pSilencer-VEGF-siRNA诱导骨肉瘤细胞系MG63凋亡并上调caspase-3表达

目的 研究pSilencer-VEGF-siRNA转染骨肉瘤细胞系MG63后诱导其凋亡以及对capase-3表达能力的影响。 方法 体外常规培养骨肉瘤细胞系MG63并构建人类特异性的pSilencer-VEGF-siRNA;分为pSilencer-VEGF-siRNA转染组和空载体组。转染后48~72 h,流式细胞仪Annexin V-FITC/PI 染色检测细胞凋亡、分析细胞周期;Western blotting法测定caspase-3表达。 结果 MG63转染pSilencer-VEGF-siRNA表达质粒后,早期出现细胞凋亡及明显的G1期阻滞以及G1期细胞数增加;pSilencer-VEGF转染组caspase-3的表达较空载体组和对照组明显上调。 结论 pSilencer-VEGF-siRNA可诱导MG63细胞早期凋亡;并可上调caspase-3的表达。     

PI3K抑制剂TGX-221对人骨肉瘤细胞增殖、周期及凋亡的影响

目的探究PI3K抑制剂TGX-221对人骨肉瘤细胞增殖、周期及凋亡的影响。方法培养人骨肉瘤U2OS和MG-63细胞株,分为对照组和实验组,实验组加入10μmol/L的TGX-221,对照组加入相同剂量的DMSO。处理48 h后,CCK8法检测TGX-221对人骨肉瘤细胞活力及增殖的影响;流式细胞术检测TGX-221对人骨肉瘤U2OS和MG-63细胞的细胞周期及凋亡的作用。结果CCK8检测结果显示,TGX-221对人骨肉瘤细胞有显著的增殖抑制作用,且具有时间和浓度依赖性;流式细胞术结果显示,TGX-221将U2OS和MG-63细胞周期阻滞在G1期,并诱导人骨肉瘤细胞凋亡,具有显著的抗肿瘤作用。结论PI3K抑制剂TGX-221能够抑制人骨肉瘤细胞的增殖、阻滞细胞周期并诱导细胞凋亡,可能成为一种潜在的抗肿瘤药物。     

沉默Cat D对骨肉瘤细胞增殖、凋亡和侵袭的影响及相关信号通路

研究组织蛋白酶D(cathepsin D,Cat D)在骨肉瘤细胞中的表达，并通过shRNA技术沉默Cat D,探讨该分子对骨肉瘤细胞MG63和U2OS增殖、凋亡及侵袭的作用和机制。构建sh-Cat D质粒及阴性对照质粒sh-NC并转染至骨肉瘤细胞MG63和U2OS中，RT-PCR和Western blotting检测Cat D在骨肉瘤细胞中的表达；CCK-8法、克隆形成实验和EdU荧光染色实验检测骨肉瘤细胞的增殖活性；Transwell实验检测肿瘤细胞的侵袭能力；FACS检测肿瘤细胞的凋亡情况；Western blotting检测细胞凋亡相关蛋白(Bax和Bcl-2)的表达及Akt/mTOR信号通路相关蛋白(Akt和mTOR)的磷酸化水平。结果显示，相较健康人成骨细胞hFOB,Cat D在骨肉瘤细胞中的表达显著升高，尤其是MG63和U2OS细胞中Cat D的高表达均显著(均P<0.001)。与sh-NC组相比，Cat D沉默显著抑制MG63和U2OS细胞的增殖和侵袭(均P<0.001)。与sh-NC组相比，Cat D沉默通过上调Bax表达和下调Bcl-2表达促进肿瘤细胞的...     

