NO ASSOCIATION OF GLUTATHIONE S-TRANSFERASE M1 GENE POLYMORPHISM WITH DIABETIC NEPHROPATHY IN JAPANESE TYPE 2 DIABETIC PATIENTS

Oxidative stress possibly contributes to the development of diabetic nephropathy. Therefore, the levels of endogenous antioxidants may be one of determinants of the susceptibility to diabetic nephropathy. Glutathione S-transferases (GSTs) can work as one of endogenous antioxidants to protect cells from oxidative stress. The M1 member of GST mu class (GSTM1) is polymorphic and only expressed in 55–60% of Caucasians because of the homozygous deletion of the gene (null genotype). Recent studies have provided evidence that the GSTM1 null genotype, i.e. lack of the GSTM1 activity, is associated with an increased susceptibility to lung cancer and colorectal cancer. The present study was conducted to determine whether the genetic polymorphism influences the development of diabetic nephropathy. We examined 105 patients with diabetic nephropathy and 69 patients without diabetic nephropathy in Japanese type 2 diabetic patients with proliferative diabetic retinopathy. GSTM1 genotyping was performed by polymerase chain reaction. The two patient groups were well matched with regard to age, body mass index and HbA1c. GSTM1 null genotype was observed in 48.6% of patients with nephropathy versus 55.1% of patients without nephropathy. The frequency of GSTM1 null genotype was not significantly higher in the patient group with nephropathy than in the patient group without nephropathy. This study is the first to investigate the association of GSTM1 gene polymorphism with the development of diabetic nephropathy. The present results suggest that GSTM1 null genotype does not contribute to the development of diabetic nephropathy in Japanese type 2 diabetic patients.     

截短型转化生长因子-βⅡ型受体在皮肤成纤维细胞的过度表达

目的　研究截短型转化生长因子 -β(TGF-β) 型受体在皮肤成纤维细胞的过度表达对 TGF-β1 自分泌的抑制作用。　方法　组织块法体外培养正常皮肤成纤维细胞 ,加入重组人 (rh) TGF- β1 (5ng/ ml)或含截短型 TGF- β 型受体基因的重组腺病毒 (50 pfu/ cell) ,并用 Northern印迹观察其对 TGF-β1 的基因调控。　结果　 rh TGF-β1 能上调TGF-β1 和 I型前胶原的基因表达。截短型 TGF-β 型受体的过度表达能减少细胞 TGF-β1 的基因表达。　结论　截短型TGF- β 型受体的过度表达可以通过阻断 TGF- β1 的信号转导来抑制皮肤成纤维细胞 TGF- β1 的自分泌。为研究瘢痕的基因治疗提供了新的策略     

TRANSFER OF PLATELET-DERIVED GROWTH FACTOR-BB GENE BY GENE GUN INCREASES CONTRACTION OF COLLAGEN LATTICE BY FIBROBLASTS IN DIABETIC AND NON-DIABETIC HUMAN SKIN

We have used an in vitro model of wound contraction, the fibroblast-populated collagen lattice, to examine the effect of platelet-derived growth factor BB (PDGF-BB) and PDGF-BB gene transfer by gene gun on the contraction of lattices composed of either diabetic or non-diabetic human fibroblasts. The area of collagen lattice and DNA synthesis were measured in 12 specimens. There were significant increases in lattice contraction with increasing doses of PDGF-BB and fibroblasts transfected with the PDGF-BB gene compared with control (p < 0.01). DNA synthesis of the non-diabetic and diabetic fibroblast lattices showed significantly increased incorporation of tritiated thymidine with increasing doses of PDGF-BB and fibroblasts transfected with the PDGF-BB compared with controls (p < 0.05). The effect of PDGFBB gene transfer on diabetic and non-diabetic fibroblasts was similar to that of 20 ng/ml or less of PDGF-BB.     

