目的 探讨初诊的弥漫大B细胞淋巴瘤(DLBCL)患者外周血Th17细胞的表达与国际预后指标(IPI)之间关系。方法 初诊DLBCL组(n=45)按照国际预后指数(IPI)积分分为4组,采用ELISA和流式细胞术检测各个DLBCL组与正常对照组(n=43)的外周血中IL-17的浓度以及Th17阳性细胞比例,比较各组数值间的差异,并分析IPI的5个指标与IL-17的浓度以及Th17阳性细胞比例的相关性。结果 DLBCL中高危组与高危组的IL-17的浓度以及Th17阳性细胞比例较正常对照组及其他IPI组降低,有显著性差异;DLBCL四组的IL-17的浓度以及Th17阳性细胞比例均低于正常对照组;且可见随着IPI分组的增高,IL-17的浓度以及Th17阳性细胞比例呈降低的趋势;IPI指标中年龄、临床分期、全身状态与Th17细胞的表达有相关性。结论 初诊DLBCL患者外周血Th17细胞的表达与国际预后指标有关系;随着IPI积分的增加,DLBCL患者Th17细胞表达下降;临床上对于年龄60岁以上、临床分期Ⅲ期以上、长期卧床及需别人照顾的患者更要注意监测其外周血Th17细胞的表达情况。

Objective To explore the relationship between the international prognosis indexes(IPI) and the Th17 cells expression in DLBCL patients. Methods DLBCL patients (n=45) were divided into 4 groups according to IPI score, peripheral blood were taken from each person in DLBCL groups and normal group. We used ELISA to test IL-17 and flow cytometry (FCM) to examine the Th17 positive cells. We compared the value of each group, and analyzed the relativity of IPI and Th17 cells' expression. Results Th17 cells' expression level in middle-high risk group and high risk group were higher than that in normal group and other IPI groups; Th17 cells' expression level in DLBCL groups were all lower than that in normal group; Th17 cells decreased while IPI score increased; Age, clinical stage and general body state have the relativity with DLBCL patients' Th17 cells expression level. Conclusion Th17 cells in DLBCL patients has the relativity with prognosis index. In our clinical diagnosis and treatment, we need to pay more attention to those patients who are over 60 years old, or whose clinical stage is above Ⅲ phase, or who need to stay on bed for a long time and need other peoples' help .

目的 构建重组pEGFP-C3-HCVc真核表达载体,并建立稳定表达HCVc基因的肝内胆管癌细胞株RBE-core。方法 采用PCR钓取目的基因HCVc,并克隆入pEGFP-C3的多克隆位点,构建pEGFP-C3-HCVc重组质粒。经过双酶切及测序验证后,采用脂质体将pEGFP-C3-HCVc质粒转染到RBE细胞中,经2周G418 (200 μg/mL) 筛选后进行单克隆挑选及扩大培养,建立稳定表达HCVc的胆管癌细胞株RBE-core。采用RT-PCR和Western blot验证HCVc在RBE-core中的表达情况。结果 PCR成功钓取HCVc基因,大小约573 bp,并插入pEGFP-C3载体HindⅢ和BamHⅠ多克隆位点;双酶切及测序证实目的基因HCVc正确连接到pEGFP-C3的多克隆位点。RT-PCR和Western blot分别在573 bp处和34 KD左右检测到相应的阳性条带。结论 成功构建重组质粒pEGFP-C3-HCVc,并在胆管癌细胞RBE中获得稳定表达。

Objective To construct a recombinant plasmid of pEGFP-C3-HCVc containing hepatitis C virus core protein, and establish the HCVc-expressing cell line RBE-core. Methods The HCVc gene was amplified by PCR and cloned into HindⅢ and BamHⅠsite of pEGFP-C3 plasmid. The recombinant plasmid of pEGFP-C3-HCVc was confirmed by sequencing. RBE cells were transfected with the recombinant plasmid by using Lipofectamine 2000, and then performed G418 (200 μg/mL) selection after 2 weeks. The expressing of HCVc gene in RBE cells was confirmed by RT-RCR and western blot. Results The recombinant plasmid of pEGFP-C3-HCVc was successfully constructed. RT-PCR and western blot detected a 573bp and 34KD bland, indicating the stably expressing of HCVc in RBE cells. Conclusion The recombinant plasmid of pEGFP-C3-HCVc is stabled expressing in RBE cells,which provides support for the further study.

