山楂过氧化物酶基因的克隆及在烟草中异位表达分析

【目的】从山楂(Crataegus pinnatifida)中克隆过氧化物酶(POD)编码基因,通过转基因验证其在木质素合成中的作用,为揭示山楂内果皮形成分子机制奠定基础。【方法】利用RT-PCR技术从山楂克隆基因,以p RI 101-AN为构建基因过量表达载体的基础载体,通过农杆菌介导的遗传转化方法将目的基因导入烟草。【结果】从山楂中克隆出编码序列长度为996 bp的POD72基因,命名为Cp POD72。山楂POD72与木本和草本植物POD72的氨基酸序列一致性均较高。构建了Cp POD72基因的过量表达载体,通过农杆菌介导的转化获得了66个烟草转化植株。RT-PCR检测结果显示Cp POD72基因在烟草转基因植物中异位表达,组织化学染色分析表明转Cp POD72基因烟草植株中木质素含量增加。【结论】Cp POD72基因可能参与木质素合成。     

Methylation and mRNA expression of TIG1 gene in esophageal squamous-cell carcinoma tissues

AIM: To investigate the expression and promoter methylation of tazarotene-induced gene-1 (TIG1) in esophageal squamous-cell carcinoma (ESCC) tissues. METHODS: The methods of methylation-specific PCR and real-time fluorescence quantitative PCR were applied to examine the methylation and mRNA expression of TIG1, respectively, in 43 cases of ESCC tissues, 20 cases of paracancerous tissues and 15 cases of normal tissues. RESULTS: The frequency of promoter methylation of TIG1 gene in ESCC tissues was 25.6% (11/43), which was significantly higher than that in the paracancerous tissues (5.0%, 1/20) and normal tissues (0/20). The hypermethylation of TIG1 gene in these tissues had no correlation with sex, age and clinical stage of the patients. However, it was correlated with the pathological stage (P<0.01) and lymph node metastasis (P<0.05). The mRNA expression of TIG1 in ESCC tissues was significantly lower than that in paracancerous tissues (P<0.05) and normal tissues (P<0.01). However, the expression level of TIG1 mRNA in methylated tissues was significantly lower than that in unmethylated tissues (P<0.01). CONCLUSION: Promoter methylation may be an important mechanism of TIG1 gene inactivation in ESCC, which was related to lymph node metastasis and TNM stage of esophageal carcinoma.     

三明野生蕉Ran基因克隆及其组织特异性与低温胁迫表达分析

【目的】探讨三明野生蕉Ran基因在低温胁迫响应过程中的作用。【方法】通过RT-PCR与RACE相结合的方法和q RT-PCR克隆Ran基因,并对其在响应低温胁迫和水杨酸处理过程中的表达进行分析。【结果】分离得到6条含有完整开放阅读框的Ran基因c DNA序列,编码的蛋白质与其他植物Ran蛋白高度同源,带有GTP水解结构域、Ran GAP结合结构域和酸性尾巴等典型结构域。q PCR分析结果表明,三明野生蕉Ran基因在根、叶片、花序轴、苞片、花、果皮和果肉中均有表达,在根中表达量最低,在叶片、花和果肉中的表达量较高。此外,低温胁迫和水杨酸处理可显著影响三明野生蕉Ran基因的表达。【结论】三明野生蕉Ran基因与细胞分裂、低温胁迫响应和水杨酸信号转导有关。     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.     

甜瓜ACC氧化酶反义基因植物表达载体的构建及对烟草的转化

应用反义基因技术抑制水果成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决水果延熟保鲜难题的可行新方法。一个来自甜瓜的ACC氧化酶cDNA全序列被反向插入到植物表达载体pBI121中,从而构建了甜瓜ACC氧化酶反义基因植物表达载体。用该反义基因载体转化烟草获得转基因植株,并且转基因在转化植株的自交子代中发生孟德尔分离,由此验证了该载体构建的成功和可用性。此项研究为下一阶段用该反义基因载体转化甜瓜栽培品种做了充分必要准备。     

