癌相关成纤维细胞在乳腺癌侵袭转移及耐药中的作用研究进展

癌相关成纤维细胞(CAF)是肿瘤微环境中主要的基质细胞。CAF可在血小板源性生生长因子、成纤维细胞生长因子、白介素6及肝细胞生长因子等多种分泌因子作用下由正常成纤维细胞转化形成,也可由间充质干细胞、脂肪细胞等多种细胞可通过上皮间质转化(EMT)过程形成,还有部分由癌症干细胞转化而来。近来有研究显示,乳腺癌中的CAF可通过分泌多种细胞因子及外泌体、参与EMT及细胞外基质重塑,进而促进乳腺癌细胞侵袭转移,也可在肿瘤缺氧微环境下通过激活相关信号通路促进乳腺癌细胞生长和侵袭。此外,CAF通过增加了乳腺癌细胞的凋亡阈值、作为抗肿瘤药物的物理屏障、分泌的谷氨酰胺增加乳腺癌细胞的存活率、激活生长因子相关的信号通路或增加线粒体功能产生抗凋亡作用等多种途径介导乳腺癌化疗耐药、内分泌治疗耐药及多药耐药。笔者总结CAF的重要来源及其在乳腺癌侵袭转移与治疗耐药中的研究进展。     

PFKFB3参与脂多糖诱导肺成纤维细胞及肺组织有氧糖酵解的观察性研究

目的:观察脂多糖(lipopolysaccharide,LPS)诱导肺成纤维细胞及肺组织有氧糖酵解关键酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)表达及其与有氧糖酵解的关系,探讨在LPS诱导肺纤维化过程中肺成纤维细胞和肺组织有氧糖酵解的潜在机制。方法:将人胚肺成纤维细胞MRC-5细胞系采用随机数字表法分为PBS对照组(PBS组)和LPS组(每组3孔),Western blot检测LPS刺激细胞6h后PFKFB3表达情况,同时免疫荧光显示PFKFB3在细胞内的定位情况;于LPS刺激后48 h采用海马细胞能量代谢仪检测细胞耗氧率(oxygen consumption rate,OCR)和产酸率(extracellular acidification rate,ECAR),并采用比色法检测有氧糖酵解产物乳酸产生情况,同时Western blot检测LPS刺激48 h后Ⅰ型胶原蛋白合成情况。将24只C57BL/6小鼠按随机数字表法分为生理盐水对照组(C组)、LPS组(L组),每组12只,L组、C组连续5d分别腹腔注射5 mg/kg LPS、等容量生理盐水;每组各6只于造模后第7天无痛处死小鼠,取血浆和肺组织,Western blot和免疫荧光检测各组肺组织中PFKFB3表达情况,比色法检测各组小鼠血浆中乳酸的含量;剩余小鼠于造模后第28天取肺组织,一侧肺通过Western blot检测肺组织Ⅰ型胶原蛋白合成情况,另一侧肺做石蜡切片进行病理学检测。结果:与PBS组比较,LPS刺激细胞6h后PFKFB3表达明显升高(P<0.05);LPS刺激细胞48 h后,与PBS组比较,LPS组细胞耗氧率降低、产酸率增加,代谢产物乳酸含量明显升高(P<0.05),同时细胞Ⅰ型胶原蛋白合成显著增加(P<0.05)。与C组比较,L组小鼠腹腔注射LPS 7 d后肺组织中PFKFB3表达明显升高(P<0.05),血浆乳酸含量明显升高(P<0.05);LPS注射28 d后,L组小鼠Ⅰ型胶原蛋白表达明显升高(P<0.05),肺组织出现明显纤维化。结论:在LPS诱导的肺纤维化过程中,LPS可诱导肺成纤维细胞和肺组织中PFKFB3蛋白表达,该过程与其有氧糖酵解过程相关,PFKFB3的表达上调可能是LPS诱导肺成纤维细胞和肺组织有氧糖酵解和肺纤维化的关键环节。     

