黄瓜CMV复制酶基因相对表达定量PCR方法的建立

为了对黄瓜感染CMV植株体内CMV复制酶基因表达水平进行测定,建立CMV复制酶基因的相对表达定量检测技术,并用该方法开展黄瓜植株CMV抗性鉴定试验,以18SrRNA基因为对照,CMV复制酶基因为目标基因,设计引物,探索SYBRGreen实时荧光定量PCR反应体系,同时对CMV复制酶基因进行均一化处理,利用荧光阈值(Q值)计算p-防御素基因的相对表达量。结果表明:利用SYBR GreenⅠ实时荧光定量PCR技术测定黄瓜CMV复制酶基因相对表达定量具有较高的准确性,耗时短,该技术可应用于CMV抗性植株的筛选。     

应用实时荧光RT-PCR检测柑橘黄化脉明病毒

为更快速准确检测柑橘黄化脉明病毒（Citrus yellow vein clearing virus,CYVCV）,根据病毒外壳蛋白基因的保守序列设计特异性引物,通过体系优化得到最佳反应条件,建立基于SYBR GreenⅠ的CYVCV实时荧光RT-PCR检测方法。该方法可特异性检测CYVCV,而测试的柑橘衰退病毒、碎叶病毒等5种柑橘病毒均不能检测出。灵敏度较普通RT-PCR提高了100倍,标准曲线循环阈值与模板浓度呈良好的线性关系,扩增效率和相关性系数分别为102%和0.999。对田间样品的检测结果表明,建立的实时荧光RT-PCR适用于检测不同柑橘品种中CYVCV的含量。     

Detection of miR-122 expression in serum by Taqman probe real-time fluorescence quantitative PCR and its clinical significance

AIM: To detect the serum level of miR-122 expression by the technique of Taqman probe real-time fluorescence quantitative PCR for identifying its clinical significance. METHODS: The stem-loop RT primer, PCR primer and Taqman probe of miR-122 and U6 snRNA were designed. The expression of miR-122 in the serum samples of 27 cases of preoperative hepatocellular carcinoma (HCC), 15 cases of hepatitis B, 15 cases of hepatitis C, 15 cases of healthy control (HC), 11 cases of postoperative hepatocellular carcinoma (PHCC) and 10 cases of recurrence after postoperative hepatocellular carcinoma was detected by Taqman probe real-time fluorescence quantitative PCR. U6 snRNA was used as the internal control. RESULTS: The results showed that the method of Taqman probe real-time fluorescence quantitative PCR could detect the amplification signal of serum miR-122. The expression level of serum miR-122 in the patients with HCC, hepatitis B, hepatitis C and recurrence was higher than that in HC and the patients with PHCC. Meanwhile, the serum level of miR-122 in the patients with hepatitis C was higher than that in the patients with HCC, hepatitis B and recurrence. However, no difference of miR-122 expression level among HCC, hepatitis B and recurrent patients was observed. The miR-122 level was lower in PHCC patients than that in HCC and recurrent patients. In hepatitis B virus surface antigen (HBsAg) and/or hepatitis B virus e antigen (HBeAg) positive patients, the miR-122 level was higher than that in the negative ones. The miR-122 level in hepatitis C antibody (HCV-Ab) positive patients was raised compared with the negative ones. The serum level of alanine aminotransferase (ALT) was positively correlated with the serum level of miR-122 (r=0.34, P<0.05). The miR-122 expression level in the patients with serum AFP≥400 μg/L was higher than that in the patients with serum AFP<400 μg/L. CONCLUSION: The method of Taqman probe real-time fluorescence quantitative PCR can detect the serum level of miR-122 expression. Serum miR-122 might be used as a new biomarker of liver diseases, especially in the early diagnosis of primary hepatocellular carcinoma, the curative effect of surgical operation and the prognosis.     

