Induced Neural Stem Cells Generated from Rat Fibroblasts

The generation of induced tissue-specific stem cells has been hampered by the lack of well-established methods for the maintenance of pure tissue-specific stem cells like the ones we have for embryonic stem （ES） cell cultures. Using a cocktail of cytokines and small molecules, we dem- onstrate that primitive neural stem （NS） cells derived from mouse ES cells and rat embryos can be maintained. Furthermore, using the same set of cytokines and small molecules, we show that induced NS （iNS） cells can be generated from rat fibroblasts by forced expression of the transcrip- tional factors Oct4, Sox2 and c-Myc. The generation and long-term maintenance of iNS cells could have wide and momentous implications.

Expression of Basic Fibroblast Growth Factor in Rat Liver Fibrosis and Hepatic Stellate Cells

Summary:The expression of basic fibroblast growth factor (bFGF) in rat liver fibrosis and hepatic stellate cells (HSCs) and the relationship between the expression of bFGF and rat liver fibrogenesis were studied. Sixty male SD rats (230-260 g) were divided into 4 groups randomly (the 0 week group, 1 week group, 4 week group and 8 week group). Liver fibrosis was induced by subcutaneous injection of carbon tetrachloride. The sections of rats' liver in each group were tested by Van-Gieson (V-G) staining and immunohistochemistry. The expression of bFGF mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). HSCs were isolated by the combined methods of collagenase IV perfusion and density gradient centrifugation. The expression of bFGF protein in cultured HSCs was detected by Western blot. Images of immunohistochemistry detection, agarose gel electrophoresis of RT-PCR and SDS-polyacrylamide gel electrophoresis of Western blot were analyzed semiquantitatively by image-analyzing system. The results were analyzed by statistics. The results showed that the fibers were gradually increased in the sections of rat liver with the prolongation of the model induction. At the end of the 8th weeks, liver fibrosis was formed. The expression of bFGF detected by immunohistochemistry showed a similar tendency of gradual increase. At the end of the 8th weeks, the bFGF expression could be observed in many regions in sections and the strongest expression was in interstitial cells including HSCs and some hepatocytes in regions around the portal area and central veins. Also there was moderate expression widely in extracellular matrix (ECM). In RT-PCR detection and Western blot detection of HSCs cultured in vitro, the similar tendency of gradual increase was evident either. It is suggested that bFGF is related with liver fibrosis of rats closely and may be a fibrogenesis factor of liver. bFGF possibly regulates liver fibrogenesis through regulating metabolism of extracellular matrix (ECM) by autocrine and paracrine stimulation.

Retinoic Aacid Diminished the Expression of MMP-2 in Hyperoxia-exposed Premature Rat Lung Fibroblasts through Regulating Mitogen-activated Protein Kinases

This study examined the effects of retinoic acid （RA）, PD98059, SP600125 and SB203580 on the hyperoxia-induced expression and regulation of matrix metalloproteinase-2 （MMP-2） and metalloproteinase-2 （TIMP-2） in premature rat lung fibroblasts （LFs）. LFs were exposed to hyperoxia or room air for 12 h in the presence of RA and the kinase inhibitors PD98059 （ERK1/2）, SP600125 （JNK1/2） and SB203580 （p38） respectively. The expression levels of MMP-2 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. MMP-2 activity was measured by zymography. The amount of p-ERK1/2, REK1/2, p-JNK1/2, JNK1/2, p-p38 and p38 was determined by Western blotting. The results showed that： （1） PD98059, SP600125 and SB203580 significantly inhibited p-ERK1/2, p-JNK1/2 and p-p38 respectively in LFs; （2） The expression of MMP-2 mRNA in LFs exposed to hyperoxia was decreased after treatment with RA, SP600125 and SB203580 respectively （P0.01 or 0.05）, but did not change after treatment with PD98059 （P0.05）. Meanwhile, RA, PD98059, SP600125 and SB203580 had no effect on the expression of TIMP-2 mRNA in LFs exposed to room air or hyperoxia （P0.05）; （3） The expression of pro- and active MMP-2 experienced no change after treatment with RA or SP600125 in LFs exposed to room air （P0.05）, but decreased remarkably after hyperoxia （P0.01 or 0.05）. SB203580 inhibited the expression of pro- and active MMP-2 either in room air or under hyperoxia （P0.01）. PD98059 exerted no effect on the expression of pro- and active MMP-2 （P0.05）. It was suggested that RA had a protective effect on hyperoxia-induced lung injury by down-regulating the expression of MMP-2 through decreasing the JNK and p38 activation in hyperoxia.