目的 研究灵芝孢子油抑制肿瘤细胞增生的作用。方法 通过“预防性”和“治疗性”喂食方法,观察并检测喂食过程中小鼠乳腺癌细胞生长速度及处死后的瘤重量。结果 灵芝孢子油喂食组肿瘤生长速度较对照组慢,且预防性喂食组抑瘤效果好于“治疗性”喂食组。结论 灵芝孢子油具有抑制小鼠乳腺癌生长的作用。

Objective To research the effect of tumor growth inhibition by Ganoderma spore oil. Methods Murine mammary cancer cells were inoculated and Ganoderma spore oil was given by preventing and therapeutic feeding respectively. Results Tumor growth speed of Ganoderma spore oil feeding group was slower than control group, and tumor weight was lighter than control group. In addition, tumor weight of preventing feeding was lighter than therapeutic feeding. Conclusion Ganoderma spore oil has the effect of anti - mammary cancer cell growth.

目的 研究前列腺癌细胞中miR-221的表达情况及其对癌细胞增殖的影响。方法 运用实时荧光定量PCR(qRT-PCR)检测miR-221在前列腺正常细胞株与前列腺癌细胞株中表达的差异情况,利用细胞转染构建miR-221过表达LNCaP和DU145细胞株,再通过CCK8细胞增殖实验检测细胞增殖情况的变化。结果 qRT-PCR检测细胞株发现miR-221在PC3、LNCaP和DU145三种前列腺癌细胞株中表达量均比前列腺正常细胞株PrEC低 (F=254.197,P<0.001),其中两两比较差异也均有统计学意义。细胞转染技术构建的miR-221过表达LNCaP和DU145细胞株,经qRT-PCR结果显示,miR-221在LNCaP和DU145细胞株中的表达水平明显升高(LNCaP,倍数变化=2.24,t=3.46,P<0.01;Du145,倍数变化=2.24,t=4.29,P<0.01)。细胞增殖实验结果显示,过表达了miR-221的LNCaP(P<0.001)和DU145(P<0.001)细胞生长速度慢于对照组。结论 实验证明miR-221表达过度能减慢前列腺癌细胞的增殖,miR-221有可能成为前列腺肿瘤治疗的生物学标志物。

Objective To investigate miR-221 expression in prostate cancer cells and its influence on prostate cancer cell proliferation. Methods miR-221 expressions in prostate normal cell lines and cancer cell lines were measured by qRT-PCR. Overexpression of the miR-221 in LNCaP and DU145 cell lines used by cell transfection. Effects of the depletion on cell proliferation were assessed in vitro with CCK8. Results qRT-PCR showed miR-221 was lower expressed in PC3, LNCaP and DU145 than in PrEC(F=254.197, P<0.001), in which pairwise comparison also had significant differences. qRT-PCR showed miR-221 expression rose significantly in LNCaP and DU145 cell lines whose miR-221 was overexpression with cell transfection(LNCaP, Fold Change=2.24,t=3.46,P<0.001;Du145, Fold Change=2.24,t=4.29,P<0.001). Cell proliferation assay showed that growth of LNCaP(P<0.001) and DU145(P<0.001) cells whose miR-221 was overexpression was slower than the control group. Conclusion This study demonstrates miR-221 overexpression can inhibited the proliferation of prostate cancer cells for the first time, it also suggests that miR-221 has the potential to serve as a biomarker for PCa therapy.

