角质细胞生长因子的研究进展

角质细胞生长因子（KGF）是成纤维细胞生长因子（FGFs）家族的成员,即FGF－7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26－28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。FGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

角质细胞生长因子的研究进展

角质细胞生长因子(KGF)是成纤维细胞生长因子(FGFs)家族的成员,即FGF-7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26—28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。KGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

H－ras在颊粘膜正常、良性、癌前及恶性变上皮中的表达：免疫组织化学定量研究

本文通过建立图象分析方法对免疫组织化学反应结果进行定量,检测观察Ｈ－ｒａｓ在口腔颊粘膜上皮在正常（Ｎ）、慢性炎症（ＩＦ）、癌旁上皮（ＥＡＣ）和鳞癌（ＳＣＣ）的变化过程中的表达并进行分析。结果显示Ｈ－ｒａｓ在ＳＣＣ组中,以中等分化的ＳＣＣ无论是Ｈ－ｒａｓ表达的量还是细胞阳性率都较高。此外,组织学观察显示,Ｈ－ｒａｓ在处于分化末期但尚未角化的正常上皮细胞中有较高的表达。本文结果显示了Ｈ－ｒａｓ的过表达与上皮细胞的会化程度密切相关。本研究还显示,所采用的阳性区域透光值、平均总透光值及阳性反应区域与阴性反应区域比值可靠并有相关性。这进一步说明了用免疫组化定量方法检测Ｈ－ｒａｓ癌基因表达的精确和可靠性。     

骨形成蛋白(BMP)-2/4，-5与IA型BMP受体在口腔正常上皮、良性病变及癌变中的表达分析

认识BMP及其受体与口腔正常上皮及其癌变的关系。有助于深入了解口腔上皮癌变的机理。本文用免疫组织化学方法对BMP-2/4,-5与BMPR-IA在口腔颊部粘膜正常上皮,良性病变和癌变中的表达进行观察和半定量分析。标本包括：9例正常上皮（normal buccal muosa,NB)。8例慢性炎症（nonspecific chronic inflammation,NCI),7例过度角化（hyperkeratosis,HK)。5例乳头状瘤（squamous cell papilloma,SCP)。29例鳞癌（squamous cell carcinoma,SCC)。10例癌旁上皮（epithelium immediately adjacent to carcinoma,EAC)以及6例硬腭粘膜上皮（normal mucosa of hard palate,NHP）。结果显示：BMP-2/4,-5与BMPR-IA在口腔粘膜的正常与良性病变上皮中有弱的和不均一的阳性表达,NB与NHP无明显差别,而除3例SCC外,其它SCC几乎均有程度不一的阳性表达,在EAC中的表达接近于SCC,二者明显高于正常与良性组,此外,转移在淋巴结中的癌细胞的BMP-2/4与BMP-5阳性程度略高于原发灶的癌细胞,本文认为;BMP-2/4,5与BMPR-IA可能参与调控口腔上皮的癌变。     

金雕肺的组织结构及KGF、c-Fos和Bax的表达

为了解金雕(Aguila chrysaetos)肺的组织结构特征,观察角质细胞生长因子(KGF)、c-Fos蛋白和Bax蛋白在肺中的表达情况,应用生物显微技术和免疫组织化学方法观察了金雕肺的组织结构,检测KGF、c-Fos和Bax蛋白在肺中的表达.结果表明,金雕的肺由各级支气管及其周围的呼吸毛细管组成,肺房呈辐射状开口于三级支气管周围,呼吸毛细管相互吻合成网状,周围有丰富的毛细血管网.KGF、c-Fos和Bax在三级支气管、肺房和呼吸毛细管的上皮细胞中呈免疫反应阳性,三者的表达强度有差异.KGF、c-Fos和Bax可能发挥不同的功能,它们的协同表达可能调控鸟肺细胞的生长、发育及成熟.     

