表皮干细胞在糖尿病创面愈合过程中的动态变化

目的 观察糖尿病（diabetes mellitus，DM）大鼠创伤后不同时期表皮厚度、表皮干细胞（epidermalste mcells，ESCs）数量变化和分布特征及创面愈合率等动态变化，探讨ESCs与DM皮肤创面难愈之间的关系。方法 48只Wistar大鼠，雄性，体重180～200g。随机分为DM组和正常对照组，各24只。DM组大鼠制备DM大鼠慢性创面愈合模型。两组大鼠同时用特制打孔器在大鼠背部脊柱两侧约1．5cm制作直径1．8cm、面积2．54cm。的全层皮肤缺损（共计96个创面），分别在致伤后第3、7、14和21天拍摄创面，计算创面愈合率；取创缘及肉芽组织，行HE染色及角蛋白19（keratin19，K19）和β1整合素抗体免疫组织化学染色，显微图像分析系统测量表皮厚度、阳性部分面积及灰度值。结果 致伤后第3、7、14和21天，正常对照组大鼠创面愈合率分别为24．48％±3．37％、50．46％±1．26％、92．82％±2．12％和99．41％±0．66％，而DM组分别为2．43％±1．02％、40．59％±1．65％、80．77％±3．57％和85．40％±0．94％，两组同时期比较差异有统计学意义（P〈0．01）。致伤后第3、7、14和21天，正常对照组表皮厚度分别为26．43±3．21、33．29±3．52、31．53±3．35和26．01±3．19p．m，DM组表皮厚度分别为23．58±2．33、31．02±3．38、33．72±5．49和21．80±4．02p．m，致伤后第3、21天，二者比较差异有统计学意义（P〈0．01）。致伤后第3、7、14和21天，正常对照组K19染色的平均阳性单位（positiveunit，PU）值分别为91．68％、93．14％、72．27％和70．31％，而DM组分别为40．29％、40．79％、29．94％和10．37％；正常对照组β1整合素染色的平均PU值分别为49．60％、91．16％、77．13％和57．17％，DM组分别为38．94％、24．16％、61．36％和38．83％，与正常对照组同时期比较，DM组K19和β1整合素染色的平均PU值都降低，且差异有统计学意义（P〈0．05）。结论 ESCs数量少和活性低可能是DM创面难愈的重要机制之一。     

创面愈合过程中创缘表皮干细胞的异位

目的观察创缘表皮干细胞在全层皮肤创面愈合过程中的分布特征,初步探讨其在创面愈合过程中的作用.方法将已行BrdU活体标记的 20只Wistar大鼠背部制备4个为2.54 cm2的全层皮肤创面,分别于伤后3、7、14和21 d(n=5)行组织学检查,动态观察创面愈合情况,并行β1整合素、角蛋白19(K19)与BrdU免疫组织化学法检测表皮干细胞在创面愈合过程中的分布情况.结果创面愈合率为83.75%(61/80).所有创面肉芽组织于各时相点均未见β1整合素、K19阳性细胞出现,但于创缘表皮的棘层或颗粒层均出现散在的β1整合素、K19和BrdU阳性细胞.且越接近创面阳性细胞越密集,组织学上与基底层的阳性细胞无直接联系;其数量随创面的缩小逐渐增加,直至创面愈合.创面上皮化后,阳性细胞逐渐减少,并随愈合创面表皮脚的出现而消失.而感染创面的阳性细胞数量明显少于未感染创面.结论表皮干细胞能主动参与创面的修复,创缘的表皮干细胞异位的主要功能可能是促进创面再上皮化.     