The effect of postconditioning on apoptosis-related gene expression of lung tissues in rat intestinal ischemia-reperfusion

AIM: To investigate the effect of postconditioning on apoptosis-related gene expression of lung tissues in rat intestinal ischemia-reperfusion(I/R). METHODS: Male Sprague-Dawley rats were divided into 3 groups which were sham operation group (sham), I/R group and ischemic postconditioning (IPOST) group. The experimental animals were sacrificed after reperfusion for 6 h. The tissue sections were stained with hematoxylin and eosin and examined under light microscope to observe lung histopathological changes. Lung W/D ratios were measured. Lung tissue samples were taken to detect Bcl-2, Bax and caspase-3 mRNA and protein expression using RT-PCR and Western blotting analysis, respectively. RESULTS: Compared with I/R group, the morphological changes of lung were alleviated and the lung W/D ratios significantly decreased in IPOST group (P<0.05). The mRNA and protein expression of Bcl-2 in IPOST group was obviously higher than that in I/R group (P<0.05), while the mRNA and protein expression of Bax and caspase-3 in IPOST group was obviously higher than that in I/R group (P<0.05). CONCLUSION: Ischemic postconditioning attenuated intestinal ischemia-reperfusion-induced acute lung injury partly by increasing Bcl-2 mRNA and protein expression, reducing Bax mRNA and protein expression.     

Study on expression of long non-coding RNA in colon cancer tissues and adjacent tissues

AIM: To screen the differentially expressed long non-coding RNA (lncRNA) in colon cancer, and to explore its expression in colon cancer tissues and adjacent tissues. METHODS: The "Colon adenocarcinoma:Person neoplasm cancer status" which consisted of 36 cases of colon cancer tissues and 29 cases of normal colonic tissues was downloaded from the lncRNAtor database. The candidate genes were selected from these differentially expressed lncRNAs based on artificial criterion (P<0.01; fold change ≥ 2 or<0.5) and then validated by real-time PCR in 60 pairs of colon cancer tissues and adjacent tissues. RESULTS: A total of 50 lncRNAs were differentially expressed in colon cancer tissues, including 28 up-regulated and 22 down-regulated (P<0.01). The verifying results displayed that HNF1A-AS1 and ZDHHC8P1 were up-regulated (P<0.01), and SUZ12P expression was down-regulated (P<0.05), but the expression of AC069513.3 was not statistically significant between colon cancer tissues and adjacent tissues. The abilities of HNF1A-AS1, ZDHHC8P1, SUZ12P and AC069513.3 to discriminate the colon cancer from normal adjacent tissue by the ROC curve with an AUC of 0.729 (sensitivity 78%, specificity 67%), 0.617 (sensitivity 68%, specificity 55%), 0.689 (sensitivity 66%, specificity 55%) and 0.518 (sensitivity 52%, specificity 48%) were observed. CONCLUSION: Long non-coding RNA HNF1A-AS1 and ZDHHC8P1 are up-regulated and SUZ12P is down-regulated in colon cancer tissues, suggesting that they may be involved in the pathogenesis of colon cancer.     

Distribution and activity of calcineurin in different tissues of rat

AIM: To investigate the activity and distribution of calcineurin (CaN) in different tissues of rat. METHODS: Using western blot and immunohistochemical staining methods to measure the amount and location of CnAα, isoform of catalytic subunit of CaN in different tissues of rat. CaN activity was measured by [³²P] labelled substrate peptide. RESULTS: 1. Western blot showed that CnAα expression was detected in brain, heart, skeletal muscle and lung tissues. There was no detectable CnAα expression in kidney and aorta. 2. In immunohistochemical staining study, there was strong immunostaining of CnAα in brain. CnAα was located in cytoplasm of cardiac cell, macrophage and connective tissue of peribronchiolar in lung tissue, aorta adventitia, connective tissue around small vessels and outer wall of renal tube. 3. CaN activity was highest in brain, the following was skeletal muscle, myocardium and lung tissue. CaN activity was lowest in aorta and kidney tissue. CONCLUSION: CaN is widely distributed in rats and might be involved in functional regulation of various organs and tissues.     

