局部注射博莱霉素诱导小鼠硬皮病模型

目的建立小鼠硬皮病动物模型.方法将0.01～1mg博莱霉素皮下注射于6周龄BALB/c雌性小鼠背部皮肤,第日1次共注射4周.取注射区皮肤行光镜和电镜观察,并用免疫组化方法检测TGFβ1、TGFβ2和α-SMA的表达.结果 0.1mg/d以上博莱霉素剂量在4周后皆可致小鼠背部皮肤硬化,表现为皮肤弹性降低、毛发脱落、表皮萎缩、真皮炎细胞浸润、毛囊减少或消失、纤维组织增生、大量肌成纤维细胞增生,真皮TGFβ1、TGFβ2和α-SMA表达增强.结论博莱霉素连续皮下注射可导致小鼠皮肤硬化,符合硬皮病皮肤基本的组织病理学改变.     

phVEGF165基因导入小鼠硬皮病皮损致血管新生和Ⅰ型前胶原mRNA表达增加

目的检测phVEGF165基因导入小鼠硬皮病皮损后Ⅰ型和Ⅲ型胶原表达的变化.方法应用电穿孔技术将phVEGF165基因导入小鼠硬化的皮肤内,以分子杂交、免疫组化等方法检测Ⅰ型和Ⅲ型胶原的表达.结果随着VEGF165mRNA表达增加,Ⅰ型前胶原mRNA表达上调,但免疫组化检测Ⅰ型及Ⅲ型胶原蛋白表达尚无明显改变.结论 VEGF可一定程度上调皮肤Ⅰ型胶原基因的表达.     

养血生发胶囊对C57BL/6小鼠毛发生长的影响及机制

目的 观察养血生发胶囊对C57BL/6小鼠毛发生长的影响,并探讨其可能的作用机制。方法 采用C57BL/6小鼠脱发实验模型,观察毛发生长情况,并于脱毛后第9、19天,做脱毛区皮肤毛囊数、毛细血管及毛囊血管内皮生长因子(VEGF)检测。结果 养血生发胶囊明显缩短小鼠皮肤变黑时间;明显增加皮肤毛囊数、毛囊毛细血管数及毛囊组织中VEGF表达积分吸光度(A)值。结论 养血生发胶囊通过增加毛囊组织中VEGF表达、毛囊血管数及毛囊数,促进毛发生长。     

支架携带pSV-VEGF165基因促进内皮修复的实验研究

目的 评估通过支架向兔颈动脉壁转染pSV-VEGF165基因促进内皮修复的疗效。方法 采用自制支架导送系统，由生物凝胶介导，将携带质粒pSV-VEGF165的支架植入兔一侧颈动脉段，对侧植入携带质粒pSV-β-gal的支架作对照。通过RT-PCR、免疫组化染色及扫描电镜，观察局部动脉壁外源VEGF基因、蛋白的表达及内皮修复情况。结果 RT-PCR检测证实，支架植入术后ld，VEGF基因转染动脉段有VEGF mRNA的表达；7d表达达高峰。免疫组化染色显示，术后3d外源VEGF蛋白表达，表达时间延迟于基因表达，但表达趋势与基因表达一致。扫描电镜显示，治疗组内皮完全修复时间提前至术后1月。结论 采用支架携带基因的方法能成功将pSV—VEGF165基因转移至血管壁，血管壁细胞能摄取基因，并表达具有生物学功能的蛋白质，促进内皮修复。     

phVEGF165原位转染缺血肌肉后hVEGF及其蛋白的表达

目的探讨慢性下肢动脉缺血的基因治疗. 方法选用血管内皮生长因子(VEGF)作为治疗基因,通过建立实验兔慢性下肢动脉缺血模型,将构建的人VEGF165cDNA真核表达载体phVEGF165注入缺血肌肉内,采用RT-PCR和原位杂交法测定肌肉内hVEGF165基因表达,采用免疫组化和Western印迹法测定其蛋白产物的表达.结果骨骼肌细胞外源性hVEGF165基因表达量于注射后1周达到高峰,持续2～3周.蛋白表达于注射后2周达到高峰,维持至注射后4周. 结论骨骼肌细胞能摄取并表达外源性hVEGF165基因,并能进一步合成分泌hVEG165蛋白.phVEGF165肌肉直接注射的作用时间有限、范围局限,使用安全性较高.     

