骨髓间充质干细胞分化为皮肤附属器细胞的初步实验研究

目的探讨骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)分化为创面皮肤附属器细胞的可能性,及其参与创面修复的可能机制。方法无菌条件下取Wistar大鼠股骨骨髓细胞,密度梯度离心分离、纯化MSCs,体外培养扩增后,用BrdU标记细胞。另于同种雄性Wistar大鼠背部正中,制备1cm×1cm全厚皮肤缺损创面模型,将BrdU标记的1×106/mlMSCs从阴茎静脉输注,术后第3天与第7天切取创面组织,行BrdU免疫组织化学单染色,以及BrdU和广谱角蛋白免疫组织化学双染色。结果BrdU阳性细胞出现在创面皮下组织、皮脂腺、毛囊和骨髓腔中。免疫组织化学双染色结果显示,皮脂腺和毛囊有BrdU阳性细胞,同时表达广谱角蛋白。结论创面愈合过程中,MSCs归巢并参与创面修复;在实验性全身皮肤缺损创面微环境下,MSCs可分化为皮肤附属器细胞。     

rhPDGF对糖尿病鼠全层皮肤缺损创面微血管形成的影响和意义

目的:观察外源性重组人血小板源性生长因子(rhPDGF)对全层皮肤缺损的糖尿病鼠创面愈合过程中微血管形成的影响及促创面愈合的作用。方法:利用大鼠全层皮肤缺损创面愈合模型,将26只非糖尿病鼠52个创面分为3组:①A组为糖尿病大鼠创伤自然愈合组;②B组为接受rhPDGF治疗的糖尿病大鼠创伤愈合组;③C组为赋性剂组。于伤后3 d、7 d和14 d采取创面皮肤标本,用组织病理HE和血管Ⅷ因子抗体免疫组织化学染色技术检测创面肉芽再生与再血管化情况进行观察,同时观察创面的愈合情况。结果:全层皮肤缺损糖尿病鼠创面肉芽组织中微血管的形成数量少,采用赋性剂对照组与糖尿病鼠自然愈合组无显著差别。但外源性使用rhPDGF后,肉芽组织的形成量明显增多,内含的微小血管数量显著增加,愈合能力明显增强。结论:糖尿病大鼠微血管形成障碍可能是其创面愈合延迟的重要因素。而应用外源性的PDGF有助于微血管的形成,可以改善糖尿病鼠的创面愈合能力。     

增生瘢痕皮肤中转化生长因子基因表达

目的探讨转化生长因子(TGF)-β1和转录因子Smad 2,Smad 3三种基因影响增生性瘢痕形成和胎儿皮肤伤口无瘢痕愈合的调节机制.方法 32份被检测标本中包括增生性瘢痕8份,其对应的正常皮肤组织8份,胎儿和成人皮肤组织各8份.用逆转录-多聚酶链反应方法(RT-PCR)检测这3种基因在不同的组织细胞内的表达变化规律.结果 TGF-β1、Smad 2 和Smad 3三种基因在增生性瘢痕、胎儿和出生后机体皮肤组织细胞内都有表达.在所检测8对增生性瘢痕和其对应正常皮肤细胞中,这3种不同基因在增生性瘢痕细胞中的表达量高于正常皮肤细胞的组数分别为TGF-β1基因有5对,Smad 2基因有8对,而Smad 3基因有5对.在胎儿皮肤细胞内,TGF-β1的mRNA含量明显低于成人皮肤细胞(t=2.204,P<0.05),差异有显著意义;Smad 3基因表达也呈相似的变化规律,mRNA含量显著低于成人皮肤细胞(t=4.269,P<0.01),差异有非常显著意义;而Smad 2基因的mRNA含量却明显高于成人皮肤细胞(t=6.685,P<0.01),差异亦有非常显著意义.结论 TGF-β1和它的下游信号分子Smad 2,Smad 3可能与增生性瘢痕形成密切相关.在增生性瘢痕细胞内,这3种基因高表达可诱导胞外基质大量沉积,加速成纤维细胞增殖,促进组织纤维化.TGF-β1和Smad 3基因在胎儿皮肤组织细胞内低表达可能是皮肤伤口无瘢痕愈合的机制之一.     

