性粒细胞第1天达到最多.2组TGF-β1表达在术后呈上升趋势,B组TGF-β1在第3天降低,随着时间延长回升,第7天2组比较差异有统计学意义(P＜0.05).胶原Ⅰ、Ⅲ蛋白表达随着时间的延长呈下降趋势,B组胶原Ⅲ蛋白表达在第7、10天差异有统计学意义(P＜0.05).A组第1、3天胶原Ⅰ、Ⅲ蛋白表达高于B组,在第7、10天则低于B组,且胶原Ⅲ蛋白在第3天显示A组高于B组,差异有统计学意义(P＜0.05).结论 皮肤溃疡创面感染铜绿假单胞菌后延迟了TGF-β1和胶原Ⅰ、Ⅲ蛋白的表达,可能影响创面的正常愈合.

Abstract：

Objective To explore the different expression of TGF-β1 and collagen during the healing process of wound infected by pseudomonas aeruginosa(PAO1).Methods 24 female Wister rats were randomly divided into pure wound group(group A)and wound+PAO1 group(group B).The reepithelial rate,shrinkage rate and neutrophils number on the wounds were observed on the 1st,3rd,7th and 10th day after operation.The expression of TGF-β1 and collage Ⅰ,Ⅲ was also detected.Results On the 7th day,the re-epithelial rate in group A was higher than that in group B,while the shrinkage rate in group A was lower than that in group B.The neutrophils number increased to peak on the 1 st day in group B,but on the 3rd day in group A.The TGF-β1 expression increased after operation in both groups,but it decreased in group B on the 3rd day and re-increased after that.The TGF-β1 expression was significantly different between the two groups on the 7th day(P＜0.05).The expression of collagen Ⅰ and Ⅲ decreased during healing.The expression of collagen Ⅲ in group A was higher on the 3rd day and was lower on the 7th and 10th day than that in group B,showing a significant difference(P＜0.05).Conclusions PAO1 infection could delay the expression of TGF-β1 and collagen Ⅰ,Ⅲon wound,which may interfere the healing process of wound.     

The effect of the microscopic and nanoscale structure on bone fragility

Bone mineral density is the gold-standard for assessing bone quantity and diagnosing osteoporosis. Although bone mineral density measurements assess the quantity of bone, the quality of the tissue is an important predictor of fragility. Understanding the macro- and nanoscale properties of bone is critical to understanding bone fragility in osteoporosis. Osteoporosis is a disease that affects more than 75 million people worldwide. The gold standard for osteoporosis prognosis, bone mineral density, primarily measures the quantity of bone in the skeleton, overlooking more subtle aspects of bone's properties. Bone quality, a measure of bone's architecture, geometry and material properties, is evaluated via mechanical, structural and chemical testing. Although decreased BMD indicates tissue fragility at the clinical level, changes in the substructure of bone can help indicate how bone quality is altered in osteoporosis. Additionally, mechanical properties which can quantify fragility, or bone's inability to resist fracture, can be changed due to alterations in bone architecture and composition. Recent studies have focused on examination of bone on the nanoscale, suggesting the importance of understanding the interactions of the mineral crystals and collagen fibrils and how they can alter bone quality. It is therefore important to understand alterations in bone that occur at the macro-, micro- and nanoscopic levels to determine what parameters contribute to decreased bone quality in diseased tissue.