EXPERIMENTAL MODEL FOR LOCAL APPLICATION OF GROWTH FACTORS IN SKIN RE-EPITHELIALISATION

We did an experimental study to assess the effects of different growth factors on re-epithelialisation of skin wounds by creating a partial-thickness defect in rats with a handle dermatome. Three different growth factors that are particularly involved in the re-epithelialisation phase of wound repair (epidermal growth factor (EGF), keratinocyte growth factor (KGF), and basic fibroblast growth factor (FGF-b) n     

细胞生长因子在美容护肤中的应用

细胞生长因子,对多种细胞生理功能和代谢活动发挥生物调节作用,他们在体内含量极微,但生物活性极高.在美容护肤、整形外科、烧伤溃疡以及各种皮肤病的伤口修复与愈合中有重要作用[1～2].     

自体富血小板凝胶的制备及其生长因子分析

目的 探讨不同离心方法 制备自体富血小板凝胶(autologous platelet-rich gel,APG)的方法 ,通过改变离心速度比较不同的离心力对PLT富集的影响分析全血和APG中5种生长因子浓度.方法 对13例糖尿病难治性皮肤溃疡患者行APG治疗.取11例自身外周静脉血,分别用3种离心速度制备富血小板血浆(platelet-rich plasma,PRP).A组(n=6)先以529×g离心4 min,再以854×g离心6 minB组(n=5)先以313×g离心4 min,再以1252×g离心6 min;C组(n=5)先以176×g离心5 min,再以1 252×g离心5 min.将离心后制得的PRP与凝血酶.钙剂以101混合凝固后制备APG,用于患者皮肤溃疡的治疗.采用全自动血细胞分析仪计数各组全血和PRP中PLT数量.采用酶联免疫吸附法测定全血和APG中PDGF.BB、VEGF、IGF.1、EGF和TGF.a1 5种生长因子浓度.结果 A组PRP中PLT数量为(779.67±352.39)×109/L,较全血的(263.50±76.63)×109/L提高(2.98±1.42)倍,差异有统计学意义(P＜0.05);PLT回收率为51.5%±22.2%.B组PRP中PLT数量最高,为(1363.80±919.74)X 109/L,较全血的(232.80±127.99)×109/L提高(5.91±2.04)倍,差异有统计学意义(P＜0.05);PLT回收率为75.2%±21.0%,明显高于A组(P＜0.05).全血和APG中PDGF-BB、EGF、IGF-1以及TGF-a.浓度分别为(145.94±133.24)、(503.81±197.86)pg/mL,(160.73±71.10)、(265.95±138.43)pg/mL,(14.54±35.34)、(110.56±84.36)ng/mL,(3.31±2.27)、(5.67±4.80)ng/mL,差异有统计学意义(P＜0.05);VEGF浓度升高,两者间差异无统计学意义(P＞0.05).对数转换后的PLT数量与PDGF-BB、TGF-a1浓度成正相关,相关系数r分别为0.627和0.437(P＜0.05).13例患者共行18次APG治疗,其中9例治疗12周溃疡愈合,愈合率为69.2%;10例窦道愈合,愈合率为83.3%.结论 以313×g离心4 min,再以1252×g离心6 min是制备PRP的最佳方法 ;APG中生长因子浓度高于全血;PLT数量与PDGF-BB、TGF-a1浓度成正相关.     

多种生长因子促糖尿病患者难愈合性创面愈合的临床研究

目的　观察多种生长因子治疗糖尿病难愈合性创面的临床效果,并探讨其可能的机制。方法　将７８ 例糖尿病患者分为三组,即生理盐水对照组、表皮细胞生长因子（ Ｅ Ｇ Ｆ）治疗组、血小板源伤口愈合因子（ Ｐ Ｄ Ｗ Ｈ Ｆ）治疗组。观察治疗后１～８ 周创面闭合指数、创面治愈率及组织形态学变化。结果　 Ｅ Ｇ Ｆ组、 Ｐ Ｄ Ｗ Ｈ Ｆ组的创面闭合指数、创面治愈率较对照组明显增加,其肉芽组织毛细血管与成纤维细胞增生、胶原沉积和表皮覆盖明显,尤以溃疡与正常组织交界处明显。组间比较发现, Ｐ Ｄ Ｗ Ｈ Ｆ的促愈合作用优于 Ｅ Ｇ Ｆ。结论　局部应用多种生长因子可治疗糖尿病难愈合性创面,生长因子单独或配伍使用是改善糖尿病患者创面愈合受阻的有效措施。     