Expression of Connexin43 in Rat Epithelial Cells and Fibroblasts

Summary: To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy . And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n=10) and electrical acupuncture group (EA group, n=10). EA ( 0.5-1.5 V, 4-16 Hz , 30 min) was applied to “Zusanli” acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected. The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43 was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43 on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91 % and 29.53 % respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

Inhibitive effect of TAK-242 on Tenon’s capsule fibroblasts proliferation in rat eyes

AIM: To study the inhibition effect of TAK-242 on the proliferation of rat eye Tenon's capsule fibroblasts via the toll-like receptor 4(TLR4) signaling pathway.METHODS: SD rat Tenon's capsule fibroblasts were extracted and cultured, then the cells were divided into normal control group, lipopolysaccharide(LPS) group(10 g/m L LPS) and TAK-242 group(1 μmol/L TAK-242, and 10 μg/m L LPS after 30 min). The expressions of TLR4, transforming growth factor-β1(TGF-β1) and interleukin-6(IL-6) in each group were detected by Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR). Cell proliferation was detected by cell counting kit-8(CCK-8).RESULTS: Double immunofluorescent labeling in the extracted cells showed negative keratin staining and positive vimentin staining. Western blot showed that the LPS group had the highest expression of TLR4 and TGF-β1(P<0.01). Enzyme linked immunosorbent assay(ELISA) also showed that the secretion of IL-6 was the highest in LPS group(P<0.01). But there was no significant difference in TLR4 and TGF-1, as well as IL-6 expressions between the TAK-242 group and the normal control group(P>0.05). RT-PCR showed that the IL-6 m RNA expression in LPS group was the highest in the three groups(P<0.01). CONCLUSION: TAK-242 inhibits the proliferation of LPSinduced Tenon's capsule fibroblasts and the release of inflammatory factors by regulating the TLR4 signalingpathway, providing a new idea for reducing the scarring of the filter passage after glaucoma filtration surgery.

Effects of basic fibroblast growth factor on beta-catenin protein and mRNA expression during the proliferation of endogenous neural stem cells following focal cerebral ischemia

BACKGROUND： The Wnt/β-catenin signaling pathway plays an important role in neural development. ,β-catenin is an important component of the Wnt/β-catenin signaling pathway. The Wnt signaling pathway has been shown to regulate the interaction of neural stem cells with the extracellular matrix. OBJECTIVE： To investigate the effects of basic fibroblast growth factor （bFGF） on β-catenin protein and mRNA expression, and on hippocampal neural stem cell proliferation in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiology experiment was performed in Shenyang Medical College between August 2006 and August 2008. MATERIALS： A total of 72 healthy male Wistar rats, aged 3 months, were used in this study. bFGF was provided by Beijing SL Pharmaceutical Co.,Ltd., China. METHODS： Rats were randomly divided into 3 groups： sham-operated, ischemia/reperfusion, and bFGF-treated （n = 24 per group）. Focal cerebral ischemia/reperfusion was induced in rats from the ischemia/reperfusion group and the bFGF-treated group by 2 hour right middle cerebral artery occlusion and 2 hour restoration of blood flow using the suture method. The ischemia/reperfusion and bFGF-treated groups were intraperitoneally administered 500 IU/mL of bFGF, or the same volume of physiological saline, once a day at postoperative days 1 3, and once every 3 days thereafter. Simultaneously, the sham-operated group underwent experimental procedures identical to the ischemia/reperfusion and bFGF-treated groups, with the exception of ischemia/reperfusion induction and drug administration. At 2 hours, 2, 6, 13, and 20 days after ischemiaJreperfusion induction, 50 mg/kg bromodeoxyuridine （BrdU） was administered to each group, twice daily, to label proliferating neural stem cells. MAIN OUTCOME MEASURES： The effects of bFGF on BrdU labeling, and ,8 -catenin mRNA and protein expression, in neural stem cells were examined by immunohistochemistry, Western blot, RT-PCR, and in situ hybridization techniques. RESULTS： In the sham-operated group, only a few BrdU-immunoreactive neural stem cells were found. In the ischemia/reperfusion group, BrdU-immunoreactive cells began to increase from 3 days after ischemia/reperfusion induction, reached a peak level at 7 days, and gradually reduced from 21 days. At 3, 7, 14, and 21 days after ischemia/reperfusion induction, the numbers of BrdU-immunoreactive cells were significantly greater in the bFGF-treated group than in the ischemia/reperfusion group. The sham-operated group exhibited slight expression of β-catenin and β-catenin mRNA. In the ischemia/reperfusion group, the expression of β-catenin and β-catenin mRNA gradually increased with reperfusion time, peaked at 14 days after reperfusion, and gradually decreased thereafter; by 21 days, the expression was markedly lower. Following bFGF injection, the expression of hippocampal BrdU, β-catenin, and β-catenin mRNA had apparently increased in each group. CONCLUSION： bFGF promotes neural stem cell proliferation, and the expression of β-catenin and β-catenin mRNA in the ischemic brain tissue. These findings indicate that bFGF promotion of neural stem cell proliferation may be mediated by Wnt/β-catenin signaling pathway.

