以cDNA为内参标RT-PCR定量检测心肌细胞内p53和Bcl-2基因mRNA表达量的方法研究

目的 :探讨 p5 3和 Bcl 2基因在急性心肌梗死时心肌细胞中 m RNA表达量的检测方法。方法 :用以c DNA为内参标的逆转录聚合酶链反应 (RT PCR)方法检测 p5 3和 Bcl 2基因的 m RNA表达量。结果 :p5 3基因 m RNA表达量〔m RNA/总 RNA(ng/ g)〕依次为 :冠脉结扎后 4小时组 (5 76 4 8.78± 19776 .96 ) ng/ g>结扎后 6小时组 (339.0 6± 10 4 .11) ng/ g>结扎后 3小时组 (16 5 .4 4± 33.36 ) ng/ g>结扎后 2小时组 (88.2 5±16 .6 5 ) ng/ g>未结扎 (0 )组 (3.16± 0 .6 9) ng/ g和结扎后 1小时组 (16 .37± 2 .73) ng/ g、8小时组 (2 3.13±7.0 3) ng/ g、 12小时组 (6 .75± 2 .86 ) ng/ g,P均 <0 .0 5 ;Bcl 2基因 m RNA表达量〔m RNA/ g总 RNA(ng/ g)〕:冠脉结扎后 3小时组 (4.5 3± 1.5 9) ng/ g、 4小时组 (0 .0 2± 0 .0 1) ng/ g和 6小时组 (3.4 6±0 .39) ng/ g<结扎后 2小时组 (5 2 .4 8± 14 .18) ng/ g、8小时组 (5 9.2 4± 2 .91) ng/ g<结扎后 1小时组 (77.2 0±12 .4 8) ng/ g和未结扎 (0小时 )组 (81.77± 9.6 2 ) ng/ g<结扎后 12小时组 (99.6 0± 4 .71) ng/ g,P均 <0 .0 5。该种方法能对不同的样本检出不同量的 m RNA含量 ,其特异性强 ,且能检出 0 .0 2 ng/ g的 m RNA含量 ,敏感性高。

人类bcl-2cDNA在NIH/3T3细胞中的表达

将０．９１ｋｂ的含有完整开放阅读框的人类ｂｃｌ－２ｃＤＮＡ以正向克隆于逆转录病毒载体ｐＬＸＳＮ的ＥｃｏＲⅠ位点，经ＰＡ３１７细胞包装后感染ＮＩＨ／３Ｔ３细胞，筛选出Ｇ４１８抗性克隆，经免疫印迹证实人类ｂｃｌ－２ｃＤＮＡ在ＮＩＨ／３Ｔ３细胞中获得了表达。这一表达人类Ｂｃｌ－２蛋白的哺乳类细胞模型的建立为探讨ｂｃｌ－２的作用机制及其与其它基因间关系的研究奠定了基础。

蒙古绵羊Bcl-2基因3’端cDNA的克隆及序列分析

为扩增蒙古绵羊Bcl-2基因3’端,根据GenBank上已公布的牛的序列,设计了2条引物。从蒙古绵羊脾中提取总RNA,采用RT-PCR技术扩增出Bcl-2的cDNA,并重组到PMD18-T载体,经限制性内切酶谱分析和DNA序列测定,证实所克隆的cDNA为Bcl-2,因为该cDNA包含由261个碱基组成的开放读码框(ORF),该ORF编码83个氨基酸。经比对,与牛的核苷酸序列的同源性为95.8%。

人类bcl-2 cDNA在哺乳类细胞中的表达及反义bcl-2对U937细胞生物学行为的影响(摘要)

人类ｂｃｌ┐２ｃＤＮＡ在哺乳类细胞中的表达及反义ｂｃｌ┐２对Ｕ９３７细胞生物学行为的影响（摘要）何凤田１朱锡华１本文借助逆转录病毒载体将人类ｂｃｌ－２ｃＤＮＡ转染于三种哺乳类细胞，建立了组成性稳定表达ｂｃｌ－２的细胞模型，观察了模型细胞生长特性及对某...

