Hsa-miR-4282对肝癌细胞系SMMC-7721生长的影响

目的  探讨Hsa-miR-4282在肝癌细胞系SMMC-7721中的表达及其对细胞生长的影响。方法 采用实时荧光定量PCR法检测Hsa-miR-4282在人正常肝上皮细胞系HL-7702和人肝癌细胞系MHCC97-H、SMMC-7721，以及 20例肝癌组织及其相应癌旁组织中的表达。采用瞬时转染法将Hsa-miR-4282 mimics（上调组）和Hsa-miR-4282 inhibitor（下调组）分别转染肝癌SMMC-7721细胞，上调组和下调组分别设置相应阴性对照组。转染后采用MTT法检测细胞增殖能力，平板克隆形成实验检测细胞克隆形成能力，流式细胞仪检测细胞凋亡能力。结果 Hsa-miR-4282在肝癌组织、肝癌细胞MHCC97-H及肝癌细胞SMMC-7721中的表达均低于癌旁组织及正常肝细胞HL-7702（P＜0.05）。MTT实验结果显示，Hsa-miR-4282上调后肝癌SMMC-7721细胞的OD值低于其阴性对照组，而下调后OD值高于其阴性对照组 （P＜0.05）。平板克隆形成实验显示，下调组的细胞克隆数高于其阴性对照组［（240±7） 个 vs （191±10） 个，P=0.005）］，而上调组细胞克隆数低于基阴性对照组［（146±10） 个 vs （193±12） 个，P=0.013）］。流式细胞仪检测结果显示，Hsa-miR-4282上调组细胞凋亡率较其阴性对照组升高［（23.89±1.89）% vs（16.6±1.14）%，P=0.009）］，下调组细胞凋亡率较期阴性对照组降低［（14.98±0.46）% vs （17.79±0.73）%，P=0.010］。结论 Hsa-miR-4282上调可抑制肝癌SMMC-7721细胞增殖，促进细胞凋亡，可能与肝癌的发病机制有关。     

健脾益肾方对非小细胞肺癌细胞体外增殖凋亡作用的实验研究

目的:探讨健脾益肾方对非小细胞肺癌(NSCLC)细胞体外增殖凋亡的作用。方法:人NSCLC细胞系A549分为四组:空白对照组(仅加入细胞培养液)、阴性对照组(加入细胞,不进行中药处理)、实验组(加细胞加中药处理)。荧光定量PCR和Western blot分别检测Survivin、Bcl-2和Caspase-3的mRNA和蛋白表达。MTT检测细胞增殖;流式细胞术检测细胞凋亡。结果:与空白对照组相比,阴性对照组细胞在24、48、72 h的吸光度值明显升高,细胞凋亡率下降,Survivin和Bcl-2 mRNA和蛋白相对表达量上调,Caspase-3 mRNA和蛋白相对表达量下调,组间比较差异有统计学意义(P<0.05);而健脾益肾方处理的实验组24、48、72 h的吸光度值均显著降低,细胞凋亡率显著上升,Survivin和Bcl-2 mRNA和蛋白相对表达量下调,Caspase-3 mRNA和蛋白相对表达量上调,与空白对照组相比差异有统计学意义(P<0.05)。结论:健脾益肾方可通过下调Survivin和Bcl-2、上调Caspase-3表达诱导NSCLC细胞凋亡,并抑制肿瘤细胞的增殖,进而抑制NSCLC的发展。     

体外转染Cyclin A2基因对原代大鼠心肌细胞增殖的影响

目的探讨体外转染细胞周期素A2(Cyclin A2)基因对原代大鼠心肌细胞增殖的影响,为心脏再生提供细胞学依据。方法 SD乳鼠12只,分离、培养、鉴定原代乳鼠心肌细胞并分为3组,实验组:转染带有强化绿色荧光蛋白(GFP)和Cyclin A2基因的重组腺病毒(Ad-Cyclin A2-GFP);空病毒组:转染不含目的基因带有GFP的重组腺病毒(Ad-Null-GFP);阴性对照组:未做转染处理,仅加入等量的培养基。利用GFP示踪技术,评估原代心肌细胞转染效率;转染后的细胞继续体外培养3~5d,利用免疫荧光技术分别检测Cyclin A2、磷酸化组蛋白H3(H3P)、心肌肌钙蛋白-T(c Tn T)。结果 1.荧光GFP示踪表明原代心肌细胞的转染效率高达(97±0.74)%;2.免疫荧光标记显示,空病毒组和对照组结果相似;心肌细胞特异性标记蛋白c Tn T分布于细胞质,原代心肌细胞纯度高达(95±0.62)%;心肌细胞Cyclin A2主要在胞核内聚集,少数分布于细胞质。H3P为核蛋白在细胞核内分布。3.转染Cyclin A2后,实验组H3P阳性率明显高于空病毒组和对照组,差异具有统计学意义(P0.05);实验组可见大量多核细胞,以双核为主,伴少量3核细胞。结论腺病毒作为转染载体对原代心肌细胞有很好的侵染效率;Cyclin A2超表达促进原代心肌细胞形成双核。     