不同来源的表皮干细胞促进皮肤创面愈合的研究

目的 观察表皮干细胞(ESCs)及去分化来源的表皮干细胞(DESCs)对皮肤创面愈合的促进作用及其异同.方法 由新鲜人包皮标本以贴壁法分离ESCs,随机分为3组,第1组作为原代ESCs:第2组传代培养至第6代时可见细胞成熟呈表皮细胞(ECs)形态,由原来的大克隆生长的小圆细胞形态变为较为肥大的不规则型细胞,无法形成克隆样增殖;其细胞表型也由β1整合素、CK19强阳性、CK10阴性变为β1整合素、CK19阴性,而CK10强阳性,予以100μg/L的碱性成纤维细胞生长因子(bFGF)培养48 h进行去分化诱导,48 h后ECs周边开始出现小圆细胞并呈克隆样生长,β1整合素、CK19、CK10等细胞表型也趋于与ESCs一致,即DESCs;第3组传代培养至第6代获得ECs.3组细胞皆以D-Hanks液重悬调其终质量浓度为2×106个/ml.将12只裸鼠每只背部左右各制备1个直径6 mm创面,共24个创面.将全部创面随机等分为4组,每组6个,分别以ESCs、DESCs、Ecs及生理盐水(NS)各0.2 ml进行创面注射.于术后0、3、7 d测量创面面积进行统计学分析,并于术后7 d行创面取材苏木素-伊红(HE)染色.结果 ESCs组与DESCs组在术后3 d时创面面积分别为(11.758±2.544)、(11.515±1.351)mm2,7 d时创面面积分别为(1.795±1.063)、(2.043±1.138)mm2,修复速度要明显快于ECs组与NS组[3 d时创面面积分别为(17.857±1.722)、(16.192±2.256)mm2,7 d时创面面积分别为(5.367±1.219)、(5.070±1.357)mm2,P＜0.01];而ESCs与DESCs组之间创面修复速度比较差异无统计学意义(P＞0.05);ECs组与NS组之间比较差异无统计学意义(P＞0.05).HE染色提示ESCs组与DESCs组的创面愈合质量优于ECs组与NS组,可见有皮肤附属腺的再生且纤维瘢痕组织较少.结论 ESCs与DESCs皆有促进创面愈合的作用,相互之间无明显差异.

Abstract：

Objective To investigate the enhancing effect of epithelial stem cells (ESCs) and dedifferentiation derived epithelial stem cells (DESCs) in the healing of epidermal wound. Methods ESCs were isolated from the fresh circumcised foreskins by using adherence method and randomly divided into three cohorts: ESCs cohort, DESCs cohort and epithelial cells (ECs) cohort. The final cell density was adjusted to 2 × 106/ml with D-Hanks in each group. The model of BALB/C mice full-thickness skin loss wounds was established. Two circular 6 mm-diameter skin loss wounds were cut on every BALB/C mouse back. Twenty-four wounds of 12 BALB/C mice were divided equally and randomly into 4 groups:DESCs, ESCs, ECs and NS. The cell suspensions of DESCs, ESCs and ECs were injected respectively into the wound edges with the density of 2 × 106/ml. And the volme was 0. 2 ml per treatment wound. The same volume of normal saline was injected in NS group. On the postoperative day 0, 3, 7, all of the wound areas were measured and analyzed statistically. On the postoperative 7 day, the sections were made and observed by using hematoxylin and eosin (HE) staining. Results The wound areas in DESCs group and ESCs group were ( 11. 758 ± 2. 544) and ( 11.515 ± 1.351 ) mm2 on the 3rd day postoperatively, and ( 1. 795 ±1. 063) and (2. 043 ± 1. 138) mm2 on the 7th day postoperatively, respectively. The wound areas in ECs group and NS group were ( 17. 857 ± 1. 722) and ( 16. 192 ±2. 256) mm2 on the 3rd day postoperatively,and ( 1. 795 ± 1. 063) and (2. 043 ± 1. 138) mm2 on the 7th day postoperatively, respectively. There was statistically significant difference in wound area between ESCs or DESCs group and ECs, NS groups on the 3rd, 7th day postoperatively ( P ＜ 0. 01 ), but there was no statistically significant difference between DESCs and ESCs groups ( P ＞ 0. 05 ). HE staining showed the repairing quality of treatment wounds was superior to that in the control wounds, and regenerating glands and less fibrous connective tissues were seen in ESCs and EDSCs groups. Conclusion Both ESCs and DESCs could promote the wound repair.     