Comparison of iNOS mRNA expression in colorectal carcinoma cell strains with different proliferation protentials

AIM: To evaluate the expression of iNOS mRNA in different invasion ability colon carcinoma cell strains. METHODS: MTT was used to detect the growth and reproduction of colon cancer cell strain CW-2 and LS174T. RT-PCR and Northern blot were used to detect expression of iNOS mRNA in colon cancer. RESULTS: MTT growth curve displayed that colon cancer cell strain LS174T grew and reproduced faster than cell strain CW-2. RT-PCR showed that iNOS mRNA expressed strongly in CW-2 cell strain, while iNOS mRNA expressed weakly in LS174T cell strain. Northern blot detected that iNOS mRNA expressed obviously in CW-2 cell strain, but cell strain LS174T have no obvious iNOS mRNA expression. All-trans retinoic acid (ATRA) had no obvious effect on iNOS mRNA expression in CW-2 cell strain of colon cancer. CONCLUSIONS: ATRA has no obvious effect on iNOS mRNA expression. iNOS has a dual effect on tumor growth. In low-metastatic colon carcinoma CW-2, iNOS may exert it's anti-tumor influence by cytotoxicity or inducing cell apoptosis. In high-metastatic colon cancer LS174T, iNOS produced low concentration of NO, which may be an important signal-transduction molecule for increasing blood supply and angiogenesis, which improve the growth, invasion and metastasis of tumor.     

几种果实不同组织总RNA提取及质量分析

以菊水梨为试验材料,通过2种改良CTAB法对果实不同成熟阶段提取的总RNA质量和产量的变化的影响,研究果实不同成熟阶段果皮和果肉总RNA提取的质量和产量,并针对梨果实不同成熟阶段采用不同的改良CTAB法,以较低的成本从果实中提取完整性好、质量高的总RNA。同时提取近成熟的红富士葡萄、富士苹果和丰香草莓3种果实总RNA。总RNA的质量和产量分析结果表明,方法Ⅰ和Ⅱ分别适用于成熟度较低和较高梨果实总RNA的提取,方法Ⅰ也适用于其它3种近成熟果实总RNA提取。并通过RT-PCR验证,所提取的总RNA可以满足基因克隆和表达等分子生物学实验的要求。     

Isolation and characterization of a new d-limonene synthase gene with a different expression pattern in Citrus unshiu Marc

We isolated a new d-limonene synthase gene (CitMTSE2) from Satsuma mandarin (Citrus unshiu Marc.) and compared its mRNA expression pattern with that of the previously isolated CitMTSE1. The predicted protein of CitMTSE2 showed 84.2% identity to that of CitMTSE1 at the amino acid level and possessed the typical structure and chemical features of general monoterpene synthases, such as transit peptides in the N-terminus, sequence motifs of RR and DDXXD, and cation-dependent reactions. A functional test demonstrated that CitMTSE2 is a d-limonene synthase gene.Under different expression regulations in response to the fruit developmental stage, the mRNA expression of CitMTSE2 indicated a specificity to fruit tissue and was different from that of CitMTSE1. Occurring mainly in peel at an early stage of fruit development, the mRNA expression profile of CitMTSE2 was similar to that of d-limonene biosynthesis in C. unshiu.     

Effect of IL-8 on mitochondrial fusion and fission gene expression in human lung adenocarcinoma cell line A549 during cell migration