丝甘蛋白聚糖促进非小细胞肺癌的侵袭与转移

丝甘蛋白聚糖(serglycin,SRGN)在肿瘤细胞的侵袭与转移中具有广泛的研究前景。本研究报道SRGN与肺癌细胞侵袭与转移能力之间的相关性。首先,通过检测SRGN在正常肺上皮细胞株BEAS-2B及不同侵袭与转移能力的肺癌细胞株95C、95D中的表达差异。利用shRNA干扰技术,在侵袭与转移能力强的95D细胞中建立稳定干扰SRGN表达的95D/shSRGN的细胞株,并通过RT-qPCR、Western印迹、酶联免疫吸附测定验证其敲除效率。结果显示：干扰SRGN可抑制侵袭与转移性强的95D细胞的侵袭与转移能力,减弱细胞迁移与侵袭等生物学特性,导致上皮标志物上皮细胞钙黏连蛋白（E-cadherin）表达上调,间质标志物纤维连接蛋白1（fibronectin1, FN1）及EMT（epithelial-mesenchymal transition, EMT）相关转录因子锌指E盒结合同源框1（zinc finger E-box binding homeobox 1, ZEB1）表达下调。进一步分析发现, SRGN与上皮细胞钙黏连蛋白表达成负相关（P=-0.25）,而与FN1(P=0.12)及ZEB1(P=0.35)表达成正相关,并且SRGN高表达的患者总生存时间明显少于SRGN低表达组（P=0.0077）,SRGN与ZEB1同时高表达的患者,总生存时间显著小于SRGN与ZEB1低表达患者（P=0.0005）。研究结果证实,SRGN促进上皮间质转化发生,增强非小细胞肺癌的侵袭与转移能力,为非小细胞肺癌预后提供参考。     

双峰驼睾丸和隐睾细胞外基质相关蛋白的组织化学特征及超微结构比较

为探索细胞外基质相关蛋白在隐睾双峰驼的分布情况及其组织化学特征,应用电镜技术和多种组织化学方法比较了隐睾和正常睾丸的超微结构,组织化学特点及层粘连蛋白（LN）、Ⅳ型胶原（Col Ⅳ）和硫酸乙酰肝素糖蛋白（HSPG）的分布特征。结果显示：(1)与正常睾丸间质结构相比,光镜下隐睾生精小管发育不全,间质内胶原纤维稀疏,网状纤维分布明显,间质血管及生精小管固有膜PAS及AB-PAS阳性反应较弱。电镜下,隐睾生精上皮基膜明显增生,外围I型胶原纤维较少,管周肌样细胞不典型;间质毛细血管及Leydig细胞周围纤维细胞多见,而正常睾丸在间质毛细血管及Leydig细胞周围多分布有成纤维细胞。(2) 免疫组织化学染色显示,正常睾丸组织的Col Ⅳ、LN及HSPG在Leydig细胞内均为强阳性表达,Col Ⅳ和LN在毛细血管内皮细胞强阳性表达,后者在Sertoli细胞的表达尤为明显,HSPG在精原细胞无表达;隐睾时Col Ⅳ、LN及HSPG在Leydig细胞内阳性表达均明显减弱,Col Ⅳ、LN在管周肌样细胞及毛细血管内皮细胞阳性表达也减弱明显,HSPG在精原细胞较强阳性表达,且在精子细胞呈强阳性表达。免疫组织化学图像分析结果显示,双峰驼正常睾丸组织中Col Ⅳ和LN的分布显著高于隐睾组织（P<0.05）,HSPG检测结果在正常睾丸与隐睾之间无统计学差异（P>0.01）。该研究表明,双峰驼隐睾生精小管发育异常,间质组织中合成胶原纤维的能力下降,睾丸细胞外基质的重要成分Col Ⅳ,LN与正常组差异显著与生精小管及Leydig细胞异常发育有关,而HSPG在隐睾生精上皮的强阳性表达与精原细胞发育不成熟密切相关。     

肝细胞生长因子诱导人肝癌细胞上皮间质化

上皮间质转化（epithelial-mesenchymal transition,EMT）与肿瘤侵袭转移密切相关.虽然肝细胞生长因子（hepatocyte growth factor,HGF）已被证实为肿瘤EMT的主要诱导剂,但是HGF诱导肿瘤EMT发生的分子机制尚不完全清楚.本研究旨在探讨Snail在HGF诱导肝癌细胞上皮间质转化中的作用.用HGF处理肝癌HepG2和Hep3B细胞,显微镜观察细胞形态变化,划痕试验及Transwell试验检测细胞迁移能力,Western印迹检测Met,AKT的磷酸化及蛋白质表达的变化,Western印迹与real-time RT-PCR检测上皮细胞表面标志E-Cadherin和间质细胞表面标志N-Cadherin、Fibronectin的表达变化,以及EMT相关转录因子的表达变化.经HGF处理的HepG2、Hep3B细胞,Met和AKT的磷酸化水平显著增强;相差倒置显微镜下观察细胞形态向间质型细胞形态转化;细胞划痕和Transwell试验检测细胞的迁移能力较对照组显著增强;Real-time RT-PCR和Western印迹实验显示HGF的诱导能上调间质标记蛋白的表达及下调上皮型标志蛋白的表达.进一步发现,HGF能上调转录因子Snail的表达,干扰Snail能逆转HGF对HepG2和Hep 3B细胞EMT发生的诱导作用.由此可见,HGF可能通过诱导Snail的表达促进肝癌细胞EMT的发生.这为阐明肝癌细胞侵袭转移机制,以及肝癌的防治提供新线索.     