目的 氧化苦参碱对视网膜母细胞瘤细胞SM-106凋亡的诱导作用及机制。方法 以不同作用时间(24 h、48 h、72 h)和不同作用浓度(12.5 μl/mL、25 μl/mL、50 μl/mL、100 μl/mL)氧化苦参碱处理视网膜母细胞瘤细胞SM-106,分别采用流式细胞仪及western blot检测视网膜母细胞瘤细胞SM-106细胞凋亡及其凋亡因子(Bax、Bcl-2)蛋白表达。结果 氧化苦参碱可促进SM-106细胞体外凋亡,上调Bax蛋白表达及Bax/Bcl-2蛋白表达比值,下调Bcl-2蛋白表达,并呈现剂量及时间依赖性。结论 氧化苦参碱可诱导视网膜母细胞瘤细胞SM-106凋亡,调控凋亡因子Bax、Bcl-2的表达是其可能作用机制。

Objective To evaluate the apoptosis and its mechanism of retinoblastoma cells SM-106 induced by oxymatrine. Methods Retinoblastoma cells SM-106 were treated with different time(24 h、48 h、72 h)and different concentrations(12.5 μl/mL, 25 μl/mL, 50 μl/mL or 100 μl/mL) of oxymatrine. The apoptosis and protein expression of apoptosis factors (Bax and Bcl-2) were respectively determined by flow cytometry and western blot. Results Oxymatrine significantly promoted the SM-106 cells apoptosis in vitro, raised Bax protein expression and Bax/Bcl-2 protein expression ratio, reduced the Bcl-2 protein expression, and showed the dose and time dependent. Conclusion Oxymatrine is able to induce the apoptosis in retinoblastoma cells SM-106. Regulating apoptosis related gene Bax and Bcl-2 expression may be the mechanism of apoptosis.

目的 观察并评估内毒素性急性肺损伤大鼠吸入一氧化氮后外周血中内皮祖细胞和炎症介质的变化情况。方法 90只SPF级健康大鼠分为3组,A组为正常对照组(n=30),B组为急性肺损伤组(ALI)(n=30), C组为一氧化氮(NO)组(n=30)。分别计算各组外周血内皮祖细胞(Endothelial progenitor cells,EPCs) 数量,同时监测肺组织中白细胞介素-10(Interleukin-10,IL-10)水平和髓过氧化物酶(Myeloperoxidase,MPO)活性。结果 我们成功建立了大鼠的ALI肺损伤模型, C组EPCs数量、MPO活性上升幅度均小于B组、而IL-10上升水平均高于B组,差异有统计学意义(P<0.05)。结论 大鼠吸入一氧化氮可减轻内毒素所致急性肺损伤程度,其机制可能与外周血中内皮祖细胞数量及MPO水平下降和IL-10水平上升有关。

Objective To investigate the effect of nitric oxide(NO) inhalation in endotoxin-induced acute lung injury mice. Methods Ninety SPF mice were randomly assigned to the normal group(group A), ALI group(group B)and ALI+NO group(group C). The number of endothelial progenitor cells was counted and the level of Interleukin-10(IL-10) and myeloperoxidase (MPO) were measured. Results Endotoxin administration resulted in pulmonary edema. The pulmonedema was lightened and the level of MPO were decreased by the inhalation of nitric oxide while the level of IL-10 increased. Conclusion NO inhalation can mitigate acute lung injure. The decline of EPCs and MPO and the increase of IL-10 may be one of the mechanism.

目的 观察鼻咽癌患者癌组织中RhoA蛋白-Rho激酶蛋白表达情况。方法 收集增城市人民医院2009年2月—2014年6月耳鼻喉科住院治疗,进行活检的鼻咽癌患者切除标本共62例,包括癌组织及癌旁组织。通过SABC免疫组织化学法检测患者癌组织及癌旁组织中RhoA蛋白-Rho激酶蛋白表达情况。结果 鼻咽癌组织RhoA蛋白阳性表达率及Rho激酶蛋白阳性率高于癌旁组织(P=0.00);鼻咽癌癌组织RhoA及Rho激酶表达在年龄及性别分布上无差异,而TNM分期中Ⅲ期+Ⅳ期RhoA及Rho激酶表达阳性率高于Ⅰ期+Ⅱ期患者,同时存在淋巴结转移患者其RhoA及Rho激酶表达阳性率高于未转移患者。结论 鼻咽癌患者癌组织中RhoA蛋白及Rho激酶表达阳性率高,TNM分期越高及存在淋巴结转移者其RhoA蛋白及Rho激酶表达阳性率越高。RhoA-Rho激酶信号通路参与了鼻咽癌发生发展的过程。