EXPRESSION OF CYCLOOXYGENASE-2 IN THE MACULA DENSA OF HUMAN KIDNEY IN HYPERTENSION,CONGESTIVE HEART FAILURE,AND DIABETIC NEPHROPATHY

Cyclooxygenase-2 (COX-2) is constitutively expressed in the macula densa of several laboratory animal species where it is considered to play a physiologic role in the regulation of basal renal function. Pertubations to normal homeostasis is shown to be associated with the upregulation of COX-2 in the macula densa of rats and dogs. In contrast, COX-2 has not been detected in the macula densa of normal adult human and non-human primate kidneys, suggesting a less prominent role of this isoform in normal renal function in these species. In this study, we characterized COX-2 expression in human kidneys collected from subjects with a clinical history indicative of compromised renal function associated with diabetic nephropathy (DN), hypertension, and congestive heart failure (CHF). COX-2 expression was evaluated by immunohistochemistry using isoform-specific antibodies and in situ hybridization. No COX-2 protein or mRNA was observed in the macula densa of normal kidneys (n = 11), whereas slight to moderate COX-2 expression was present in the macula densa of 7/15 subjects (46%) with DN, 5/11 (46%) subjects with hypertension, and 3/10 subjects (30%) with CHF. These results indicate that COX-2 is variably induced in the macula densa of the human kidney in compromised renal conditions and that COX-2-mediated prostaglandins may be involved in maintaining adequate renal functions in some patients with DN, hypertension, and CHF. This variability may be related to individual clinical status or synthesis of vasodilatory prostaglandins by cyclooxygenase-1 (COX-1).     

热损伤角质形成细胞培养上清对真皮成纤维细胞Ⅰ、Ⅲ型胶原及基质金属蛋白酶1表达的影响

目的探讨角质形成细胞(keratinocytes,KC)热损伤后对真皮成纤维细胞(fibroblasts,Fb)Ⅰ、Ⅲ型胶原及基质金属蛋白酶1(matrix metalloproteinase 1,MMP-1)表达的影响。方法分离培养人正常Fb及KC,分别建立KC、Fb热损伤模型;收集正常及热损伤12 h后细胞培养上清,并配制成浓度为50%的细胞条件培养液。根据培养液不同将第3～5代Fb分为3组,分别采用含50%热损伤KC培养上清(A组)、含50%正常KC培养上清(B组)的条件培养液及单纯DMEM(C组)培养,24 h后收集3组细胞;另于培养0、1、2、6、12、24、48 h分别收集A组细胞。采用含50%热损伤Fb培养上清的条件培养液培养Fb,于0、1、2、6、12、24、48 h收集细胞。采用实时荧光定量PCR检测各时间点KC热损伤条件培养上清对FbⅠ、Ⅲ型胶原及MMP-1 mRNA表达影响,以及Fb热损伤条件培养上清对Fb MMP-1mRNA表达影响。结果 KC热损伤条件培养上清培养24 h,A组Ⅰ、Ⅲ型胶原及MMP-1 mRNA相对表达量均显著高于B、C组,差异有统计学意义(P<0.05)。培养2、6、12、24、48 h,A组Ⅰ、Ⅲ型胶原及MMP-1 mRNA相对表达量均高于0 h(P<0.05),1 h与0 h差异无统计学意义(P>0.05);随培养时间延长相对表达量逐渐增高,2 h后各时间点间比较差异均有统计学意义(P<0.05)。Fb热损伤条件培养上清培养1、2、6、12、24、48 h,MMP-1 mRNA相对表达量与0 h比较,差异均有统计学意义(P<0.05);培养2 h后相对表达量逐渐降低,各时间点间差异均有统计学意义(P<0.05)。结论热损伤后KC培养上清对FbⅠ、Ⅲ型胶原及MMP-1的表达具有调控作用。     

成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL、MCL体外增殖作用

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor，aFGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)以及复合因子对兔前交叉韧带(anterior cruciate ligament，ACL)和内侧副韧带(medial collateral ligament，MCL)的促增殖作用。方法分离传代培养10周龄新西兰大白兔骨关节韧带的ACL和MCL的成纤维细胞，接种96孔板，并加入浓度为0(对照组)、1、5、10、50、100ng／ml的aFGF或bFGF，浓度为0(对照组)、1．56、3．13、6．25、12．50、25、50ng／ml的EGF，单独或aFGF EGF两种因子联用与细胞(n=4)共同培养48h，以XTT方法测定其促细胞增殖作用。结果3种生长因子单独应用对ACL和MCL都有促进作用，aFGF对两种细胞均存在量效关系；bFGF 1ng／ml，EGF 5ng／ml对ACL作用最好，而对MCL则是bFGF和EGF均存在量效关系。浓度为5ng／ml的aFGF与50ng／ml的EGF联合1ng／ml aFGF与3．13ng／mlEGF作用于ACL或MCL均有协同作用。结论3种生长因子对ACL和MCL均有促进作用，单独应用aFGF或联用EGF优于单一因子促进兔ACL和MCL细胞的增殖，并且提示低浓度的aFGF联用EGF优于单一生长因子。     

RELEVANCE OF ALDO-KETO REDUCTASE FAMILY MEMBERS TO THE PATHOBIOLOGY OF DIABETIC NEPHROPATHY AND RENAL DEVELOPMENT

Aldo-keto reductases (AKRs) are a family of monomeric oxido-reductases with molecular weight ranging from 35–40 kDa and currently includes upwards of 60 members. They are expressed in a wide variety of tissues, where they catalyze the NADPH-dependent reduction of various aliphatic and aromatic aldehydes and ketones. The functions of most of the family members are not well defined. But two members, aldehyde reductase (AKR1A) and aldose reductase (AKR1B), have been extensively studied. The latter has received the most attention since being relevant to the complications of diabetes mellitus. It is up regulated during hyperglycemia, and at the same time there is an increased activity of the sorbitol pathway and non-enzymatic glycation of proteins with ensuing damage in various tissues. It is developmentally regulated in the ocular lens, and is believed to modulate lens fiber morphogenesis during fetal life. Unlike the other AKR family members that are ubiquitously expressed, recently a renal-specific oxio-reductase has been described that is expressed exclusively in the proximal tubules. Although, it has no homology with other AKR members, it binds to NADPH with high afinity and is up-regulated in streptozotocin-induced diabetes in mice. It is also developmentally regulated and seems to selectively modulate renal tubulogenesis during embryonic life.     

病理性瘢痕中热休克蛋白47的表达与胶原沉积相关性研究

目的　探讨热休克蛋白 4 7(heatshock protein 4 7,HSP4 7)在病理性瘢痕中的表达与胶原沉积的相关性。　方法　取经病理科确诊的正常皮肤 (10名 )、增生性瘢痕 (19例 )和瘢痕疙瘩 (16例 )组织标本 ,应用免疫组织化学方法、苦味酸 -天狼星红偏振光分析法检测组织中 HSP4 7的表达及胶原纤维含量。　结果　增生性瘢痕平均 IOD值5 2 115 9.5 0± 2 72 994 .13,瘢痕疙瘩组织平均 IOD值 4 0 74 4 0 .30± 2 95 780 .6 3中 HSP4 7表达量显著高于正常皮肤组织平均 IOD值 130 5 0 .17± 4 789.4 1,差异有统计学意义 (P<0 .0 1) ;HSP4 7的表达量与病理性瘢痕总胶原纤维含量呈正相关关系 (r=0 .386 ,P<0 .0 5 )。　结论　 HSP4 7在病理性瘢痕中异常高表达 ,并可能在病理性瘢痕胶原沉积的过程中发挥重要作用。