WIF-1在鼻咽未分化癌中表达的研究

目的 分析WIF-1在鼻咽慢性炎性组织、鼻咽癌中表达的特点,探寻其在鼻咽未分化癌中表达与相关临床资料的关系,以及在鼻咽癌发病机制中的作用.方法 应用免疫组化技术SP法检测65例鼻咽未分化癌鼻咽癌标本、20例鼻咽部慢性炎症中WIF-1的蛋白水平.结果 ① WIF-1在正常鼻咽上皮细胞中存在表达,在鼻咽假复层纤毛柱状上皮的纤毛柱状上皮细胞中呈强阳性表达.其表达呈周期性.② 鼻咽癌标本表达的平均百分数为（31.0±8.0）%,与非癌上皮组织中WIF-1在柱状细胞中全部呈强阳性相比,有显著差异.WIF-1在部分淋巴细胞中表达阳性.在鼻咽癌和淋巴细胞中表达两者无相关性.③ WIF-1在鼻咽未分化癌细胞中的表达与患者的年龄、性别、原发灶大小（T分期）、淋巴转移、远处转移均无关系.结论 WIF-1在正常鼻咽上皮细胞中呈阳性表达;WIF-1在鼻咽未分化癌细胞中出现表达缺少和（或）下调;WIF-1表达缺少和（或）下调的出现可能是鼻咽未分化癌发生的早期事件,WIF-1表达异常可能在鼻咽未分化癌的发病机制中发挥一定作用.     

单克隆来源的小鼠胎肝HPP-CFC肝上皮分化潜能的研究

为研究胎肝中造血和肝上皮发育的关系,建立了小鼠胎肝高增殖潜能集落形成细胞（HPP-CFC）培养体系,并进行了单克隆培养以及诱导分化实验.在造血和肝诱导因子的共同作用下,对单克隆来源的HPP集落细胞向造血和肝上皮细胞进行诱导分化,采用透射电镜(TEM)、巢式RT-PCR、细胞免疫荧光检测,从细胞形态、超微结构、上皮细胞分化标志等方面对分化后的细胞进行检测.检测结果显示诱导后的部分细胞具有肝细胞特异性的超微结构并不同程度的表达白蛋白（ALB）、甲胎蛋白（AFP）、细胞角蛋白（CK8,CK18）等肝上皮分化标志,同时还表达间质标志α-SMA和血管内皮细胞标志Flk-1.免疫磁珠分选表明：胎肝来源的HPP-CFC主要来自于CD45⁺细胞,CD45^-细胞不具有形成造血克隆的能力.在肝上皮细胞分化潜能上,流式分选获得的CD49f⁺/Sca-1⁺细胞与未分选细胞无明显差异.该模型的克隆源性通过细胞混合实验进行证明.研究结果表明,改进的胎肝来源的HPP-CFC可能代表了一个新的造血向肝上皮细胞分化的单克隆模型,为研究胎肝中造血和非造血细胞的发育关系提供了一个新的切入点.     