皮肤源祖细胞-透明质酸复合物对糖尿病大鼠创面愈合的影响

目的研究皮肤源祖细胞（SKP）-透明质酸（HA）复合物的构建方法，观察其对糖尿病（DM）大鼠创面愈合的影响。方法分离培养SD大鼠SKP，以HA为载体构建复合物，观察复合物中SKP的分化特性。选取60只SD大鼠腹腔注射链脲菌素诱导成DM模型．背部对称制作2个直径1cm全层皮肤缺损创面，随机分为SKP-HA组，创面涂布100μl SKP-HA；HA组，创面涂布100μl HA；对照组，创面涂布DMEM／F12培养基。每组20只。各组大鼠于伤后1、2、3、4周检测创面愈合率，留取创面组织标本检测羟脯氨酸（Hyp）含量，并观察SKP在创面愈合过程中的迁移。结果大鼠SKP与HA共培养后生长良好，复合物中的SKP可保持其特性：向神经元细胞、神经胶质细胞、脂肪细胞分化。伤后2周SKP-HA组、HA组的创面愈合率分别为（72．1±2．8）％、（53．7±2．9）％，均明显高于对照组的（42．5±1．5）％（P＜0．05）；伤后3周SKP—HA组高于HA组及对照组（P＜0．05或0．01）；伤后4周SKP—HA组、HA组创面完全愈合，与对照组比较，差异有统计学意义（P＜0．01）。伤后1周各组Hyp含量差异无统计学意义（P＞0．05）；伤后2～4周，SKP-HA组、HA组Hyp含量均高于对照组（P＜0．01）；伤后3、4周SKP-HA组高于HA组（P＜0．01）。SKP在创面得以成活并随时间的延长逐渐向真皮层迁移。结论SKP-HA复合物可促进DM大鼠创面愈合。     

不同传代次数大鼠骨髓间充质干细胞移植促进糖尿病大鼠创面愈合的研究

目的：探讨骨髓间充质干细胞（BMSCs）在体外培养扩增过程中引起的老化对其修复糖尿病慢性创面功能的影响。方法：从大鼠股骨骨髓中分离培养BMSCs,流式细胞分析仪鉴定细胞表型,成骨、成脂诱导鉴定其分化能力。取第5、15、25、35和45代（P5、P15、P25、P35和P45）BMSCs,0.25%的胰酶消化后制备1×10^6/m1单细胞悬液。腹腔注射65 mg/kg链脲佐菌素建立糖尿病SD大鼠模型,8周后,在SD大鼠背部左右两侧用打孔器制备直径1.2 cm的圆形创面。将70只糖尿病慢性创面模型大鼠随机分为7组,分别经尾静脉注射1 ml的P5、P15、P25、P35和P45 BMSCs单细胞悬液（1×10^6/m1）,1 ml成纤维细胞（Fb）悬液（1×10^6/m1）和1 ml生理盐水。分别于伤后3、7、14、21 d计算创面愈合率,并取创面组织制备切片,苏木精-伊红（HE）染色,光学显微镜下观察组织病理学变化及血管生成情况。结果：糖尿病SD大鼠创面较正常大鼠创面愈合延迟（P〈0.05）。伤后14 d,各BMSCs移植组与Fb组和生理盐水组比较,创面收缩与再上皮化明显,镜下可见成纤维细胞生长活跃、数量多,各BMSCs移植组间无明显差异。伤后14 d,各BMSCs移植组创面愈合率较Fb组和生理盐水组增加,以P5 BMSCs移植组变化最显著（P〈0.05）。伤后7 d,各BMSCs移植组微血管密度均显著高于Fb组和生理盐水组（P〈0.01）,以P5 BMSCs移植组效果最为显著。结论：全身注射异体BMSCs可促进糖尿病创面愈合。BMSCs在体外长期扩增传代可减少其创面修复能力,其对糖尿病创面的修复作用以P5以内干细胞较好。     