携hVEGF165基因的重组腺病毒载体的构建及鉴定

目的　构建携带人血管内皮生长因子 16 5 (hVEGF165)基因的重组腺病毒载体。方法　将hVEGF165cDNA亚克隆到腺病毒中间载体pACCMV·pLpA ,再与pJM17共转染人胚肾 2 93细胞 ,获得载hVEGF165基因的复制缺陷型重组腺病毒 ,感染体外培养的兔主动脉血管平滑肌细胞 (VSMC) ,通过RT PCR和Westernblot检测VEGF表达情况 ,并观察表达产物VEGF对人脐静脉内皮细胞 (HUVEC)增殖的影响。结果　重组腺病毒感染VSMC 4 8h后有VEGFmRNA转录及蛋白质的表达 ,并呈剂量依赖性地促进HUVEC增殖。结论　构建的重组腺病毒载体在VSMC中能够有效表达目的基因 ,且能有效促进HUVEC增殖 ,为hVEGF165基因的实际应用奠定了基础。     

肝细胞生长因子在肺纤维化模型中的表达及意义

目的:研究肝细胞生长因子(HGF)在大鼠肺纤维化模型中的表达及意义,探讨肺纤维化可能的发病机制。方法:40只Wistar大鼠随机分成模型组及对照组。前者经气管内灌注博莱霉素诱导肺纤维化,后者灌注生理盐水。两组于气管内灌注后第7,14,28,42d各处死5只,原位杂交法检测HGFmRNA在肺内的表达,酶联免疫吸附法检测HGF蛋白在肺组织及血清中的表达。结果:模型组肺组织HGFmRNA表达及蛋白含量在第7d达高峰,之后下降,第28,42d时低于正常对照(P<0.05,P<0.01)。模型组血清HGF在第7d开始升高达峰,后降低,但仍高于正常对照(P<0.05)。结论:HGF在肺纤维化组织中的表达早期升高,中晚期降低,而血清中的含量始终高于正常组。HGF参与了肺纤维化的发病过程。     

人血管内皮细胞生长因子165基因真核表达载体构建

目的获得人血管内皮生长因子（hVEGF165）cDNA,构建其真核表达载体。方法采用PCR技术,从HL60细胞中扩增出hVEGF165 cDNA,将其克隆至pcDNA3真核表达载体上,构建成为pCD-hVGEF165重组质粒。结果经酶切鉴定,克隆的基因片段为人hVGEF165 cDNA;建立了pCD-hVGEF165重组质粒。结论成功地克隆和表达出了人VEGF165基因,为大量制备重组质粒的研究奠定了基础。     

pAdtrack-CMV-VEGF165活体转染动脉粥样硬化兔再狭窄模型的实验研究

[目的]克隆人VEGF165基因,构建其真核表达质粒pAdtrack-CMW-VEGF165,活体定向转染动脉粥样硬化兔再狭窄血管,检测其表达情况.[方法]PCR法从人心脏cDNA文库钓取人WEGF基因,插入pUC19载体测序后与真核表达质粒pAdtrack-CMV连接构建pAdtrack-CMV-VEGF165,PTCA球囊导管介导基因转移至动脉粥样硬化兔再狭窄血管局部,检测基因在局部及远离部位表达情况,以及对肝肾功能的影响.[结果]①转染pAdtrack-CMW-VEGF165的动物血管荧光显微镜下观察再狭窄局部可见黄绿色荧光条带,并检测到人VEGF165基因的表达;②远离脏器无转染基因表达;③对肝肾功能无明显影响.[结论]成功将目的基因定向转移至靶部位并获有效表达,对重要脏器功能无明显影响.     