Effect of basic fibroblast growth factor and danshen on bcl-2 and p53 mRNA expression in the brain of rats exposed to repeated, high, positive acceleration (+Gz)

BACKGROUND： Both animal experiments and clinical studies have shown that basic fibroblast growth factor （bFGF） and danshen （Salvia miltiorrhiza） can exhibit protective effects on ischemia-reperfusion cerebral injury. OBJECTIVE： To test whether bFGF and danshen can protect cerebral injury induced by exposure to repeated, high, positive acceleration （＋Gz） in an animal model and to analyze the possible mechanisms. DESIGN, TIME AND SETTING： Randomized controlled animal study. The experiment was performed at the Research Center for Molecular Biology, Air-force General Hospital of Chinese PLA from April to August 2000. MATERIALS： A total of 20 clean grade, healthy, Sprague Dawley rats of both genders, weighing （200 ± 15） g, were provided by our experimental animal center. Rats were randomly divided into 5 groups： the control group, ＋Gz exposure group, bFGF group, danshen group, and saline group, with 4 animals per group. bFGF （Beijing Bailuyuan Biotechnology Co. Ltd.） and danshen solution （Shanghai Zhongxi Pharmaceutical Co. Ltd.） were used. METHODS： All rats were fixed on a rotary arm of a centrifugal apparatus （2 m in radius） with their heads oriented towards the center of the apparatus. Except for rats in the control group, the value of ＋Gz exposure was ＋14 Gz with an acceleration rate of 1.5 G/s. The peak force lasted for 45 seconds. ＋Gz exposure was performed three times with intervals of 30 minutes. Rats in the control group received the same ＋Gz procedure, but the G value was ＋1 Gz. Rats in bFGF group and danshen group were intraperitoneally injected with 100 μg/kg bFGF or 15 g/kg danshen solution, respectively, at 30 minutes prior to centrifugation and immediately after centrifugation. Rats in saline group were injected with the same volume of saline. Six hours after exposure, rats were decapitated. One hemisphere was preserved in liquid nitrogen for RNA extraction and the other was processed for apoptosis detection. MAIN OUTCOME MEASURES： mRNA levels of bcl-2 and p53 were measured by semi-quantitative reverse-transcription polymerase chain reaction. Apoptotic cell death was detected by terminal deoxynuleotidyl transferase-mediated dUTP nick end labeling. RESULTS： Changes in mRNA expression of bcl-2 and p53 and apoptotic cells were observed in rat brain six hours after repeated ＋Gz exposures, bFGF and danshen were able block the changes of bcl-2 and p53 expression and inhibit apoptotic cell death. CONCLUSION： The data suggest that apoptosis and changes in bcl-2 and p53 expression in the rat brain can be induced by repeated ＋Gz exposures. Apoptosis is, therefore, one of the molecular mechanisms of brain damage induced by repeated ＋Gz exposures, bFGF and danshen were of the equal potency in preventing brain injury induced by repeated ＋Gz exposures.

An in vivo Study of Basic Fibroblast Growth Factor on Activation and Proliferation of Retinal Progenitor Cells in RCS Rat