神经肽P物质与烫伤创面愈合关系的实验研究

目的　探讨神经肽P物质(SP)与烫伤创面愈合之间的关系。　方法　(1)制作大鼠不同深度烫伤模型,分别于伤后1、3、7、14d致死,用放射免疫法测定创面SP含量。(2)将大鼠肉芽组织成纤维细胞(GTF)用不同培养液培养,分为空白对照组、SP组及SP SP受体拮抗剂(Spantide)组。体外检测SP及Spantide对GTF增殖活性[以吸光度(A)值表示]及凋亡率的影响。　结果　(1)伤后1d,浅Ⅱ、深Ⅱ、Ⅲ度烫伤创面SP含量分别为(145±78)、(94±48)、(53±27)ng/g,深Ⅱ度创面与其余两者比较,差异有统计学意义(P<0 01).浅Ⅱ度创面伤后3、7dSP含量显著增高;深Ⅱ度创面伤后7、14dSP含量显著增高;Ⅲ度创面伤后SP含量无显著变化。(2)SP增强GTF增殖活性(空白对照组A为0. 21±0. 05,SP组A为0. 36±0 07,P<0. 01)并抑制其凋亡,Spantide可抑制SP对GTF的作用。　结论　SP可促进GTF增殖,创面SP含量与创面损伤程度及愈合能力关系密切。     

表皮生长因子对大鼠深Ⅱ度烧伤创面愈合的影响

目的　进一步观察重组人表皮生长因子 (rhEGF)对深Ⅱ度烧伤创面愈合的促进作用。方法　采用大鼠深Ⅱ度烫伤模型 (以下称烧伤 ) ,创面分别外用rhEGF、肝素加rhEGF及等渗盐水。比较不同方式处理创面后的愈合时间 ,测定创面愈合率、创面含水量、羟脯氨酸 (OHP)含量及Ⅰ /Ⅲ型胶原比例 ,进行细胞DNA周期分析和组织学检查。　结果　外用rhEGF可使烧伤创面愈合时间缩短 2d,增加创面OHP含量 (P <0.0 5),降低Ⅰ /Ⅲ型胶原比例 (P <0.0 5),促进肉芽组织形成 ,加速细胞DNA复制 (P <0.0 5 )。加用肝素后可使创面愈合时间进一步缩短 2d,促进肉芽组织生长。伤后第 7天用药各组间比较 ,差异无显著性意义 (P >0.0 5)。　结论　外用rhEGF能明显促进深Ⅱ度烧伤创面愈合 ,加用肝素后效果更加显著 ,但早期使用rhEGF效果不明显     

SDF-1对创缘表皮干细胞定向趋化及促创面愈合效应的研究

目的：观察干细胞趋化因子SDF-1在创面愈合过程中调控表皮干细胞定向迁移促进创面愈合的过程。方法：制作大鼠背部全层皮肤缺损模型,随机分为三个实验组：①SDF-1组;②AMD3100（CXCR4受体的拮抗剂）组;③空白对照组。分别于致伤后1天、3天、5天、7天和12天照相计算伤后不同时间创面占初始创面的百分比并取材。运用HE染色和免疫组化技术观察创缘表皮干细胞定向迁移分化的特点,寻找其规律。结果：与其他两个处理组相比,SDF-1处理组愈合时间缩短,其创缘皮表皮基底层细胞分裂增殖旺盛,创缘表皮干细胞数目明显多于其他两组。结论：在创面愈合过程中,SDF-1可以调控表皮干细胞向创缘迁移,加速创面上皮化。     

重组人表皮生长因子促进大鼠皮肤创面愈合的研究

目的观察重组人表皮生长因子(rhEGF)对皮肤创面愈合的作用。方法制作大鼠背部创伤模型,采用自身平行对照,将34只大鼠背部的68个创面分成rhEGF治疗组与盐水对照组,观察大体形态和组织学改变、创面愈合时间和愈合率,测定伤后不同时间创面羟脯氨酸(OHP)含量和Ⅰ型Ⅲ型胶原比例,进行细胞DNA周期分析。结果经rhEGF治疗的创面愈合速度较盐水对照明显加快,2组平均愈合时间为(17.2±1.3)d和(20.5±1.6)d(P<0.01);外用rhEGF使创面肉芽组织生成增多,再上皮化明显,显著增加创面中OHP含量,降低Ⅰ型Ⅲ型胶原比例,加速细胞DNA复制。结论外用rhEGF可缩短创面愈合时间,增加肉芽组织及OHP含量,降低Ⅰ型Ⅲ型胶原比例,加速细胞DNA复制,明显促进皮肤创面的修复。     

