动物皮肤,肌腱,软骨和骨的ECM制作

细胞外基质研究是组织工程学的重要部分，即是重要基础研究内容，又是研究细胞外基质的替代物课题。针对目前临床上大量应用人工合成的ＥＣＭ存在的问题（炎性反应，排斥反应、相容性等），本研究试图用动物的组织（自然的）ＥＣＭ替代人工合成的ＥＣＭ。用组织工程学方法处理动物组织，获得的产物经ＨＥ、奥新蓝染色、光镜观察和不染色切片偏光显微镜观察，证实经处理后的动物ＥＣＭ仅剩下肌腱、皮肤、软骨和骨的胶原框架。     

动物皮肤、肌腱、软骨和骨的ECM制作

细胞外基质（ＥＣＭ）研究是组织工程学的重要部分，即是重要基础研究内容，又是研究细胞外基质的替代物课题。针对目前临床上大量应用人工合成的ＥＣＭ存在的问题（炎性反应、排斥反应、相容性等），本研究试图用动物的组织（自然的）ＥＣＭ替代人工合成的ＥＣＭ。用组织工程学方法处理动物组织，获得的产物经ＨＥ、奥新蓝染色、光镜观察和不染色切片偏光显微镜观察，证实经处理后的动物组织ＥＣＭ仅剩下肌腱、皮肤、软骨和骨的胶原框架。     

皮肤组织工程

目的 综述近阶段皮肤组织工程研究领域中的进展。方法 广泛查阅国内外近期在皮肤组织工程研究的文献,着重阐述表皮替代物,真皮替代物,培养的表皮真皮复合皮片的研究进展和重要问题。结果 多数学者认为理想的皮肤替代物应及时重建已人的表皮和真皮结构。目前的研究主要集中在如何尽早移植表皮细胞,并保护移植后细胞的活性和功能,以及研制能更有效地促进细胞功能,诱导创床血植入,移植后可降解,无毒性,无病原携带风险的细胞     

细胞外基质与下肢静脉曲张关系研究

静脉壁的细胞外基质维持了静脉壁正常的结构和功能。曲张静脉壁的细胞外基质中各种成分的分布和含量发生了不同程度的改变，且对静脉曲张的形成具有重要的意义。本文就曲张静脉壁的细胞外基质的改变作一综述。     

口服胶原蛋白水解产物对皮肤的作用

胶原蛋白是结缔组织主要的结构蛋白,占全身总蛋白的25%～30%。皮肤中胶原蛋白主要分布于真皮层,含量约为70%,主要为I(85%)、III和Ⅴ型[1]。胶原蛋白纤维的网状架构为皮肤提供了弹性,纤维间分布着大量的水分、细胞外基质和细胞,是皮肤重要的生化反应场所[2]。随着人体衰老,皮肤中胶原     

皮肤扩张条件下真皮中细胞外基质变化的研究进展

皮肤软组织扩张术是整形外科重要的治疗手段之一,主要通过机械张力作用牵张皮肤,或借助牵拉组织皮肤缺氧促进皮肤细胞增殖,获得“额外”皮肤,以满足体表皮肤软组织缺损修复的需要。但扩张后真皮变薄,严重限制了其临床应用。而真皮主要由细胞外基质和成纤维细胞构,因此,探明缺氧或机械张力作用条件下细胞外基质的合成与降解规律具有重要意义。现就扩张条件下细胞外基质的变化规律作一综述。     

紫外线致皮肤光老化及光癌变机制的研究进展

日光的累计照射可加速皮肤老化与癌变,使皮肤的生物学及临床反应发生改变,包括急性损伤(日晒伤)和慢性损伤(光老化、光癌变或色素沉着等)。文章概述了近年来紫外线对皮肤光老化及光癌变影响的研究进展,揭示光老化和光致癌机制是通过紫外线照射产生活性氧和DNA损伤,以及由此引起的细胞损伤、炎症、免疫抑制、细胞外基质重塑及血管生成改变所致,为临床防治光老化和光癌变提供帮助。     