人S100A6对人骨肉瘤细胞系β-catenin的作用

目的 研究人S100A6对人骨肉瘤细胞系MG63和U2OS中β-catenin的作用。方法 分别用携带人S100A6及其siRNA基因的重组腺病毒AdS100A6和AdSiS100A6感染MG63和U2OS,分别使细胞中S100A6表达上调和下调;RT-PCR、蛋白印迹和免疫细胞化学法分别检测细胞中β-catenin mRNA和蛋白表达。结果 上调骨肉瘤细胞系MG63和U2OS中S100A6后, β-catenin mRNA和蛋白表达均增加（P＜0.05）,蛋白的增加以胞核更明显。下调S100A6则使细胞中β-catenin mRNA及蛋白表达减少（P＜0.05）,蛋白的减少也以胞核更甚。结论 增加Wnt信号途径活性可能是S100A6参与肿瘤发生发展的机制之一。     

CD44对骨肉瘤细胞增殖、黏附和侵袭性的影响

目的研究细胞表面黏附分子CD44对骨肉瘤细胞增殖、黏附和侵袭特性的影响,探讨骨肉瘤细胞生长侵袭的机制。方法流式细胞术和Westernblot检测3株骨肉瘤细胞系MG63、HOS和U2-OS中CD44的阳性表达率和相对蛋白含量;逆转录聚合酶链反应(RTPCR)法研究3株细胞系之间CD44mRNA的表达差异;同时应用MTT法、美蓝硼酸盐法和微孔迁移技术研究阻断CD44的作用前后3株细胞的增殖、黏附和侵袭能力的变化。结果HOS和U2-OS中CD44的阳性百分率均高于99%,但HOS中CD44的平均荧光强度显著高于U2-OS(P<0.01);而MG63中CD44阳性百分率仅为(2.10±0.46)%;Westernblot亦证明HOS中CD44的蛋白含量显著高于U2-OS(P<0.05),而MG-63中CD44表达为阴性;CD44mRNA在HOS和U2OS中的表达也显著高于MG-63(P值均<0.05)。HOS的侵袭性显著高于MG63和U2-OS(P值均<0.01);HOS与MG63的增殖速率和黏附性差异无统计学意义,但均显著高于U2OS(P值均<0.01)。应用中和抗体阻断CD44的作用之后,HOS和MG-63的黏附性均显著降低(P值均为0.03),U2-OS的黏附性无明显变化(P=0.93);HOS和U2-OS的侵袭力显著降低(P值均<0.01),而MG-63的侵袭力无明显改变(P=0.18);3株细胞的增殖速率均无显著变化(P值均>0.05)。结论CD44能促进骨肉瘤细胞系HOS的黏附和侵袭,并参与U2-OS的侵袭和MG-63的黏附过程,但是不影响骨肉瘤细胞的增殖速率。     

低渗刺激对成骨样细胞MG63功能的影响

目的本试验拟通过研究低渗透压的静牵张作用,对成骨样细胞MG63的增殖能力、碱性磷酸酶活性以及[Ca2 ]i的影响,探讨该应力形式下成骨样细胞MG63的力学响应特征。方法采用人成骨肉瘤来源的成骨样细胞MG63作为细胞源进行细胞培养传代。用不同渗透压的低渗透液,对成骨样细胞MG63分别进行2 h、4 h、8h、12 h和24 h持续牵张作用后,用免疫组化法检测ALP表达的情况,用ALP试剂盒检测ALP活性的变化,用钙试剂盒检测[Ca2 ]i含量波动情况。结果成骨样细胞MG63的ALP免疫细胞化学染色为阳性。随着作用时间的延长,成骨样细胞MG63在277 mOsm和240 mOsm低渗透液的持续性膨胀作用下,其细胞的[Ca2 ]i、ALP活性缓慢增高,但240 mOsm组ALP活性始终低于对照组(p<0.01);而细胞在163 mOsm低渗液作用下,8 h时出现[Ca2 ]i急剧升高(11.383±0.111),ALP活性(0.326±0.002)明显高于其对照组(p<0.01)。结论结果提示不同水平的低渗膨胀对MG63的增殖分化、ALP活性以及Ca2 -ATPase都有一定的影响作用,且Ca2 内流与ALP活性之间存在一定的相关性。低渗膨胀法作为一种应力形式有一定的实验意义。     