重组人表皮生长因子对肛周脓肿术后创面愈合的影响

目的观察重组人表皮生长因子对肛周脓肿患者术后创面愈合的影响。方法随机将112例肛周脓肿手术患者分为治疗组(n=56)和对照组(n=56)。治疗组:创面用生理盐水消毒,以浸有重组人表皮生长因子凝胶纱条填塞。对照组:常规消毒,用甲硝唑纱条或雷佛奴尔纱条填塞。手术后3、7、10 d观察创面水肿情况及愈合时间。结果治疗组和对照组手术后3、7、10 d创面水肿评分分别为[(1.20±0.70)、(1.00±0.67)、(1.05±0.69)]和[(1.70±0.66)、(1.90±0.74)、(1.70±0.73)],两组比较差异有统计学意义(P0.05),两组创面愈合时间比较,差异有统计学意义(P0.05)。结论重组人表皮生长因子能明显促进创面愈合。     

Effect of substance P released from peripheral nerve ending on endogenous expression of epidermal growth factor and its receptor in wound healing

Objective: To explore the relationship between substance P (SP) released from peripheral nerve endings and the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) during wound bealing. Methods: Fifty Wistar rats were randomly divided into 2 groups, injury group and capsaicin group. In the injury group, a full-thickness skin wound on the back of the rat was taken. The wound edge and granulation tissues were taken on the 1st, 3rd, 6th, 9th, 12th days after injury, respectively. In the capsaicin group, capsaicin was injected subcutaneously on the back of the rats to destroy the sensory nerve to prevent the secretion of SP, then a wound and sample was made in the same way.Immunohistochemistry and in situ hybridization were employed to detect the expression of SP, EGF/EGFR, and EGF mRNA/EGFR mRNA in the granulation tissues.Results: In the injury group,immunohistochemical stain of SP and EGF/EGFR was located on the hair follicles and sebaceous glands at the 1st day. And the stain of SP was obvious at the 3rd day in the granulation tissues, then decreased gradually. EGF/EGFR was at low level at the 3rd day, then increased gradually and reached the peak at the 9th day, then declined. In the capsaicin group, the stain of SP and EGF/EGFR was faint and without obvious change during the wound healing process. The tendency of the EGF mRNA/EGFR mRNA expression was similar to that of EGF/EGFR. Conclusions: During wound healing, SP may promote the healing process by affecting the expression of EGF/EGFR in the erunuation tissues.     

骨髓间充质干细胞促进“创面”愈合的实验研究

目的：探讨骨髓间充质干细胞（MSCs）促进创面愈合的作用和用于创面修复的可行性。方法：密度梯度离心法分离培养正常人骨髓间充质干细胞（hMSCs），流式细胞仪检测培养第二代细胞CD14、CD29、CD34、CD44、CD45和CD106抗原表达以鉴定MSCs。实验分3组：直接共培养组（n=8）为hMSCs（1×10^5/ml）与人表皮角质形成细胞（HEKa）直接接触共培养；间接共培养组（n=7）为在transwell透明聚碳酸酯膜上层加入hMSCs细胞悬液[（2-3）×10^5个细胞）]，下层接种HEKa，间接共培养；单纯HEKa培养组（n=7）单纯培养HEKa。在倒置显微镜下刮擦生长融合成片的HEKa细胞以制备宽度为100μm的“表皮创面”模型。模型制备后24、48、72h，倒置相差显微镜下计数每高倍视野越过创缘迁移到创面的HEKa数，并用SigmaScan Pro5软件计算创面愈合率，检测HEKa细胞吸收脱氧胸腺嘧啶核苷的放射活性，判定HEKa细胞的分裂增殖活性。结果：流式细胞仪检测第二代hMSCs CD29、CD44和CD106抗原阳性（分别为94.81%、95.38%、97.40%），CD14、CD34、CD45抗原阴性（分别为1.23%、3.05%、2.64%）；直接培养组、间接培养组和单纯HEKa培养组越过创缘进入创面的细胞伤后24h分别有（10.00±0.25）、（5.50±0.20）和（5.20±0.35）个/高倍视野；48h分别有（55.30±0.22）、（27.00±0.34）和（26.50±0.25）个/高倍视野；72h分别有（110.50±0.45）、（42.50±0.50）、（40.20±0.38）个/高倍视野。3组伤后72h创面愈合率分别为（60.00±0.04）%，（35.00±0.01）%、（33.00±0.05）%。脱氧胸腺嘧啶苷掺入培养基法表明hMSCs对体外共培养HEKa细胞的增殖作用分别为（1650±270）cpm/10^5 cell、（1240±210）cpm/10^5 cell、（1180±220）cpm/10^5 cell。上述各指标直接共培养组与单纯HEKa培养组比较，差异有显著性（P〈0.01），而间接培养组与单纯HEKa培养组间差异无显著性（P〉0.05）。     