17β雌二醇对人皮肤成纤维细胞胶原和弹性蛋白合成的影响

目的探讨不同浓度17β雌二醇（17β-estrogen,17β-E2）在不同时间点对体外培养的人皮肤成纤维细胞（Human skin fibroblast,h SFB）合成胶原及弹性蛋白的影响。方法体外培养hSFB,分别加入不同浓度的17β-E2(10^-7、10^-8、10^-9、10^-10、10^-11mol/L),继续培养24 h、48 h、72 h,在不同时间点分别提取相应的RNA,RT-PCR检测经17β-E2处理后的hSFB的Ⅰ、Ⅲ型前胶原及原弹性蛋白mRNA的表达情况。结果 RT-PCR结果显示,17β-E2处理组较空白对照组前胶原及原弹性蛋白表达水平在24 h没有变化,48 h时明显上调,72 h时呈现下降;在48 h时,17β-E2对前胶原与原弹性蛋白的影响,10^-10 mol/L组上调作用较其他浓度组明显;在48 h时,同一浓度17β-E2对前胶原蛋白的促进作用较原弹性蛋白明显,尤其是对Ⅲ型的促进作用更强。结论 17β-E2对胶原及弹性蛋白的合成有一定的促进作用,不同时间、不同浓度对其影响不同,在10^-10 mol/l、48 h时作用最强。     

异体颗粒状脱细胞真皮基质与自体刃厚皮复合移植修复大鼠皮肤缺损创面效果观察

目的 观察异体颗粒状脱细胞真皮基质(PADM)与自体刃厚皮复合移植修复大鼠皮肤缺损创面的效果.方法 采用随机数字表法将12只SD大鼠分为实验组和对照组,每组6只.于2组大鼠背部制作全层皮肤缺损创面,实验组创面复合移植SD大鼠异体PADM(扩张比10:5)及厚度0.20 mm的自体刃厚皮,对照组创面仅移植厚度0.20 mm自体刃厚皮.术后2周起打开敷料观察大鼠创面愈合情况.术后2、3、4、6、8、12、20周计算2组创面移植皮片成活率、收缩率(或扩张率).术后20周取2组创周正常皮肤及创面皮肤标本,采用HE染色法观察胶原纤维束结构,测量胶原纤维束直径和间隙率;用天狼星红染色法观察Ⅰ、Ⅲ型胶原分布情况,测量Ⅰ、Ⅲ型胶原含量及其比值.对实验数据行独立样本t检验、Levene检验、t'检验.结果 (1)术后2周,实验组大鼠创面移植皮片成活率[(76.1±13.1)%]低于对照组[(94.5±1.3)%,t'=3.440,P=0.018].术后3周,实验组创面移植皮片收缩率[(34±8)%]明显大于对照组[(16±12)%,t=-3.211,P=0.009];术后8周,2组移植皮片扩张率接近一致.(2)HE染色和天狼星红染色显示,与大鼠创周正常皮肤比较,对照组移植皮片胶原纤维束呈均质化改变,胶原纤维纤细,排列紊乱;实验组移植皮片胶原纤维束结构、排列更接近创周正常皮肤,可见未完全降解的PADM.与对照组创面皮肤胶原纤维束直径[(7.3±1.4)μm]、间隙率[(17±4)%]、Ⅰ型胶原含量[(68.1±8.4)%]、Ⅲ型胶原含量[(32.0±8.4)%]以及Ⅰ、Ⅲ型胶原比例(2.3±1.0)比较,实验组胶原纤维束更粗[(9.6±0.8)μm,t=-3.562,P=0.005],间隙率更大[(24±5)%,t=-2.760,P=0.020],Ⅰ型胶原含量更高[(80.2±5.4)%,t=-2.981,P=0.014],Ⅲ型胶原含量更低[(19.8±5.4)%,t=2.981,P=0.014],Ⅰ、Ⅲ型胶原比例更高(4.3±1.2,t=-3.204,P=0.009).实验组创面皮肤上述胶原相关指标更接近于创周正常皮肤水平.结论 异体PADM在体内作为真皮再生模板,有助于改善自体刃厚皮所修复的大鼠皮肤缺损创面中真皮胶原纤维束的结构,提高再生真皮组织的成熟度.