<Abstract>Purpose: To investigate the effect of intravitreal injection of basic fibroblast growth factor(bFGF) on activation and proliferation of endogenous retinal progenitor cells in the Royal College of Surgeons(RCS) rat. Methods: Twenty-four rats were studied after the 30th postnatal day(≥30). Eighteen RCS-p+/LAV rats were divided into 3 groups: bFGF-treated, vehicle-treated and untreated groups randomly, and 6 RCS-ray+p+/Lav respectively rats were used as normal controls. 6 μl of bFGF (5 μg/10 μl) or vehicle was injected into the vitreous on day 31, 33 and 35 after birth (P31, P33, P35) in the bFGF group and vehicle group respectively, and no injections were administered in the untreated and control groups. All the rats were euthanized, and their eyes were enucleated, hemisected and fixed at 50 d ays after birth for immunohistochemistry and measurement of outer nuclear layer thickness. Results:Nestin and Chx10 were positive in all retinal layers, intravitreal injection of bFGF in retina-dystrophic RCS(RCS-p+/Lav) rats induced intense labeling for the retinal progenitor cell markers Chx10 and Nestin, which were highly colocalized. Fluorescence intensity for both labels was somewhat less in the control rats, and much less in the vehicle-injected rats as well as in the untreated RCS rats. The outer nuclear layer(ONL) was significantly thicker in bFGF group than that in vehicle-treated or untreated group(P<0.01), but thinner than that of the control group(P<0.01). No significant difference was observed in the ONL thickness between the vehicle group and untreated group(P>0.05). Conclusion:bFGF may contribute to the activation of retinal progenitor cells in RCS rats,thus counteract degeneration by promoting the proliferation of the progenitor cells.

Effect of the implant composite of poly lactide-co-glycolide and bone mesenchymal stem cells modified by basic fibroblast growth factor on injured spinal cord in rats

Objective To investigate the effect of the implant composite of poly lactide-co-glycolide(PLGA)and bone mesenchymal stem cells (BMSCs) modified by basic fibroblast growth factor (bFGF) on injured spinal cord in rats.Methods Two hundred and

新生与成年大鼠心脏成纤维细胞来源外泌体蛋白质组学差异分析

目的幼体心脏具有再生修复功能,通过分析新生大鼠与成年大鼠心脏成纤维细胞来源外泌体的差异蛋白,从分子水平上探讨心肌受损后可能的治疗靶点。方法分别从新生和成年大鼠心脏成纤维细胞中分离出外泌体,并提取蛋白质进行高效液相色谱法(high per-formance liquid chromatography,HPLC)和液相色谱串联质谱法(liquid chromatography coupled to tandem mass spectrometry,LC-MS/MS)分析,鉴定差异表达的蛋白质。使用DAVID数据库对差异表达蛋白基因进行基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路分析。并进一步使用STRING数据库和Cytoscape软件进行蛋白质相互作用网络(protein-protein interaction,PPI)分析,以筛选出关键基因。结果在新生大鼠中共发现241个差异表达蛋白,其中上调表达的蛋白有117个,下调表达的蛋白有124个。GO分析显示上调表达基因主要涉及整合素介导的信号通路和对缺氧的反应,而对内肽酶活性负调控的基因下调表达。KEGG分析结果表明上调表达基因主要富集于Hippo信号通路,而下调表达基因主要涉及细胞外基质与受体相互作用。通过PPI分析得出有6个关键基因(Lamc1、Agrn、Itih3、Tfrc、Bgn和Atp5pd)下调,有1个关键基因(Rp17a)上调。结论通过蛋白组学和生物信息学方法,筛选出了新生大鼠和成年大鼠心脏成纤维细胞来源外泌体的差异蛋白,以及相关的信号通路和关键基因,为心肌受损精准治疗提供了潜在的靶点。