补骨脂素抗增生性瘢痕作用机制研究

目的探讨补骨脂素抗增生性瘢痕的作用机制。方法体外培养成纤维细胞,按随机数字表法分为正常组(培养正常成纤维细胞)、瘢痕组(培养增生性瘢痕成纤维细胞)、TGF-β1组(10 ng/ml TGF-β1处理增生性瘢痕成纤维细胞5 min^12 h)、Smurf2 RNA干扰组[Smad泛素化调节因子2(Smad ubiquitin regulatory factor2,Smurf2)siRNA转染增生性瘢痕成纤维细胞72 h]、补骨脂素组(10μmol/L补骨脂素处理增生性瘢痕成纤维细胞继续培养72 h)、补骨脂素+TGF-β1组(增生性瘢痕成纤维细胞加入补骨脂素培养72 h后加入TGF-β1培养6 h)。采用Western blot法检测Smurf2、α-平滑肌肌动蛋白(α-actin SMA,α-SMA)蛋白表达;RT-PCR法检测Ⅰ型胶原蛋白mRNA表达;ELISA法检测TGF-β1蛋白分泌。结果与正常组比较,瘢痕组Smurf2蛋白[(0.83±0.08)比(0.38±0.07)]表达增加(P<0.05);与瘢痕组比较,Smurf2 RNA干扰组TGF-β1[(2.2±0.18)比(4.2±0.47)]表达降低(P<0.05);TGF-β1组Smurf2[(0.71±0.06)比(0.42±0.04)]、α-SMA[(1.42±0.12)比(0.91±0.09)]蛋白表达增加(P<0.05),Ⅰ型胶原蛋白mRNA[(0.72±0.09)比(0.41±0.07)]表达增加(P<0.05);补骨脂素组Smurf2[(0.05±0.01)比(0.42±0.04)]、α-SMA[(0.71±0.07)比(0.91±0.09)]蛋白表达降低(P<0.05),Ⅰ型胶原蛋白mRNA表达[(0.12±0.04)比(0.41±0.07)]降低(P<0.05)。结论补骨脂素可能通过TGF-β1/Smurf2信号通路抑制α-SMA蛋白表达,从而降低Ⅰ型胶原蛋白表达,起到抑制瘢痕形成的作用。     

黑素细胞及相关细胞群参与白癜风发病的研究进展

【摘要】 白癜风发病机制复杂，黑素细胞缺失是核心。目前认为除黑素细胞自身缺陷外，表皮和真皮细胞群的功能异常及其与黑素细胞的交互作用对白癜风的发病同样重要，充分而准确地了解不同病期白癜风皮肤全层细胞、组织的病理生理状态，对认识和治疗白癜风有重要作用。本文旨在综述黑素细胞及相关细胞群在白癜风发病中作用的研究进展。     

Cancer progression is associated with increased expression of basement membrane proteins in three-dimensional in vitro models of human oral cancer

Background

Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front.

Objective

This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial-mesenchymal interactions play on it, by using an in vitro oral cancer progression model.

Material and methods

In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin.

Results

Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium-biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin.

Conclusions

These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial-mesenchymal environment, respectively the existence of fibroblasts.     

以胚鼠脱细胞真皮构建口腔黏膜固有层的实验研究

目的:探讨以胚鼠脱细胞真皮构建组织工程化口腔黏膜固有层的可行性。方法:取胚鼠皮肤制备脱细胞真皮支架,将体外培养的人口腔黏膜成纤维细胞复合入胚鼠脱细胞真皮支架中,分别于接种后的1、4、7 d行HE染色、荧光显微镜及电镜检测。结果:胚鼠脱细胞真皮支架行HE染色显示未见细胞残留,表皮-真皮结合面保存波浪状基底膜样结构,HE、荧光显微镜及电镜检测均显示成纤维细胞复合入胚鼠脱细胞真皮支架后可黏附、生长、增殖。结论:以胚鼠脱细胞真皮体外构建的口腔黏膜固有层具有较好的组织构架,为进一步研究成纤维细胞-上皮细胞相互作用机制和构建全层口腔黏膜提供良好的实验模型。     