Abstract：

Objective To evaluate the effects of mixed grafting of allogeneic PADM and autologous STS on wound healing of full-thickness defect in rats. Methods Full-thickness defects with size of 6 cm×4 cm were produced on the back of 12 SD rats, and they were divided into E group(n =6) and C group ( n = 6) according to the random number table. The wounds in E group were grafted with a mix of allogeneic PADM (expansion rate 10: 5) and autologous STS with thickness of 0.2 mm, while those in C group were grafted with autologous STS in the same thickness. The wound healing rate, survival rate, contraction rate,and expansion rate of transplanted skin were observed at post operation week (POW) 2, 3, 4, 6, 8, 12,20. Tissue samples form wounds and surrounding normal skin were harvested at POW 20 for histopathological observation as follows. The structure of collagen fiber bundle was observed by HE staining, the diameter and gap rate of collagen fiber bundle were also measured. The distribution of type Ⅰ and Ⅲ collagen was observed by sirsus red staining, and the contents of type Ⅰ , Ⅲ collagen and their ratio were also examined.Data were processed with independent samples t test, Levene test, and t' test. Results Survial rate of transplanted skin in E group at POW 2 [(76. 1 ± 13. 1)%] was obviously lower than that in C group [(94.5 ± 1.3)%, t' =3.440, P =0.018]. Contraction rate of transplanted skin in E, C groups at POW 3 showed significant difference [(34±8)%vs. (16 ±12)%, t = -3.211, P =0.009]. Compared with those in peri-wound normal skin, collagen fiber bundles in C group showed signs of homogenization, and collagen fibers were thin with irregular arrangement. Collagen fiber structure and arrangement of composite skin in E group were similar to those surrounding normal skin with incomplete degradation of PADM. Diameter of collagen fiber bundle [( 9.6 ± 0.8) μm] , gap rate between collagen bundle [( 24±5) %] , content of type Ⅰ collagen [( 80.2 ± 5.4) %] and the ratio of typeⅠto type Ⅲ collagen(4.3 ± 1.2) in E group were all increased as compared with those inC group [(7.3±1.4) μm (t = -3.562, P =0.005), (17±4)%( t =-2.760, P =0.020), (68.1 ±8.4)%(t = -2.981, P =0.014), 2.3±1.0(t = -3.204, P =0. 009)], while content of type Ⅲ collagen [( 19.8 ± 5.4) %] in E group was lower than that in C group [(32.0 ±8.4)% , t = 2. 981, P = 0. 014]. Above-mentioned indexes of collagen in wound of E group were similar to those of normal skin surrounding the wound. Conclusions Allogeneic PADM used as dermal regeneration template is beneficial in improving collagen fiber bundle structure in dermis layer of rats with fullthickness skin wounds when repaired with autologous STS, and it accelerates maturation of regenerative dermal tissue.     

Effects of a Topical Anti-aging Formulation on Skin Aging Biomarkers

ObjectiveFollowing previous clinical trials, an antiaging product (Restorative Skin complex [RSC]; Alastin Skin Care Carlsbad, a Galderma company), was investigated for its effects on Klotho gene regulation, telomere length, and histological biopsy changes to provide a comprehensive picture of the mechanism and efficacy of its anti-aging effect.MethodsNeonatal human fibroblasts were used for telomere length studies to examine the effect of the full RSC formulation and the amino acid components Tripeptide-1 and Hexapeptide-12 (TriHex™) on these cellular aging mechanisms. In addition, RNA sequencing was conducted using human keratinocytes specifically investigating Klotho and related genes. This was supplemented by a clinical study using biopsy samples.ResultsTriHex™ significantly upregulated the Klotho gene and related FGF23, FGFR1 and FOXO3B anti-aging genes. Significant telomere shortening reduction over control was demonstrated with the RSC formulation at four weeks and with TriHex™ at six weeks for all percentiles tested. Previous clinical studies demonstrated that the use of the antiaging regimen for 12 weeks produced a statistically significant improvement in scores for all evaluated parameters. Restaining of previous biopsy blocks from the clinical trial revealed positive ECM changes, stimulation of collagen, fibrillin, CD44 and elastin.LimitationsThe study was limited by a relatively small numbers of patients in the clinical trial and the non-competitive nature of the trial.ConclusionRSC anti-aging formulation and its TriHex™ components demonstrated significant reduction in telomere shortening, upregulation of Klotho and FOXO3 genes and biopsy validation of anti-aging efficacy. This new science supplements previous trials that demonstrated clinical efficacy of the formulation.     

大鼠自体静脉移植后内膜增生与细胞外基质堆积的研究

目的:研究静脉移植术后内膜增生(IH)中细胞基质(ECM)堆积的发生、发展及与平滑肌细胞(SMC)增殖的关系.方法:将大鼠一段颈外静脉桥接移植入颈总动脉,运用组织切片的特殊染色及免疫组化方法,观察术后1天至24周移植静脉的IH中,Ⅰ型胶原(ColⅠ)、Ⅱ型胶原(ColⅢ型)及SMC结蛋白(D_m)的变化.结果及结论:术后2周,IH中开始出现胶原的堆积,术后12周,IH中胶原含量达最大值并自此保持稳定不变,术后24周IH主要由细胞外基质组成;术后2周,IH中SMC的D_m开始表达,表明SMC的表型开始由收缩型向分泌型改变,术后6周,D_m达最大值,表明此时分泌型SMC最多,SMC可能是分泌细胞外基质的间质效应细胞.     