炙甘草汤通过miR-181a-5p靶向SPHK2调控心肌纤维化改善糖尿病心肌病

目的探究炙甘草汤对糖尿病心肌病(DCM)模型大鼠心肌纤维化的影响以及调控机制。方法雄性Sprague Dawley(SD)大鼠30只,10只大鼠作为对照组;20只通过高糖高脂饮食联合腹腔注射链脲佐菌素(STZ)构建DCM模型大鼠,造模后按随机数字表法分成DCM组、炙甘草汤组。炙甘草汤组予2 mL/100 g炙甘草汤(1 g/mL)灌胃,对照组及DCM组予等量生理盐水灌胃,连续给药4周,每天1次。灌胃给药结束后,HE染色观察DCM模型大鼠心肌组织的病理改变,Masson染色观察DCM模型大鼠的心肌纤维化情况。提取大鼠乳鼠心肌成纤维细胞,分为对照组、高糖组、炙甘草汤组、miRNA-NC组、miR-181a-5p模拟物组、miR-181a-5p拮抗剂组、炙甘草汤+miRNA-NC组和炙甘草汤+miR-181a-5p拮抗剂组并进行相应处理。通过高糖(30 mmol/L)诱导心肌成纤维细胞增殖模型。蛋白免疫印迹法(Western blot)检测大鼠心肌组织和心肌成纤维细胞中的α-SMA、Ⅰ型胶原蛋白(CollagenⅠ)、Ⅲ型胶原蛋白(CollagenⅢ)和鞘氨醇激酶2(SPHK2)蛋白表达。实时荧光定量聚合酶链式反应(qRT-PCR)检测miR-181a-5p、CollagenⅠ和CollagenⅢ的相对表达水平。细胞计数试剂盒8(CCK-8)检测心肌成纤维细胞的细胞活力。TargetScan数据库预测miR-181a-5p与SPHK2的结合序列。双荧光素酶报告基因实验验证miR-181a-5p与SPHK2的靶向关系。结果与对照组比较,DCM组大鼠血糖水平显著升高[(30.40±3.48)mmol/L比(5.08±0.17)mmol/L,P<0.01],心肌细胞肥大、排列紊乱、成纤维细胞增生伴炎症细胞浸润,炙甘草汤组大鼠上述情况均得到改善。与对照组比较,DCM组大鼠心肌组织α-SMA、CollagenⅠ、CollagenⅢ、SPHK2蛋白水平升高,miR-181a-5p水平显著下降[(0.45±0.20)比(1.00±0.12),P<0.01];而炙甘草汤喂养能够逆转上述表达水平变化。与对照组比较,高糖组心肌成纤维细胞的细胞活力[(194.03±25.59)%比(100.00±11.00)%,P<0.01]、CollagenⅠ[(3.73±0.81)比(1.00±0.17),P<0.01]、CollagenⅢ[(3.93±0.97)比(1.00±0.24),P<0.01]、α-SMA蛋白、SPHK2蛋白水平升高,miR-181a-5p水平显著下降[(0.41±0.18)比(1.00±0.21),P<0.01];与高糖组比较,炙甘草汤组心肌成纤维细胞的细胞活力[(147.63±25.80)%比(194.03±25.59)%,P<0.01]、CollagenⅠ[(1.91±0.58)比(3.73±0.81),P<0.01]、CollagenⅢ[(2.02±0.45)比(3.93±0.97),P<0.01]、α-SMA蛋白、SPHK2蛋白水平降低,miR-181a-5p水平显著升高[(0.69±0.17)比(0.41±0.18),P<0.01]。与miRNA-NC比较,心肌成纤维细胞转染miR-181a-5p模拟物后miR-181a-5p表达显著升高[(2.82±0.86)比(1.00±0.28),P<0.01],SPHK2表达下降;而转染miR-181a-5p拮抗剂后miR-181a-5p表达显著下降[(0.29±0.09)比(1.00±0.28),P<0.01],SPHK2表达升高。TargetScan数据库分析发现,miR-181a-5p与SPHK2的3’UTR存在结合序列。双荧光素酶报告基因实验证实,miR-181a-5p和SPHK2存在靶向作用。与炙甘草汤+miRNA-NC组比较,炙甘草汤+miR-181a-5p拮抗剂组心肌成纤维细胞CollagenⅠ[(2.95±0.67)比(1.89±0.26),P<0.01]、CollagenⅢ[(2.99±0.74)比(1.85±0.63),P<0.01]、细胞活力[(78.57±10.64)%比(59.75±8.08)%,P<0.01]、α-SMA蛋白、SPHK2蛋白水平升高,miR-181a-5p水平显著降低[(0.77±0.17)比(2.89±0.57),P<0.01]。结论炙甘草汤可能通过miR-181a-5p靶向SPHK2调控心肌纤维化来实现对DCM的改善作用。

鼠脂肪间充质干细胞对大鼠肛瘘创面愈合的促进作用及机制研究

目的探讨脂肪间充质干细胞(ADSCs)对大鼠肛瘘创面愈合的作用及相关分子机制。方法选择雌性SD大鼠30只,随机分为ADSCs组(A组)、ADSCs条件培养基组(B组)、磷酸盐溶液治疗对照组(C组),每组10只。建立肛瘘动物模型后进行实验。于实验后7、14 d观察各组大鼠治疗后创面愈合情况,并取创面肉芽组织;免疫组织化学法检测各组大鼠7、14 d创面肉芽组织血管内皮细胞生长因子(VEGF)、血管内皮细胞生长因子受体2(VEGFR-2)、白介素-2(IL-2)及成纤维生长因子(FGF)的表达。结果(1)治疗后14 d,创面愈合率比较,A组[(95.14±4.93)%]、B组[(94.94±3.19)%]较C组[(88.10±3.12)%]更高,差异有统计学意义(P<0.05);(2)治疗后14 d,A、B组创面肉芽组织VEGF、VEGFR-2和FGF较C组明显升高,且A组较B组表达升高,组间比较差异有统计学意义(P<0.05);(3)治疗后14 d,A、B组创面肉芽组织IL-2表达较C组明显降低,A组IL-2表达较B组更低,组间差异有统计学意义(P<0.05)。结论ADSCs可有效促进肛瘘模型大鼠创口愈合,其机制可能与调节创面IL-2炎症因子表达、并促进创面血管新生有关。