用原位末端标记方法观察细胞凋亡在正常喉粘膜、炎症、癌前和癌变中的作用

为了观察凋亡与喉粘膜癌变的关系,本实验应用原位末端标记（ＩＳＥＬ）方法检测喉粘膜正常（ｎｏｒｍａｌｌａｒｙｎｇｅａｌｍｕｃｏｓａ,Ｎ）、炎症（ｉｎｆｌａｍｍａｔｉｏｎ,ＩＦ）、不典型增生（ｄｙｓｐｌａｓｉａ,ＤＹＳ）及鳞癌（ｓｑｕａｍｏｕｓｃｅｌｃａｒｃｉｎｏｍａ,ＳＣＣ）中细胞凋亡情况。标记后的凋亡细胞通过计数以凋亡指数（ＡＩ）表示。结果表明：ＤＹＳ组的凋亡指数（ＡＩ）在所有病变中最高;ＤＹＳ及ＳＣＣ组的ＡＩ值较正常Ｎ及ＩＦ组明显增多,且有显著性差异（Ｐ＜００５）;对ＳＣＣ的计数显示,ＡＩ值随着ＳＣＣ的分化程度下降有逐渐减少的趋势。ＳＣＣ转移组与非转移组的ＡＩ值间无显著性差异（Ｐ＞００５）。结论：细胞凋亡在喉粘膜癌前病变和癌变中发挥着重要作用,与喉癌的发生发展密切相关     

Keratinocyte growth factor induces gene expression signature associated with suppression of malignant phenotype of cutaneous squamous carcinoma cells

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes.     

Attenuation and loss of hormonal modulation of KGF (FGF-7)/KGF receptor expression and mitogenesis during mammary tumor progression

Keratinocyte growth factor (KGF), alone and in synergism with progesterone (P) and prolactin (PRL), is mitogenic for normal mammary epithelium (ME) in vitro. In addition, P can upregulate ME sensitivity to KGF by slowing KGF receptor (KGFR) mRNA turnover in vitro. These hormonal interactions with KGF in vitro raise the possibility that alterations in these interactions can play a role in hormone-dependent mammary tumor growth and progression. The effect of hormones on KGF mitogenesis and the regulation of KGFR expression was examined in pregnancy-dependent (PDT) and ovarian-independent (OIT) mouse mammary tumors. In serum-free, collagen gel cell culture, dose/response (2-20 ng/ml) and time course studies showed that KGF stimulated the proliferation of PDT (not OIT) cells but synergism with P or PRL was not observed. The level of KGFR mRNA in PDT cells was not significantly different from normal ME but in OIT it was reduced more than 90%. P did not affect KGFR mRNA turnover in cultured PDT cells. However, KGFR mRNA was more stable in PDT cells compared to normal ME; after 6 days culture in basal medium, KGFR mRNA levels declined 40% vs. 85% previously shown for normal ME. Determination of KGF mRNA levels in tissues showed that it was lower in PDT compared to normal mammary gland and not detectable in OIT. These data show that in PDT both KGF-stimulated mitogenesis and the regulation of KGFR expression are independent of hormones. OIT has progressed to independence from any KGF influence. Thus, a subset of hormonally regulated pathways related to epithelial/stromal cell interactions can be lost in hormone-dependent mammary tumors during tumor progression.     

Heparin-binding keratinocyte growth factor is a candidate stromal-to-epithelial-cell andromedin.

The growth of isolated epithelial and stromal cells from both androgen-dependent normal rat prostate and an androgen-responsive model rat prostate tumor is androgen-independent. When added to co-cultures of epithelial and stromal cells separated by a semipermeable membrane, androgen stimulated epithelial cell growth without an effect on stromal cell growth. Northern blot and nuclease protection analysis of mRNA revealed that stromal cells specifically expressed an androgen-sensitive secreted member of the heparin-binding fibroblast growth factor family [keratinocyte growth factor (KGF)/fibroblast growth factor-7]. KGF was mitogenic for epithelial cells, but not for stromal cells. Epithelial cells expressed specifically a splice variant of the bek receptor gene that specifically binds KGF. Expression of the bek receptor gene in stromal cells was undetectable by Northern blot and nuclease protection analyses. The results suggest that stromal cell-derived KGF has the properties of an andromedin, which mediates the indirect control of epithelial cell proliferation by androgen through a directional stromal-to-epithelial cell paracrine mechanism.     

Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P- labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P- induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P- dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.     

Keratinocyte growth factor‐2 intratracheal instillation significantly attenuates ventilator‐induced lung injury in rats

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor‐2 (KGF‐2), a potent epithelial cell mitogen, may have a role in preventing ventilator‐induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF‐2 in VILI in rats. Forty healthy adult male Sprague‐Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high‐volume zero positive end‐expiratory pressure) and HVZP+KGF‐2 were given intratracheally equal PBS and 5 mg/kg KGF‐2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF‐2 were administered in the same manner in Groups Control and KGF‐2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor‐α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre‐treatment with KGF‐2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF‐2 might be considered a promising prevention for human VILI or other acute lung injury diseases.