和解聚散方对单侧输尿管梗阻肾间质纤维化大鼠细胞外基质表达的影响

目的:研究和解聚散方对单侧输尿管梗阻(unilateral ureteral obstruction,UUO)大鼠肾间质纤维化(renal interstitial fibrosis,RIF)、细胞外基质(extracellular matrix,ECM)、纤维连接蛋白(FN)和Ⅳ型胶原(Col Ⅳ)表达的影响.方法:...     

组织工程学研究中偏光显微镜的应用

偏光显微镜是地质界用来观察晶体矿物的重要仪器。其理论基础是结晶光学，特点是即可观察结构，又可根据光学性质（折光率值不同）来判断成分。通过在研究ＮＥＣＭ和注射性胶原制作的过程中，用偏光显微镜和光镜观察同一部位的组织片，加以对比分析。因为胶原是生物体内的特殊“晶体”，故认为偏光显微镜应用在组织工程研究中，不仅开阔了观察领域，而且是重要的观测手段和方法。用偏光显微镜观察了猪骨ＥＣＭ胶原框架，以及注射性胶原制作过程中胶原变化特点。认为本方法简单不需染色，并可特异的观察到胶原的分布和走行。     

雌激素对皮肤老化的影响及其机制的研究进展

皮肤是雌激素在非生殖器官中最大的靶器官,绝经后雌激素水平的下降加速了皮肤老化。雌激素替代治疗(HRT)或局部应用雌激素,在治疗女性更年期症状的同时,可改善皮肤状况,使真皮中胶原量、弹性纤维和细胞外基质增加,进而增加皮肤厚度、弹性及保湿能力,减少皱纹,但其作用机制还有待于进一步的研究。我们就雌激素对皮肤老化的影响及其作用机制的研究进展进行综述。     

高糖对系膜细胞诱生型一氧化氮合酶表达及细胞外基质合成的影响

目的:探讨高糖对体外培养的系膜细胞诱生型一氧化氮合酶(iNOS)mRNA表达的调节作用及NO合成的变化,以及上述变化对细胞外基质(ECM)合成的影响.方法:利用脂多糖(LPS)诱导系膜细胞表达iNOS,观察5.6、10、25、40 mmol/L不同糖浓度,在0、4、24、48 h不同时间点培养的系膜细胞iNOS mRNA表达的变化及培养上清中NO、Ⅳ型胶原、纤维连接蛋白含量的变化,以相同渗透压的甘露醇为对照组.细胞增殖测定采用MTT法,iNOS表达采用RT-PCR的方法,NO的检测采用硝酸还原法,Ⅳ型胶原、纤维连接蛋白的测定采用ELISA法.结果:高糖抑制系膜细胞增殖;LPS刺激后系膜细胞可表达iNOS;高糖浓度时iNOS mRNA表达增加,呈浓度和时间依赖性,相同渗透压的甘露醇无类似作用(P<0.01);随着糖浓度的升高和时间延长,实验组上清中NO含量增加,Ⅳ型胶原、纤维连接蛋白含量增加.结论:高糖可上调系膜细胞iNOS mRNA的表达和促进NO、Ⅳ型胶原、纤维连接蛋白的合成.     

Culture of subconfluent human fibroblasts and keratinocytes using biodegradable transfer membranes

This study aims to assess the suitability of biodegradable membranes as transfer matrix materials for the culture of subconfluent fibroblasts and keratinocytes. The materials investigated were based on collagen, chitosan and enzyme-digestible cellulose. The proliferation and growth behaviour of human keratinocytes and dermal fibroblasts were analysed and morphology and distribution determined. Cultured fibroblasts exhibited no significant differences in proliferation for the different membrane types, whereas keratinocytes revealed significantly higher proliferation on collagen membranes compared with membranes based on cellulose and chitosan. Co-cultured fibroblasts and keratinocytes from the same donor on collagen membranes showed more homogenous cell distribution, but they segregated in heterologous co-cultures; this effect must be further investigated. Thus, collagen and collagen-coated chitosan membranes are suitable for the subconfluent transfer of human fibroblasts and keratinocytes.