肺微血管内皮细胞表型改变与博菜霉素所致大鼠肺纤维化的关系

目的：观察博莱霉素所致肺纤维化大鼠微血管内皮细胞（LMVECs）超微结构及血清CTGF的变化，探讨肺微血管内皮细胞表型及与博莱霉素损伤肺纤维化间的关系．方法：SD大鼠45只，随机分为3组：博莱霉素（BLM）组，肺炎链球菌组及正常对照组，每组15只，分别气管滴注博莱霉素及肺炎链球菌造模，对照组滴注生理盐水．电镜观察外周肺组织内皮细胞超微结构，同时测定血清CTGF含量．结果：光镜及电镜显示肺炎组与BLM组的不同改变．肺炎组组织学及肺微血管内皮细胞超微结构表现为典型的急性炎症损伤修复，之后肺组织结构恢复正常．BLM组表现为肺组织损伤的持续进行，肺微血管内皮细胞7d开始增殖并出现形态学的改变，且贯穿实验各个阶段，最终胶原明显沉积于肺泡间隔．血清CTGF测定：肺炎组3d开始增高，第7d骤降至（5．04&#177;0．82）ng／L，第21d恢复至（12．06&#177;0．43）ng／L．BLM组全程增高，第7d至（12．53&#177;2．36）ng／L，第21d增至高峰（19．45&#177;0．41）ng／L．结论：肺微血管内皮细胞功能的转变及其分泌CTGF时相和量的异常可能是博莱霉素所致肺纤维化发生发展的重要环节．     

他克莫司对小鼠毛囊生长周期的影响

目的探讨他克莫司对小鼠毛囊生长周期的影响及其作用机制。方法应用苏木素-伊红（HE）染色及RT-PCR法研究他克莫司对小鼠毛囊生长周期影响。结果脱毛后第5天他克莫司组及米诺地尔组小鼠背部毛囊呈生长期Ⅴ状态,而凡士林组毛囊呈生长期Ⅲ状态。在脱毛后第5天他克莫司组及米诺地尔组小鼠背部皮肤检测到血管内皮细胞生长因子及肝细胞生长因子表达,而凡士林组未能检测到。结论他克莫司可通过促进毛囊提前进入生长期Ⅴ而达到促进毛发生长的作用,这种促进作用可能与血管内皮细胞生长因子及肝细胞生长因子作用有关。     

活血补肾合剂对C57BL/6小鼠皮肤血管新生及毛囊中血管内皮细胞生长因子表达的影响

目的:通过观察C57BL/6小鼠皮肤局部血管新生及毛囊中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)的表达,探讨活血补肾合剂促进小鼠毛发生长的可能机制。方法:以热松香和石蜡混合物脱毛法诱导C57BL/6小鼠休止期毛囊进入生长期,建立动物模型。90只小鼠随机分为活血补肾合剂组、养血生发胶囊组和模型组。造模后第1天起开始灌药,每天观察各组小鼠皮肤毛发生长情况,并分别于造模后第4、11、17天每组分批处死10只小鼠,病理切片计数拔毛部位的血管数,并以免疫组织化学方法检测毛囊中VEGF表达情况。结果:活血补肾合剂组局部新生血管数第4天时多于模型组(P<0.05),第11天时明显多于养血生发胶囊组和模型组(P<0.05);活血补肾合剂组毛囊中VEGF的表达在第11天和第17天时均明显高于养血生发胶囊组和模型组(P<0.05)。结论:活血补肾合剂可能是通过调节细胞因子水平,起到促进血管新生和毛发生长的作用。     

新生小鼠不同部位皮肤毛囊早期发育差异的比较

目的：本研究旨在观察出生后小鼠不同部位皮肤毛囊早期发育生长差异及细胞色素C的表达分布。方法：对新生1-9天的昆明白系小鼠背部、尾部和触须部皮肤取材,进行HE染色,用二步法免疫组织化学对组织进行细胞色素C进行表达分布检测。结果：新生小鼠不同部位皮肤毛囊发育差异很大,这种差异不仅体现在形态差异上,而发育时间的差异也十分明显。小鼠出生后背部皮肤和尾部皮肤的毛囊发育都经过了一个非线性的发育和生长期,过了非线性的发育和生长期才开始快速生长,相比较尾部发育略迟于背部。触须部毛囊发育特征和背部尾部差异很大,一出生便可看到较成熟的触毛,没有经过稳定期便开始发育。结论：通过形态学比较,结合CytC表达分布水平,发现新生小鼠不同部位皮肤毛囊早期发育存在形态和时间上的差异。     

毛乳头在毛囊生长周期中的变化和作用

目的 了解人毛乳头结构在毛发生长周期中的变化及其对毛发生长的调控作用.方法 将整形手术后取下的头皮在无菌条件下分离成游离的毛囊,在Williams E培养基中培养;通过横断毛囊和完整毛囊的培养来观察毛囊的生长状况和处于不同生长周期的毛囊的毛乳头结构变化.结果 分离后的完整毛囊在Williams E培养基中平均可继续生长12 d,平均生长速度为每天0.2～0.3 mm;静止期或生长初期毛囊的毛乳头呈圆形贴在毛球底部,快速生长的毛囊毛乳头变长呈锥形,结构疏松,与毛根紧密相接,当毛囊进入衰退期直至停止生长后毛乳头萎缩变小,并与毛根逐渐分离.在低位横断毛囊,毛囊可再生毛球并继续生长,但生长速度减慢;在高位横断毛囊,毛囊不能继续生长.结论 毛乳头在毛发生长周期中不断变化,生长期毛乳头体积增大,与毛母质联系紧密,它在毛囊的生长和生长周期中起着不可缺少的调控作用.     