Changes of miR-155-5p in serum of cervical cancer patients and effects of miR-155-5p on cervical cancer cells

AIM:To detect the serum level of miR-155-5p in the patients with different cervical diseases, and to analyze its effects on the proliferation, cell cycle and apoptosis of cervical cancer cells. METHODS:SYBR Green I real-time quantitative PCR was used to detect the level of miR-155-5p in the serum of the patients with different cervical diseases. miR-155-5p mimic or inhibitor was used to increase or decrease the expression of miR-155-5p in cervical cancer cells. The proliferation, cell cycle and apoptosis were measured by CCK-8 assay and flow cytometry. RESULTS:The serum level of miR-155-5p in cervical cancer group was higher than that in cervicitis group and healthy group. No statistical difference of the serum miR-155-5p level between cervical intraepithelial neoplasia group and cervical cancer group was observed. Compared with blank group, liposome group and negative control group, the proportion of S-phase cells increased and apoptotic cells decreased in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p mimic. The proportion of G2/M-phase cells increased significantly in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p inhibitor. CONCLUSION: Compared with healthy controls, the serum level of miR-155-5p in the cervical cancer patients increases, and may act as a novel tumor molecular marker for diagnosis of cervical cancer. miR-155-5p has no significant effect on the proliferation, cell cycle and apoptosis of HeLa cell. miR-155-5p may promote SiHa cells to enter S phase and inhibit the apoptosis of SiHa cells.     

转基因甜橙定量PCR分析体系的建立

以转pthA-nls基因甜橙45d叶龄叶片、枳橙的根、茎、叶不同器官以及非转基因枳、枸橼、温州蜜柑、沙田柚的45d叶龄叶片为试材,采用SYBR Green Ⅰ荧光染料法,检验不同柑橘类型以及枳橙不同器官中β-actin基因表达的稳定性,并以β-actin基因内参基因对pthA-nls庙基因在转pthA—nls基因甜橙中的表达量进行分析。结果表明：β-actin基因在柑橘的不同类型及同一类型的不同组织中表达均较恒定,可作为内参基因用于柑橘基因的定量PCR分析;同时,试验还应用建立的方法体系分析转dthA-nls基因甜橙,基本明确各转基因株系中目的基因表达水平。     

Effects of Yigu capsule on the level of cbf α-1 mRNA in bone of ovariectomized osteoporosis rats

AIM: To analyze the regulatory effect of Yigu capsule on core binding factor alpha 1 (cbf α-1) gene expression in bone of ovariectomized osteoporosis (OP) rats. METHODS: Thirty-six 10-month old Sprague-Dawley female rats were randomized into three groups: sham-operated group, model group and drug group. After intervention by the corresponding methods, the femurs were collected, SYBR green Ⅰ fluorescence quantitative PCR technique was applied with the internal control of GAPDH according to the relative quantitative formula (2-ΔΔCt), the differentially expressed multiples between the model group or drug group and sham-operated group were calculated. RESULTS: Quantitative formula analysis showed that the level of cbf α-1 gene expression in bone of model groups was decreased than that in sham-operated rats ［compared with sham-operated group, P<0.01, it was (9.9×105)-(1.6×104) times］. While in drug groups the level of cbf α-1 gene expression was 0.19 to 0.92 times than that in the sham-operated group, no significant difference was observed (P>0.05). CONCLUSION: The results of this study show that cbf α-1 gene expression in bone tissue of ovariectomized OP rat is decreased, and Yigu capsule increases the level of cbf α-1 gene expression in bone of OP, indicating that Yigu capsule induces bone marrow mesenchymal stem cells into osteoblasts.     

Ovarian carcinoma cells inhibit ζ chain expression and the secretion of Tc1/Tc2 type cytokines in CD8+ T cells

AIM: To investigate the effect of ovarian carcinoma cells on ζ chain expression and the secretion of Tc1/Tc2 type cytokine in CD8+ T cells, and its role in the ovarian carcinoma induced immunosuppression.METHODS: The supernatants of human ovarian carcinoma cell lines of OVCAR3, CAOV3 and SKOV3 and RPMI-1640 were added into CD8+ T cells (groups I, II, III, and control), which were isolated from the peripheral venous blood of healthy persons. The expression of ζ chain was analyzed by Western blotting. Thiazolyl blue(MTT) method was used to detect the effects of those cell line supernatants on the growth of CD8+ T cells. The secretion of the Tc1 type cytokine interferon (IFN)-γ mRNA and the Tc2 type cytokine interferon (IL)-10 mRNA were detected by RT-PCR. RESULTS: The expression of ζ chain was significantly lower in groups I, II, and III in comparison with that in control group. The absorbance at the wavelength 570 nm of CD8+ T cells culture in the group I, II, and III was all significantly lower than that in the control group. The IFN-γ expression was significantly lower in groups I, II, and III in comparison with that in control group, while the expression of IL-10 was significantly higher. CONCLUSION: Ovarian carcinoma may suppress CD8+ T cell proliferation and secretion of the Tc1/Tc2 type cytokine through inhibition of ζ chain, which may play an important role in the ovarian carcinoma induced immunosuppression.     

运用实时荧光RT-PCR技术检测柑橘碎叶病毒

柑橘碎叶病是由橘碎叶病毒(Citrus tatter leaf virus,CTLV)引起的一种重要的柑橘病害,为更快、更准确的检测CTLV,合成了一对特异性引物ASG-Pf和ASG-Pr,建立了运用SYBRGreenI荧光染料法检测CTLV的实时荧光RT-PCR体系,并对该体系的特异性、灵敏性和适用性进行了测试。结果表明,该检测体系能特异的检出CTLV,对测试的衰退病毒、温州蜜柑萎缩病毒和鳞皮病毒都不能检出;灵敏度比常规PCR高100倍;适用性广,可检测出多种柑橘类植物中的CTLV。实时荧光RT-PCR检测整个过程完全闭管,无需PCR后处理,且SYBR GreenI荧光染料成本较低,适用于检测柑橘体内含量较低的CTLV病毒。     

莲藕中甘薯潜隐病毒实时荧光定量PCR检测技术的建立及应用

甘薯潜隐病毒莲藕分离物（sweet potato latent virus-lotus,SPLV-lotus）在江苏省莲藕产区普遍引起发病,并且该分离物序列与已报道的SPLV甘薯分离物存在较大差异。为进一步监测SPLV-lotus在莲藕上的发生情况,针对SPLV-lotus的CP基因设计并优化特异性引物QSPLV-lotus3-F/QSPLV-lotus3-R,通过优化引物浓度和退火温度条件,构建标准曲线,建立了SPLV-lotus实时荧光定量PCR（Real-time fluorescent quantitative polymerase chain reaction,RT-qPCR）检测技术。该方法特异性强,可以快速检测出SPLV-lotus,灵敏度为普通PCR的100倍,可广泛应用于脱毒莲藕SPLV-lotus的精确检测。     

The inducement,restricted usage and clonal expansion of TCR Vβ subfamily T cells related to chronic myelogenous leukemia

AIM: To investigate the anti-leukemia effect, the restricted usage and clonal expansion of TCR Vβ subfamily T cells from donor peripheral blood induced by chronic myelogenous leukemia(CML) cells, K562 cells and bcr-abl peptide, respectively. METHODS: T cells in donor's peripheral blood were stimulated with CML cells, K562 cells and bcr3-abl2 peptide and amplified by MLTC, to induce the CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the restricted usage and clonal expansion of TCR Vβ subfamilies by using RT-PCR and genescan analysis, and the detection of specific cytotoxicity in CML by LDH release assay. RESULTS: 10-13 Vβ subfamilies were expressed in T cells from donor peripheral blood which were induced with CML cells, K562 cells and bcr-abl peptide in 1-2 weeks by MLTC. Oligoclonal T cell in Vβ16, Vβ21 and oligoclonal tendency T cells in Vβ5, Vβ13 subfamilies were identified in induced T cells, which have the ability of specific cytoxicity to CML cells and K562 cells. CONCLUSION: The anti-CML cytotocity T cells were induced by CML cells, K562 cells and bcr-abl peptide. These induced T cells with specific cytoxicity effect may come from the clonal expansion TCR Vβ subfamily T cells.     

葡萄蚕豆萎蔫病毒实时荧光定量RT-PCR检测方法及应用

建立了葡萄蚕豆萎蔫病毒（Grapevine fabavirus，GFabV）的实时荧光定量RT-PCR（RT-qPCR）检测技术。该技术标准曲线循环阈值与模板浓度呈良好的线性关系（扩增效率103.8%，相关系数0.998），灵敏度是常规RT-PCR的1 000倍，并具有特异性。采用RT-qPCR和常规RT-PCR方法对葡萄不同发育时期及不同部位的316个样品进行检测，结果表明RT-qPCR方法对GFabV的检出率（96.8%）明显高于RT-PCR（70.8%）。RT-qPCR对休眠枝条中GFabV的检出率达100%，对其他部位样品的检出率均在92%以上，明显高于RT-PCR（42% ~ 97%）。各个发育时期样品GFabV的RT-qPCR检出率（85% ~ 95%）也普遍高于RT-PCR（70% ~ 90%）。该技术对于田间样品的检测适用范围广。     

Changes in TCR Vβ subfamily nave T cells in peripheral blood of patients with multiple myeloma

AIM: To detect the existence of signal joint T-cell receptor excision DNA circles (sjTRECs) of 23 TCR Vβ subfamilies in mononuclear cells of patients with multiple myeloma (MM), and to evaluate the recent thymic emigrants of corresponding Vβ subfamily nave T cells in MM patients. METHODS: 23 TCR Vβ subfamily sjTRECs were amplified in genomic DNA from 5×104 PBMCs of 12 cases in MM patients by using semi-nest PCR.10 normal individuals served as controls. RESULTS: The number of detectable Vβ subfamily sjTRECs was 5.00±2.45 from MM patients, as compared with 9.60±5.48 from normal individuals, the difference was significant (P<0.05). The frequencies of Vβ2-, Vβ10-, Vβ16-, Vβ17-, and Vβ21-Dβ1 sjTRECs were significantly lower than those from normal individuals. 2-9 Vβ subfamily sjTRECs were detected from 12 cases of MM patients. It was negative correlation between age and the number of detectable Vβ subfamily sjTRECs in MM patients (r=-0.892; P<0.01). CONCLUSION: It has been found that some of 23 Vβ subfamily nave T cells are absent or lower level of recent thymic output function in MM patients, suggesting that MM patients have severe cellular immunodeficiency and the capacity and potential of long-term TCR Vβ repertoire reconstitution are dramatically lowered.     

Analysis of the distribution and clonality of TCR Vβ T cells in cord blood

AIM:To investigate the distribution and clonality of TCR Vβ subfamily T cells in cord blood. METHODS:The CDR3 of TCR Vβ 24 subfamily genes were amplified in mononuclear cells from 13 cases of cord blood. To observe the usage of TCR Vβ repertoire, the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Vβ T cells. Peripheral bloods from 10 cases of normal individuals and T cell line Molt-4 and Jurkat served as controls. RESULTS:Only 38.78%±16.26% of 24 Vβ subfamily T cell were selectively expressed in cord blood, predominantly in Vβ 3, 5, 8, 9 and 13, whereas all 24 Vβ subfamilies could be detected in T cells from peripheral blood of normal individuals. Genescan analysis showed that all PCR products of TCR Vβ subfamilies from cord blood or normal individual peripheral blood displayed multi-peaks. CONCLUSION:Some TCR Vβ subfamily T cells were absent in cord blood. All TCR Vβ subfamily T cells in cord blood displayed polyclonality.     

The feature of TCR Vα repertoire and clonal expansion of T cells from peripheral blood in patients with acute monoblastic leukemia

AIM: To investigate the distribution and clonal expansion of 29 T cell receptor (TCR) Vα subfamily T cells in patients with acute monoblastic leukemia (AML-M5)．METHODS: The CDR3 of TCR Vα 29 subfamily genes were amplified in peripheral blood mononuclear cells (PBMCs) from 8 cases with AML-M5 using RT-PCR，and the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size,to evaluate clonality of the detectable TCR Vα subfamily T cells.9 normal individuals served as control.RESULTS: Most Vα subfamily genes could be detected in PBMCs from normal individuals,whereas only 1-10 subfamily T cells were identified in 8 cases with AML-M5,the highest frequently expressed Vα subfamily was Vα3 (75%),and Vα12 was the second (62.5%),15 Vα subfamilies (Vα1,4,5,7,9,14-18,20,21,26,28 and Vα29) were absent.Genescan analysis showed that clonal expanded T cells were found in T cells from 6 out of 8 AML-M5 cases.The highest frequency of clonal expanded T cells predominated in Vα12 (3 out of 5 positive samples).In PBMCs from two cases,clonal expanded Vα3 T cells were the unique detectable Vα subfamily T cells.CONCLUSION: The selected usage and clonal expansion of TCR Vα subfamily T cells from peripheral blood could be found in patients with AML-M5.It may be a specific immune response which the host T cells are activated by the M5 leukemia cells.The distribution pattern of Vα subfamily clonal expansion displays individual specificity．     

Quantitative analysis of RNA levels from single hepatocytes in vivo: combined use of real-time RT-PCR and laser microdissection

AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed.To better understand cellular functions,the isolation of single cells and subsequent quantification of the expressed genes is essential.METHODS: Normal liver tissue was obtained from operation,snap-frozen in liquid nitrogen and sectioned in crystat.Individual hepatocytes were microdissected.RNA was extracted,then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR).RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide.In this way,cells were collected,RNA was extracted,reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR.The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells.CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR.These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.