AIM: To investigate the changes of migration of lung adenocarcinoma cells promoted by IL-8 and the inner and outer mitochondrial membrane dynamic changes during this process.METHODS: Human lung adenocarcinoma cell line A549 was divided into control group and IL-8 group. Cell migration was analyzed by scratch detection and Transwell assay. The secretion of endogenous IL-8 was detected by ELISA. The protein levels of mitochondrial cytochrome C (Cyt C) and mitochondrial outer membrane protein Tom20 was detected by Western blot. The mRNA expression of mitochondrial fusion genes Mfn1, Mfn2 and OPA1 and fission genes Fis1, Drp1 and MTP18 was detected by RT-PCR. The morphological changes of mitochondria were observed by MitoTracker Red CMXRos dye staining and confocal microscopy.RESULTS: The migratory rate of A549 cells and endogenous secretion of IL-8 in A549 cells were higher than those in SPC-A-1 cells. The migratory rate of A549 cells was improved by IL-8 in a time-dependent manner. Compared with control group, the Tom20 protein expression was increased (P<0.05), and the Cyt C protein expression was decreased (P<0.05). The expression of mitochondrial outer membrane fusion genes Mfn1 and Mfn2 was increased (P<0.05), and the expression of mitochondrial inner membrane fusion gene OPA1 was decreased (P<0.05). The expression of fission genes Drp1 and MTP18 were decreased (P<0.05), while the expression of Fis1 was no change (P>0.05). Under confocal microscope, the punctate aggregates in the mitochondria of the A549 cells treated with IL-8 were observed.CONCLUSION: The migratory rate of A549 cells is increased by IL-8, which is related to the changes of mitochondrial fusion genes and the fission genes.     

Relationship between STEAP expression and hydrogen peroxide in human prostate tissues

AIM:To observe the STEAP expression and its function in human prostate tissues.METHODS:The expressions of STEAP mRNA and protein were detected by RT-PCR and Western blotting. H₂O₂ and SOD levels were detected by molybdic acid reduction and xanthine oxidation methods respectively.RESULTS:STEAP mRNA and protein were highly expressed in prostate cancer tissues. H₂O₂ and SOD levels in prostate cancer were obviously higher than those in normal prostate tissue.CONCLUSION:The function of STEAP gene is possibly to induce intracellular H₂O₂ and promote cell growth.     

Cross-species comparison of Smad3 expression in hepatocellular carcinoma tissues

AIM： To study the expression of Smad3 and its significance in hepatocellular carcinoma (HCC) in different species including human, rat and tree shrew, and to verify the feasibility of cross-species oncogenomic approach. METHODS：Real-time fluorescent quantitative polymerase chain reaction and Western blotting were applied to detect the expression of Smad3 at mRNA and protein levels in HCC tissues, corresponding HCC adjacent liver tissues and normal liver tissues collected from different species including human, rat and tree shrew. RESULTS：The mRNA expression of Smad3 in HCC tissues of human, rat and tree shrew was lower than those in the corresponding HCC tissues adjacent liver tissues. The mRNA level of Smad3 in HCC tissues of rat was lower than those in its normal liver tissues, while that in HCC adjacent tissues of tree shrew appeared higher than those in the normal liver tissues. No significant difference of Smad3 expression in other tissues was observed. The protein levels of Smad3 in HCC of human and rat were lower than those in the corresponding HCC adjacent liver tissues and the normal liver tissues. However, the protein expression of Smad3 was at a low level in HCC tissues of tree shrew and was lower than that in the HCC adjacent liver tissues and the normal liver tissues, although without statistical difference. The differences of Smad3 expression between HCC adjacent liver tissues and normal liver tissues in all the 3 species were not statistically significant. CONCLUSION：Smad3 is lowly expressed in HCC tissues of different species, suggesting that it might play a pivotal role in hepatocarcinogenesis and be applied as a key molecular target in HCC prevention and treatment.     