蒙古绵羊Bcl-2基因cDNA的克隆及序列分析

为扩增蒙古绵羊bcl-2基因全序列,根据Gen Bank上已公布的牛的序列,设计了3条引物。从蒙古绵羊脾中提取总RNA,采用RT-PCR,技术扩增出bcl-2的c DNA,并重组到p Blueselect T载体,经限制性内切酶谱分析和DNA序列测定,证实所克隆的c DNA为bcl-2,因为该c DNA包含由687个碱基组成的开放读码框(ORF),该ORF编码229个氨基酸。经比对,与牛的核苷酸序列和氨基酸序列的同源性分别为95.5%和93.4%。

蒙古绵羊Bcl-2基因5′端cDNA的克隆及序列分析

为扩增蒙古绵羊bcl-2基因5′端,根据Gen Bank上已公布的牛的序列,设计了2条引物。从蒙古绵羊脾中提取总RNA,采用RT-PCR技术扩增出bcl-2的c DNA,并重组到p Blueselect T载体,经限制性内切酶谱分析和DNA序列测定,测出5′RACE产物434个核苷酸序列,该序列包括C端144个氨基酸编码序列、翻译起始密码子ATG。

Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a doseand timedependent manner. It did notaffect the cell cycle, but induced apoptosis. PE significantly decreased ΔΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a doseand time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

Therapeutic effects of human umbilical cord-derived mesenchymal stem cells against acute tubular necrosis quantified through measures of iNOS, BMP-7 and Bcl-2

Introduction: Acute tubular necrosis (ATN) is the most prevalent cause of acute renal failure (ARF). Mesenchymal stem cell transplantation has been studied as a potential treatment for renal dysfunction due to ATN. Inducible nitric oxide synthase (iNOS), bone morphogenetic protein-7 (BMP-7) and B-cell lymphoma 2 (Bcl-2) are surrogate markers of renal tubular epithelial regeneration and subsequent recovery of renal function following ATN. Methods: Serum creatinine (Scr) and blood urea nitrogen (BUN), as well as expression of iNOS, BMP-7 and Bcl-2 in gentamycin-induced ATN rat kidneys was investigated after human umbilical cord-derived mesenchymal stem cell (HUC-MSC) transplantation. Immunohistochemical staining was performed in 3 groups of rats: gentamycin-induced ATN treated with HUC-MSC, gentamycin-induced ATN without HUC-MSC, and untreated rats not receiving any treatments. Results: HUC-MSC transplantation led to a reduction in Scr and BUN in the kidneys of rats with gentamycin-induced ATN. Expression of iNOS in the HUC-MSC treated group occurred later and the expression levels were much lower during gentamycin-induced ATN compared to rats with ATN that were not treated with HUC-MSC. The expression of BMP-7 and Bcl-2 in the MSC-transplanted group was significantly increased compared to both control groups of rats with injured and healthy renal tubules. Conclusions: HUC-MSCs induce renal protection in a rat model of gentamycin-induced ATN, which is associated with reduced iNOS expression and up-regulation of Bcl-2 and BMP-7.

人白细胞介素2受体α链cDNA导入哺乳细胞及其克隆与稳定表达的研究

按DNA—磷酸钙共沉淀法将人白细胞介素2受体α链cDNA转染中国仓鼠卵巢细胞(CHOdhfr^-)及小鼠成纤维细胞(L929),获得稳定表达人IL-2Rα链的CHOdhfr^+细胞克隆。经RNA点渍杂交分析、荧光标记IL-2染色法、特异性ELISA和玫瑰花环试验检测证明,哺乳细胞表达的重组IL-2Rα链具有结合IL-2和抗Tac McAb的能力。还报道了T细胞白血病Jukat及Molt-4等细胞系异常表达IL-2R的结果,并作了分析讨论。