重组人血小板源性生长因子增加细胞外信号调节激酶磷酸化促进糖尿病大鼠全层皮肤缺损创面愈合

目的 在体观察重组人血小板源性生长因子（recombinant human platelet—derived growth factor，rhPDGF）促进糖尿病大鼠全层皮肤缺损创面修复可能涉及的细胞和分子机制，研究其可能涉及的信号通路。方法 26只糖尿病大鼠，每只动物背部制备4个全层皮肤缺损创面，选取其中52个创面，随机分成3组，即对照组，创面自然愈合；rhPDGF治疗组，创面rhPDGF用量为7．0μg／cm^2；赋形剂组，创面用等量赋形剂凝胶。观察治疗后3、7和14d创面肉芽形成、胶原沉积、再上皮化速率以及炎性细胞浸润情况，并采用免疫荧光和免疫组织化学技术观察创面周围和创面修复细胞内细胞外信号调节激酶1／2（extracellular signal—regulated kinase1／2，ERK1／2）磷酸化和增殖细胞核抗原（proliferative cell nuclear antigen，PCNA）的表达。结果 组织学观察，rhPDGF治疗组创面可见大量炎性细胞浸润，毛细血管胚芽及成纤维细胞明显多于另两组（P〈0．05）；胶原沉积明显，肉芽组织生长活跃，创面收缩显著，与对照组比较差异有统计学意义（P〈0．05）。免疫学研究显示，应用rhPDGF7～14d后，rhPDGF治疗组ERK1／2明显强于对照组和赋形剂组（P〈0．05）；且损伤后3～7d rhPDGF治疗组修复细胞PCNA的表达明显高于对照组和赋形剂组（P〈0．05）。结论 rhPDGF促糖尿病大鼠刨面愈合的作用部分是通过ERK1／2信号通路的磷酸化来完成的。     

人表皮角质细胞系培养过程中细胞生物学行为和表型的变化

目的:获得较高比例的已分化表皮细胞,为表皮细胞去分化研究奠定基础.方法:采用常规表皮细胞培养方法对人表皮角质细胞系(HEK)进行培养和传代.每一代细胞均通过免疫细胞化学染色和Western blot 方法进行表皮干细胞和表皮细胞相应标志物的检测,包括β1整合素、角蛋白19(K19)、角蛋白10(K10)等指标.结果:第1代HEK呈克隆样生长,表皮干细胞标志物β1整合素、K19均有高表达,而K10的表达为阴性.经过数次传代,细胞克隆形成数目减少,细胞逐渐呈分散分布,K10的表达逐渐增高,而β1整合素、K19的表达呈下降趋势,比例减少.细胞培养至第5～6代时,为其生长的一个转折点,即此时细胞增殖能力开始明显降低,但形态良好,K10表达阳性细胞比例明显增高,而β1整合素、K19表达阳性细胞比例迅速降低.细胞培养至10～11代时,为其生长的第二个转折点,此时细胞几乎丧失增殖能力,数量显著减少,体积变大,核浆比明显减小,完全见不到β1整合素、K19表达阳性细胞,K10染色均为阳性.结论:可以选取适当代数的HEK用于去分化研究,为增加HEK的新用途提供了实验基础.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats

Objective: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. Methods: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term =21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5 705 probes representing 5 705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. Results: Among 5 705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 ( FGF2 ) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. Conclusions: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.     

高分辨MRI对颈动脉粥样硬化斑块成分显示的病理对照研究

目的分析和评价高分辨MRI对颈动脉粥样硬化斑块不同成分的显示效果,为颈动脉内膜切除术术前判断斑块稳定性提供参考。方法对26例行颈动脉内膜切除术的颈动脉粥样硬化性狭窄患者术前高分辨MRI 4种不同序列的影像(T1WI、T2WI、PDWI和3D TOF)与斑块标本病理进行逐层对照,分析斑块内不同成分的MRI影像特点。结果获得斑块28块,切为238段,主要分布于颈总动脉和颈内动脉,以复杂斑块为特征的Ⅳ～Ⅴ型58段(24.37%)和Ⅵ型79段(33.19%)为主;斑块内纤维帽主要表现为TOF序列的带状低信号,钙化和纤维化组织分别表现为在各序列影像上的不规则低信号和不特定信号,脂质池和坏死核呈T1WI、PDWI和3D TOF序列的等至稍高信号,近期出血表现为T1WI、T2WI和PDWI序列的明显高信号。结论高分辨MRI不仅可以清晰显示动脉粥样硬化斑块,进行动脉管腔狭窄程度的测定,通过多序列影像联合分析还可以分辨斑块内部不同成分,有助于术前对斑块稳定性的判断。     

低氧诱导因子-1的病毒载体制备及感染效果

目的 制备低氧诱导因子(HIF)-1腺病毒载体并评价其对表皮角质细胞的感染效果.方法 Ad/HIF-1质粒经酶切鉴定后,以4μg质粒转染1.2×106个HEK293细胞,2 d后荧光显微镜下观察转染效率.1周后收集细胞及上清并感染HEK293细胞进行病毒扩增,待扩增的细胞及上清量达300 ml时进行病毒纯化及滴度测定.最后用制备的病毒载体感染表皮角质细胞,流式测定其感染效果.结果 Ad/HIF-1质粒酶切鉴定正确;转染或感染HEK293细胞后有绿色荧光蛋白(GFP)表达;获得的腺病毒滴度为1.8×1011PFu/ml.病毒以MOI=50感染表皮细胞,2 d后GFP阳性细胞的比例为97.46%.结论 成功制备了HIF-1腺病毒载体,且感染表皮角质细胞后能得到较高的感染效率,从而为腺病毒介导的基因功能研究提供理论基础.     

成纤维细胞生长因子-10及其受体对胎儿皮肤附件形成的诱导作用

目的:探讨成纤维细胞生长因子-10(FGF10)及其受体(FGFR2b或Bek)在胎儿皮肤附件(SA)形成中的表达特征及其生物学意义.方法:对130块分别来自26例不同胎龄(EGA 8～31周)胎儿,5个不同部位(头、下颌、耳垂、肩和胸骨柄)的皮肤标本,采用病理学、免疫组化方法和计算机图像扫描技术检测FGF10、Bek、细胞角蛋白-19(CK19)、Bcl-2和细胞增殖核抗原(PCNA)在胎儿皮肤组织的表达水平.结果:除EGA8周胎儿缺乏典型的皮肤组织学结构和FGF10和Bek蛋白表达外,所有蛋白在E11周以后的皮肤内都有阳性表达.在EGA11周,表皮下成团分布的间质细胞过表达FGF10、PCNA和Bcl-2,表皮细胞和周皮细胞则所有蛋白均呈强阳性表达;在EGA13周时表皮细胞局灶性增殖形成SA胚芽,并逐渐向真皮内生长、分化和迁移;在EGA11～31周的皮肤组织内,真皮内间质细胞表达FGF10、PCNA和Bcl-2以及SA上皮细胞表达FGF10、Bek、PCNA、CK19和Bcl-2蛋白呈现与生长发育同步的表达特征,但单个细胞表达的平均光密度(A)值则随胎龄增加而逐渐下降(P＜0.05～0.001).结论:间质细胞旁分泌的FGF10与上皮细胞膜Bek特异性结合,是诱导胚胎SA上皮细胞生长及其形态发生的重要信号,但有关SA的形成部位和种类及其与FGF10蛋白表达的相互关系仍有待深入研究.