miR-30c Impedes Glioblastoma Cell Proliferation and Migration by Targeting SOX9

miR-30c has been acknowledged as a tumor suppressor in various human cancers, such as ovarian cancer, gastric cancer, and prostate cancer. However, the role of miR-30c in glioblastoma (GBM) needs to be investigated. In our study, we found that the expression of miR-30c was significantly downregulated in GBM tissues and cell lines. We found that overexpression of miR-30c inhibited cellular proliferation of GBM cells in vitro and in vivo. More GBM cells were arrested in the G₀ phase after miR-30c overexpression. Moreover, we showed that miR-30c overexpression suppressed the migration and invasion of GBM cells. Mechanistically, we found that SOX9 was a direct target of miR-30c in GBM cells. Overexpression of miR-30c inhibited the mRNA and protein levels of SOX9 in GBM cells. Moreover, there was a negative correlation between the expression of miR-30c and SOX9 in GBM tissues. Finally, we showed that restoration of SOX9 in GBM cells reversed the proliferation, migration, and invasion of GBM cells transfected with miR-30c mimic. Collectively, our results demonstrated that miR-30c suppressed the proliferation, migration, and invasion of GBM cells via targeting SOX9.     

The oncogenic impact of RNF2 on cell proliferation,invasion and migration through EMT on mammary carcinoma

Mammary carcinoma (MC) is one of most common malignancy in women, and ring finger protein 2 (RNF2) possesses various roles in vast human tumors. In MC tissues as well as in cell lines RNF2 exhibited high expression, had significant association with tumor size, lymph node status, TNM stage, patients’ poor survival, and promoted cell proliferation, colony formation, cell migration and invasion of MC cell lines which was mediated by downregulation of E-cadherin protein. These data reveal that RNF2 protein plays a vital role in the development of MC and may be a potential therapy target of MC.     

蓝莓花青素对人胃腺癌BGC-823细胞增殖、侵袭的影响及机制研究

目的:探讨蓝莓花青素(BA)对人胃腺癌BGC-823细胞增殖、侵袭的影响及相关机制。方法:低、中、高剂量实验组与对照组人胃腺癌BGC-823细胞分别以25、50、100μg/mL BA与等量生理盐水处理,分别处理12 h、24 h、48 h。采用噻唑蓝(MTT)与流式细胞仪检测人胃腺癌BGC-823细胞增殖与凋亡;采用Transwell小室实验与免疫细胞化学法检测人胃腺癌BGC-823细胞侵袭与P53、半胱氨酸蛋白酶-3(Caspase-3)蛋白表达。结果:干预48 h时,对照组、低、中、高剂量实验组人胃腺癌BGC-823细胞P53与Caspase-3蛋白阳性细胞百分比分别为(11.12±1.01)%,(24.31±2.18)%,(47.56±2.91)%,(66.49±5.14)%与(15.04±1.23)%,(28.22±3.09)%,(69.36±5.42)%,(86.52±6.15)%。实验组胃腺癌BGC-823细胞增殖抑制率随BA浓度增大而逐渐升高;与对照组比较,低、中、高剂量实验组人胃腺癌BGC-823细胞凋亡率、P53与Caspase-3蛋白阳性细胞百分比均显著升高,侵袭率降低,呈浓度依赖性(均P0.05)。结论:BA可有效抑制胃腺癌BGC-823细胞增殖,诱导其凋亡,并降低侵袭能力,其机制可能与上调P53与Caspase-3蛋白表达有关。     