敲低3-磷酸甘油酸脱氢酶靶向能量代谢逆转骨肉瘤恶性生物学的行为

目的 探讨敲低3-磷酸甘油酸脱氢酶(PHGDH) 靶向能量代谢对人骨肉瘤143B细胞恶性生物学行为及成骨分化的影响。 方法 Real-time PCR及Western blotting检测PHGDH在成骨细胞hFOB1.19和不同恶性程度骨肉瘤细胞TE85、MG63、143B中的表达。采用脂质体转染法将短发夹RNA（shRNA）-PHGDH重组质粒转染至143B细胞中, Real-time PCR和Western blotting检测PHGDH的表达变化；结晶紫染色法、细胞计数法、CCK-8实验检测细胞增殖；划痕实验检测细胞平行迁移能力，Transwell实验检测细胞垂直迁移及侵袭能力；Annexin V-FITC/PI双染法、DAPI染色法检测细胞凋亡；碱性磷酸酶（ALP）染色和茜素红S染色检测成骨分化作用,Western blotting检测成骨分化指标Runt相关转录因子2(Runx2)、骨钙素(OC)的表达情况；Real-time PCR检测能量代谢相关基因葡萄糖转运蛋白1（(GLUT1）、6-磷酸果糖激酶1（PFK1）、M2型丙酮酸激酶（PKM2）、乳酸脱氢酶A（LDHA)的表达，乳酸检测试剂盒测定乳酸分泌量，三磷酸腺苷（ATP）检测试剂盒检测ATP产生量。 结果 PHGDH在143B细胞中的表达明显高于在hFOB1.19、MG63和TE85细胞中(P<0.01)；转染shRNA-PHGDH重组质粒使143B细胞中的PHGDH表达量降低(P<0.01)、增殖能力降低(P<0.01)、细胞迁移及侵袭能力减低(P<0.01)、凋亡率增高(P<0.01)、ALP染色阳性率增加(P<0.01)、茜素红染色阳性率增加(P＜0.05)、Runx2(P<0.05)和OC的表达增高(P<0.01)、能量代谢相关基因(GLUT1、PFK1、PKM2、LDHA)的表达下调(P<0.01)、乳酸减少(P<0.01)、ATP增多(P<0.05)。 结论 敲低PHGDH可通过能量代谢抑制人骨肉瘤143B细胞增殖、迁移、侵袭, 促进其凋亡, 并促进其成骨分化。     

增殖细胞核抗原短发夹状RNA对人骨肉瘤细胞生长的影响

目的构建增殖细胞核抗原(PCNA)短发夹状RNA表达载体,检测其对人骨肉瘤细胞PCNA表达、细胞增殖及细胞凋亡的影响。方法体外构建PCNAshRNA表达载体pSilence2.1neo-PCNA,转染人骨肉瘤细胞系MG63,逆转录聚合酶链反应(RT-PCR)法检测转染前后PCNAmRNA表达,免疫组织化学(SP)法检测转染前后PCNA蛋白的表达,四甲基偶氮唑盐(MTT)比色法及克隆形成试验检测细胞增殖活性,3H胸腺嘧啶核苷(3H-TdR)细胞掺入试验检测细胞DNA合成,流式细胞仪检测细胞周期分布,吖啶橙染色法检测细胞凋亡情况。结果pSilence2.1neo-PCNA载体转染能显著抑制PCNAmRNA及蛋白的表达,转染后mRNA表达抑制率为80.51%、增殖指数值为空白组的25.68%。转染组48h细胞增殖抑制率为61.78%,转染组3H-TdR掺入率明显低于空白组(P<0.01)。细胞周期分析显示,siRNA转染组G0G1期细胞含量显著增加,而S期细胞含量显著减低。转染组凋亡率为16.54%。结论pSilence2.1neo-PCNA可显著抑制MG63细胞PCNA的表达及细胞增殖活性,促进细胞凋亡。     