新生血管抑制分子IP-10/Crg-2的cDNA克隆及序列鉴定

目的： 拟获得编码ＩＰ１０ （ＩＦＮγｉｎｄｕｃｉｂｌｅ ｐｒｏｔｅｉｎ１０） 和Ｃｒｇ２ （ｃｙｔｏｋｉｎｅ ｒｅｓｐｏｎｓｉｖｅ ｇｅｎｅ２） 的ｃＤＮＡ 序列。方法： 针对ＩＰ１０ 和Ｃｒｇ２ 的ｃＤＮＡ序列设计相应引物， 通过反转录ＰＣＲ技术， 分别从用ＩＦＮγ及ＴＮＦα处理的人成纤维细胞及Ｂａｌｂ／ｃ 小鼠肝脏中， 扩增出编码ＩＰ１０ 和Ｃｒｇ２ 的全基因序列， 并将其克隆入载体ｐＵＣ１９及ｐＧＥＭ３Ｚｆ （＋ ） 中， 通过酶切及序列测定鉴定重组载体。结果： 经序列测定证实， 重组载体含有正确地分别编码ＩＰ１０ 及Ｃｒｇ２ 的ｃＤＮＡ序列。结论： 获得的阳性克隆分别含有３０６ｂｐ 和３１４ｂｐ 的重组片段， 为进一步对其生物学活性和配体进行研究奠定了基础。

人类bcl-2蛋白在CHOdhfr^-细胞中的表达

目的：建立表达人类bcl－2蛋白的哺乳类细胞模型，为探讨bcl－2的作用机制及其与其它基因间的关系奠定基础。方法：通过将910bp的含有完整开放阅读框的人类bcl－2cDNA以正向克隆于逆转录病毒载体pLXSN的EcoRI位点而构建重组真核表达载体pLXSN／s－bC12。利用电穿孔技术将pLXSN／s－bC12转染于CHOdhfr-细胞，筛选出G418抗性克隆，经免疫印迹检测人类bcl－2蛋白在CHOdhfr-细胞中的表达，观察在正常培养条件下，bcl～2蛋白的表达对该细胞的增殖与存活能力的影响。结果：①得到了人类bcl－2cDNA以正向插人的重组真核表达载体pLXSN／S－bC12。②免疫印迹表明转染有pLXSN／s-bc12的CHOd－hfr-细胞表达了人类bcl－2蛋白（相应细胞命名为CHO／s－bclZ）。③在正常培养条件下，CH（）／s－bcl2细胞的增殖与存活能力正常。结论：①表达人类bcl－2蛋白CHO／S－bc12细胞模型的建立，为深入研究奠定了基础。②在正常培养条件下，人类bcl－2蛋白的表达对CHOdhfr-心细胞的增殖与存活表型无明显影响。

人白细胞介素2受体(IL-2R)cDNA在哺乳类细胞中表达的初步研究

按DNA-磷酸钙共沉淀法将人白细胞介素2受体(IL-2R)cDNA转染小鼠成纤维细胞L929,经RNA点渍杂交分析、荧光标记IL-2染色和抗Tac(人IL-2受体α链)特异性玫瑰花环试验,均证明转入的IL-2R cDNA在L929细胞中表达,其产物具有结合IL-2和抗Tac抗体的能力。本文还报道了T细胞白血病Jukat细胞和Molt-4等细胞系异常表达IL-2R的结果,并对此作了分析和讨论。

Apoptosis of human colon carcinoma HT-29 cells induced by ceramide

AIM： To investigate the effect of exogenous ceramideinduced apoptosis on human colon carcinoma HT-29 cells. METHODS： Light microscope, transmission electron microscope and fluorescence microscope were used to observe the morphology change of apoptosis in HT-29 cells. Agarose gel electrophoresis was performed to detect the DNA fragment. Mitochondrial function was detected by MTT assay, mRNA expression of Bcl-2 family gene members was determined by reverse transcription polymerase chain reaction （RT-PCR） assay. RESULTS： After C2-ceramide treatment, typical characteristics of apoptosis, such as nuclear chromatin breakage, apoptotic body and DNA ladder, could be observed. After exposure to 50μmol/L C2-ceramide for 12 and 24 h, cell apoptosis was 64.1% and 81.3% respectively, which had a time-and dose-effect relationship. Mitochondrial function started to decrease from 6 h after exposure to ceramide. Meanwhile, ceramide up-regulated or down-regulated the mRNA expression of Bcl-2 family gene members. CONCLUSION： Ceramide induces apoptosis of human colon carcinoma HT-29 cells by affecting the expression of Bcl-2 family gene members and impacting the mitochondrial function.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the 'ladder pattern' revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

Objective： To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma MDA-MB-231 cells. Methods： Light microscopy was used to determine the effects of amlodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen （PCNA） and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results： Amlodipine concentration of 8.25umol/L （1/2 of ICs0） affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion： The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.