人脂肪来源间充质干细胞条件培养基对病理性瘢痕成纤维细胞生物学行为的影响

目的探讨人脂肪间充质干细胞条件培养基(human adipose-derived mesenchymal stem cells conditioned media,hADSCs-CM)对病理性瘢痕成纤维细胞增殖、迁移能力的影响。方法分离培养hADSCs和病理性瘢痕成纤维细胞,取第3代细胞用于后续实验。于培养12、24、48 h收集hADSCs条件培养基作为实验组条件培养基(12 h-CM、24 h-CM、48 h-CM),无hADSCs的空白无血清低糖DMEM培养基置于培养箱内12、24、48 h作为对照组条件培养基(12 h-CC、24 h-CC、48 h-CC)。选取增生性瘢痕成纤维细胞(HSFs)、瘢痕疙瘩成纤维细胞(KFs)以及正常皮肤成纤维细胞(NFs),分别以实验制备的培养基进行处理,每组培养基3个复孔,以CCK-8实验检测HSFs及KFs的增殖能力,以细胞划痕实验检测条件培养基处理后细胞迁移能力的变化,流式细胞仪分析细胞的周期分布、凋亡及乳酸脱氢酶(LDH)水平变化情况。实验数据以Graphpad 7.0软件进行分析,多组样本均数比较应用One-Way ANOVA,P<0.05为差异有统计学意义。结果CCK-8结果显示,24 h-CM、48 h-CM处理后24 h KFs、HSFs的增殖速度较对照组开始出现明显减慢,HSFs增殖趋势也出现了类似的变化趋势,NFs无明显的增殖趋势变化;实验组条件培养基对HSFs增殖的抑制作用在48 h达到峰值:24 h-CM KFs增殖活性为1.81±0.10,24 h-CC KFs为2.36±0.05(t=4.24,P<0.001);24 h-CM HSFs增殖活性为1.52±0.10,24 h-CC HSFs为1.96±0.15(t=8.98,P=0.001);48 h-CM KFs增殖活性为1.65±0.10,48 h-CC KFs为2.57±0.10(t=26.64,P<0.001);48 h-CM HSFs增殖活性为1.29±0.20,48 h-CC HSFs为1.94±0.10(t=11.14,P<0.001);之后抑制作用渐弱。细胞划痕实验结果表明,与对照组相比,24、48 h收集的hADSCs-CM处理后HSFs和KFs的迁移能力下降。细胞周期检测结果显示,在KFs中,12 h-CM、24 h-CM、48 h-CM组细胞处于G0/G1期的细胞比例较对照组升高,而处于S期的细胞比例则较对照组下降,在HSFs中也观察到了类似的现象。细胞凋亡检测结果显示各组NFs、KFs、HSFs未见明显凋亡。LDH漏出检测结果显示各组间无明显差异。结论hADSCs条件培养基可能通过其中含有的分泌因子抑制病理性瘢痕成纤维细胞的增殖、迁移,但不诱导其凋亡。     

PDCD4在肺纤维化模型中的表达及意义

目的:检测程序性细胞死亡蛋白4(PDCD4)在肺纤维化模型中的表达水平及对细胞活力和肺纤维化指标的影响及其机制。方法:Western blot和RT-qPCR检测PDCD4、α-平滑肌肌动蛋白(α-SMA)和I型胶原蛋白(COL-I)在人胚肺成纤维细胞系HFL-1组和HFL-1+TGF-β1组的表达水平。将质粒pEZ-M03-PDCD4和空质粒pEZ-M03转染进肌成纤维细胞(MB),应用Western blot检测PDCD4的蛋白表达水平;应用Western blot检测空白对照组、pEZ-M03-PDCD4组、pEZ-M03组和LY294002组PI3K/AKT信号通路相关分子p-AKT和AKT的蛋白水平及周期相关蛋白c-Myc和cyclin D1的表达情况; CCK-8法检测PDCD4对MB活力的影响;羟脯氨酸消化法检测HFL-1细胞和转染后MB上清液羟脯氨酸含量。Western blot检测小鼠模型组与对照组中肺组织PDCD4的表达水平。结果:与HFL-1组比较,HFL-1+TGF-β1组α-SMA和COL-I的mRNA及蛋白表达明显上调(P 0. 01),PDCD4mRNA表达无明显变化,PDCD4的蛋白表达明显下调(P 0. 01)。与空白对照组及pEZ-M03组比较,pEZ-M03-PDCD4组的PDCD4蛋白表达水平明显上调(P 0. 05),c-Myc和cyclin D1的蛋白表达都显著减少(P 0. 05),细胞活力明显受到抑制(P 0. 01),上清液中羟脯氨酸的含量显著减少(P 0. 05)。与空白对照组相比,pEZ-M03-PDCD4组及LY294002组中的p-AKT蛋白水平显著减少(P 0. 05),pEZ-M03组的差异无统计学显著性。在小鼠体内模型实验中,与对照组相比,模型组的PDCD4表达减少(P 0. 01)。结论:PDCD4在肺纤维化过程中低表达。过表达PDCD4可抑制MB活力,减少羟脯氨酸含量,抑制PI3K/AKT信号通路。     