葡萄ZTL、COP1基因的鉴定及其在不同光质和转光条件下的表达分析

【目的】在葡萄试管苗离体培养下,研究不同波长对基因ZTL、COP1表达的影响。【方法】以‘红地球’葡萄(Vitis vinifera‘Red Globe’)试管苗为材料,经RT-PCR法克隆获得葡萄ZTL、COP1基因全长cDNA,并利用生物信息学分析了以上基因的理化性质、蛋白质二级结构、亚细胞定位以及系统进化树。利用q RT-PCR技术分析了以上基因在不同光质处理下表达量的变化情况。利用荧光参数系统分析不同光质对葡萄试管苗生长的影响。【结果】ZTL的分子式为C1972H3107N553O576S20,是亲水性稳定蛋白,脂溶性较差;COP1的分子式为C832H1291N209O225S9,是疏水性稳定蛋白,脂溶性较好。ZTL和COP1的二级结构均由α-螺旋、β-转角、无规则卷曲和延伸链结构组成;亚细胞定位分析结果显示,ZTL、COP1均位于细胞质中;系统进化分析表明,ZTL与桃的亲缘关系最近,COP1与其他物种的亲缘关系均较远,单独聚为一个亚族。在不同光质处理后ZTL均为上调表达,其中白光转蓝光、红光、红光转白光、红光转蓝光和蓝光转红光处理后分别上调达11.2、22.3、12.0、32.9和19.6倍。不同光质处理后COP1基因的表达呈现不同的变化趋势:红光转蓝光处理后,COP1上调1.97倍,红光处理后表达量无显著变化,其他光质处理后均为下调表达,其中蓝光转白光和蓝光转红光处理后下调达18倍。荧光参数分析表明,qP、qN、NPQ和F_v/F_m在转光培养中表达活跃,均在红光转蓝光的转光培养下表达量最高。【结论】ZTL在葡萄转光培养中表达活跃,均呈显著上调趋势,而COP1总体表现为下调趋势。     

Expression of melusin in myocardial tissues with different degrees of coronary atherosclerosis

AIM: To observe the pathological changes of myocardial tissues with different degrees of coronary atherosclerotic lesions, and to detect the level of melusin in myocardial tissue for exploring the role of melusin in the deve-lopment of coronary heart disease. METHODS: The cases of coronary heart disease were confirmed by autopsy and histopathological examination, and were classified into 3 groups: coronary atherosclerotic stable plaques (B group); atherosclerotic unstable plaques without thrombosis, plaque rupture, plaque hemorrhage and other secondary changes (C group); atherosclerotic unstable plaques with any of the secondary changes mentioned above (D group). The control group (A group) had no lesion of coronary artery. The coronary and myocardial tissues were collected separately, and routine HE staining of paraffin sections was performed to observe the histological structure of myocardium. The expression of melusin was detected by immunohistochemical staining, Western blot and real-time PCR. Logistic regression analysis was used to analyze the relationship between melusin expression changes and the development of coronary heart disease. RESULTS: Compared with A group, human cardiac myocytes showed pathological changes such as hypertrophy, atrophy, necrosis and adipose tissue infiltration in the other 3 groups. The expression of melusin in C and D groups was significantly lower than that in control group, while the expression of melusin was increased in B group (P<0.05). The expression of melusin in myocardial tissues was negatively correlated with the development of coronary heart disease (OR=0.3; 95% CI: 0.2~0.4; P<0.01). CONCLUSION: Melusin in human myocardial tissue may be a protective factor of heart in coronary heart disease. It plays an important role in the development of coronary heart disease.     

Alteration of AQP2 expression in kidney tissues of emphysema model rats

AIM: To observe the expressions of aquaporin 2 (AQP2) in kidney tissues and the contents of endotoxin (ET), interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α) in serum in emphysema model rats, and to investigate the relationship between lungs and kidney in humoral metabolism. METHODS: The rats of emphysema were treated by injecting lipopolysaccharide into the trachea with cigarette smoking. Immunohistochemistry and Western blotting analysis were used to observe the expression of AQP2 in kidney tissues. RT-PCR was applied to detect the expression of AQP2 mRNA in kidney tissues. Blood sample and lung tissue were taken and the levels of ET, IL-1β and TNF-α were measured by radioimmunoassay. RESULTS: AQP2 expression in the kidney tissue in model group was greater than that in control group, and the expression of AQP2 mRNA showed the same results (P<0.01). ET, IL-1β and TNF-α levels in serum and lung tissue in model group were markedly higher than those in control group (P<0.01). CONCLUSION: In the emphysema model rats, AQP2 expression is up-regulated in the kidney tissue. The mechanism of emphysema may be related to increasing the levels of ET, IL-1β and TNF-α in the serum and lung tissue obviously.