A NEW METHOD TO CONSTRUCT A FULL-LENGTH cDNA LIBRARY OF HUMAN NORMAL BLADDER TISSUE

Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switching activity of powerscript reverse transcriptase to synthesize and anchor first strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full length cDNA population with 1.0 g human normal bladder poly(A) + RNA, then double strand cDNA was synthesized. After digestion with sfiI and size fractionation by CHROMA SPIN 400 columns, double strand cDNA was ligated into λ TripIEx 2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×10 6 pfu·mL -1 , and the amplified cDNA library was 6×10 11 pfu·mL -1 , the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.

Experimental study of piperlongumine inducing apoptosis of human breast adenoma MDA-MB-231 cells

Objective： The aim of the study was to investigate the apoptosis induced by piperlongumine on human breast adenoma MDA-MB-231 cells and the mechanism involved. Methods： Human breast adenoma MDA-MB-231 cells line was cultured in vitro. The inhibitory effect of piperlongumine on the proliferation of human breast adenoma MDA-MB-231 cells was measured by CCK-8 assay. Distribution of cell cycle was analyzed by flow cytometry. The apoptosis rates of MDA-MB- 231 cells were measured using Annexin V/PI staining. The flow cytometry with the probe of DCFH-DA was used to detect the intracellular reactive oxygen species levels. Western blot was used to explore the protein expression of Bcl-2 and Bax. Results： The CCK-8 assay showed that piperlongumine had an inhibiting effect on the proliferation of MDA-MB-231 cells in a concentrationand time-dependent manner. MDA-MB-231 cells were markedly arrested at G0/G1 phase after treatment of piperlongumine. Piperlongumine induced apoptosis of MDA-MB-231 cells obviously. The level of intracellular reactive oxygen species was increased in a dose-dependent manner. The antioxidant N-acetyI-L-cystein inhibited the apoptosis of cells and the level of intracellular reactive oxygen species was also decreased. By Western blot analysis, we found the expression of Bax was up-regulated whereas that of Bcl-2 was down-regulated in a concentration-dependent manner. Conclusion： PiperIongumine possesses a significant function for inhibiting proliferation, arresting cells at G0/G1 phase and inducing apoptosis of MDA-MB-231 cells, which seems to be associated with the increased generation of intracellular reactive oxygen species as well as the down-regulation of Bcl-2 and up-regulation of Bax.

Expression of Bcl-2 and Survivin in uterine cervical carcinogenesis and correlation between the expression and infection of HPV_(16/18)

To investigate the expression of Bcl-2 and Survivin and HPV16/18 infection in uterine cervical carcinogenesis, formalin-fixed, paraffin-embedded specimens from cervical carcinomas, cervical intraepithelial neoplasia （C1N） and normal cervical tissues were studied. Using streptavidin-biotin peroxidase （S- P） immunohistochemical technique, the authors examined the expression of Bcl-2 and Survivin in these specimens. The number of apoptosis cells was assessed in situ by TdT-mediated dUTP-biotin end labeling （TUNEL） method. The infection of HPV type 16, 18 DNA were determined by PCR. It was found that there were significant differences in Bcl-2, Survivin and apoptotic index （AI） between cervical carcinomas, CIN and normal cervical tissues, respectively. Expression of Bcl-2 and AI were correlated with tu- mor grades, clinical stages and lymph node metastasis and expression of Survivin was associated with tu- mor grades and lymph node metastasis. There were different positive rate of HPV^s between cervical car- cinomas, C1N and normal tissues and were not associated with tumor grades, clinical stages and lymph node metastasis. The infection of HPV16/18 was associated with the expression of Bcl-2, Survivin and AI, respectively. It was concluded that the abnormal expression of Bcl-2, Survivin and infection of HPV16/18 were associated with cervical carcinomas. They possibly can be useful indexes for the primary screening and prognosis of cervical carcinomas.