分化抑制因子Idl对骨肉瘤细胞增殖的影响

目的探讨分化抑制因子Idl对骨肉瘤细胞增殖的影响及可能的途径。方法采用四甲基偶氮唑蓝（MTY）法检测Idl表达调节后MG．63细胞增殖变化情况;采用流式细胞术检测Idl下调表达后,MG．63细胞凋亡及细胞周期变化情况;Westemblotting技术检测Idl下调表达后对p-AKT水平的影响。结果本实验利用RNA干扰技术下调骨肉瘤细胞系Idl表达后,可以明显抑制MG．63细胞的增殖,p-AKT的水平也显示下降,流式细胞术证明下调后Idl的水平可以一定程度地调节细胞凋亡水平,而对细胞周期无明显影响,另外进一步构建了Idl的真核表达质粒,转染Idl过表达质粒后。并未见骨肉瘤细胞明显地增殖加速。结论分化抑制因子Idl可能通过影响AKT信号通路进而影响了骨肉瘤细胞的增殖。     

沉默长链非编码RNA CCAT2对人骨肉瘤细胞MG-63增殖与凋亡的影响

目的探讨靶向沉默长链非编码RNA肠癌相关转录子2(lncRNA CCAT2)对人骨肉瘤细胞MG-63增殖与凋亡的影响。方法实时荧光定量PCR(qPCR)方法检测骨肉瘤MG63细胞与人成骨hFOB1.19细胞中lncRNA CCAT2表达水平;转染小干扰RNA沉默MG63细胞中lncRNA CCAT2的表达,qPCR方法验证沉默效率。沉默MG63细胞中lncRNA CCAT2表达后,MTT方法检测MG63细胞增殖能力的变化,流式细胞术检测MG63细胞的凋亡率,Western blot方法检测MG63细胞p53及Bcl-2蛋白的表达变化。结果lncRNA CCAT2在骨肉瘤MG63细胞中的表达显著高于人成骨hFOB1.19细胞(P<0.01);转染小干扰RNA能够显著降低MG63细胞中lncRNA CCAT2的表达水平(P<0.01)。沉默lncRNA CCAT2后,MG63细胞的增殖能力显著降低,凋亡率显著上升,Bcl-2蛋白表达显著降低p53蛋白表达显著升高(P<0.01)。结论靶向沉默lncRNA CCAT2能够抑制人骨肉瘤细胞MG-63的增殖并诱导凋亡。     

3-Hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglycemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

3-Hydroxy-3-Methylglutaric Aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglecemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

γδ cells involved in contact sensitivity preferentially rearrange the Vγ3 region and require interleukin-7

Ptak and Askenase showed that both αβ and γδ cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of γδ cells fail to transfer CS to trinitrochlorobenzene (TNP-Cl) systemically and demonstrates that administration of anti-γδ monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, γδ cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8α⁺, CD8β^-, IL-4 R⁺ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of γδ cells increases in the regional lymph nodes with a peak at 4 days. Of the γδ cells, 80%, both in the lymph nodes of TNP-Cl-immune mice and accumulating at the antigen challenge site are Vγ3⁺. The γδ cells expressing Vγ3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)-7, but only poorly to IL-2 and IL-4. They also respond to concanavalin A and immobilized anti-γδ mAb, but not to haptens or heat-shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL-7 inhibits accumulation of Vγ3⁺ cells both in the lymph nodes after skin sensitization and at the antigen-challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the γδ cells found in the lymph node after skin sensitization and at the site of challenge, and that IL-7 is implicated in these phenomena.     

Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.     

γδ cells regulate autoimmunity

γδ cells are attractive candidates for mediators of autoimmune disease. They can expand in germ-free mice, probably through recognition of autoantigens, and γδ-cell-deficient mice, unlike mice deficient in αβ T cells or B cells, show no severe defects in the immune response to foreign antigen challenge. A capacity of γδ cells to effect or regulate tissue damage is also plausible, given their ready localization to tissues, and their myriad of effector functions. Added to this, attempts to reconstruct the physiological course of autoimmune diseases with only autoreactive αβ T cells seem invariably to fall short for lack of other unidentified players. γδ cells and their putative ligands have been linked to autoimmune conditions, and recent experiments confirm that γδ cells play a significant role in autoimmune disease in vivo.