电针调控lncRNA NEAT1减轻类风湿关节炎滑膜成纤维细胞炎症反应的研究

目的:观察电针对调控lncRNA NEAT1减轻类风湿关节炎大鼠滑膜成纤维细胞炎症反应的影响。方法:将16只2月龄大鼠应用随机数字表法分为空白对照组和电针组,每组8只。空白对照组大鼠未接受干预,电针组大鼠予以电针干预,分别制备空白对照组、电针组血清。予以连续酶消化法获取3周龄SD大鼠滑膜成纤维细胞,波形蛋白免疫组化染色进行鉴定;再将6孔板中已培养的滑膜成纤维细胞随机分为空白对照组、模型对照组、电针组成纤维细胞。空白对照组成纤维细胞予以空白对照组血清干预,模型对照组成纤维细胞予以含10 ng·mL^(-1)白细胞介素-1β(IL-1β)的空白对照组血清诱导滑膜成纤维细胞致炎,电针组成纤维细胞予以含10 ng·mL^(-1)IL-1β的电针组血清干预。Real-time PCR检测各组滑膜成纤维细胞中lncRNA NEAT1水平变化;Western blot检测各组滑膜成纤维细胞基质金属蛋白酶-3(MMP-3)、V-Rel网状内皮增生病毒癌基因同源物A(RELA)、肿瘤坏死因子-α(TNF-α)蛋白表达含量。结果:经波形蛋白免疫组化染色后,大鼠滑膜成纤维细胞波形蛋白染色为阳性,证实成纤维细胞提取成功。与空白对照组比较,模型对照组中lncRNA NEAT1的相对表达水平显著升高(P<0.05);与模型对照组比较,电针组中lncRNA NEAT1相对表达水平显著降低(P<0.05)。Western blot结果显示,与模型对照组比较,电针组MMP-3、RELA、TNF-α蛋白表达含量显著降低(P<0.05)。结论:电针可通过调控lncRNA NEAT1减轻类风湿关节炎大鼠滑膜成纤维细胞炎症反应。

Functional electrical stimulation-facilitated proliferation and regeneration of neural precursor cells in the brains of rats with cerebral infarction

Previous studies have shown that proliferation of endogenous neural precursor cells cannot alone compensate for the damage to neurons and axons. From the perspective of neural plastici- ty, we observed the effects of functional electrical stimulation treatment on endogenous neural precursor cell proliferation and expression of basic fibroblast growth factor and epidermal growth factor in the rat brain on the infarct side. Functional electrical stimulation was performed in rat models of acute middle cerebral artery occlusion. Simultaneously, we set up a placebo stimulation group and a sham-operated group. Immunohistochemical staining showed that, at 7 and 14 days, compared with the placebo group, the numbers of nestin （a neural precursor cell marker）-positive cells in the subgranular zone and subventricular zone were increased in the functional electrical stimulation treatment group. Western blot assays and reverse-transcription PCR showed that total protein levels and gene expression of epidermal growth factor and basic fibroblast growth factor were also upregulated on the infarct side. Prehensile traction test results showed that, at 14 days, prehension function of rats in the functional electrical stimulation group was significantly better than in the placebo group. These results suggest that functional electrical stimulation can promote endogenous neural precursor cell proliferation in the brains of acute cerebral infarction rats, enhance expression of basic fibroblast growth factor and epidermal growth factor, and improve the motor function of rats.