Stromal vascular fraction promotes fibroblast migration and cellular viability in a hyperglycemic microenvironment through up-regulation of wound healing cytokines

Diabetic wounds have impaired healing and a propensity for further morbidity, which may result in amputations. Stromal vascular fraction (SVF) is an autologous source of heterogeneous cell population obtained from adipose tissue, which is rich in stem cells and presents little immunogenicity to the host. In this study, we hypothesized that murine fibroblasts subjected to hyperglycemic conditions co-treated with SVF exhibit greater functional activity through the colorimetric MTT assay and a cell-monolayer in-vitro scratch assay. We sought to establish the underlying mechanism of action via the utility of an ELISA chemiluminescence array on the supernatant medium of the cells. Our results demonstrate that the mean percentage gap closure at 24?h in the hyperglycemia?+?SVF group was significantly greater at 41.1%?±?1.6% compared to the hyperglycemia alone group 16.6%?±?1.5% (post-hoc Bonferroni test p?<?0.001, n?=?3) although there was no difference between the SVF and normoglycemia group. Further, this SVF group exhibited a significantly greater 2.4 fold increase in fibroblastic cell viability as compared to the hyperglycemia alone group (p?=?0.001, n?=?3). The supernatant medium of the cells upon testing with ELISA indicated that early phase wound healing cytokines including platelet-derived growth factor (p?=?0.012, n?=?3), interleukin-1 (p?=?0.003, n?=?3), basic fibroblast growth factor (p?=?0.003, n?=?3) and interleukin-10 (p?=?0.009, n?=?3) were expressed in significantly greater relative luminescent units in SVF as compared to hyperglycemia alone groups (Student t-test). Taken together and for the first time, our study shows that SVF is a promising therapeutic agent for up-regulating fibroblastic activity in a hyperglycemic microenvironment, and this result can be explained in part by the stimulation of wound-healing cytokines.     

Proteomic Analyses of Human Gingival and Periodontal Ligament Fibroblasts

Background: Although human gingival fibroblasts (hGFs) and human periodontal ligament fibroblasts (hPDLFs) exhibit numerous phenotypic similarities, it has been suggested that the secretory and behavioral differences, which exist between these cell types, are a result of the membrane protein composition of these cells. Methods: Four matched pairs of hGFs and hPDLFs were cultured. Before confluence, membrane‐bound and ‐associated proteins from cells of the fourth passage were extracted. The processed protein samples were evaluated using capillary‐liquid chromatography‐nanospray tandem mass spectrometry. Global protein identification was performed on an orbitrap mass spectrometer equipped with a microspray source operated in positive ion mode. Proteome software was used to validate protein identifications derived from tandem mass spectrometry sequencing results. Results: Four hundred fifty proteins were common to both hGFs and hPDLFs. Of the proteins identified, 214 were known membrane‐bound or ‐associated proteins, and 165 proteins were known nuclear‐associated proteins. Twenty‐seven proteins, identified from the 450 proteins, common to both hGFs and hPDLFs, were detected in statistically significant greater quantities in either hGFs or hPDLFs. More specifically, 13 proteins were detected in significantly greater quantities in hGFs, whereas 14 proteins were detected in significantly greater quantities in hPDLFs. Conclusions: Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGFs in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDLFs in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization.