小鼠睾丸引带细胞的培养及己烯雌酚对培养睾丸引带细胞的影响

目的:探讨小鼠睾丸引带细胞体外培养的有效方法,并进行形态学观察。研究经典的外源性雌激素己烯雌酚(DES)体外对小鼠睾丸引带发育的影响。方法:手术放大镜解剖出3日龄雄性昆明小鼠的睾丸引带组织并进行细胞培养。台盼蓝染色检测细胞存活率,HE染色观察细胞形态。传代培养并随机分为空白对照组、溶剂对照组(DMSO组)和实验组(DES 1~4组)共6组。溶剂对照组加入DMSO,实验组分别加入0.01、0.10、1.00以及10.00μg/ml DES。分别于培养12、24、48 h后进行细胞形态学观察,CCK-8法检测睾丸引带细胞的生长情况。结果:培养睾丸引带细胞大部分为成纤维细胞型,有少数的上皮样细胞。原代细胞的存活率为85%~90%。在不同剂量DES作用后的3个不同时间段里,细胞增殖性的检测结果存在时间-剂量效应的显著差异(P<0.01)。结论:睾丸引带细胞可经一定的方法进行体外培养,外源性雌激素对睾丸引带细胞的生长有抑制作用,且呈现一定的时间-剂量效应。对培养睾丸引带细胞影响的研究是外源性雌激素影响生殖系统发育研究的一条有效的睾丸外途径。     

己烯雌酚影响新生小鼠睾丸引带相关基因表达的初步研究

目的:通过不同剂量己烯雌酚(DES)孕期染毒,了解其对新生仔鼠睾丸引带中雄激素受体(AR)、雌激素受体α(ERα)、增殖细胞核抗原(PCNA)和肌动蛋白α1(ACTα1)mRNA表达的影响,以期窥探DES影响睾丸引带的作用途径。方法:将发现雌性昆明小鼠阴道栓当天记为孕第0天(GD0),在GD9~GD17分别给予不同剂量DES[0.02、0.1、0.5、10、50μg/(kg·d)]作为实验组,给予等体积二甲基亚砜及生理盐水分别作为溶剂和空白对照组。生后第1天处死雄性仔鼠获取睾丸引带,通过RT-PCR检测AR、ERα、PCNA和ACTα1 mRNA的表达量。结果:随着DES浓度的增加,ERαmRNA的表达逐渐增加,各实验组与溶剂对照组比较差异有统计学意义(RE~2=0.825,P0.05);AR、PCNA和ACTα1 mRNA的表达都呈下降趋势,高剂量组[10、50μg/(kg·d)DES组]与溶剂对照组比较差异有统计学意义(RA~2=0.713,RP~2=0.946,RT2=0.960,P0.01)。结论:正常新生昆明小鼠睾丸引带中存在ERα、AR、PCNA及ACTα1 mRNA的表达。孕期不同剂量DES染毒对新生小鼠睾丸引带细胞受体蛋白(ERα和AR)及功能蛋白(PCNA和ACTα1)mRNA表达的影响不同。外源性雌激素可能通过影响ER和或AR的代谢而影响睾丸引带细胞的增殖和收缩等功能,从而影响睾丸甚至整个雄性生殖系统的正常发育。     

己烯雌酚对小鼠睾丸引带中LGR8表达的影响

目的:通过体内实验研究己烯雌酚(DES)对小鼠睾丸引带中胰岛素样因子3受体LGR8的影响,从而探讨外源性雌激素对小鼠睾丸下降的影响。方法:8～10周龄KM孕鼠随机分为正常组、空白对照组和实验组(0.1、1.0、10、100μg/kg DES 4个剂量组),共6组,每组20只。于孕9～17 d每天分别给予实验组妊娠小鼠不同剂量的DES(0.1、1、10、100μg/kg),空白对照组给予等体积DMSO+生理盐水,正常组不给药。应用免疫组化和RT-PCR分别检测胎鼠和幼鼠睾丸引带中LGR8蛋白和mRNA的表达。结果:HE染色可见胎鼠正常组、对照组睾丸引带发育良好,中间的间叶组织和外周的肌源细胞之间分界清楚;实验组可见睾丸引带发育不良,间叶组织和肌源细胞之间无明显分界,组织结构紊乱。幼鼠正常组和实验组睾丸引带发育未见明显不同。LGR8表达于睾丸引带肌源细胞和间叶组织细胞,以肌源细胞表达为主。实验组LGR8阳性表达较正常组弱,胎鼠各实验组和幼鼠DES 1、10、100μg组与同发育阶段的正常组相比均有统计学意义(P<0.05)。DES高剂量(10、100μg)组与同发育阶段的正常组相比LGR8 mRNA表达增加(P<0.05)。各实验组PCR产物测序均未见明显突变。结论:DES可影响小鼠睾丸引带LGR8 mRNA和蛋白的表达,DES可能通过影响INSL3-LGR8信号系统干扰睾丸引带的发育,进而影响睾丸正常下降。     

邻苯二甲酸二(2-乙基)己酯对小鼠胎鼠睾丸及睾丸引带的影响

目的:研究邻苯二甲酸二(2-乙基)己酯(DEHP)对小鼠胎鼠睾丸与睾丸引带形态结构及功能的影响,探讨隐睾发生的可能机制。方法:30只健康KM孕鼠随机均分为3组:空白对照组、玉米油对照组、DEHP组。DEHP组以剂量500mg/(kg.d)的DEHP灌胃作用于怀孕12～19d(GD12～GD19)的KM母鼠,观察DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重、睾丸重量、睾丸引带形态结构、位置、睾丸到膀胱颈之间的相对距离(TBD)、颅侧悬韧带残留情况、引带内雄激素受体(AR)、雌激素受体(ER)、肌动蛋白、增殖细胞核抗原(PCNA)表达水平的影响。结果:DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重无明显影响;DEHP可影响胎鼠睾丸重量及TBD;DEHP组睾丸均有一定程度的下降不全,睾丸引带形态结构无明显差异;光镜下见DEHP组睾丸生精小管、支持细胞存在明显的发育障碍,睾丸Leydig细胞明显增生;DEHP组睾丸引带AR阳性表达率降低(P<0.01)。结论:DEHP可通过抗雄激素效应导致睾丸引带功能障碍,使睾丸下降发生异常而诱导小鼠隐睾;同时造成睾丸Ser-toli细胞、睾丸Leydig细胞和生精细胞的发育障碍和功能改变。     

保留睾丸引带在腹腔镜小儿腹腔型隐睾手术中的应用

目的:探讨保留睾丸引带在腹腔镜小儿腹腔型隐睾手术中的疗效.方法:回顾分析2007年6月至2010年8月为24例腹腔型隐睾患儿行腹腔镜睾丸阴囊肉膜囊固定术的临床资料,术中保留睾丸引带和精索双向血供.结果:24例均成功完成腹腔镜Ⅰ期睾丸下降固定术,无一例中转手术,手术时间45～60 min,平均55 min,无并发症发生....     

新生小鼠睾丸引带形态结构及其异常的显微连续研究

目的:应用新生小鼠生殖系统整体原位固定、横断面连续组织切片显微观察的研究方法,整体展现正常及异常状态下新生小鼠睾丸引带的形态结构,以探讨应用整块组织固定、连续切片显微研究的方法在判定生殖系统形态异常中的适用性。方法:正常及孕期(9~17d)接触不同剂量外源性雌激素己烯雌酚(DES)的新生雄性昆明小鼠,切取其腹部(自膈下)固定、包埋,作连续切片、染色。显微镜下顺序摄像,分析睾丸引带的形态结构,并测算其相应位置和大小。结果:显微连续观察显示新生小鼠睾丸引带的结构清晰,形态结构各段变化较大且左右不对称,DES对睾丸引带形态结构发育的影响各段不尽相同,而且明显影响整个引带的长度。结论:孕期接触DES对新生小鼠睾丸引带的发育有明显影响,其影响是整体性的。整块组织切片显微连续研究对睾丸引带或及其它相关生殖系统器官的形态结构评价能够比较全面和精确。     

LC调节人成骨样MG-63细胞表达OPG、RANKL分子机制的研究

目的 探讨大黄酸哌嗪雌酚酮（rhein-piperizinyl-estrone,命名为 LC）调节人成骨样MG-63细胞表达骨保护素(osteoprotegerin, OPG)、NF-κB激活受体配体(receptor activator of NF-κB ligand, RANKL)的分子机制。方法 在原工作基础上,选择兼有两种雌激素受体(estrogen receptor, ER)亚型表达的人成骨样MG-63细胞为研究模型,采用RT-PCR、免疫印迹及小RNA干扰等技术,探讨LC对人成骨细胞产生的骨吸收调节因子OPG、RANKL表达的作用及作用机制。结果 LC可上调MG-63细胞OPG表达及下调RANKL表达,该作用可被纯ER阻断剂ICI 182,780完全阻断,应用小RNA干扰技术进一步证实LC对成骨细胞OPG、RANKL表达的调节作用主要是由ERα介导的。结论 LC调节成骨细胞表达OPG、RANKL是经ER途径、主要是由ERα介导的。     

Ang/Tie2体系与VEGF、bFGF在血管瘤增生退化过程中的协同作用

目的:探讨促血管生成素家族(Ang/Tie2)与血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)在血管瘤增生退化过程中的协同作用。方法:采用免疫组化SP法,检测增生期血管瘤32例、退化期血管瘤10例及小儿正常皮肤10例标本中促血管生成素1(Ang1)、促血管生成素2(Ang2)及其受体Tie2、VEGF及bFGF的表达情况。结果:增生期血管瘤组织Ang2、Tie2、VEGF及bFGF表达明显高于消退期血管瘤(P<0.01);消退期血管瘤组织Ang2、Tie2、VEGF及bFGF表达明显高于小儿正常皮肤(P<0.01);Ang1在血管瘤和小儿正常皮肤表达均较弱,各组之间的比较无显著性差异(P﹥0.05);血管瘤中Ang2、Tie2分别与VEGF、bFGF标记指数间具有强的正相关性(P<0.01)。结论:Ang/Tie2体系与VEGF、bFGF在血管瘤增生退化过程中可能起协同的作用。     

TNP2基因在小鼠睾丸组织中的表达研究

目的 筛选与精子发生相关的基因.方法 将4d、9d、18d、35d、54d和6月龄小鼠睾丸组织cDNA探针与Affymetrix全基因组芯片进行杂交,筛选出差异表达的基因.通过RT-PCR分析差异表达基因在小鼠睾丸不同发育阶段中的表达.结果 对Affymetrix全基因组芯片杂交结果分析后,筛选得到1个差异表达杂交点,通过NCBI网站与小鼠全基因组序列Blast分析可知,该差异表达基因是TNP2基因.小鼠TNP2基因全长724bp,其编码框大小为375bp.RT-PCR结果表明TNP2基因在小鼠21d龄及之前的睾丸中没有表达,在35d龄睾丸开始高表达.结论 TNP2基因为小鼠年龄依赖性表达基因,小鼠TNP2基因的表达与小鼠精子发生过程有很强的一致性,因此,可以推测该基因在精子发生中可能起重要作用.     

水通道蛋白AQP2在原发性肝细胞肝癌多柔比星耐药机制中的作用研究

目的探索原发性肝细胞肝癌(HCC)多柔比星(doxorubicin,DOX)耐药的机制,为进一步提高HCC患者介入治疗的疗效提供前期探索。方法应用Western blotting方法检测DOX处理后的上皮间质化转变(EMT)的相关蛋白E-Cadherin和Vimentin表达情况,使用流式细胞仪观察细胞干性标记CD133的变化情况,使用基因芯片技术观察DOX处理后的细胞机制变化。结果 DOX能诱导HCC细胞发生上皮间质化转变,并且能提升HCC细胞CD133的比例(3.5%vs 35.5%,P0.05);基因芯片提示HCC细胞内水通道蛋白AQP2基因显著上调;敲降AQP2基因后,能显著减弱DOX诱导HCC细胞产生EMT的效果。结论水通道蛋白AQP2在肝细胞肝癌多柔比星耐药机制中具有重要作用,降低AQP2的表达可以减弱多柔比星诱导肝癌细胞上皮间质化转变的效果。     

Expression of RXFP2 receptor on human spermatozoa and the anti-apoptotic and antioxidant effects of insulin-like factor 3

Insulin-like factor 3 (INSL3) has an important role in the human reproductive system; however, its detailed function is still mysterious. We aimed to investigate the possibility of expression of RXFP2 receptor on human spermatozoa and to determine the anti-apoptotic and antioxidant mechanism derived the binding of INSL3 and RXFP2. In this experimental study, the expression/location of the RXFP2 receptor was determined on the spermatozoa of fertile and infertile men. Twenty samples from 20 fertile men were collected and divided into 6 parts (control group, and five groups treated with INSL3 10, 100, 250, 500, 1,000 ng/ml). DNA damage, active caspase, reactive oxygen species (ROS) and sperm parameters were evaluated by TUNEL, flow cytometry, optical microscope and computer-assisted sperm analysis. The expression of RXFP2 was confirmed by Western blot. Immunocytochemistry illustrated that this receptor is expressed in the posterior half of the spermatozoa's head. The INSL3 at concentrations of 500 and 1,000 ng/ml reduced the active caspase and mitochondrial ROS, and also reduced DNA fragmentation at 1,000 ng/ml. Besides, INSL3 500 and 1,000 ng/ml significantly increased the sperm motility. This study confirmed the presence of RXFP2 receptor in fertile and infertile men's spermatozoa, indicating the highly dose-dependent efficacy of the INSL3, which may have promising impacts on the in-vitro fertilisation outcomes.     

Expression and localisation of RXFP3 in human spermatozoa and impact of INSL7 on sperm functions

Insulin‐like peptide 7 (INSL7) or relaxin‐3 is a member of the insulin superfamily that is recently discovered. This hormone interacts with relaxin family peptide receptor 3 (RXFP3). Although recent studies of INSL7 have focused on its function in the brain as a neuropeptide, spermatozoa may be a candidate target of INSL7 due to its detection in testes and contains binding sites. Therefore, this study aims to analyse the expression and localisation of RXFP3 on human spermatozoa and to assess the effect of INSL7 on human sperm motility. We have incubated normal semen samples in different doses of INSL7. Sperm motility was analysed by Computer Assisted Sperm Analysis. Moreover, localisation and expression of RXFP3 were assessed in human spermatozoa by immunofluorescence and RT‐PCR respectively. This study indicated that RXFP3 mainly localised in the post‐acrosomal region of sperm head and neck. However, we did not observe expression of RXFP3 mRNA in human spermatozoa. This study showed that INSL7 alleviated the natural decline in sperm motility after a 4‐hr incubation period. This was particularly observed in the 1.8 pmol/L treated samples. These data suggested that most likely expression of RXFP3 arrested in spermiogenesis, but the RXFP3 peptide existed on the surface of mature spermatozoa.     

FOXA2在胰腺癌组织中的表达及其对胰腺癌细胞增殖和侵袭的影响

目的 探讨FOXA2在胰腺癌组织中的表达,以及特异性沉默FOXA2基因表达对PANC-1细胞 增殖、迁移和侵袭能力的影响。方法 选取2010年1月至2013年1月在郑州市中心医院择期行手术切除的 胰腺癌患者71例,所有患者从手术时间开始进行随访,截止时间为2018年1月31日或患者死亡时间,采用免疫组化SP法检测胰腺癌和癌旁组织中FOXA2蛋白表达,生存分析采用Kaplan-Meier法和Log-Rank检 验,影响患者预后的危险因素分析采用Cox比例风险回归模型。培养人胰腺癌PANC-1细胞,分为siRNAFOXA2组、siRNA-NC组和对照组,实时荧光定量PCR技术和Western blotting法检测细胞中FOXA2 mRNA和蛋白表达,CCK-8法检测细胞增殖活性,Transwell法检测细胞迁移和侵袭能力。结果 胰腺癌组 织中FOXA2蛋白阳性表达率（32.39%）低于癌旁组织（83.10%,P<0.001）;胰腺癌组织中FOXA2蛋白阳性 表达与TNM分期、淋巴结转移及脉管癌栓有关（P<0.05）;Kaplan-Meier生存分析结果显示,阳性组患者平 均生存时间高于阴性组,Log-Rank检验差异有统计学意义（χ2=11.135,P=0.001）;Cox比例风险回归模型 结果显示,淋巴结转移、脉管癌栓和FOXA2蛋白阴性表达是影响患者预后的风险因素（P<0.05）;siRNAFOXA2组细胞中FOXA2 mRNA和蛋白表达量低于siRNA-NC组和对照组（P<0.05）;siRNA-FOXA2组24 h、 48 h、72 h和96 h时OD值均高于siRNA-NC组和对照组（P<0.05）;siRNA-FOXA2组PANC-1和AsPC-1细胞 中迁移细胞数和侵袭细胞数均高于siRNA-NC组和对照组（P<0.05）。 结论 胰腺癌组织中FOXA2蛋白呈 低表达,是影响患者预后的风险因素;沉默FOXA2后会促进胰腺癌细胞增殖、迁移和侵袭。     

Calcitonin gene-related peptide stimulates mitosis in the tip of the rat gubernaculum in vitro and provides the chemotactic signals to control gubernacular migration during testicular descent

Background/aims

We investigated whether calcitonin gene-related peptide (CGRP) released from sensory genitofemoral nerve branches could stimulate rodent gubernacular growth and provide chemotactic signals for directing inguinoscrotal gubernaculum migration in vitro.

Materials and Methods

Neonatal rat gubernacula containing a developing cremaster sac (n = 60) were removed at days 0, 2, 4, 6, 8, and 10 (n = 10 per age; n = 5 per experimental group) and placed in organ culture for 24 hours with or without added CGRP (720 nmol/L). The gubernacula were stained for bromodeoxyuridine (BrdU) immunohistochemistry. Cells were counted (3 × 100 cells) in the mesenchymal tip of the gubernaculum to find the percentage of BrdU uptake. A further group of neonatal rat gubernacula (n = 21 per group) were placed in organ culture on an agar platform with 5 agarose beads soaked in either PBS or 10⁻⁶ mol/L CGRP placed approximately 0.8 to 1 mm on each side of the tip of the cremaster sac. After 72 hours, the position of the gubernaculum was compared with its starting position and any deviation measured.

Results

Exogenous CGRP caused a significant increase in BrdU uptake in the tip of the gubernaculum in 0-day-old rats compared with control cultures. Two-way analysis of variance in the cellular proliferation pattern between gubernacula cultured ± CGRP between 0 and 10 days showed a significant difference (P < .001). The cultures containing CGRP-impregnated beads caused significant (P < .01) deviation of the tip of the gubernaculum toward the beads, whereas the controls demonstrated no net movement of the tip.

Conclusions

These studies demonstrate that mitosis in the tip of the rat gubernaculum is significantly increased in response to CGRP in vitro. Also, CGRP may provide chemotactic signals to control inguinoscrotal gubernacular migration in the rat.     

Inhibition of SULT2B1b expression alters effects of 3beta-hydroxysteroids on cell proliferation and steroid hormone receptor expression in human LNCaP prostate cancer cells

BACKGROUND: Sulfation is an important steroid inactivation in human tissues. Sulfotransferase (SULT) 2B1b selectively conjugates 3beta-hydroxysteroids and is expressed in epithelial cells of normal and cancerous prostate tissues. Dehydroepiandrosterone (DHEA) and Delta(5)-androstenediol (Delta(5)-Adiol) sulfation prevents their conversion to more potent androgens and estrogens in tissues although both compounds may also be biologically active. METHODS: SULT2B1b expression and activity were inhibited >85% in human LNCaP prostate adenocarcinoma cells using short interference RNA (siRNA). The effects of treating control and SULT2B1b-deficient LNCaP cells with DHEA, Delta(5)-Adiol, and 5alpha-androstane-3beta-17beta-diol (Anstane-diol) on cellular proliferation, estrogen receptors (ERs), androgen receptor (AR), and prostate specific antigen protein levels were examined. RESULTS: Physiological concentrations of DHEA and Delta(5)-Adiol increased proliferation of control cells and the proliferative effects were significantly increased in SULT2B1b-siRNA cells. DHEA, but not Delta(5)-Adiol increased AR levels at concentrations >/=1,000 nM in SULT2B1b-siRNA cells but not in control LNCaP cells. ER-alpha levels were not affected with any of the compounds tested. Physiological concentrations of DHEA and Delta(5)-A-diol decreased ER-beta levels in control cells and had significantly greater effects in SULT2B1b-siRNA cells. In contrast, Anstane-diol had no effect on AR or ER-alpha levels but induced more elevation of ER-beta levels in SULT2B1b-siRNA cells at concentrations >/=1,000 nM. CONCLUSIONS: SULT2B1b is involved in regulating prostate cell responsiveness to DHEA and Delta(5)-Adiol. Inhibition of SULT2B1b increased cell proliferation and ER-beta repression after treatment with physiological levels of DHEA and Delta(5)-Adiol indicating that SULT2B1b has an inhibitory effect on DHEA and Delta(5)-Adiol activity.     

胰高血糖素样肽-2对烧伤大鼠肠粘膜细胞增殖的影响

目的　探讨胰高血糖素样肽 2 (GLP 2 )对烧伤大鼠肠粘膜细胞增殖及肠粘膜结构的影响。　方法　 5 5只Wistar大鼠随机分为烧伤组、GLP 2组 (烧伤后经GLP 2处理 ,2 0 0 μg/kg ,2次 /d腹腔注射 )与正常对照组。前两组动物于 30 %TBSAⅢ度烧伤后 6、12h及 1、3、5d分别处死 ,另处死正常对照组大鼠。检测各组增殖细胞核抗原 (PCNA)、细胞周期蛋白CyclinD的表达情况以及血浆二胺氧化酶 (DAO)的活性 ,并行肠粘膜组织学观察。　结果　与正常对照组比较 ,烧伤组伤后 6、12hPCNA表达稍有增强 ,伤后 1d减弱 ,3d时最低 ,5d时仍低于正常 ;GLP 2组PCNA表达的变化在伤后早期与烧伤组基本一致 ,但伤后 3、5d时强于烧伤组。烧伤组大鼠肠粘膜CyclinD蛋白在伤后 6、12h略有升高 ,但 1d时迅速下降至伤前的 4 0 % ,而GLP 2组CyclinD蛋白表达在伤后 1、3、5d高于烧伤组。大鼠烧伤后血浆DAO活性明显升高 ,经GLP 2治疗 5d后该指标明显降低 (P <0 .0 1)。组织学观察见GLP 2组肠绒毛排列较为规则 ,长短较一致 ,未见明显的上皮脱落。　结论　大鼠烧伤后腹腔给予外源性GLP 2能减轻肠粘膜损伤 ,其机制可能与GLP 2使PCNA、ClyclinD表达增加、促进受损肠粘膜细胞增殖有关。     

性激素对烧伤血清处理后IEC-6细胞增殖与凋亡的影响

目的观察雌激素和雄激素对烧伤后小肠上皮细胞增殖和凋亡的影响。方法用30%体表面积Ⅲ度烫伤大鼠的血清处理大鼠源性IEC-6细胞,构建烧伤后小肠上皮的体外细胞模型。观察己烯雌酚或睾酮处理后的细胞增殖速率（MTT法和流式细胞仪检测）、细胞周期分布（流式细胞仪检测）、凋亡比率（DAPI染色法检测）、生长曲线（细胞分析仪检测）及Claudin-1表达（免疫组化法检测）。结果烧伤血清可促进IEC-6细胞增殖（0.777vs0.415,P〈0.001）,促细胞凋亡（10.5%vs1.59%,P〈0.001）和Claudin-1表达,减少IEC-6细胞总数。己烯雌酚和睾酮可同时抑制烧伤组细胞的增殖和凋亡,己烯雌酚处理后细胞的增殖指数（12.3%vs14.6%;P〈0.001）和凋亡比率（1.04%vs2.33%,P〈0.005）均低于睾酮处理后的细胞。结论烧伤后小肠上皮细胞凋亡速度超过了增殖速度,己烯雌酚和睾酮可同时抑制烧伤后小肠上皮细胞的增殖和凋亡,其中己烯雌酚的作用较睾酮更明显。     

血管内皮生长因子对人骨髓基质细胞增殖的影响及其形态学观察

目的 了解血管内皮生长因子(VEGF)基因对体外生长的人骨髓基质干细胞(hBMSCs)增殖以及组织学形态的影响. 方法 实验用骨髓取自早产死胎,采用全骨髓法培养.原代培养至第8～10天后传代培养.实验分为三组:VEGF和绿色荧光蛋白(GFP)基因转染hBMSCs组、空腺病毒载体转染hBMSCs组以及空白hBMSCs组.相差显微镜和光镜下进行HE染色组织学观察细胞生长形态变化.进行MTT检测.流式细胞仪分析细胞周期. 结果 倒置相差显微镜和HE染色后光镜下观察,三组细胞外形无明显差异,形态基本一致.随培养时间的延长,各组细胞数量都有所增加,各时间点经VEGF基因转染的hBMSCs吸光度值差异无统计学意义(P>0.05).转染VEGF基因的hBMSCs与转染空载体、未转染的hBMSCs相比较,DNA合成前期细胞所占百分比差异无统计学意义,反应增殖活力的增殖指数PrI(包括DNA合成期、DNA合成后期和有丝分裂期)差异无统计学意义(P>0.05).结论 转染VEGF对hBMsCs体外增殖和形态无明显影响.     

Effects of interleukin 2 receptor b chain (P75)-specific monoclonal antibody on the generation of cytotoxic T lymphocytes and suppressor T cells in mixid lymphocyte culture

The interaction of interleukin 2 (IL-2) with its receptor (IL-2R) plays an essential role in the proliferation and differentiation of T cells. The IL-2R β-chain is considered to function directly in the intracellular signal transduction. In this study, we investigated using a newly established IL-2R β-chain-specific monoclonal antibody (MAb) (TU-25) and an IL-2R α-chain-specific MAb (H-31). The IL-2-induced proliferation of concanavalin blasts and the mixed lymphocyte reaction (MLR) were suppressed by TU-25 in combination with H-31. This combination had a greater suppressive effect than each of them alone. The generation of cytotoxic T lymphocytes (CTL) using a cell-mediated lympholysis (CML) assay, was not inhibited by TU-25 alone. TU-25 in combination with H-31 suppressed the generation of CTL completely in this assay even if recombinant IL-2 (rIL-2) was added. Although the CTL generation was inhibited, cells that suppressed a fresh MLR were preserved. Our study suggests that the combination of TU-25 with H-31 completely blocks the functional high-affinity binding site of IL-2 but does not inhibit the generation of suppressor cells. This may lead to immunosuppressive therapy using an IL-2R 13-chain-specific MAb in combination with an IL-2R α-chain-specific MAb in clinical organ transplantation.     

缓激肽B2受体基因多态性对膝关节骨关节炎易感性的影响

目的明确BDKRB2基因多态性与OA易感性的相关性,并检测OA患者基因多态性对滑膜组织中BDKRB2表达的影响。方法共有278名膝关节骨关节炎患者和291名健康志愿者参与实验。测定实验对象的BDKRB2基因多态性,在OA患者滑膜组织中通过RT-PCR和Western Blot检测BDKRB2的m RNA和蛋白水平。结果 OA组和对照组中+9/-9 bp多态性的基因型分布和等位基因频率差异明显。-9/-9 bp多态性和+9/+9 bp相比,与OA的罹患风险和严重程度存在明显相关性(OR=2.35,95%CI:1.409~3.937,P0.001)。-9 bp等位基因的表达,与OA的Kellgren-Lawrence分型相关(OR=1.545,,P=0.011)。另外,+9/-9 bp多态性显著影响OA患者滑膜组织中BDKRB2的m RNA和蛋白表达。+9/-9和-9/-9基因型在滑膜组织中BDKRB2的表达水平显著升高,同时KL4型OA患者平均BDKRB2 m RNA和蛋白水平明显高于其他对象(2.502±1.319 vs.1.471±1.002和1.895±1.174,P0.001)。结论本研究提示BDKRB2基因+9/-9bp多态性可能成为OA易感性和严重性筛查的分子标记。