感觉神经肽P物质在表皮干细胞分化中作用的实验研究

目的以体外培养表皮干细胞(ESC)为实验平台，观察感觉神经肽P物质(SP)在ESC分化中的作用。方法以黏附分离法分离、纯化新生Wistar大鼠的ESC，在出现ESC克隆生长时加入SP刺激，分别于刺激前及刺激后24、48、72、96、144、192、240、288、336、384、432h取样进行角蛋白14(K14)免疫组织化学染色并用流式细胞仪(FCM)，鉴定细胞分群及比例。结果ESC在加入SP后可以继续呈片状聚集生长，细胞K14染色阳性，提示为短暂扩充细胞(TAC)。FCM检测结果提示，ESC经SP刺激后出现TAC细胞群落，细胞比例随时间的延长上升。结论SP可以诱导体外培养ESC分化为TAC，在这个过程中ESC数量仍保持一定的水平。     

High glucose inhibits human epidermal keratinocyte proliferation for cellular studies on diabetes mellitus

In order to more clarify the delayed wound healing in diabetes mellitus, we cultured the human epidermal keratinocytes in both 6 mM (control group) and 12 mM glucose (high-glucose group) of "complete" MCDB 153 medium. Hyperglycaemia slowed the rate of their proliferation and inhibited their DNA synthesis and the production of total proteins. By 1 month after primary seeding in high-glucose group, the cells ceased their proliferation, whereas the cells in control group grew for more than 40 days. Mean population doublings in high-glucose group was 5.27 (vs. 7.25 in control, P = 0.001), and mean population doubling time during 1 month in high glucose group was 5.43 days (vs. 3.65 days in control, P = 0.02). They indicate that prolonged exposure to high glucose decreases the replicative life span of human epidermal keratinocytes in vitro. Furthermore, analysis of fatty acid contents in membrane phospholipids with thin-layer and gas chromatography showed no difference between the cultured keratinocytes in both conditions. Immunocytochemical staining of glucose transporter 1 shows that 28.1% of cells in high-glucose group were almost twice positive of those in control group (13.2%, P = 0.008). The mechanism of the ill effects of high glucose on epidermal keratinocytes is not so far clear, but it indicates the possibility of any direct effect of hyperglycaemia on glucose metabolism without changing lipid metabolism on cell membrane. The high-glucose group presented in this report can be available as an in vitro valuable study model of skin epidermal condition on diabetes mellitus.     

The combined effect of recombinant human epidermal growth factor and erythropoietin on full‐thickness wound healing in diabetic rat model

Diabetic wound is a chronic wound in which normal process of wound healing is interrupted. Lack of blood supply, infection and lack of functional growth factors are assumed as some of the conditions that lead to non‐healing environment. Epidermal growth factor (EGF) acts primarily to stimulate epithelial cell growth across wound. Erythropoietin (EPO) is a haematopoietic factor, which stimulates the production, differentiation and maturation of erythroid precursor cells. This study hypothesised combining these two factors, non‐healing process of diabetic wound will be compensated and eventually lead to acceleration of wound healing compared with single growth factor treatment. A total of 30 diabetic Sprague–Dawley rats were divided into three treatment groups (single treatment of rh‐EPO or rh‐EGF or combined treatment on a full‐thickness skin wound). To assess the wound healing effects of the components, the wound size and the healing time were measured in each treatment groups. The skin histology was examined by light microscopy and immunohistochemical analysis of proliferating markers was performed. The combined treatment with rh‐EPO and rh‐EGF improved full‐thickness wound significantly (P < 0·05) accelerating 50% healing time with higher expression of Ki‐67 compared with single growth factor‐treated groups. The combined treatment failed to accelerate the total healing time when compared with single growth factor treatments. However, the significant improvement were found in wound size reduction in the combined treatment group on day 4 against single growth factor‐treated groups (P < 0·05). This study demonstrated that the combined treatment of rh‐EPO and rh‐EGF improved the wound healing possibly through a synergistic action of each growth factor. This application provides further insight into combined growth factor therapy on non‐healing diabetic wounds.     

糖基化终末产物对糖尿病大鼠烧伤创面愈合的影响

目的 了解糖基化终末产物(AGE)蓄积对糖尿病大鼠烧伤创面愈合的影响. 方法 将75只SD大鼠按完全随机化方法 分为对照组、糖尿病组及氨基胍干预组,每组25只.将大鼠造成深Ⅱ度烫伤(以下称烧伤)后,后2组制作成糖尿病模型,氨基胍干预组给予管饲氨基胍100 mg·kg~(-1)·d~(-1).于伤后0(伤后当天)、3、7、14、21 d处死大鼠.描取创面形状,并取全层皮肤组织待测.同时取大鼠背部表皮行KC培养及鉴定.观察各组创面愈合率及皮肤组织糖含量,创面组织形态学变化,皮肤组织中AGE分布.观察不同浓度AGE对KC增殖,凋亡的影响,以仅加表皮细胞培养液的KC为对照组. 结果 伤后7、14、21 d糖尿病组创面愈合率显著低于对照组(P<0.01),而氨基胍干预组却明显高于前2组(P<0.01).糖尿病组皮肤组织含糖量为(2.62±0.19)mmol/g,氨基胍干预组为(2.58±0.07)mmol/g,均高于对照组(1.04±0.09)mmol/g(P<0.01).对照组大鼠创面炎性细胞浸润强烈而局限,坏死组织形成、脱落及时,创面愈合无明显延迟;糖尿病组大鼠炎性细胞浸润缓慢、弥散而持久,坏死组织形成、脱落较迟,创面愈合明显延迟;氨基胍干预组大鼠炎性细胞浸润及时、强烈,坏死组织形成、脱落以及创面愈合时间较糖尿病组早.对照组中有少量散在的AGE沉积,糖尿病组AGE蓄积显著增加,而氨基胍干预组AGE含量显著降低.AGE作用48 h后,KC的增殖显著降低,呈现浓度依赖性,各剂量AGE干预组的吸光度值均低于对照组(P<0.01).100μg/mLAGE干预组KC早期凋亡连接素V阳性细胞比例为(15.1±2.3)%,明显高于对照组[(11.2±1.2)%,P<0.05];终未期凋亡双阳性细胞比例为(14.3±3.5)%,与对照组(15.2±2.4)%比较,差异无统计学意义(P>0.05). 结论 高血糖可能是通过AGE蓄积,抑制KC等修复细胞增殖导致创面难愈;减少AGE蓄积,可改善糖尿病创面愈合延迟现象.     

MSCs静脉移植对周围神经再生的作用

目的检测间充质干细胞(mesenchymal stem cells,MSCs)经静脉移植后在周围神经损伤模型内的迁移、分布情况,并评价其对轴突、靶器官的保护作用。方法经尾静脉注射BrdU标记的MSCs入大鼠坐骨神经损伤模型内(实验组)。术后自神经断端、肌肉组织取材检测BrdU阳性细胞,评价坐骨神经指数(SFI)、肌纤维横截面积及肌细胞凋亡数量。结果术后4、8周从神经断端、肌肉组织内检测到BrdU阳性细胞。实验组的SFI、肌纤维横截面积明显高于对照组(不注入MSCs),细胞凋亡数量低于对照组,两组差异均有统计学意义(P<0.05,P<0.01)。结论MSCs静脉移植至体内能归巢到损伤的神经断端、肌肉组织,具有促进轴突再生、延缓肌萎缩的作用。