阿托品对大鼠巩膜成纤维细胞增殖和凋亡的影响及其机制

目的 探讨不同浓度阿托品对大鼠巩膜成纤维细胞增殖和凋亡的影响及其机制。方法 取出生24 h的健康雄性SD大鼠眼巩膜组织(均为右眼)进行原代培养。取细胞进行分组:对照组(正常巩膜成纤维细胞),0.1 g·L^(-1)、1.0 g·L^(-1)及5.0 g·L^(-1)阿托品处理组(正常巩膜成纤维细胞分别加入三种不同浓度阿托品溶液),每组5只大鼠原代培养细胞。加药后24 h, CCK-8法检测各组巩膜成纤维细胞活力,流式细胞仪检测各组巩膜成纤维细胞凋亡率,Western blot法检测各组巩膜成纤维细胞光蛋白聚糖、基质金属蛋白酶(MMP)-2、MMP-14和MMP抑制剂(TIMP)-2蛋白表达情况。结果 CCK-8实验结果显示:与对照组相比,0.1 g·L^(-1)、1.0 g·L^(-1)及5.0 g·L^(-1)阿托品处理组细胞活力均增高,其中以1.0 g·L^(-1)阿托品处理组增高最显著,差异均有统计学意义(均为P<0.05)。流式细胞仪检测结果表明:阿托品处理组各组的巩膜成纤维细胞凋亡率与对照组相比,差异均无统计学意义(均为P>0.05)。与对照组相比,阿托品处理组巩膜成纤维细胞中光蛋白聚糖、TIMP-2蛋白相对表达量均升高,其中以5.0 g·L^(-1)阿托品处理组上升最明显(均为P<0.05);与对照组相比,阿托品处理组巩膜成纤维细胞中MMP-2、MMP-14蛋白相对表达量均降低,其中以5.0 g·L^(-1)阿托品处理组下降最明显(均为P<0.05)。结论 阿托品处理能增强体外培养大鼠巩膜成纤维细胞活力,这种效应的产生可能与光蛋白聚糖、MMPs、TIMPs等细胞因子有关。

SMYD2特异性抑制剂对高糖环境下大鼠肾成纤维细胞活化的影响及其机制

目的:明确赖氨酸甲基转移酶SMYD2(SET and MYND domain containing 2)特异性抑制剂对体外高糖(HG)环境下大鼠肾成纤维细胞(NRK-49F)活化的影响并探索其可能机制。方法:采用CCK-8法检测SMYD2特异性抑制剂LLY507对体外培养的NRK-49F细胞活力的影响。实验分组为正常糖(NG;含5.5 mmol/L葡萄糖)组、HG(含30 mmol/L葡萄糖)组、NG+LLY507(1.5μmol/L)组和HG+LLY507(1.5μmol/L)组,37℃培养48 h后收集各组细胞蛋白。采用Western blot实验检测SMYD2、组蛋白H3第4位赖氨酸三甲基化(H3K4me3)、纤维连接蛋白(fibronectin)、I型胶原蛋白(Col I)、α-平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)、p-Smad3、Smad3、肿瘤坏死因子α(TNF-α)、核因子κB(NF-κB)p65、NF-κB p-p65、白细胞介素6(IL-6)、信号转导及转录激活因子3(STAT3)及p-STAT3蛋白水平。结果:LLY507浓度低于1.6μmol/L对NRK-49F细胞的活力无显著影响;与NG组相比,HG组SMYD2、H3K4me3、fibronectin、Col I、α-SMA、TGF-β1、p-Smad3,TNF-α、NF-κB p-p65、NF-κB p65、IL-6、p-STAT3和STAT3蛋白水平均显著升高(P<0.05);与HG组相比,HG+LLY507组SMYD2、H3K4me3、fibronectin、Col I、α-SMA、TGF-β1、p-Smad3、TNF-α、NF-κB p-p65、NF-κB p65、IL-6、p-STAT3和STAT3蛋白水平均显著降低(P<0.05)。结论:SMYD2特异性抑制剂LLY507可明显抑制HG诱导的NRK-49F细胞活化和细胞外基质分泌,其机制可能与阻断TGF-β1/Smad3、NF-κB和STAT3细胞信号通路激活相关。