毛囊细胞移植法诱导毛囊的初步研究

目的 构建一个可靠有效的移植毛囊细胞诱导毛发发育的模型,以治疗脱发.方法 取自愿捐献的成人头皮标本,联用显微分离与免疫磁珠法获得人毛囊干细胞;消化法获得毛乳头细胞.培养后混合植入裸鼠皮下,观察毛囊形成情况.结果 在裸鼠的皮肤切片中可以看到较为完整的毛囊结构形成.结论 毛囊细胞移植法可以在体内诱导出毛囊样结构,为将来治疗脱发奠定了基础.

Abstract：

Objective To establish a convenient and reliable method for inducing hair regeneration by follicular cell implantation for the treatment of alopecia. Methods The human hair follicle stem cells were separated and purified by micromanipulation and magnetic cell sorting, and human scalp dermal papilla cells were isolated by enzyme digestion. The two cells were mixed and implanted subcutaneously in nude mice to observe the regeneration of the hair follicles. Results Formation of intact hair follicle-like structures was observed in the skin sections of the recipient nude mice. Conclusion Follicular cell implantation can induce hair follicle-like structures in nude mice, which provides a means for efficient hair regeneration for treatment of hair loss.     

TPA促进小鼠毛囊中TCF3的表达

目的 检测12-O-十四烷酰佛波醋酸酯-13 (TPA)处理C57 BL/6( B6)小鼠背部皮肤后TCF3表达的变化情况.方法 采用免疫荧光染色、RT-PCR和Western blot等技术,检测TPA和丙酮分别处理7周大小的B6鼠背部皮肤1、2和4周后TCF3的表达情况.结果 与丙酮处理组相比,TPA处理组中B6鼠...     

Expression of stem cell factor in hair follicl epithelium

Thedermalepidernlalinteractioninthehairfollicleisahotspotincytobiologlcalstudies.anytoklnesplayimportantrolesinin(luctionofbiologicafactivitiesbetweentilegermalandepidermal].Stemcellfactor(St'F).whichwasfoundinrecentyears.isacyloklnerelatedicthestir\-tval.growl11anddevelopmentofthehemop()leticstemcellsandcanexertimportanlbiologicaleffectsonthedevelopmentofkeratlnocytesandnlelanocytes"/'.Inthisstudy.theexpressionofSCFInhumanhairfollicleepitheliumwasin\'estigateclwithimmunohistochemistryandI)…     

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells (DPC) are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor (bFGF), endothelin-1 (ET-1) and stem cell factor (SCF) in different passages of cultured DPC and their effects on the biological behaviour of DPC.Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in situ hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice.Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages (>6 passages). Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration.Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.     

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells （DPC） are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor （bFGF）, endothelin-1 （ET-1） and stem cell factor （SCF） in different passages of cultured DPC and their effects on the biological behaviour of DPC. Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in sire hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice. Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages （〉6 passages）. Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration. Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.     

phVEGF165原位转染缺血肌肉后hVEGF及其蛋白的表达

目的：探讨慢性下肢动脉缺血的基因治疗。方法：选用血管内皮生长因子（VEGF）作为治疗基因，通过建立实验兔慢性下肢动脉缺血模型，将构建的人VEGF165cDNA真核表达载体phVEGF165注入缺血肌肉内，采用PT-PCR和原位杂交法测定肌肉hVEGF165基因表达，采用组化和Western印迹法测定其蛋白产生的表达。结果：骨骼肌细胞外源hVEGF165基表达量于注射后1周达到高峰，持续2-3周。蛋白表达于注射后2周达到高峰，维持至注射后4周。结论：骨骼肌细胞能摄取并表达外源性hVEGF165基因，并能进一步合成分泌hVEGF165蛋白。PhVEGF165肌肉直接注射的作用时间有限、范围局限、使用安全性高。