microRNA-613调控CDK14来抑制老年胶质瘤患者瘤细胞的增殖和侵袭作用

目的 探讨microRNA-613调控细胞周期蛋白依赖性激酶14(CDK14)通路来抑制老年胶质瘤患者瘤细胞增殖和侵袭的机制。方法 购自上海北诺生物科技有限公司的人脑胶质瘤细胞株U251共24株,分为shNC组、shmicroRNA-613组、Vector组、microRNA-613组,每组各6株; Western Blotting法和qRT-PCR法检测敲减microRNA-613和过表达microRNA-613后的人脑胶质瘤细胞株U251中CDK14蛋白表达水平,CCK-8细胞增殖实验、Transwell小室实验验证敲除microRNA-613和过表达microRNA-613后U251细胞的增殖、侵袭能力。结果 人胶质瘤细胞株U251敲减microRNA-613后CDK14蛋白表达增加(P<0.05); 人胶质瘤细胞株U251过表达microRNA-613后CDK14蛋白表达减少(P<0.05); 转染第24、36、48 h microRNA-613组U251细胞增殖率P<0.05); microRNA-613组U251细胞侵袭能力>Vector组,shNC组>shmicroRNA-613组(P<0.05)。结论 microRNA-613可通过调控CDK14信号转导通路来抑制人脑胶质瘤细胞U251增殖、转移。     

透明质酸钠对大鼠成肌细胞增殖和分化的影响

目的 研究透明质酸钠对成肌细胞增殖和分化的影响。方法 采用酶消化法将新生SD大鼠骨骼肌组织分离、纯化、原代培养及传代培养；取第3代成肌细胞，分别加入浓度为0．05%、0．1％及0．2％的透明质酸钠溶液作为生长培养基行体外培养为实验A、B及C组，加入常规生长培养基为对照D组，观察各组成肌细胞增殖情况，并采用细胞计数及MTT法绘制生长曲线。另选择0．1%透明质酸钠融合培养基行体外成肌细胞培养，常规融合培养基作对照，观察透明质酸钠对成肌细胞分化功能的影响。结果A、B及C组成肌细胞增殖表现相似，2d时进入对数生长期，4d时均达顶峰；D组成肌细胞于3d时进入对数生长期，5d时细胞数倍增，6d时达顶峰。MTT法所测吸光度（A）值的变化反映成肌细胞的增殖情况，与细胞计数的结果一致，以B组细胞增殖作用最明显，B组A值在2～8d时均高于C、D组（P〈0．05），8d时高于A组（P〈0．05）。0．1％透明质酸钠融合培养基中的成肌细胞融合率较低且上升缓慢，7d时融合率最高，为11．7％；常规融合培养基成肌细胞融合率于6d时达到峰值，约为35．0%。结论 透明质酸钠与成肌细胞的细胞相容性较好，可以作为良好的成肌细胞培养基。     

持续低剂量率照射后HepG2细胞的ATM磷酸化

目的 探讨持续低剂量率照射下HepG2细胞共济失调毛细血管扩张症突变基因(ATM)磷酸化的变化规律。方法 应用间接免疫荧光、Western blot技术检测持续低剂量率(8.28 cGy/h)照射下HepG2细胞ATM磷酸化蛋白的表达;采用集落形成法观察持续低剂量率照射对HepG2细胞增殖活性的影响。 结果持续低剂量率照射30 min后,ATM即已发生磷酸化,持续照射6 h时,ATM磷酸化蛋白表达量最多,以后逐渐减弱。使用Wortmannin抑制ATM磷酸化后,降低了持续低剂量率照射下肝癌细胞的存活分数。结论在持续低剂量率照射中后期ATM磷酸化减弱,提示持续低剂量率照射具有增加HepG2细胞辐射敏感性的潜能。     

Role of retinol in protecting epithelial cell damage induced by Clostridium difficile toxin A

Vitamin A (retinol), a fat-soluble vitamin, is an essential nutrient for the normal functioning of the visual system, epithelial cell integrity and growth, immunity, and reproduction. Our group has investigated the effect of high doses of oral vitamin A on early childhood diarrhea in our prospective community-based studies from Northeast Brazil and found a beneficial role in reducing the mean duration but not incidence of diarrheal episodes. In this study, we explored the role of retinol supplementation in intestinal cell lines following Clostridium difficile toxin A (TxA) challenge. C. difficile is the most common anaerobic pathogen borne with antibiotic-borne diarrhea and pseudomembranous colitis. Since retinol is critical for the integrity of tight junctions and to modulate the cell cycle, we have focused on changes in transepithelial electrical resistance (TEER) in Caco-2, a more differentiated intestinal cell line, and on models of cell proliferation, migration and viability in IEC-6 cells, an undifferentiated crypt cell line, following TxA injury. In this model, retinol therapy reduced apoptosis, improved cell migration and proliferation, and prevented the reduction in TEER, following C. difficile TxA challenge in a glutamine-free medium. These results suggest the role of retinol in protecting intestinal epithelial barrier function from C. difficile TxA enterotoxic damage.