二甲双胍对糖尿病大鼠创面肌成纤维细胞纤维化的影响及其机制研究

目的探讨二甲双胍对糖尿病大鼠创面修复的促进作用及对真皮层肌成纤维细胞纤维化的影响及其机制研究。方法将18只雄性SD大鼠随机分为正常血糖对照(normal control,NC)组、糖尿病对照(diabetic control,DC)组及糖尿病二甲双胍干预(diabeitic metformin intervention,DM)组。使用高剂量链脲佐菌素(streptozotocin,STZ)腹腔注射构建糖尿病大鼠模型,在大鼠背部制备直径5 mm全层皮肤创面,DM组大鼠创面用二甲双胍霜剂处理,收集创面及创周组织,通过Western blot和免疫组化染色检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、转化生长因子β1(transforming growth factor-β1,TGF-β1)和肝激酶B1(liver kinase B1,LKB1)的表达水平。结果Western blot和免疫组化结果表明,与NC组相比,DC组中α-SMA蛋白表达量下调(P<0.05);与DC组相比,DM组中α-SMA蛋白表达量高于DC组(P<0.05)。与NC组相比,LKB1在DC组中的表达水平降低(P<0.05),与DC组相比,DM组中LKB1的表达高于DC组(P<0.05)。与NC组相比,DC组中TGF-β1的表达低于NC组(P<0.05);与DC组相比,DM中组TGF-β1的表达高于DC组(P<0.05)。结论二甲双胍可以减轻创面真皮层肌成纤维细胞的纤维化,从而促进糖尿病大鼠急性创面的愈合。

超极化激活环核苷酸门控阳离子通道4在大鼠心肌成纤维细胞和心肌细胞中的表达

目的:探讨超极化激活环核苷酸门控阳离子通道4(HCN4)在大鼠体内和体外心肌成纤维细胞、心肌细胞中的表达分布及其增龄变化。方法:SD大鼠分为新生组(1~3 d),成年组(17个月)和老年组(22个月),每组8只,其中4只用于冰冻切片,另4只用于PCR;取新生SD大鼠心,酶消化法分离培养原代心肌成纤维细胞和原代心肌细胞。用免疫荧光、免疫组织化学检测原代心肌成纤维细胞和原代心肌细胞HCN4的表达;免疫印迹、实时荧光定量PCR检测原代心肌成纤维细胞HCN4的表达,用实时荧光定量PCR检测不同月龄大鼠心肌组织HCN4的表达。结果:体外细胞培养显示,成纤维细胞和原代心肌细胞中均表达HCN4。组织切片显示,新生、成年和老年大鼠心肌成纤维细胞和部分心肌细胞表达HCN4。P1~P4代成纤维细胞的免疫印迹和实时荧光定量PCR结果显示HCN4随着成纤维细胞代次增加,表达量逐渐降低;不同月龄大鼠左心室的实时荧光定量PCR结果显示随着大鼠月龄增加,HCN4的表达量逐渐降低,其结果与培养的细胞表达一致。结论:HCN4在心肌成纤维细胞和部分心肌细胞中表达。随增龄变化,心肌细胞和成纤维细胞HCN4的表达量逐渐下降。

外源性碱性成纤维细胞生长因子对大鼠阴茎肌源性干细胞增殖的影响

目的探讨碱性成纤维细胞生长因子对大鼠阴茎肌源性干细胞体外增殖的影响。方法本试验自2020年12月至2021年6月在滕州市中心人民医院精准实验室完成。取健康雄性Wister大鼠5只,体质量(200±50)g,取其阴茎海绵体并采用酶消化法分离肌源性干细胞,应用密度梯度离心法和差速贴壁法进行纯化。取第2代肌源性干细胞分为两组进行培养,实验组:200μl生长培养基+肌源性干细胞,分别用终浓度为10μg/L、30μg/L、50μg/L、70μg/L、90μg/L外源性碱性成纤维细胞生长因子进行干预;对照组:加入200μl生长培养基+肌源性干细胞。分别于培养24 h、48 h、72 h、96 h、120 h、144 h加入四甲基偶氮唑蓝(MTT)溶液。应用免疫细胞化学法鉴定大鼠阴茎肌源性干细胞,MTT比色法检测细胞增殖情况。统计学方法采用独立样本t检验、Tukey检验。结果(1)肌源性干细胞多呈Sca-1阳性反应[表达量为(79.90±1.82)%],少数呈Desmin阳性反应[表达量为(68.60±0.72)%]。(2)两组肌源性干细胞均随培养时间的延长而明显增殖(均P<0.01),出现显著促增殖效应的培养时间均为96 h。(3)与对照组比较,实验组各浓度碱性成纤维细胞生长因子对肌源性干细胞均有明显的促增殖效应;当浓度从10~70μg/L递增时,促增殖作用也随之均显著增强(均P<0.01),至70μg/L时其促增殖作用接近顶峰。结论外源性碱性成纤维细胞生长因子可促进体外培养的大鼠阴茎肌源性干细胞增殖,且呈浓度-时间依赖性增加,96 h与70μg/L为最佳的体外培养时间和浓度。