Objective To observe the expression of RhoA-Rho kinase protein in patients with nasopharyngeal carcinoma tissue. Methods Collecting 62 cases of patients with nasopharyngeal carcinoma who hospitalized and hadbiopsyof specimens, including carcinoma tissues and adjacent tissues in department of otolaryngology-head and neck surgery from February 2010 to October 2014. The expression of RhoA-Rho kinase protein in carcinoma tissues and adjacent tissues was detected with SABC immune histochemical method. Results Positive expression rate of nasophryngeal carcinoma tissue RhoA protein and Rho kinase protein was significantly higher than that of adjacent tissues, and there was no obvious difference in age and sex between the expression of nasopharyngeal carcinoma tissue RhoA and Rho kinase(P=0.00), while patients being in the third and fourth periods of TNM stage had a higher positive expression rate of RhoA and Rho kinase than those being in the first and second periods. At the same time, RhoA and Rho kinase positive expression rate in patients with lymph node metastasis was higher than that in those without lymph node metastasis. Conclusion Positive expression rate of RhoA protein and Rho kinase in patients with nasopharyngeal carcinoma. The higher TNM stage is, the higher positive expression rate of RhoA protein and Rho kinase in lymph node metastasis is. RhoA-Rho kinase signaling pathways involved in the occurrence and development of nasopharyngeal carcinoma.

目的 评价实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达时12个候选内参基因表达的稳定性,获得最适合的内参基因。方法 以低氧性肺动脉高压大鼠肺动脉平滑肌为研究对象,选择文献报道的常用12种内参基因为候选内参基因,利用geNorm和NormFinder程序分析实时荧光定量PCR数据,筛选出最适内参基因。结果 12个候选内参基因在低氧性肺动脉高压大鼠肺动脉平滑肌表达稳定性由强到弱顺序为:TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1,平均表达稳定度(M值)均<0.5,geNorm和NormFinder评估后推荐使用TBP和B2M一起作为该研究时的内参基因。结论 同时使用TBP和B2M是实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达的最适合内参基因,为低氧性肺动脉高压相关基因研究提供最优内参基因。

Objective To compare and select the suitable reference genes in real-time quantitative PCR analysis of rat pulmonary artery smooth muscle cells mRNA expression level of pulmonary hypertension. Methods To choose appropriate reference gene, the expression of twelve commonly use housekeeping genes were examined in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension by using geNorm and NormFinder programs. Results The expression consistency of 12 genes was (from high to low): TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1. The average expression stability(M) values of them were low than 0.5. TBP and B2M reference genes were recommended to use in the same condition. Conclusion TBP and B2M reference genes were the most suitable combination of the reference genes for real-time quantitative PCR analysis in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension.

目的 构建抑癌基因SEMA3B真核表达载体pcDNA3.1-SEMA3B,并检测其对肺癌A549细胞恶性生物学行为的影响。方法 应用PCR扩增SEMA3B全长cDNA片段,构建真核表达载体pcDNA3.1-SEMA3B。克隆PCR、双酶切法、基因测序验证过表达载体构建成功。将pcDNA3.1-SEMA3B真核表达载体和空载体pcDNA3.1分别转染入A549细胞中,应用qRT-PCR、Western blot检测SEMA3B mRNA、蛋白表达水平的变化;MTS法检测细胞增殖;流式细胞仪检测细胞凋亡、细胞周期;克隆形成实验检测细胞集落形成能力。结果 SEMA3B基因扩增片段与预测片段一致,克隆成功,且测序鉴定证实真核表达载体构建成功。转染pcDNA3.1-SEMA3B真核表达载体可上调SEMA3B mRNA、蛋白表达水平,且可抑制A549细胞的增殖,诱导凋细胞亡,细胞被阻滞在G1期,抑制细胞集落形成能力。结论 成功构建了SEMA3B基因真核表达载体,抑癌基因SEMA3B在肺癌恶性生物学进程中可能发挥重要作用。

Objective To construct the eukaryotic expression vector of the cancer suppressor gene, SEMA3B, and research the effects on malignant biological behavior of lung cancer A549 cells. Methods By reverse transcriptase-polymerase chain reaction (RT-PCR), the full length SEMA3B gene was amplified and then was inserted into pcDNA3.1. The recombinant plasmid pcDNA3.1-SEMA3B was confirmed correctly through double enzyme digestion and PCR identification, which was transfected into lung cancer A549 cells by lipid media transfection. The untransfected A549 and A549 transfected with pcDNA3.1 were used as controls. SMEA3B gene was detected by qRT-PCR and western blot. MTS assay, flow cytometry, and colony formation test were performed to evaluate the effect of overexpression of SEMA3B gene on A549 cell proliferation, apoptosis, cell cycle, and colony forming ability. Results The amplied fragment of SEMA3B gene by PCR was consistent with the anticipated result, the SEMA3B gene was cloned successfully. And the recombinant plasmid pcDNA3.1-SMEA3B was constructed successfully through gene sequence identification. After transfection of pcDNA3.1-SEMA3B, SEMA3B mRNA and protein expression levels were raised, and overexpression of SEMA3B gene in A549 cells significantly inhibited the proliferation of A549 cells, induced apoptotic cell death, blocked cell cycle in the G1 phase, and suppressed cell colony-forming ability. Conclusion The recombinant pcDNA3.1-SEMA3B is constructed successfully. SEMA3B gene can significantly inhibit the malignant biological behavior of lung cancer A549 cells.

目的 分析乳腺癌细胞中Snail与MTDH基因的作用,明确Snail是否通过结合于MTDH的启动子区域促进乳腺癌转移。方法 克隆、转染Snail基因至乳腺癌细胞,观察过表达Snail的乳腺癌细胞中MTDHmRNA及蛋白表达的变化;再使用免疫共沉淀法检测Snail与MTDH基因的共作用。结果 转染Snail基因进入乳腺癌MDA-MB-435细胞后,转染组、空白组和对照组中MTDHmRNA的表达水平分别为1.61±0.22、1.02±0.18、0.99±0.20,转染组高于空白组和对照组,差异有统计学意义(P<0.05),而后两组表达无差异(P>0.05);Westren blot检测结果显示,Snail可促进MTDH蛋白的表达;免疫共沉淀显示,Snail与MTDH在细胞内存在相互结合作用。结论 Snail在乳腺癌细胞中可通过结合于MTDH基因的启动子区域,促进MTDHmRNA转录及相关蛋白的表达,从而导致乳腺癌转移。

Objective To investigate the function of Snail to MTDH gene in breast cancer cells. Methods We observed the changement of MTDHmRNA and protein expression in breast cancer cell line MDA-MB-435 after transfected with Snail gene. Then, we used co-immunoprecipitation to determine the domain of Snail and MTDH binding in vitro. Results After transfected with Snail gene into MDA-MB-435 cell, the expression levels of MTDHmRNA in transfection group, blank group and control group were 1.61±0.22,1.02±0.18,0.99±0.20. The level of transfection group was significantly higher than the other groups(P< 0.05). Western blot showed that the expression of MTDH protein can be promoted by Snail. Co-immunoprecipitation showed that Snail and MTDH are binding interactions in breast cancer cell line MDA-MB-43. Conclusion Snail can promote transcription and expression of MTDH in breast cancer cells by binding to the promoter region of the MTDH gene resulting in metastasis of breast cancer.